CN110117272B

CN110117272B - Salts of cyclin dependent kinase inhibitors and crystalline forms thereof

Info

Publication number: CN110117272B
Application number: CN201910106868.2A
Authority: CN
Inventors: 李宗斌; 呙临松
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2018-02-05
Filing date: 2019-02-02
Publication date: 2021-12-10
Anticipated expiration: 2039-02-02
Also published as: CN110117272A

Abstract

The present invention relates to salts of cyclin-dependent kinase inhibitors and crystalline forms thereof. The invention relates to (4- (cyclopropylamino) piperidin-1-yl) (6- ((5-fluoro-4- (1-isopropyl-2-methyl-1H-benzo [ d) having a structure of general formula (I)]Methods for the preparation and use of imidazol-6-yl) pyrimidin-2-yl) amino) -2-methylpyridin-3-yl) methanone and salts thereof and pharmaceutical compositions containing a therapeutically effective amount of the salts. CDK4/6 highly selective inhibitor is clinically useful for treating HR +/HER 2-breast cancer patients. In addition, the compound can also be used for treating non-small cell lung cancer, head and neck cancer, brain tumor, melanoma and the like, wherein each substituent in the general formula (I) is defined as the specification.

Description

Salts of cyclin dependent kinase inhibitors and crystalline forms thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to a preparation method and application of (4- (cyclopropylamino) piperidine-1-yl) (6- ((5-fluoro-4- (1-isopropyl-2-methyl-1H-benzo [ d ] imidazole-6-yl) pyrimidine-2-yl) amino) -2-methylpyridin-3-yl) methanone and salts thereof, which are used as CDK4/6 high-selectivity inhibitors and used for treating diseases including cancers, myelodysplastic syndromes, Alzheimer's disease, autoimmune diseases, depression, anxiety, cataract, psychological disorders and AIDS.

Background

Cyclin-dependent kinases (CDKs) are a class of serine (Ser)/threonine (Thr) kinases, a family of 13 members, each classified as a-L for cyclins. Different CDKs and cyclins (cyclins) form a CDK-cyclin complex, different substrates are catalyzed to be phosphorylated through CDK kinase activity, DNA synthesis is started, and propulsion and conversion of different phases of a cell cycle are realized; regulating gene transcription, and participating in cell growth, proliferation, dormancy or apoptosis. Thus, CDKs have important functions in the regulation of proliferation and death of all cells, including tumor cells and normal cells. Among them, CDK4/6-Cyclin D complex plays an important role in the transformation of cells from G1 phase to S phase. After CDK4/6 binds to cyclin D, a series of substrates including Retinoblastoma protein (Rb) are phosphorylated in stage G1. Rb is phosphorylated to release proteins which are combined with Rb and inhibited by Rb, mainly transcription factors E2F and the like, and E2F activates and transcribes a plurality of genes which are necessary for entering an S phase, thereby promoting the transformation of G1/S of cells. The specific activation of CDK4/6 is closely related to the proliferation of some tumors, and the abnormal ubiquitous presence of cyclinD-CDK 4/6-INK 4-Rb pathway. The expression is as follows: (1) p16INK4a gene deletion, point mutation, or DNA methylation results in p16INK4a inactivation; (2) CDK4 gene amplification or point mutation (R24C), loss of binding ability to p16INK4 a; (3) cyclinD1 was overexpressed due to gene rearrangement or gene amplification. The change of the pathway accelerates the G1 phase process, so that the tumor cell proliferation is accelerated to obtain the survival advantage. Therefore, intervention thereof becomes a therapeutic strategy, and CDK4/6 therefore becomes one of the antitumor targets. Palbociclib from pyroxene (PD0332991) is the first FDA-approved CDK4/6 small molecule inhibitor on the market for the treatment of breast cancer. In addition, several pharmaceutical companies, including Norwalk (LEE011), Li-lai (LY2835219), etc., have compounds in clinical studies and have shown good therapeutic results. Besides breast cancer, research shows that the selective CDK4/6 inhibitor has good antitumor activity in various tumors such as ovarian cancer, non-small cell lung cancer, B cell lymphoma, liver cancer, glioma, colon cancer, multiple myeloma and the like. Therefore, the development of new small molecule inhibitors of CDK4/6, which are new effective methods for treating these tumors, motivates the continuous efforts of scientists.

Disclosure of Invention

The invention relates to a compound shown in a general formula (I), a stereoisomer thereof or a mixture form thereof,

wherein:

m is an inorganic or organic acid, wherein the inorganic acid is selected from the group consisting of hydrochloric acid, sulfuric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, formic acid, fumaric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, Glutaric acid, 2-oxoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, and L-malic acid; and is

x is selected from 1,2, 3 or 4.

In a preferred embodiment of the present invention, the compound of formula (I) may form a hydrate form as shown in formula (II),

wherein:

y is 0 to 3, preferably 0, 1,2 or 3;

x is selected from 1,2, 3 or 4.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), or a stereoisomer, a hydrate or a mixture thereof is preferably methanesulfonic acid, sulfuric acid, phosphoric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid or L-malic acid; most preferred is p-toluenesulfonic acid.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), or a stereoisomer, a hydrate or a mixture thereof, is characterized in that: x is 1.

In a preferred embodiment of the present invention, the compound of formula (I), a stereoisomer thereof or a mixture thereof, in free base crystalline form, has an XRPD exhibiting peaks at diffraction angles 2 θ (± 0.2 °) of 4.2, 13.8, 15.9, 17.0 and 18.1 ± 0.2 °; further preferred is an XRPD showing peaks at diffraction angles 2 θ (+ -0.2 °) of 8.7, 9.6, 12.4, 19.7, 24.6 and 25.8 + -0.2 ℃ with a melting peak at 193.4 + -0.5 ℃ on DSC.

In a preferred embodiment of the present invention, when M is p-toluenesulfonic acid, i.e. p-toluenesulfonate, the compound of formula (I), its stereoisomer or its mixture is characterized in that: XRPD having diffraction angles 2 θ (± 0.2 °) exhibiting peaks at 7.5, 9.7, 10.7, 11.1, 15.3, 16.2, 17.8, 20.5, 20.9, 21.5, 22.7, 25.9 and 27.9 ± 0.2 °; further preferred is XRPD showing peaks at diffraction angles 2 theta (+ -0.2 DEG) of 12.1, 12.8, 13.6, 14.0, 16.8, 24.0, 26.8, 29.3, 31.5, 33.6, 39.4 + -0.2 DEG,

form II is characterized by: XRPD having diffraction angles 2 θ (± 0.2 °) at 7.4, 9.6, 11.4, 12.6, 14.5, 17.1, 17.7, 19.7, 21.9, 22.8 and 27.5 ± 0.2 ° showing peaks; further preferred is XRPD showing peaks at diffraction angles 2 theta (+ -0.2 DEG) of 5.7, 12.1, 13.6, 20.9, 24.1, 24.8, 28.7, 30.3, 32.4, 34.6, 38.2 + -0.2 DEG,

form III is characterized by: XRPD having peaks at diffraction angles 2 θ (± 0.2 °) of 7.1, 7.8, 11.4, 12.1, 12.8, 13.3, 15.1, 18.2, 20.6, 21.5, 21.8 and 23.5 ± 0.2 °, further preferably XRPD having peaks at diffraction angles 2 θ (± 0.2 °) of 9.0, 9.4, 13.9, 14.1, 14.4, 15.8, 16.9, 17.2, 17.4, 19.2, 19.4, 21.2, 22.0, 25.7, 26.1, 26.7, 27.1, 27.3, 27.5, 28.1, 28.8, 29.0, 29.2, 31.7, 33.1, 35.5, 37.3 and 39.3 ± 0.2 °, XRPD having peaks at diffraction angles 2 θ (± 0.2 °) of 185.8 ± 0.5 on DSC, and a crystalline form III monohydrate of methyl benzene at 185.8 ± 0 ℃.

The invention also relates to a method for preparing the compound shown in the general formula (I), the stereoisomer, the hydrate or the mixture form thereof, which comprises the following steps:

1) preparation of stock solution: taking free alkali of a compound with a general formula, adding an organic solvent for dissolving to obtain a clear stock solution, wherein the concentration of the solution is preferably 50 mg/mL;

2) preparation of a counter-ion acid solution: adding a counter-ion acid into an organic solvent or water to obtain a clarified counter-ion acid solution; the organic solvent is preferably ethanol, and the concentration is preferably 0.25-2 mol/L;

3) preparation of compound salt: adding a counter-ion acid solution into the stock solution to obtain a clear salt solution, naturally volatilizing, and drying in vacuum to obtain a salt of the compound shown in the general formula (I); the vacuum temperature is preferably 40 ℃, and the amount of the counter ion acid is preferably 1-1.2 equivalent;

wherein:

the organic solvent is selected from methanol, ethanol, ethyl acetate, dichloromethane, acetone, N-hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tert-butyl ether, isopropyl ether, 1, 4-dioxane, tert-butyl alcohol or N, N-dimethylformamide; preferably 88% acetone, methanol or tetrahydrofuran;

the counter-ionic acid is selected from hydrochloric acid, sulfuric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid, and the organic acid is selected from 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, formic acid, fumaric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, glutaric acid, 2-oxoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, and L-malic acid; preferably methanesulfonic acid, sulfuric acid, phosphoric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid or L-malic acid; most preferred is p-toluenesulfonic acid.

The invention also relates to a preparation scheme, a method for preparing the compound shown in the general formula (I), the stereoisomer, the hydrate or the mixture form thereof, which comprises the following steps:

1) weighing a proper amount of free alkali, and dissolving the free alkali by using a benign solvent; the benign solvent is preferably dichloromethane, methanol or tetrahydrofuran;

2) weighing a proper amount of counter ion acid, and dissolving the counter ion acid by using an organic solvent; the organic solvent is preferably ethanol; the amount of the counter-ionic acid is preferably 1.2 equivalents;

3) mixing the two solutions, stirring for several hours, dripping a poor solvent until turbidity appears, and stirring overnight; the poor solvent is preferably isopropyl ether;

4) quickly centrifuging or standing and blow-drying to obtain a salt of the compound shown in the general formula (I);

wherein:

the benign solvent is selected from acetone, 2-butanone, tetrahydrofuran, 1, 4-dioxane, 3-pentanone, 2-methyltetrahydrofuran, ethyl acetate or acetonitrile; preferably 88% acetone, methanol or tetrahydrofuran.

The organic solvent is selected from methanol, ethanol, ethyl acetate, dichloromethane, acetone, N-hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tert-butyl ether, isopropyl ether, 1, 4-dioxane, tert-butyl alcohol or N, N-dimethylformamide; preferably 88% acetone, methanol or tetrahydrofuran; the benign solvent and the organic solution need to be mutually soluble when in use;

the poor solvent is selected from heptane, dichloromethane, water, methyl tert-butyl ether, toluene, isopropyl ether or ethanol; preferably tert-butyl ether and isopropyl ether;

The invention also relates to a preparation scheme, a method for preparing the compound shown in the general formula (I), the stereoisomer, the hydrate or the mixture thereof, wherein the solvent is ethyl acetate or ethanol.

The invention also relates to a method for preparing the compound shown in the general formula (I), the stereoisomer, the hydrate or the mixture thereof, wherein the counter ion acid is benzene sulfonic acid and p-methyl benzene sulfonic acid.

The invention also relates to a pharmaceutical composition which contains a therapeutically effective amount of the compound shown in the general formula (I), the stereoisomer, the hydrate or the mixture form thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

In a preferred embodiment of the present invention, the compound of formula (I), its stereoisomers, hydrates or mixtures thereof, or the pharmaceutical composition is used for the preparation of a medicament for the treatment and/or prevention of cancer or tumor-related diseases mediated by CDK4 and/or 6.

In a preferred embodiment of the invention, the compound of the general formula (I), its stereoisomers, hydrates or mixtures thereof or the composition is used for the production of a medicament for the prophylaxis and/or treatment of diseases which are characterized by HR +/HER 2-related diseases.

The use as described above, wherein said disease is selected from the group consisting of cancer, myelodysplastic syndrome, alzheimer's disease, autoimmune diseases, depression, anxiety, cataracts, psychological disorders and aids; the cancer is preferably selected from breast cancer, cervical cancer, colon cancer, lung cancer, stomach cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, glioma, glioblastomas, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer; preferably non-small cell lung cancer, head and neck cancer, brain tumor, and melanoma

The present invention also relates to a method for the treatment and/or prophylaxis of a disease having the pathological characteristics of the tryptophan metabolic pathway mediated by a highly selective inhibitor of CDK4/6, which comprises administering to a patient a therapeutically effective dose of a compound of formula (I) or a tautomer, hydrate, meso, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. These diseases include infection by viruses such as AIDS, cell infections such as lyme disease and streptococcal infection, neurodegenerative disorders (e.g., alzheimer's disease, huntington's disease, and parkinson's disease), autoimmune diseases, depression, anxiety, cataracts, psychological disorders, AIDS, cancer (including T cell leukemia and colon cancer), ocular disease states (e.g., cataracts and age-related yellowing), and autoimmune diseases.

Another aspect of the present invention relates to a method for treating cancer, which comprises administering to a patient a therapeutically effective amount of a compound of formula (I) of the present invention, or a tautomer, hydrate, meso, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof. The method shows outstanding therapeutic effects and fewer side effects, wherein the cancer may be selected from breast cancer, cervical cancer, colon cancer, lung cancer, stomach cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer, preferably fallopian tube tumor, peritoneal tumor, stage IV melanoma, myeloma, and breast cancer, more preferably breast cancer.

Drawings

Fig. 1-3 are XRPD, DSC-TGA diagrams of free base.

Figures 4-6 are XRPD, DSC-TGA graphic representations of p-toluenesulfonate form III.

Fig. 7-9 are XRPD, DSC-TGA representations of succinate salts.

Figures 10-12 are XRPD, DSC-TGA diagrams of fumarate salts.

Figure 13 is a schematic XRPD of the hydrochloride salt.

Figure 14 is an XRPD pattern of tartrate salt.

Figure 15 is a schematic XRPD of maleate.

Figure 16 is a schematic XRPD of the mesylate salt.

Figure 17 is a schematic XRPD pattern of citrate.

Figure 18 is an XRPD pattern of phosphate.

Figure 19 is an XRPD pattern of sulfate.

FIG. 20 is an XRPD pattern for the hydrobromide salt.

Figure 21 is a graphical XRPD pattern of adipate.

FIG. 22 is an XRPD pattern for nitrate.

Figure 23 is a schematic XRPD pattern of benzenesulfonate salts.

Figure 24 is a graphical representation of XRPD of p-toluenesulfonate form I.

Figure 25 is a graphical representation of XRPD of p-toluenesulfonate form II.

Figure 26 is an infrared spectrum of crystalline form III of p-toluenesulfonate.

FIG. 27 is a single crystal structure diagram of crystalline form III of p-toluenesulfonate.

Detailed Description

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

Different terms such as "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", "X is A, B and C" and the like all express the same meaning, that is, X can be any one or more of A, B, C.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not.

"stereoisomerism" encompasses geometric isomerism (cis-trans isomerism), optical isomerism, conformational isomerism, and the like.

All hydrogen atoms described in the present invention can be replaced by deuterium, which is an isotope thereof, and any hydrogen atom in the compound of the embodiment related to the present invention can also be replaced by a deuterium atom.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

"TGA" refers to a thermogravimetric analysis (TGA) experiment.

"DSC" refers to a Differential Scanning Calorimetry (DSC) experiment.

"XRPD" refers to X-ray powder diffraction (XRPD) experiments.

"IR" refers to infrared spectroscopy (IR) experiments.

"HPLC" refers to High Performance Liquid Chromatography (HPLC) experiments.

"PK" refers to Pharmacokinetic (PK) experiments.

The present invention is further described below with reference to examples, which are not intended to limit the scope of the present invention.

1.1 Experimental instruments

1.1.1 some parameters of the physicochemical measuring instrument

1.2 conditions of liquid phase analysis

1.2.1 instruments and devices

1.2.2 chromatographic conditions

A chromatographic column: waters X-Bridge (C18, 3.5 μm, 4.6X 100mm)

Flow rate: 1.1mL/min

Column temperature: 40 deg.C

Detection wavelength: 266nm

Sample introduction volume: 5.0. mu.L

Operating time: 11min

Diluent agent: methanol-water (v/v, 4:1)

Mobile phase: a: water (0.1% trifluoroacetic acid); b: methanol (0.1% trifluoroacetic acid)

T(min)	B(％)
		0.00	10
6.00	90
		8.00	90
8.01	10
		11.00	10

The first embodiment is as follows: natural volatilizing dry process for preparing

1.1 purpose of experiment:

different counter ion acids are selected, and a natural volatilizing method is adopted to detect which counter ion acids can form compound salts.

1.2 Experimental procedures:

1) apparatus and device

Name (R)	Model number	Source
			Analytical balance	BSA224S-CW	Sartorius
Ultrasonic cleaning instrument	SK5200LHC	Shanghai department leads ultrasonic instrument
			Liquid-transfering gun	Eppendorf(5mL,1000μL)	Eppendorf

2) Operating procedure

Preparation of stock solution

Taking 156mg of free base of the compound shown in the general formula, adding 2X 3.25mL of tetrahydrofuran (using a 5mL pipette), and carrying out ultrasonic dissolution to obtain a clear solution, wherein the concentration of the solution is as follows: 24 mg/mL.

② salt solution preparation (free alkali with counter ion acid addition of 1-1.2 equivalent)

Adding salt solution of II into 0.42mL of stock solution I, naturally volatilizing, and vacuum drying at 60 ℃ to obtain the salt of the corresponding compound.

Example two: preparation by solution crystallization or anti-solvent method

2.1 purpose of experiment:

and (4) selecting a benign solvent to dissolve the compound, and crystallizing the compound out by using a poor solvent if no precipitate is separated out.

Benign solvent: acetone, 2-butanone (MEK), Tetrahydrofuran (THF), 1, 4-dioxane, 3-pentanone, 2-methyltetrahydrofuran, ethyl acetate, acetonitrile;

poor solvent: heptane, dichloromethane, water, methyl tert-butyl ether, toluene, isopropyl ether, ethanol;

counter ion acid: methanesulfonic acid, sulfuric acid, phosphoric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid, and L-malic acid.

2.2 Experimental procedures

1) Apparatus and device

Name (R)	Model number	Source
			Analytical balance	BSA224S-CW	Sartorius
Ultrasonic cleaning instrument	SK5200LHC	Shanghai department leads ultrasonic instrument
			Circulating waterPump and method of operating the same	SHB-III	Zhengzhou great wall science, industry and trade, Inc

2) Operating procedure

The specific implementation method for selecting the good solvent and the anti-solvent for salt preparation comprises the following steps:

1) weighing free alkali 200mg or 100mg, and dissolving with 2 or 4mL of methanol or 8mL of ethanol;

2) weighing 1.2 equivalents of counter ion acid, and dissolving with ethanol or water;

3) the free alkali solution and the counter ion acid solution are mutually soluble;

4) if no precipitate exists, adding a poor solvent dropwise until turbidity appears, and stirring overnight;

5) solid salt was obtained by rapid centrifugation.

The formulation of the free base and counter-ionic acid solutions is as follows:

2.2.3 physical characterization test

Fig. 1-3 are XRPD, DSC-TGA diagrams of free base.

Fig. 7-9 are XRPD, DSC-TGA representations of succinate salts.

Figures 10-12 are XRPD, DSC-TGA diagrams of fumarate salts.

Figure 13 is a schematic XRPD of the hydrochloride salt.

Figure 14 is an XRPD pattern of tartrate salt.

Figure 15 is a schematic XRPD of maleate.

Figure 16 is a schematic XRPD of the mesylate salt.

Figure 17 is a schematic XRPD pattern of citrate.

Figure 18 is an XRPD pattern of phosphate.

Figure 19 is an XRPD pattern of sulfate.

FIG. 20 is an XRPD pattern for the hydrobromide salt.

Figure 21 is a graphical XRPD pattern of adipate.

FIG. 22 is an XRPD pattern for nitrate.

Figure 23 is a schematic XRPD pattern of benzenesulfonate salts.

2.3 conclusion of the experiment

Analysis of the data by XRPD, DSC-TGA (differential thermal-TGA) and comparison with the data for the free base indicated that all of the above counter-ionic acids were able to form salts of the compounds.

2.4 XRPD diffraction data for free base with various salts

1. XRPD diffraction data for free base:

	2θ(±0.2°)	d value	Peak height	Proportion (I%)	Area of	Proportion (I%)
							1	4.202	21.0113	2168	100.0	38156	100.0
2	8.695	10.1613	42	1.9	592	1.6
							3	9.618	9.1881	54	2.5	1004	2.6
4	12.442	7.1085	74	3.4	950	2.5
							5	13.788	6.4170	155	7.1	3781	9.9
6	15.853	5.5855	121	5.6	3351	8.8
							7	16.978	5.2180	51	2.4	848	2.2
8	18.061	4.9074	73	3.4	1899	5.0
							9	19.706	4.5015	75	3.5	1168	3.1
10	24.576	3.6193	60	2.8	1700	4.5
							11	25.833	3.4460	50	2.3	1017	2.7

2. XRPD radiation diffraction data for mesylate salt:

3. XRPD radiation diffraction data for sulfate:

4. XRPD radiation diffraction data for citrate:

5. XRPD radiation diffraction data for phosphate:

6. XRPD radiation diffraction data for succinate:

7. XRPD radiation diffraction data for sulfate:

8. XRPD radiation diffraction data for hydrobromide:

9. XRPD radiation diffraction data for adipate:

10. XRPD radiation diffraction data for nitrates:

11. XRPD radiation diffraction data for benzenesulfonate salts:

12. XRPD radiation diffraction data for maleate:

13. XRPD radiation diffraction data for tartrate:

14. XRPD radiation diffraction data for fumarate salt:

15. XRPD radiation diffraction data for hydrochloride salt:

16. XRPD ray diffraction data for crystalline form I of p-toluenesulfonate:

17. XRPD radiation diffraction data for crystalline form II p-toluenesulfonate:

18. XRPD radiation diffraction data for p-toluenesulfonate form III:

example three: experiment for simulating solubility in artificial intestines and stomach

3.1 purpose of experiment:

the solubility of different salts of the compound in simulated artificial gastrointestinal fluid is compared, and a basis is provided for the evaluation of the druggability of the salt.

3.2 Experimental protocol:

approximately 2mg of the compound was suspended in 1mL of artificial Simulated Gastric Fluid (SGF), fasted artificial simulated intestinal fluid (FaSSIF), non-fasted artificial simulated intestinal fluid (FeSSIF) and purified water for 24 hours, and thermodynamic solubility of the compound at room temperature was determined by HPLC, external standard method.

3.3 Experimental results:

as can be seen from the results of the solubility of the compound and its salts in water or biologically relevant media, the solubility of the compound in water after salt formation is improved to various degrees.

Example four: solid stability test

4.1 purpose of experiment:

and (3) investigating the physical and chemical stability of the free alkali or the candidate salt form of the candidate compound under the acceleration condition or the influence factor condition, and providing a basis for salt form screening and compound salt storage.

4.2 protocol:

taking 5mg of free alkali or salt, sealing the free alkali or salt in a 70 ℃ oven and a lighting box (5000lx +/-500 lx), placing the free alkali or salt in a 50 ℃ and RH 75% (saturated NaCl aqueous solution) open for 5 days and 10 days, measuring the content of the salt by using HPLC and an external standard method, and calculating the change of substances related to the salt by using a chromatographic peak area normalization method. The 10 day salt samples were also examined and their XRPD determined and compared to the 0 day XRPD.

4.3 Experimental results:

1) physicochemical stability results of free base and salt:

the free alkali of the compound is extremely unstable at a high temperature of 70 ℃, the impurities are increased by more than 20 percent after the compound is placed for 10 days, the impurities are increased by 37 percent after the compound is placed for 10 days under the illumination condition, and therefore, the stability of the compound needs to be increased by salifying

The p-toluenesulfonate in the salt form of the compound is stable under high temperature and high humidity illumination conditions, and almost no impurities are increased, while other salt forms have related substances increased at a high temperature of 70 ℃.

The XRPD result shows that when the p-toluenesulfonate is inspected for 10 days at 50 ℃/RH 75% and 70 ℃ under the illumination condition, the crystal form of the p-toluenesulfonate has no transformation compared with a 0-day sample.

4.4 summarize:

the compound fixation stability results show that the compound free alkali is extremely unstable under the conditions of high temperature and strong light, and the impurities of the p-toluenesulfonate after salification are hardly increased under the conditions of high temperature, high humidity and strong light and are more stable than other salts, so the p-toluenesulfonate is the most preferable salt type.

Example five: hygroscopicity test

5.1 purpose of the experiment

And the hygroscopicity of the compound under different relative humidity conditions is inspected, so that a basis is provided for screening and storing compound salts.

5.2 protocol:

and (3) placing the compound salt in saturated water vapor with different relative humidity to enable the compound and the water vapor to reach dynamic equilibrium, and calculating the percentage of moisture absorption weight gain of the compound after equilibrium.

5.3 Experimental results:

the p-toluenesulfonate absorbed moisture and increased by about 0.93% under the condition of RH 80%, and was slightly hygroscopic, the fumarate absorbed moisture and increased by 5% under the condition of RH 80%, and was hygroscopic, and the hydrochloride absorbed moisture and increased by 16% under the condition of RH 80%, and was very hygroscopic. And the p-toluenesulfonate is subjected to moisture absorption and desorption for 1 time under the condition of 0-95% relative humidity, and XRPD is not changed, namely, no crystal form transformation exists;

from the viewpoint of hygroscopicity, p-toluenesulfonate is superior to other salt forms.

Example six: polycrystal screening experiment

6.1 purpose of experiment: and finding out a relatively stable crystal form of the p-toluene sulfonate through polycrystal screening.

6.2 protocol: selecting organic solvent and water with certain solubility, suspending the compound in a solvent system, stirring and pulping at room temperature for 1 week, centrifuging, removing supernatant, vacuum drying the solid at 40 deg.C (-0.1Mpa) overnight, measuring XRPD of the solid, and comparing with XRPD of compound salt.

6.3 Experimental results:

1) the preparation method of the p-toluenesulfonate crystal form I comprises the following steps:

weighing 50.0mg of free base, adding 1.0mL of ethanol for dissolving, adding 1.1 equivalent of 1.0M p-toluenesulfonic acid ethanol solution under the stirring condition, separating out precipitate, filtering and drying to obtain a p-toluenesulfonic acid salt crystal form I sample, wherein XRPD of the methylbenzenesulfonic acid salt crystal form I is shown in figure 24.

2) The preparation method of the p-toluenesulfonate crystal form II comprises the following steps:

weighing 100.0mg of free alkali, adding 2.0mL of methanol for dissolving, adding 1.1 equivalent of 1.0M p-toluenesulfonic acid ethanol solution under the stirring condition, gradually dropwise adding an anti-solvent methyl tert-butyl ether, stopping dropwise adding after a cloud point appears, continuously stirring, separating out a precipitate, filtering and drying to obtain a p-toluenesulfonic acid salt crystal form II sample, wherein XRPD of the methylbenzenesulfonic acid salt crystal form II is shown in an attached figure 25.

3) The preparation method of the p-toluenesulfonate crystal form III comprises the following steps:

weighing 100.0mg of free base, adding 2.0mL of tetrahydrofuran to dissolve, heating the sample to 50 ℃, adding 1.1 equivalent of 1.0M p-toluenesulfonic acid aqueous solution under the stirring condition, separating out a precipitate, filtering and drying to obtain a p-toluenesulfonic acid salt crystal form III sample, wherein XRPD of the methylbenzenesulfonic acid salt crystal form III is shown in figure 4, and an infrared spectrogram is shown in figure 26.

4) Polymorphism competition test

And mixing the crystal form I and the crystal form III, the crystal form II and the crystal form III in equal mass, adding an ethyl acetate solution saturated by free alkali, and testing XRPD after pulping for two days at room temperature.

The crystal form competition test result shows that the crystal form III of the p-toluenesulfonate is the most stable crystal form at the present stage.

Example seven: moisture testing of p-toluenesulfonate form III

7.1 purpose of the experiment: the moisture content in crystalline form III of p-toluenesulfonate was tested by the karl fischer method.

7.2 embodiment: weighing 15-20mg of p-toluene sulfonate crystal form III sample, and testing the moisture content by Karl Fischer coulomb method.

7.3 Experimental results: the karl fischer moisture test results are respectively 2.483%, which is consistent with a theoretical value of 2.425% of one molecule of water, and prove that the p-toluenesulfonate crystal form III contains one molecule of water.

Example eight: single crystal growth

8.1 purpose of the experiment: and (3) verifying and confirming whether the p-toluenesulfonate crystal form III is a monohydrate or not through single crystal culture.

8.2 protocol: selecting an organic solvent with certain solubility, dissolving the compound in a solvent system, preferably N, N dimethylformamide, preferably ethyl acetate as an anti-solvent, and diffusing at room temperature by adopting a gas phase diffusion method to obtain the single crystal of the p-toluenesulfonate.

8.3 Experimental results:

single crystal data show that compound p-toluenesulfonate form III is a monohydrate.

The single crystal data are shown in the following table:

the specific structure is shown in FIG. 27: structure of crystal form III single crystal of compound p-toluenesulfonate

Example nine: animal PK Studies

9.1 purpose of experiment: the differences in exposure of the free base and the three salts of the compound in animals were compared by animal PK studies.

9.2 protocol: after fumarate, p-toluenesulfonate (crystal form III) and succinate of the compound are uniformly suspended by CMC-Na (0.5%) aqueous solution containing 0.1% Tween80, the mixture is perfused into the stomach, and the administration is carried out on rats which are parallel to two rats, wherein the administration dose is 50mg/kg, and the total amount of the compound is converted into the same amount of free alkali.

After suspending the free base of the compound uniformly in an aqueous solution containing CMC-Na (0.5%), the stomach was gavaged, and the compound was administered to rats in parallel with two rats at a dose of 5 mg/kg.

9.3 results of the experiment:

rat PK results show: all three salts had good exposure compared to the free base.

Example ten: reaction stoichiometry study

10.1 purpose of experiment: adding counter ions with different molar reaction ratios, and investigating the stability of the metering ratio of the generated p-toluenesulfonate under the given crystallization process conditions.

10.2 protocol: the charged molar ratio of the counter ions was changed to 0.55 and 2.10 (the molar ratio is the ratio of the number of moles of counter ions to the number of moles of free base), and the feasibility of the salt crystallization process and the salt reaction stoichiometric ratio were examined. And (3) measuring the mass percentage content of the free alkali in the crystallized product by adopting an HPLC (high performance liquid chromatography) and external standard method, and comparing the mass percentage content with the mass percentage content of the theoretical reaction stoichiometric ratio.

10.3 results of the experiment:

the amount of counter ions fed was varied and the solids crystallized out were quantified for free base, still forming a 1: 1 reaction molar ratio, and the charging amount molar ratio of the p-toluenesulfonic acid salt is preferably 1.0-1.2.

Claims

1. A compound of the general formula (I),

wherein:

m is an inorganic acid selected from the group consisting of hydrochloric acid, sulfuric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid, or an organic acid selected from the group consisting of 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfuric acid, dibenzoyl tartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, and mixtures thereof, Galactaric acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, lauric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, p-toluenesulfonic acid and L-malic acid; and is

x is selected from 0, 1,2, 3 or 4.

2. A compound of formula (I) according to claim 1, characterized in that: x is 1.

3. A free base crystalline form of a compound, the specific structure of the compound being as follows:

it has an XRPD showing peaks at diffraction angles 2 theta (. + -. 0.2 deg.) of 4.2, 13.8, 15.9, 17.0 and 18.1 + -0.2 deg..

4. The free base crystalline form of claim 3, further exhibiting a peak XRPD at diffraction angles 2 θ (+ 0.2 °) of 8.7, 9.6, 12.4, 19.7, 24.6 and 25.8 ± 0.2 °.

5. The free base crystalline form of claim 4, characterized by a melting peak on DSC of 193.4 ± 0.5 ℃.

6. The p-toluenesulfonate crystal form I of the compound has the following specific structure:

characterized by an XRPD exhibiting peaks at diffraction angles 2 θ (± 0.2 °) of 7.5, 9.7, 10.7, 11.1, 15.3, 16.2, 17.8, 20.5, 20.9, 21.5, 22.7, 25.9 and 27.9 ± 0.2 °.

7. The crystalline form I of the p-toluenesulfonate salt of claim 6 further having an XRPD exhibiting peaks at diffraction angles 2 θ (+ 0.2 °) of 12.1, 12.8, 13.6, 14.0, 16.8, 24.0, 26.8, 29.3, 31.5, 33.6, 39.4 ± 0.2 °.

8. The p-toluenesulfonate crystal form II of the compound has the following specific structure:

characterized by an XRPD exhibiting peaks at diffraction angles 2 θ (± 0.2 °) of 7.4, 9.6, 11.4, 12.6, 14.5, 17.1, 17.7, 19.7, 21.9, 22.8 and 27.5 ± 0.2 °.

9. The crystalline form II of the p-toluenesulfonate salt of claim 8, further having an XRPD exhibiting peaks at diffraction angles 2 Θ (± 0.2 °) of 5.7, 12.1, 13.6, 20.9, 24.1, 24.8, 28.7, 30.3, 32.4, 34.6, 38.2 ± 0.2 °.

10. The crystal form III of the compound p-toluenesulfonate monohydrate has the following specific structure:

the method is characterized in that: XRPD showing peaks at diffraction angles 2 θ (± 0.2 °) of 7.1, 7.8, 11.4, 12.1, 12.8, 13.3, 15.1, 18.2, 20.6, 21.5, 21.8 and 23.5 ± 0.2 °.

11. Form III of the tosylate monohydrate of claim 10, further having an XRPD exhibiting peaks at diffraction angles 2 Θ (± 0.2 °) of 9.0, 9.4, 13.9, 14.1, 14.4, 15.8, 16.9, 17.2, 17.4, 19.2, 19.4, 21.2, 22.0, 25.7, 26.1, 26.7, 27.1, 27.3, 27.5, 28.1, 28.8, 29.0, 29.2, 31.7, 33.1, 35.5, 37.3, and 39.3 ± 0.2 °.

12. Crystalline form III of the p-toluenesulfonate monohydrate of the compound of claim 11, characterized by a melting peak on DSC of 185.8 ± 0.5 ℃.

13. A process for the preparation of a compound of formula (I) according to claim 1, comprising the steps of:

1) preparation of stock solution: taking free alkali of a compound of a general formula (I), adding an organic solvent for dissolving to obtain a clear stock solution, wherein the concentration of the solution is 50 mg/mL;

2) preparation of a counter-ion acid solution: adding a counter-ion acid into an organic solvent or water to obtain a clarified counter-ion acid solution;

3) preparation of compound salt: adding the stock solution into a counter-ion acid solution to obtain a clear salt solution, naturally volatilizing, and drying in vacuum to obtain a salt of the compound shown in the general formula (I); the vacuum temperature is 40 ℃, and the amount of the counter ion acid is 1-1.2 equivalent;

wherein:

the organic solvent is 88% of acetone, methanol, ethanol or tetrahydrofuran; in the step (2), the organic solvent is ethanol, and the concentration is 0.25-2 mol/L; the counter ion acid is methanesulfonic acid, sulfuric acid, phosphoric acid, benzenesulfonic acid, maleic acid, adipic acid, fumaric acid, succinic acid, p-toluenesulfonic acid, citric acid, malonic acid or L-malic acid.

14. The method of claim 13, wherein the counter-ionic acid is fumaric acid, succinic acid, or p-toluenesulfonic acid.

15. A process for the preparation of a compound of formula (I) according to claim 1, comprising the steps of:

1) weighing a proper amount of free alkali, and dissolving the free alkali by using a benign solvent;

2) weighing a proper amount of counter ion acid, and dissolving the counter ion acid by using an organic solvent; the amount of the counter-ionic acid was 1.2 equivalents;

3) mixing the two solutions, stirring for several hours, dripping a poor solvent until turbidity appears, and stirring overnight;

wherein:

the benign solvent is selected from 88% of acetone, methanol, ethanol, dichloromethane, acetone and tetrahydrofuran;

the organic solvent is selected from 88% acetone, methanol, ethanol or tetrahydrofuran;

the benign solvent and the organic solution need to be mutually soluble when in use;

the poor solvent is selected from heptane, ethyl acetate, methyl tert-butyl ether and isopropyl ether;

the counter ion acid is selected from methanesulfonic acid, sulfuric acid, phosphoric acid, benzenesulfonic acid, maleic acid, adipic acid, fumaric acid, succinic acid, p-toluenesulfonic acid, citric acid, malonic acid or L-malic acid.

16. The method of claim 15, wherein the counter-ionic acid is fumaric acid, succinic acid, or p-toluenesulfonic acid.

17. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 2 or a therapeutically effective amount of a crystalline form according to any one of claims 3 to 12, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

18. Use of a compound according to any one of claims 1 to 2 or a crystalline form according to any one of claims 3 to 12 or a pharmaceutical composition according to claim 17 in the manufacture of a medicament for the treatment and/or prevention of cancer or a tumour-associated disease mediated by CDK4 and/or 6.

19. Use of a compound according to any one of claims 1 to 2 or a crystalline form according to any one of claims 3 to 12 or a pharmaceutical composition according to claim 17 for the preparation of a medicament for the prophylaxis and/or treatment of diseases characterized by HR +/HER 2-related diseases.

20. The use according to claim 18 or 19, wherein the disease is selected from the group consisting of cancer, myelodysplastic syndrome, alzheimer's disease, depression, anxiety, cataracts, psychological disorders and aids; the cancer is selected from breast cancer, cervical cancer, colon cancer, stomach cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, peritoneal tumor, stage IV melanoma, glioma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma and non-small cell lung cancer.

21. The use according to claim 20, wherein the cancer is selected from the group consisting of non-small cell lung cancer, head and neck cancer, brain tumor, melanoma, glioblastoma, and hepatocellular carcinoma.