WO2020140934A1 - Salt of selective inhibitor of egfr tyrosine kinase and crystal form thereof - Google Patents

Abstract

The present invention relates to a pharmaceutically acceptable salt of N-(5-((4-(7-cyano-1,3-dimethyl-1H-indol-5-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide as shown in formula (I), specifically, a tartrate and crystal forms I and II of a tartrate of the compound of formula (I), a preparation method therefor, a pharmaceutical preparation containing the above-mentioned salt and crystal forms, and the use thereof in the preparation of a drug for treating and/or preventing diseases mediated by EGFR.

Description

Salts and crystal forms of selective inhibitors of EGFR tyrosine kinase

This application claims the priority of the Chinese patent application filed on January 05, 2019 in the Chinese Patent Office with the application number 201910009657.7 and the invention titled "Salts and Crystal Forms of Selective Inhibitors of EGFR Tyrosine Kinases". The entire contents are incorporated by reference in this application.

Technical field

The invention relates to a selective inhibitor salt of EGFR tyrosine kinase and its mutant, its polymorphic form, preparation method, pharmaceutical composition containing the inhibitor salt and its crystalline form, and its preparation Use in medicine for treating and/or preventing abnormal cell proliferation diseases.

Background technique

Protein kinase (PK) is a very important signaling entity in intracellular communication, where it modifies many proteins by catalyzing the transport of phosphate groups from ATP, which acts as a phosphorus donor, to the phenolic hydroxyl group on the protein tyrosine side chain. Sometimes, tyrosine kinase is incorporated into the intracellular domain of a very large transmembrane protein with a homologous ligand binding domain in the extracellular domain, whereby ligand binding activates tyrosine kinase within the cell. Such molecules are receptor tyrosine kinases (RTK).

More than a decade ago, two 4-anilinoquinazoline inhibitors, gefitinib and erlotinib, of the epidermal growth factor RTK (EGFR, erbB-1) were approved for lung cancer. EGFR is one of the most common dysregulated kinases in solid tumors, where overexpression or mutations are often seen in 50% or more of tumor types including non-small cell lung cancer (NSCLC).

When following the responders of gefitinib and erlotinib, it was found that the onset of resistance may be related to several different genetic changes. Although in rare cases, tumors seem to pick completely different signaling systems to drive tumors, resistance usually involves distortion of the original system. EGFR is a member of the erbB (type I) subfamily of RTK, along with erbB-2, erbB-3, and erbB-4. These receptors are activated by ligands that induce their dimerization, and although EGFR-EGFR homodimers are commonly used for signaling, the more common process in this family is that ligands induce heterodimerization, making signaling entities It will be, for example, EGFR:erbB-2 or erb-B2:erbB-3 and appropriate ligands. The easiest way to reactivate the system is to increase the expression of one of the other erbBs, and even before treatment, this is often seen, and can help explain why many tumors that overexpress wt EGFR do not respond to EGFR inhibition. A somewhat related mechanism involves RTK HGFR, which, although not a member of the erbB family, has been shown to form tumorigenic heterodimers with erbB family members, especially erbB-3, when overexpressed, and the overexpression of HGFR is A common mechanism of resistance to EGFR inhibitors. At least in the laboratory setting, the addition of HGFR inhibitors to these cells will restore sensitivity to EGFR inhibitors. The third and most common pattern of resistance is the further mutation of EGFR, resulting in a double mutant receptor (dm-EGFR), which reduces its sensitivity to EGFR inhibitors. The most common of these is the so-called "gatekeeper" mutation T790M, and NSCLCs with double mutants such as L858R/T790M are common among initial responders who subsequently develop resistance to EGFR inhibitors. Although it is not known whether such subclones always exist or only appear after treatment, it seems most likely that mutations already exist in short-term responders and may appear as remutations in long-term responders who develop resistance later.

N-(5-((4-(7-cyano-1,3-dimethyl-1H-indol-5-yl)pyrimidin-2-yl)amino) described in the following formula (I) 2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide can selectively modulate protein kinases that are resistant to current EGFR-based inhibitor therapy , Especially type I receptor tyrosine kinase (RTK) family or erbB family, and more particularly active novel compounds of certain mutant forms of EGFR receptor, thereby effectively inhibiting cell proliferation and cell invasion, inhibiting metastasis, and inducing cells Apoptosis or inhibit angiogenesis.

The study of crystal form plays an important role in the process of drug development. Different forms of compounds have different bioavailability, solubility, stability and other physical and chemical properties. In order to meet the requirements of preparations, production, transportation, etc., we I) The crystal form of the compound was studied in order to find a crystal form with good properties.

Summary of the invention

The present invention provides tartrates of compounds of formula (I), specifically L-(+)-tartrates of compounds of formula (I).

The present invention also provides an EGFR inhibitor N-(5-((4-(7-cyano-1,3-dimethyl-1H-indol-5-yl)pyrimidine-2 represented by formula (I) -Yl)amino)-2-((2-(dimethylamino)ethyl)methylamino)-4-methoxyphenyl)acrylamide L-(+)-tartrate crystal form, including crystal Form I and Form II.

The present invention also provides L-(+)-tartrate crystal form I (hereinafter referred to as crystal form I) of the compound of formula (I) and L-(+)-tartrate crystal form II (hereinafter referred to as crystal form I) of the compound of formula (I) The preparation method of crystal form II) for short, includes the pharmaceutical composition of the crystal form, and the application of the crystal form in the preparation of a medicine for preventing and/or treating abnormal cell proliferation diseases.

The present invention provides tartrate of the compound represented by formula (I),

In some embodiments, the molar ratio of the compound of formula (I) to tartaric acid in the tartrate salt of the compound of formula (I) is 1:3-1:1, preferably 1:1.

In some embodiments, the tartaric acid in the tartaric acid salt of the compound of formula (I) is L-(+)-tartaric acid.

The present invention also provides L-(+)-tartrate crystal form I of the compound represented by formula (I),

Among them, the molar ratio of the compound of formula (I) to L-(+)-tartaric acid is 1:1, and Cu-Kα radiation is used to express the X-ray powder diffraction pattern at an angle of 2θ, at 8.9±0.2°, 11.2± There are characteristic peaks at 0.2°, 12.0±0.2°, 12.5±0.2°, 13.1±0.2°, 14.7±0.2°, 17.5±0.2°, 19.1±0.2°.

In some embodiments, in the X-ray powder diffraction pattern expressed at a 2θ angle using Cu-Kα radiation, the crystalline form I, on the basis of including the above characteristic peaks, is also 16.3±0.2°, 16.6±0.2 There are characteristic peaks at °, 20.0±0.2°, 20.6±0.2°, 21.1±0.2°, 21.6±0.2°.

In some embodiments, the crystalline form I has substantially the same X-ray powder diffraction pattern as in FIG. 1.

In some embodiments, the Form I differential scanning calorimetry (DSC profile) has an endothermic peak in the range of 190°C to 230°C. In some embodiments, the DSC pattern of Form I has an endothermic peak in the range of 200°C to 225°C. In some embodiments, the maximum endothermic transition temperature of the DSC pattern of Form I is 215.5±5°C. In some embodiments, the crystalline form I has a differential scanning calorimetry curve substantially as shown in FIG. 2.

In some embodiments, the TGA pattern of Form I shows no significant weight loss at 0°C to 200°C. In some embodiments, the crystalline form I has a TGA pattern substantially as shown in FIG. 3.

In another aspect, the present invention also provides a method for preparing L-(+)-tartrate crystal form I of the compound of formula (I), the steps of which include: combining the compound of formula (I) with 1 equivalent of L-( +)-Tartaric acid is added to an organic solvent, water or N-methylpyrrolidone is added under stirring conditions, filtered after the reaction is completed, and dried to obtain the L-(+)-tartrate crystal form I of the compound of formula (I); the formula ( I) Compound N-(5-((4-(7-cyano-1,3-dimethyl-1H-indol-5-yl)pyrimidin-2-yl)amino)-2-((2- (Dimethylamino)ethyl)methylamino)-4-methoxyphenyl)acrylamide can be obtained by the preparation method in the embodiment of the present invention.

In some preferred embodiments, the organic solvent is selected from one or more combinations of one or more solvents selected from lower nitriles, lower alcohols containing more than 2 carbon atoms, and oxygen-containing heterocyclics .

In some preferred embodiments, the organic solvent is selected from one or both of acetonitrile, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol and 1,4-dioxane Any combination of the above solvents.

In some preferred embodiments, the ratio of organic solvent to water or the ratio of organic solvent to N-methylpyrrolidone in the above preparation method is 1:1-20:1, preferably 1:1-10:1, further preferably 2:1-10:1.

The present invention also provides another method for preparing L-(+)-tartrate crystal form I of the compound of formula (I). The steps include: adding the compound of formula (I) to a single solvent or a mixed solvent and heating to a certain level After the temperature, add a certain amount of L-(+)-tartaric acid, maintain the temperature to continue the reaction until the end of the reaction, lower the temperature and crystallize, separate, and dry to obtain L-(+)-tartrate crystal form I of the compound of formula (I).

In some preferred embodiments, the single solvent described in the above preparation method is selected from lower alcohols, lower ketones, lower esters, oxygenated heterocyclics, lower nitriles, lower alkanes or lower halogenated alkanes; The mixed solvent is selected from a mixed solvent of alcohols and water, a mixed solvent of nitriles and water, a mixed solvent of ketones and water, or a mixed solvent of oxygen-containing heterocyclics and water.

In some preferred embodiments, the single solvent in the above preparation method is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, t-butanol, acetone, butanone, methyl ethyl Ketone, acetonitrile, ethyl formate, ethyl acetate, methyl acetate, tetrahydrofuran, 1,4-dioxane, n-pentane, isopentane, n-hexane, isohexane, cyclohexane, cycloheptane, toluene Or dichloromethane; the mixed solvent is selected from ethanol/water, propanol/water, isopropanol/water, butanol/water, sec-butanol/water, tert-butanol/water, acetonitrile/water, 1,4 -Dioxane/water or acetone/water.

In some preferred embodiments, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1-20:1, further preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 1: 1-10:1, more preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 2:1-10:1.

In some preferred embodiments, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1, 2:1, 3:1, 3:2, 4:1, 5:1, 5:2, 5:3, 6:1, 8:1, 8:3, 8:5, 10:1 or 10:3;

In some preferred embodiments, the certain temperature described in the above preparation method is selected from 40°C-80°C, preferably 50°C-70°C.

In some preferred embodiments, certain equivalents described in the above preparation method are selected from 0.9-1.2 equivalents, preferably 1.0-1.1 equivalents, more preferably 1.0 equivalents.

The present invention also provides a third method for preparing L-(+)-tartrate crystal form I of the compound of formula (I). The steps include: adding the compound of formula (I) to a single solvent or a mixed solvent, and slurry washing at room temperature Next, add a certain equivalent of L-(+)-tartaric acid, maintain the temperature and continue the reaction until the end of the reaction. Place the crystal at room temperature, separate, and dry to obtain L-(+)-tartrate crystal form I of the compound of formula (I).

In some preferred embodiments, the single solvent described in the above preparation method is selected from lower alcohols, lower ketones, lower esters, oxygenated heterocyclics, lower nitriles, lower alkanes or lower halogenated alkanes; The mixed solvent is selected from a mixed solvent of alcohols and water, a mixed solvent of nitriles and water, a mixed solvent of ketones and water, or a mixed solvent of oxygen-containing heterocyclics and water.

In some preferred embodiments, the single solvent in the above preparation method is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, t-butanol, acetone, butanone, methyl ethyl Ketone, acetonitrile, ethyl formate, ethyl acetate, methyl acetate, tetrahydrofuran, 1,4-dioxane, n-pentane, isopentane, n-hexane, isohexane, cyclohexane, cycloheptane, toluene Or dichloromethane; the mixed solvent is selected from ethanol/water, propanol/water, isopropanol/water, butanol/water, sec-butanol/water, tert-butanol/water, acetonitrile/water, 1,4 -Dioxane/water or acetone/water.

In some preferred embodiments, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1-20:1, further preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 1: 1-10:1, more preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 2:1-10:1.

In some preferred embodiments, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1, 2:1, 3:1, 3:2, 4:1, 5:1, 5:2, 5:3, 6:1, 8:1, 8:3, 8:5, 10:1 or 10:3;

In some preferred embodiments, certain equivalents described in the above preparation method are selected from 0.9-1.2 equivalents, preferably 1.0-1.1 equivalents, more preferably 1.0 equivalents.

In another aspect, the present invention also provides L-(+)-tartrate crystal form II of the compound represented by formula (I),

Wherein, the molar ratio of the compound of formula (I) to tartaric acid is 1:1, and the Cu-Kα radiation is used to express the X-ray powder diffraction pattern at an angle of 2θ, at 9.0±0.2°, 11.3±0.2°, 12.0±0.2 There are characteristic peaks at °, 12.7±0.2°, 13.9±0.2°, 17.9±0.2°, 19.1±0.2°, 20.3±0.2°, 21.2±0.2°.

In some embodiments, in the X-ray powder diffraction pattern expressed at a 2θ angle using Cu-Kα radiation, the crystalline form II, on the basis of including the above characteristic peaks, is also 16.3±0.2°, 17.3±0.2 °, 23.5±0.2°, 24.4±0.2°, 25.5±0.2°, 25.9±0.2° have characteristic peaks; preferably, the X-ray powder diffraction pattern of the crystalline form II is basically shown in FIG. 4.

The invention also provides a method for preparing crystalline form II. The steps include: adding the compound of formula (I) and 1 equivalent of L-(+)-tartaric acid to an organic solvent, adding water under normal temperature stirring conditions, and continuing to stir at normal temperature At the end of the reaction, filter and dry to obtain Form II.

In some preferred embodiments, the organic solvent described in the above preparation method is selected from methanol.

The present invention also provides a pharmaceutical composition of L-(+)-tartaric acid crystal form I or crystal form II of the compound of formula (I) and one or more pharmaceutically acceptable carriers and/or diluents, the pharmaceutical composition may be It is prepared into any pharmaceutically acceptable dosage form and administered to patients in need thereof by oral, parenteral, rectal, or pulmonary administration. When used for oral administration, it can be made into conventional solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be made into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When making an oral preparation, a suitable filler, binder, disintegrant, lubricant, etc. may be added. When used for parenteral administration, it can be made into injections, including injections, sterile powders for injection and concentrated solutions for injection. When it is prepared as an injection, it can be produced by a conventional method in the existing pharmaceutical field. When preparing an injection, an additional agent may not be added, or an appropriate additional agent may be added according to the nature of the drug. When used for rectal administration, it can be made into suppositories. When used for pulmonary administration, it can be made into inhalants or sprays.

The present invention also provides the use of L-(+)-tartrate crystal form I or crystal form II of the compound of formula (I) in the preparation of a medicament for the treatment and/or prevention of EGFR-mediated hyperproliferative diseases.

The present invention also provides a method for treating and/or preventing hyperproliferative diseases mediated by EGFR, which comprises administering to a subject in need thereof an effective amount of the compound of formula (I) L-( +)-Step of tartrate form I or form II.

In some preferred embodiments, the hyperproliferative disease described above is selected from cancer.

In some preferred embodiments, the EGFR described above includes wild-type EGFR and EGFR mutants.

In some preferred embodiments, the above-mentioned EGFR mutants include one or any combination of one or more of EGFR 19 exon mutation, EGFR 20 exon mutation and EGFR 21 exon mutation .

In some preferred embodiments, the aforementioned exon 20 mutation of EGFR is selected from NPG, ASV or T790M.

In some embodiments, the cancer described above is selected from lung cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, liver cancer, gastric cancer, prostate cancer, glioblastoma Tumor and epithelial cell carcinoma.

In some preferred embodiments, the cancer described above is caused by mutations in exon 20 of EGFR.

In some preferred embodiments, the above-mentioned cancer is caused by the T790M mutation in EGFR 20 exon mutation, and there is also an EGFR 19 exon insertion mutation or EGFR 21 exon point mutation.

The present disclosure also provides the use of the L-(+)-tartrate crystalline form I or crystalline form II of the compound of formula (I) in the preparation of reagents that can be used to inhibit the level of EGFR enzymes in cells. In some preferred embodiments, the agent is used in an in vivo or in vitro method.

The present disclosure also provides a method of inhibiting the level of EGFR in a cell, which includes the step of administering to the cell an effective amount of L-(+)-tartrate salt form I or form II of the compound of formula (I). In some preferred embodiments, the method is performed in vivo or in vitro.

Unless otherwise stated in the invention, the scientific and technical terms used herein have the meaning commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, definitions and explanations of some terms are provided below.

In the present invention, the term "lower alcohols" refers to straight-chain or branched-chain fatty alcohols containing 1 to 6 carbon atoms, or cyclic fatty alcohols, examples of which include but are not limited to: ethanol, propanol, isopropanol, Butanol, sec-butanol, tert-butanol and cyclopentanol.

In the present invention, the term "lower nitrile" contains 1-6 carbon atoms, and examples thereof include, but are not limited to, acetonitrile, propionitrile, and adiponitrile.

In the present invention, the term "lower ketones" contains fatty ketone compounds with 1-6 carbon atoms, and examples thereof include, but are not limited to: methyl ethyl ketone, methyl isopropyl ketone, acetone, methyl butanone, and methyl isobutyl ketone.

In the present invention, the term "lower esters" contains fatty ester compounds of 1-6 carbon atoms, examples of which include but are not limited to: methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, Propyl acetate, isopropyl acetate, methyl propionate, ethyl propionate, propyl propionate and isopropyl propionate.

In the present invention, the term "lower alkanes" contains straight-chain or branched-chain aliphatic alkanes with 1-7 carbon atoms, or cyclic aliphatic alkanes, or aromatic alkanes, examples of which include but are not limited to: n-pentane, isopentane , N-hexane, isohexane, cyclopentane, cyclohexane and toluene.

In the present invention, the term "lower halogenated alkane" contains a linear or branched aliphatic alkane or cyclic aliphatic alkane containing 1 to 6 carbon atoms and substituted with at least one halogen atom. Examples include, but are not limited to: methylene chloride, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, and 1,1,1-trichloroethane.

In the present invention, the term "separation" is used to separate solids by conventional methods, such as filtration, centrifugation and discarding the supernatant.

In the present invention, the term "filtration" includes but is not limited to atmospheric pressure filtration, reduced pressure filtration and the like.

As used herein, the terms "room temperature" and "normal temperature" refer to indoor ambient temperature, usually 10-30°C, such as 20-25°C.

In the present invention, the term "drying" includes but is not limited to natural drying at room temperature, infrared lamp drying, oven drying, dryer drying, and drying under vacuum conditions. The drying temperature is room temperature or 30-60°C, preferably 40-50°C.

In the present invention, the term "cooling crystallization" refers to cooling the reaction system to 0-35°C, preferably to 10-30°C, and more preferably 15-°C by cooling in an ice-water bath, cooling by autothermal cooling, or cooling by refrigeration equipment. At 25°C, it is more preferable to lower the temperature until most crystals precipitate. Compounds can exist in two or more crystalline states, molecules with the same structure, crystallized into different solid forms, called polymorphs or polymorphs. When referring to a specific crystalline form, it is often called "crystal", which is the term "crystal form" used herein.

In the present disclosure, the position of the absorption peak in the X-ray powder diffraction pattern of each crystal form may be in the range of ±0.2° of the specific value of the above invention, for example, in the range of ±0.1°, the absorption measured by differential scanning calorimetry The thermal transition temperature may be within the range of the above specific numerical value ±5.0°C (for example, ±3.0°C or ±2.0°C).

It should be understood that using different types of equipment or using different test conditions may give slightly different XRPD patterns and peaks. The patterns, peaks and relative intensity of different diffraction peaks of different crystal forms will be affected by the purity of the compound, sample preparation, scanning speed, particle size, and calibration and maintenance of the test equipment. The value provided cannot be used as an absolute value.

It should be understood that using different types of equipment or using different test conditions may give slightly different DSC patterns and endothermic transition temperature readings. This value will be affected by compound purity, sample weight, heating rate, particle size, and calibration and maintenance of test equipment. The maximum endothermic transition temperature of the crystal form may be within the range of the specific values disclosed above ±5.0°C.

The present disclosure also uses thermal weight loss analysis (TGA) to analyze the relationship between the degree of crystal form decomposition or sublimation and evaporation (loss of weight) and temperature. It should be understood that the same crystal form is affected by sample purity, particle size, different types of equipment, different test methods, etc., and there are certain errors in the obtained values. The temperature at which the crystal form decomposes, sublimates, or evaporates may be within the range of the specific values disclosed above ±3.0°C, for example, ±2.0°C.

In the present invention, the term "subject" refers to an animal or a human, preferably a mammal or a human.

In the present invention, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic or preventive effect, for example, an amount that achieves relief of symptoms related to the disease to be treated.

In the present invention, the term "treatment" aims to reduce or eliminate the targeted disease state or disorder. If a subject receives a therapeutic amount of the crystalline form or its pharmaceutical composition according to the methods described herein, the subject's one or more indications and symptoms exhibit observable and/or detectable Decrease or improve, the subject is successfully "treated". It should also be understood that the treatment of the disease state or disorder includes not only complete treatment but also incomplete treatment, but has achieved some biological or medical related results.

The main advantages of the compound of formula (I) of the present invention, its tartrate and its L-(+)-tartrate crystal form include:

(1) The compound of formula (I) of the present invention, its tartrate and its L-(+)-tartrate crystal form have excellent EGFR inhibitory activity, especially with a higher selection of certain mutant forms of EGFR receptor Sexuality and inhibitory activity can effectively inhibit cell proliferation and cell invasion, inhibit metastasis, induce apoptosis or inhibit angiogenesis;

(2) The compound of formula (I) of the present invention, its tartrate and its L-(+)-tartrate crystal form have less toxic and side effects, especially in the aspects of diabetes and cardiotoxicity;

(3) The compound of formula (I) of the present invention, its tartrate and its L-(+)-tartrate crystal form have good pharmacokinetic properties, excellent half-life, high exposure, etc. good.

(4) The L-(+)-tartrate salt crystal form of the compound of formula (I) of the present invention has good stability, and has good properties, fluidity and compressibility, which is convenient for detection, preparation, transportation and storage;

(5) The L-(+)-tartrate salt crystal form of the compound of formula (I) of the present invention has high purity, less residual solvent, higher solubility, good dissolution, good stability and easy quality control.

BRIEF DESCRIPTION

FIG. 1 is an X-ray powder diffraction pattern of L-(+)-tartrate salt crystal form I of the compound of formula (I). The ordinate represents the diffraction intensity, and the abscissa represents the diffraction angle (2θ);

Fig. 2 is a differential scanning calorimetry (DSC) thermal analysis diagram of L-(+)-tartrate salt crystal form I of the compound of formula (I), the ordinate represents heat flow (W/g), and the abscissa represents temperature (°C) ;

Fig. 3 is a thermogravimetric analysis (TGA) curve of L-(+)-tartrate crystal form I of the compound of formula (I). The ordinate represents the mass percentage (%), and the abscissa represents the temperature (°C);

4 is a hydrogen spectrum of L-(+)-tartrate crystal form I of the compound of formula (I);

Fig. 5 is an X-ray powder diffraction pattern of L-(+)-tartrate crystalline form II of the compound of formula (I). The ordinate indicates the diffraction intensity, and the abscissa indicates the diffraction angle (2θ).

detailed description

The above content of the present invention will be further described in detail below through specific implementations in the form of examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

Example 1: Preparation of compound of formula (I)

(1) Preparation of 5-bromo-1,3-dimethyl-1H-indole-7-carbonitrile

5-Bromo-3-methyl-1H-indole-7-carbonitrile (1.17 g, 5 mmol) was dissolved in tetrahydrofuran (20 mL), cooled to 0°C, and NaH (260 mg, 6.5 mmol) was added in portions. After the addition, the mixture was stirred at 0°C for 30 minutes, and then MeI (781 mg, 5.5 mmol) was slowly added dropwise. After the completion of the drop, the mixture was naturally raised to room temperature and stirred for two hours. The reaction was quenched carefully with water, extracted with ethyl acetate (20mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. 694 mg, yield: 56%).

¹ HNMR (300 MHz, CDCl ₃ ): 7.87 (d, J = 1.5 Hz, 1H), 7.60 (d, J = 1.5 Hz, 1H), 6.88 (s, 1H), 4.06 (s, 3H), 2.28 (s ,3H).

(2) 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1H-indole- Preparation of 7-carbonitrile

The compound 5-bromo-1,3-dimethyl-1H-indole-7-carbonitrile (523mg, 2.1mmol, 1.0eq) was dissolved in dioxane (5mL), and bisboronic acid pinacol was added Ester (592 mg, 2.3 mmol, 1.1 eq), potassium acetate (125 mg, 6.3 mol, 3 eq), Pd (dppf) Cl ₂ (124 mg, 0.168 mmol, 0.08 eq) replaced nitrogen 3 times, then heated to 85° C., reaction 6 hour. TLC and LCMS detection. After the reaction was completed, suction filtration, mother liquor concentration, and column chromatography purification to obtain the target compound (350 mg, yield: 56%).

¹ HNMR (300MHz, CDCl ₃ ): 8.23 (s, 1H), 7.99 (s, 1H), 6.85 (s, 1H), 4.09 (s, 3H), 2.33 (s, 3H), 1.39 (s, 12H ).

(3) Preparation of 5-(2-chloropyrimidin-4-yl)-1,3-dimethyl-1H-indole-7-carbonitrile

Under nitrogen, the compound 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1H- Indole-7-carbonitrile (296mg, 1mmol, 1.0eq) was dissolved in dioxane and water (5/1mL), 2,4-dichloropyrimidine (162mg, 1.1mmol, 1.1eq), Pd were added respectively (PPh ₃ ) ₄ (115 mg, 0.1 mmol, 0.1 eq), potassium carbonate (411 mg, 3 mmol, 3 eq) was added and the temperature was raised to 100° C. for 3 hours. TLC and LCMS detection. After the reaction was completed, suction filtration was carried out, and the filtrate was concentrated and purified by column chromatography to obtain the target compound (164 mg, yield: 58%).

¹ HNMR (300 MHz, DMSO-d ₆ ): 8.65 (d, J = 5.1 Hz, 1H), 8.55 (s, 1H), 8.29 (s, 1H), 7.70 (d, J = 5.1 Hz, 1H), 6.95(s, 1H), 4.13(s, 3H), 2.35(s, 3H).

(4) Preparation of compound of formula (I)

Compound 5-(2-chloropyrimidin-4-yl)-1,3-dimethyl-1H-indole-7-carbonitrile (164mg, 0.58mmol, 1.0eq) and compound N-(5-amino- 2-{[2-(Dimethylamino)ethyl](methyl)amino}-4-methoxyphenyl)acrylamide (170mg, 0.58mmol, 1.0eq) dissolved in 2-pentanol (4mL) Medium, p-toluenesulfonic acid monohydrate (123mg, 0.64mmol, 1.1eq) was added in batches at room temperature. After the addition, the reaction was heated to 120°C for 5 hours. TLC, LCMS detection. After the reaction was completed, it was cooled to room temperature and water (10mL) was added. ), dichloromethane/methanol=10:1 (10 mL×3) extraction, the organic phase was washed with brine (10 mL), dried, concentrated, and purified by column chromatography to obtain the compound of formula (I) (48 mg, yield: 15%) .

¹ HNMR (300MHz, DMSO-d ₆ ): □10.19 (br, 1H), 9.07 (s, 1H), 8.71 (s, 1H), 8.51-8.49 (m, 2H), 8.20 (s, 1H), 7.59 -7.57(m,1H),7.32(s,1H),7.04(s,1H),6.40-6.34(m,1H),6.27-6.21(m,1H),5.75-5.72(m,1H),4.04 (s,3H),3.85(s,3H),2.89-2.87(m,2H),2.71(s,3H),2.34-2.32(m,2H),2.23(s,3H),2.17(s,6H ). LCMS: (M + H) ⁺ : 538.8. HPLC: 94.8%.

Example 2: Preparation of L-(+)-tartrate crystal form I of the compound of formula (I)

Preparation method one:

Take the compound of formula (I) (50 mg, 0.093 mmol) in a 10 mL centrifuge tube, add 1 equivalent of L-(+)-tartaric acid (13.9 mg, 0.093 mmol), add ethanol (2 mL) to the centrifuge tube, and stir at room temperature for 0.5 After h, add water (0.5 mL) and continue stirring. After the reaction, it was filtered and dried. The obtained solid was tested to form I by XRPD.

Preparation method two

Take the compound of formula (I) (3.0g, 5.57mmol) in a 100mL round bottom flask, add the mixed solvent of acetone and water (the volume ratio of acetone and water is 2:1), stir and heat to 50 At ℃, add L-(+)-tartaric acid (836mg, 5.57mmol), stir at 50℃ until the end of the reaction, naturally cool down, filter, and vacuum dry at 50℃. The obtained solid (3.25g, yield 84.7%) was tested to form I by XRPD.

Preparation method three

Take acetone (3mL), add water (0.5mL) to configure an acetone/water mixed solvent with a volume ratio of 6:1, take the compound of formula (I) (0.1g, 0.19mmol) in a 10mL round bottom flask, add the above Acetone/water mixed solvent (2mL) was stirred, and the temperature was raised to 50°C. L-(+)-tartaric acid (27.9mg, 0.19mmol) was added. The temperature was maintained and stirred until the reaction was completed. The obtained solid (93.8 mg, yield 73.3%) was tested as Form I by XRPD.

Preparation method four

Take L-(+)-tartrate salt form I (50mg, 0.072mmol) of the compound of formula (I) in a 10mL round bottom centrifuge tube, add a mixture of acetone (5mL) and water (0.5mL) to the centrifuge tube The solvent was washed at a constant temperature of 50°C. After the reaction was completed, the stirring condition was lowered to room temperature, centrifugation (4000rpm, 10min), the supernatant was discarded, and the lower solid was dried at 50°C under vacuum, and the crystal form I was tested by XRPD.

Preparation method five

Take 4 parts of L-(+)-tartrate crystal form I (50×5mg, 0.072mmol) of the compound of formula (I), put them into 10mL centrifuge tubes, and add acetonitrile (2mL) and acetic acid to the 4 centrifuge tubes respectively Ethyl ester (2mL), 1,4-dioxane (2mL) and n-heptane (2mL), sequentially numbered 1-4, and placed in a constant temperature slurry wash at 20 ℃, after the reaction is completed, centrifuged (4000rpm, 10min), The supernatant was discarded, and the lower solid was dried in vacuum at 50°C, and tested to form I by XRPD.

For Form I prepared by the above method, determine:

X-ray powder diffraction measurement

The crystal structure of the present invention is not limited to providing a crystal structure of an X-ray powder diffraction pattern exactly the same as the X-ray powder diffraction pattern drawn in the drawings disclosed in the present application, and X-ray powder diffraction patterns substantially the same as those disclosed in the drawings Any crystal structure of the ray powder diffraction pattern is included in the scope of the present invention.

Conditions for X-ray powder diffraction measurement: Cu palladium, Kα1

1.54060, step size 0.0203, 0.3 seconds per step.

Using Cu-Kα radiation, the X-ray powder diffracted in a crystal form expressed at a 2θ angle (°) has a characteristic peak.

The X-ray powder diffraction pattern of the L-(+)-tartrate salt form I of the compound of formula (I) is shown in FIG. 1, and this form I has a peak at the following diffraction angle 2θ (°):

8.9±0.2°, 11.2±0.2°, 12.0±0.2°, 12.5±0.2°, 13.1±0.2°, 14.7±0.2°, 17.5±0.2°, 19.1±0.2°, 16.3±0.2°, 16.6±0.2°, 20.0±0.2°, 20.6±0.2°, 21.1±0.2°, 21.6±0.2°.

Differential Scanning Calorimetry

The solid-state thermal properties of L-(+)-tartrate crystal form I of the compound of formula (I) were studied by differential scanning calorimetry (DSC). The DSC curve of Form I is shown in FIG. 2.

Measurement conditions: Purge with nitrogen at 50 ml/min, collect data at a heating rate of 10°C/min between room temperature and 225°C, and plot with the endothermic peak facing down.

In DSC measurement, the actual measured start temperature and maximum temperature have a certain degree of variability based on the measurement parameters and heating rate.

Thermogravimetric analysis

Test conditions: Purge with nitrogen at 60 ml/min and collect data at a heating rate of 10°C/min between room temperature and 300°C.

The TGA curve of L-(+)-tartrate salt form I of the compound of formula (I) is shown in FIG. 3.

NMR analysis

¹ HNMR (400MHz, DMSO-d6): δ 9.84 (s, 1H), 8.94 (s, 1H), 8.67 (d, 1H), 8.47 (s, 1H), 8.46 (s, 1H), 8.15 (s , 1H), 7.56 (d, 1H), 7.29 (s, 1H), 7.00 (s, 1H), 6.55 (m, 1H), 6.27 (m, 1H), 5.74 (m, 1H), 4.08 (s, 2H), 4.02(s, 3H), 3.86(s, 3H), 3.06(t, 2H), 2.73(t, 2H), 2.64(s, 3H), 2.48(s, 3H), 2.47(s, 3H ), 2.27(s, 3H).

The hydrogen spectrum of L-(+)-tartrate salt form I of the compound of formula (I) is shown in FIG. 4.

Example 3: Preparation of the compound of formula (I) L-(+)-tartrate crystal form II

Take the compound of formula (I) (50 mg, 0.093 mmol), place it in a 10 mL round bottom flask, add L-(+)-tartaric acid (13.9 mg, 0.093 mmol), and then add methanol (2 mL). After stirring at room temperature for 1 h, add Water (0.5 mL), continue stirring for 23 h, then filter, dry at 50° C. in vacuo, and the solid was detected by XRPD. The result showed that it was Form II.

X-ray powder diffraction measurement

Conditions for X-ray powder diffraction measurement: Cu palladium, Kα1

1.54060, step size 0.0203, 0.3 seconds per step.

Using Cu-Kα radiation, the X-ray powder diffracted in a crystal form expressed at a 2θ angle (°) has a characteristic peak.

The X-ray powder diffraction pattern of the L-(+)-tartrate salt form II of the compound of formula (I) is shown in FIG. 5, which has a peak at the following diffraction angle 2θ (°):

9.0±0.2°, 11.3±0.2°, 12.0±0.2°, 12.7±0.2°, 13.9±0.2°, 17.9±0.2°, 19.1±0.2°, 20.3±0.2°, 21.2±0.2°, 16.3±0.2°, 17.3±0.2°, 23.5±0.2°, 24.4±0.2°, 25.5±0.2°, 25.9±0.2°.

Example 4: Investigation of EGFR and tumor inhibitory activity of the compound of formula (I)

abbreviation:

DMSO

DTT dithiothreitol

ATP adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

Ki enzyme inhibition constant

DMEM Dulbecco's Modified Eagle's Medium

NCS Newborn Calf Serum

PBS phosphate buffered saline

PMSF phenylmethanesulfonyl fluoride

ELISA

IgG immunoglobulin G

FBS fetal bovine serum

BDNF brain-derived neurotrophic factor

4.1. EGFR kinase inhibition test

The inhibition of kinases by compounds of the invention is measured using commercially available assay kits and services well known to those skilled in the art. These kits and services are used to measure the inhibition of various kinases including (but not limited to): ALK, ABL, AXL, Aur B and C, BLK, erbB-2, erbB-4.EGFR, mutant EGFR, HPK, IRAK1, RON, ROS1, SLK, STK10, TIE2, TRK, c-Met, Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, ErbB2, Kdr, Flt-1, Flt- 3. Tek, c-Met, InsR and Atk. Commercial suppliers of these assay kits and services include Promega and Reaction Biology, EMD Millipore and CEREP. In addition to commercially available assay kits and services, the kinase inhibitory activity of compounds of formula (I-VIII) is measured by means of the following assay.

4.1.1. Purification of epidermal growth factor receptor tyrosine kinase

Human EGF receptor tyrosine kinase was isolated from A431 human squamous cell carcinoma cells overexpressing EGF receptor by the following method. Cells were grown in 50% Dulbecco's modified Eagle's and 50% HAM F-12 nutrient medium (Gibco) containing 10% fetal bovine serum in roller bottles. Approximately 109 cells were dissolved in two volumes of buffer containing the following: 20 mM 2-(4N-[2-hydroxyethyl] piperazin-1-yl)ethanesulfonic acid (hepes) pH 7.4, 5 mM ethylene glycol bis (2-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1% Triton X-100, 10% glycerin, 0.1mM sodium orthovanadate, 5mM sodium fluoride, 4mM pyrophosphate, 4mM Benzamide, 1 mM dithiothreitol, 80 μg/mL aprotinin, 40 μg/mL leupeptin, and 1 mM benzylsulfonyl fluoride. After centrifugation at 25,000×g for 10 minutes, the supernatant was equilibrated with 10 mL of wheat pre-equilibrated with 50 mM Hepes, 10% glycerol, 0.1% Triton X-100 and 150 mM NaCl pH 7.5 (equilibration buffer) at 40°C. The embryo agglutination agarose was equilibrated for 2 hours. The contaminated protein was washed from the resin with an equilibrium buffer containing 1M NaCl, and the enzyme was eluted with an equilibrium buffer containing 0.5M N-acetyl-1-D-glucosamine, followed by 1 mM urea. The enzyme was eluted with 0.1 mg/ml EGF. As assessed by Coomassie blue stained polyacrylamide electrophoresis gel, the receptor appears to be homogeneous.

Using the same technique as described in the previous paragraph, various mutant forms of epidermal growth factor receptor can be isolated from appropriate cell lines containing epidermal growth factor receptor. For example, the EGFRdel746-750 mutant protein can be extracted from PC-9 cells, and the L858R/T790M double mutant EGFR protein can be isolated from H1975 cells.

4.1.2. Determination of IC ₅₀ value of single mutant EGFR_d746-750

The enzyme assay used to determine IC ₅₀ was performed in a total volume of 25 μL. All compounds were diluted to a 500 μM stock solution in 100% DMSO and serially diluted 4 times to achieve 10 doses. The "Max" and "Min" controls contain 100% DMSO. "Max" represents the DMSO control without enzyme, and "Min" represents the low control without compound. 10 μl of the compound was transferred to 90 μl of 1x kinase base buffer for intermediate dilution. 5 μl of the intermediate dilution compound was transferred to a 384-well assay plate, and then 10 μl of 2.5x enzyme buffer containing (12.5nM EGFR_d746-750, 5mMDTT, 1x kinase base buffer) was added to the assay plate. Incubate for 10 minutes at room temperature and add 10 μl of 2.5x substrate buffer containing (7.5 μM peptide, 35 μM ATP, 25 mM MgCl ₂ , 1x kinase base buffer) to start the reaction. Incubate for 1 hour at room temperature and add 25 μl stop buffer to end the reaction. Collect conversion data from Caliper, and collect conversion data from the Caliper program. Data fitting to obtain _IC50 values XLfit IC.

4.1.3. Determination of IC ₅₀ value of double mutant EGFR (EGFR_T790M/L858R)

The enzyme assay used to determine IC ₅₀ was performed in a total volume of 25 μL. All compounds were diluted to a 500 μM stock solution in 100% DMSO and serially diluted 4 times to achieve 10 doses. The "Max" and "Min" controls contain 100% DMSO. "Max" represents the DMSO control without enzyme, and "Min" represents the low control without compound. 10 μl of the compound was transferred to 90 μl of 1x kinase base buffer for intermediate dilution. 5 μl of the intermediate dilution compound was transferred to a 384-well assay plate, and then 10 μl of 2.5x enzyme buffer containing (25 nM EGFR_T790M/L858R, 5 mM DTT, 1x kinase base buffer) was added to the assay plate. Incubate at room temperature for 10 minutes and add 10 μl of 2.5x substrate buffer containing (7.5 μM peptide, 47.5 μM ATP, 25 mMMgCl ₂ , 1x kinase base buffer) to start the reaction. Incubate for 1 hour at room temperature and add 25 μl stop buffer to end the reaction. Collect conversion data from Caliper, and collect conversion data from the Caliper program. Data fitting to obtain _IC50 values XLfit IC.

4.1.4. Determination of IC ₅₀ value of wt EGFR

The enzyme assay used to determine IC ₅₀ was performed in a total volume of 25 μL. All compounds were diluted to a 500 μM stock solution in 100% DMSO and serially diluted 4 times to achieve 10 doses. The "Max" and "Min" controls contain 100% DMSO. "Max" represents the DMSO control without enzyme, and "Min" represents the low control without compound. 10 μl of the compound was transferred to 90 μl of 1x kinase base buffer for intermediate dilution. 5 μl of the intermediate dilution compound was transferred to a 384-well assay plate, and then 10 μl of 2.5x enzyme buffer containing (20 nM EGFR, 5 mM DTT, 1x kinase base buffer) was added to the assay plate. Incubate at room temperature for 10 minutes and add 10 μl of 2.5x substrate buffer containing (7.5 μM peptide, 5.75 μM ATP, 25 mM MgCl ₂ , 25 mM MnCl ₂ , 1x kinase base buffer) to start the reaction. Incubate for 1 hour at room temperature and add 25 μl of stop buffer to end the reaction. Conversion data is collected from Caliper, and conversion data is collected from the Caliper program. Data fitting to obtain _IC50 values XLfit IC.

4.2 Anti-proliferation test of EGFR cells

testing sample

The compound of formula (I) is prepared according to the method in the examples; the structure of compound AZD9291 is as follows and prepared according to the prior art method.

experiment procedure

4.2.1, H1975 inhibition assay (cell proliferation)

The H1975 cells were cryopreserved in liquid nitrogen. Before thawing the cells, 15 mL of cell culture medium (RPMI 1640 medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin) was placed in a T75 flask and incubated in a humidified 37°C/5% CO ₂ incubator Incubate the flask in advance for 15 minutes to equilibrate the medium to the proper pH and temperature. The vial was removed from the liquid nitrogen and quickly thawed by placing in a water bath at 37°C for 1-2 minutes with gentle stirring, then decontaminated by wiping with 70% ethanol, and then opened in a Class II biosafety cabinet. The contents of the vial were transferred dropwise to 10 mL of cell culture medium in a sterile 15 mL conical tube. The tube was then centrifuged at 200×g for 5 minutes, and the supernatant was aspirated. The cell pellet was resuspended with 1 mL of fresh cell culture medium, and transferred to a T75 flask containing cell culture medium.

To passage H1975 cells, first, adherent cells were washed with trypsin/EDTA. Then trypsin/EDTA (3 mL in T75 flask) was added to the flask and swirled to ensure that the cells were evenly coated with trypsin. The flask was then incubated at 37°C until the cells detached. Add an equal volume of cell culture medium to stop the reaction. The detached cells were collected and centrifuged at 200×g for 5 minutes, and then resuspended in fresh medium. Next, the cells were transferred to a new T75 flask containing cell culture medium. The cells were subcultured in the medium at a ratio of 1:2 or 1:4 three times a week.

The test compound was dissolved in DMSO at 30 mM. 45 μL of compound was transferred to a 384-well compound source plate (LABCYTE catalog number P-05525) and serially diluted at a ratio of 1:3, resulting in a 13-point dilution. Take the same volume of DMSO as a high control. 20 nL of these compound DMSO dilutions (10 points, from 1.11 mM to 0.056 μM) were dispensed into new 384-well assay plates by Echo 550.

The cells were harvested from the flask to the cell culture medium as described above and the cell count was calculated using an automated cell counter (Thermo Fisher Scientific, Countess ^™ ). Dilute the cells with culture medium to 25,000 cells/ml and add 40 μL of cell suspension to each well of the 384-well cell culture plate as indicated. The final concentration is 1,000 cells/well. Add medium alone as a low control. The plate was covered with a lid and placed in a 37°C/5% CO ₂ incubator for 72 hours.

After 72 hours of incubation, the plate was removed from the incubator and equilibrated at room temperature for 15 minutes. CellTiter Glo reagent (Promega, G9243) was incubated at 37°C before the experiment. The buffer was equilibrated to room temperature and used to dissolve the matrix. To determine cell viability, add 40 μL of CellTiter-Glo reagent to each well to be tested (1:1 with culture medium). The plate was then placed at room temperature for 30 minutes, and then read on EnSpire (PerkinElmer).

To estimate the IC ₅₀ , the luminescence reading is converted to% inhibition by applying the following equation: (Lum _HC -Lum _Cpd )/(Lum _HC -Lum _LC ). Logarithmic curve, then IC ₅₀ was calculated by four-parameter fit to the XLFit.

4.2.2, PC-9 growth inhibition assay (cell proliferation)

The inhibition of PC-9 cells was measured in the same manner as described for H1975 cells.

4.2.3, A431 inhibition assay (cell proliferation).

The medium for A431 cells was Dulbecco's modified Eagle's medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin. The DMSO dilution used for A431 determination is 10 points from 30nM to 1.52uM. The rest of the procedure was performed in the same manner as described for H1975 cells.

test results

Table 1 Cell proliferation test results

PC9 cells contain EGFR d746-750 single mutant (SM). H1975 cells contain EGFR L858R/T790M double mutant (DM). A431 cells contain unmutated EGFR wild type (WT).

Test Conclusions

The compound of formula (I) of the present invention has excellent EGFR inhibitory activity and anti-tumor proliferation activity, especially high inhibitory activity and selectivity for EGFR mutants.

4.3. Cell EGFR autophosphorylation test

testing sample

The compound of formula (I) is prepared according to the method in the examples; the compound AZD9291 is prepared according to the prior art method.

experiment procedure

L858R/T790M double mutant H1975 autophosphorylation inhibition assay (ELISA)

The H1975 cells were cryopreserved in liquid nitrogen. Before thawing the cells, 15 mL of cell culture medium (RPMI 1640 medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin) was placed in a T75 flask and incubated in a humidified 37°C/5% CO ₂ incubator Incubate the flask in advance for 15 minutes to equilibrate the medium to the proper pH and temperature. The vial was removed from the liquid nitrogen and quickly thawed by placing in a water bath at 37°C for 1-2 minutes with gentle stirring, then decontaminated by wiping with 70% ethanol, and then opened in a Class II biosafety cabinet. The contents of the vial were transferred dropwise to 10 mL of cell culture medium in a sterile 15 mL conical tube. The tube was then centrifuged at 200×g for 5 minutes, and the supernatant was aspirated. The cell pellet was resuspended with 1 mL of fresh cell culture medium, and transferred to a T75 flask containing cell culture medium.

To passage H1975 cells, first, adherent cells were washed with trypsin/EDTA. Then trypsin/EDTA (3 mL in T75 flask) was added to the flask and swirled to ensure that the cells were evenly coated with trypsin. The flask was then incubated at 37°C until the cells detached. Add an equal volume of cell culture medium to stop the reaction. The detached cells were collected and centrifuged at 200×g for 5 minutes, and then resuspended in fresh medium. Next, the cells were transferred to a new T75 flask containing cell culture medium. Cells were subcultured in the medium at a ratio of 1:4 three times a week.

The cells were harvested from the flask to the cell culture medium and the number of cells was calculated using an automated cell counter (ThermoFisher Scientific, Countess ^™ ). The cells were diluted with culture medium to 250,000 cells/ml, and 40 μL of cell suspension was added to each well of the 384-well cell culture plate as indicated. The final concentration is 10,000 cells/well. The plate was covered with a lid and placed in a 37°C/5% CO2 incubator overnight to adhere the cells.

The next day, the test compound was dissolved in DMSO at 10 mM. 45uL of compound was transferred to a 384-well compound source plate (LABCYTE catalog number P-05525) and serially diluted at a ratio of 1:3, resulting in a 13-point dilution. Take the same volume of DMSO as a high control. 40 nL of these compound DMSO dilutions (11 points, from 1.11 mM to 0.019 uM) were dispensed into H1975 cell plates by Echo 550.

Return the incubation plate to the 37°C/5% CO ₂ incubator for 2 hours. Replace the medium in each well with ice cold HBSS. Next, HBSS was removed, 30 μL of cell lysis buffer was added to each well and the plate was shaken on a plate shaker for 30 minutes. Centrifuge at 1,000 rpm for 5 minutes to remove air bubbles and transfer 25 uL of lysate supernatant to perform p-EGFR assay by using a commercial ELISA (R&D, DYC1095B-5).

To estimate the IC50, the absorption reading is converted to relative activity% by applying the following equation:% inhibition = (Abs _HC- Abs _cpd )/Abs _HC . Logarithmic curve, then IC ₅₀ was calculated by four-parameter fit to XLFit (IDBS, Guildford, Surrey) are.

4.3.1. Wild type EGFR A431 autophosphorylation inhibition assay (ELISA)

The medium for A431 cells was Dulbecco's modified Eagle's medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin. The DMSO dilution used for the A431 measurement is 11 points from 10 mM to 0.17 uM. After 2 hours of treatment with the test compound, 4.5 μL of EGF (1 μg/mL) was added to each well and stimulated for 10 minutes. The rest of the procedure was performed in the same manner as described for H1975 cells.

4.3.2 Exonl9 deletion EGFR (activated single mutant) PC-9 cell autophosphorylation assay

The human lung cell line PC9 (Exon 19 lacks EGFR) was obtained from the American Type Culture Collection. PC 9 cells were maintained in RPMI1640 containing 10% fetal bovine serum and 2 mM glutamine. The cells were grown in a humidified incubator at 37°C and 5% CO ₂ . The assay for measuring cell phosphorylation of endogenous p-EGFR in cell lysates was performed according to the protocol described in the R&D Systems DuoSet IC Human Phosphorylation-EGF R ELISA (R&D Systems catalog number #DYCI095). 40 μL of cells were seeded (10,000 cells/well) in growth medium in a Coming black transparent bottom 384-well plate, and incubated at 37° C. and 5% CO ₂ overnight. The cells were acoustically administered using Echo 555, where the compound was serially diluted in 100% DMSO. The plate was incubated for another 2 hours, then after aspirating the medium, 40 μL x lysis buffer was added to each well. Greiner black high-binding 384-well plates were coated with capture gas and then blocked with 3% BSA. After removing the block, 15 μL of lysate was transferred to Greiner black high binding 384-well plate and incubated for 2 hours. After aspirating and washing the plate with PBS, 20 μL of detection antibody was added and incubated for 2 hours. After aspirating and washing the plate with PBS, 20 μL of QuantaBlu fluorescent peroxidase substrate (Thermo Fisher Scientific Cat. No. 15169) was added and incubated for 1 hour. 20 μL of QuantaBlu stop solution was added to the plate and fluorescence was read on the Envision plate reader using excitation wavelength of 352 nm and emission wavelength of 460 nm. The data obtained from each compound is input into a suitable software package (eg Origin) to perform curve fitting analysis. From this data, the concentration of the compound required for 50% action was calculated to determine the IC ₅₀ value.

test results

Table 2 Cell EGFR autophosphorylation test results

Experimental results

The compound of formula (I) of the present invention can effectively inhibit the autophosphorylation of EGFR, and can effectively inhibit the autophosphorylation of EGFR mutants, thereby inhibiting the overexpression of EGFR and its mutants, and thereby inhibiting tumor proliferation.

Example 5: Investigation of the stability of the crystalline form I of compound L-(+)-tartrate of formula (I)

testing sample:

The compound L-(+)-tartrate crystal form I of formula (I) is prepared according to the method in the examples.

Inspection conditions:

Investigation condition 1: Put the test products under the conditions of 60℃-opening, 40℃-opening, 25℃RH75% opening, ultraviolet light-opening, respectively, on the 5th and 10th day and satisfy the total illuminance ≥1.2×10 ⁶ Lux·h, near ultraviolet energy ≥200w·h/m ² samples were taken to determine the content of related substances, XRD inspection was added on the 10th day, and compared with the 0 day sample.

Determination of related substances: According to the "Chinese Pharmacopoeia" 2015 edition of the four general rules 0512 high performance liquid chromatography.

XRD measurement: According to the "Chinese Pharmacopoeia" 2015 edition of four general principles 0981 second method X-ray powder diffraction method.

test results:

Table 3 Results of the investigation of the stability of Form I

*: Total illuminance ≥1.2×10 ⁶ Lux·h, near ultraviolet energy ≥200w·h/m ²

Test Conclusions:

The L-(+)-tartrate crystalline form I of the compound of formula (I), when placed under the above high-temperature and high-humidity conditions for 10 days and under conditions satisfying ultraviolet illuminance, the related substances and crystalline form XRD patterns of the samples are not obvious Variety. Crystal form I has good stability, which is beneficial to the preparation, transportation and storage of medicines, and basically meets the needs of medicines.

Example 6: Investigation of the hygroscopicity of Form I

Test article: L-(+)-tartrate crystal form I of the compound of formula (I).

experimental method:

1. Take a dry glass weighing bottle with a stopper, open the weighing bottle, and place it in the thermostat drier at 25°C±1°C with the cap (the lower part is a saturated ammonium chloride solution, the relative humidity is 80%±2 %), place for 24 hours, close the weighing bottle cap, and accurately weigh the weight (m ₁ ).

2. Take an appropriate amount of this product, lay it flat in the above weighing bottle, and cover with a precision weighing weight (m ₂ ).

3. Open the weighing bottle and place it under the constant temperature and constant humidity (25℃±1℃, RH80%±2%) condition for 24 hours with the bottle cap.

4. Close the weighing bottle cap and accurately weigh the weight (m ₃ ).

Experimental results:

Table 4 Results of the investigation of the hygroscopicity of Form I

testing sample	Placement conditions	Weight gain percentage (%) *
Form I	25℃±1℃, RH80%±2%, leave for 1 day	0.11

*:

Experimental results:

The L-(+)-tartrate crystal form I of the compound of formula (I) of the present invention has almost no hygroscopicity and good properties, which is convenient for the production of medicines, preparation, transportation and storage of preparations, which is conducive to ensuring the stability and safety of medicines .

Example 7: In vivo pharmacokinetic experiment of SD female rats of the compound of the present invention

Test article: L-(+)-tartrate crystal form I of the compound of formula (I).

Test animals: female SD rats, 6/administration route, body weight 204-233 g/animal.

Preparation of test solution:

Dissolution scheme:

Compound IV administration: 5% DMSO + 10% PEG400 + 85% (28% HP-β-CD);

Compound PO administration: 2% HPC + 0.1% Tween 80;

Preparation of blank solvent 1 (the preparation volume can be adjusted according to actual needs):

Preparation method of 28% HP-β-CD solution: Weigh about 28g of HP-β-CD, add sterile water for injection to a final volume of 100mL, and stir to dissolve.

Preparation of blank solvent 2 (the preparation volume can be adjusted according to actual needs):

Preparation of 2% HPC + 0.1% Tween 80: Weigh about 2 g of HPC (hydroxypropyl cellulose), add sterile water for injection to a final volume of 100 mL, then add 100 μL of Tween 80, and mix.

Specific preparation method:

① Weigh an appropriate amount of test sample (the actual sample size of the test sample = conversion factor × theoretical requirement of the test sample), add an appropriate amount of 2% HPC + 0.1% Tween 80, use a dispersing emulsifier to disperse it uniformly, set Rong, as an SD female rat by intragastric administration of the drug solution.

② Weigh an appropriate amount of test sample (the actual sample size of the test sample = conversion factor × theoretical requirement of the test sample), add the appropriate amount of DMSO and PEG400, vortex, and then add the appropriate amount of 28% HP-β-CD To the final volume, the volume ratio of DMSO: PEG400: 28% HP-β-CD in the final volume was 5:10:85, and the temperature was kept in a 50°C water bath for 30 minutes, filtered with a 0.22 μm PTFE filter, and administered intravenously as SD female rats Liquid medicine.

experimental method

Administration:

The test drug solution is administered according to the method in Table 5 below:

table 5

Blood sample collection: Animals collect whole blood (about 0.3 mL) in the jugular vein for pharmacokinetic analysis.

Time point of blood collection for IV administration animals: before administration, 2min, 10min, 30min, 1h, 2h, 4h, 8h, 24h after administration.

Time point of blood collection for PO-administered animals: before administration, 15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h, 72h after administration.

The centrifuge tube was coated with K2-EDTA and stored in an ice-water bath; after the blood sample was collected, it was transferred to the above-mentioned centrifuge tube, mixed manually, and the centrifugation was completed within 1 hour. The whole blood sample was stored in an ice-water bath before centrifugation. 10min. After centrifugation, the collected plasma was divided into 2 tubes and stored below -70℃.

Plasma sample analysis

The plasma samples of the compounds were analyzed by protein precipitation method, and the validated LC-MS/MS method was used for detection and analysis.

Table 6 PK evaluation results of female SD rats

AUC _last represents the area under the drug-time curve 0→t; AUC _inf represents the area under the drug-time curve 0→∞; CL represents the clearance rate; V _ss represents the steady-state apparent volume of distribution; T _max represents the time when the blood drug concentration reaches the peak; C _max represents the peak concentration of the blood drug concentration; t _1/2 represents the half-life; MRT represents the average residence time; F% represents the absolute bioavailability

According to the experimental results in Table 6, the pharmacokinetic properties of the compound of the present invention are good. After administration by intragastric administration of 5 mg/kg intravenously and 10 mg/kg, t _1/2 is 4.21 and 9.09 h, respectively; the exposure is 7038.37 and 8202.03h*ng/mL, oral bioavailability is 58.27%.

The above is only a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims (17)

1. The tartrate of the compound represented by formula (I),

   
2. The tartrate salt of the compound according to claim 1, wherein the molar ratio of the compound of formula (I) to tartaric acid is 1:3 to 1:1, preferably 1:1.
3. The tartrate according to claim 1 or 2, wherein the tartaric acid is L-(+)-tartaric acid.
4. L-(+)-tartrate crystal form I of a compound of formula (I),

   

   It is characterized in that the molar ratio of the compound of formula (I) to L-(+)-tartaric acid is 1:1, and using Cu-Kα radiation, the X-ray powder diffraction pattern expressed at a 2θ angle, at 8.9±0.2°, 11.2±0.2°, 12.0±0.2°, 12.5±0.2°, 13.1±0.2°, 14.7±0.2°, 17.5±0.2°, 19.1±0.2° have characteristic peaks.
5. The crystalline form I of tartrate according to claim 4, characterized in that, using Cu-Kα radiation, the X-ray powder diffraction pattern expressed at a 2θ angle is also 16.3±0.2°, 16.6±0.2°, 20.0±0.2 There are characteristic peaks at °, 20.6±0.2°, 21.1±0.2°, and 21.6±0.2°.
6. The tartrate crystalline form I according to claim 5, characterized in that its X-ray powder diffraction pattern is substantially as shown in FIG.
7. The crystalline form I of tartrate according to claim 4, wherein the differential scanning calorimetry analysis chart has an endothermic peak in the range of 190°C to 230°C; preferably, the maximum endothermic transition temperature is 215.5±5 °C; more preferably, the crystalline form A has a differential scanning calorimetry curve substantially as shown in FIG. 2.
8. The method for preparing crystalline form I of tartrate according to claim 4, characterized in that the compound of formula (I) and 1 equivalent of L-(+)-tartaric acid are added to an organic solvent, and water or N- is added under stirring Methylpyrrolidone, filtered after the reaction is completed, and dried to obtain L-(+)-tartrate crystal form I of the compound of formula (I);

   Preferably, the organic solvent is selected from lower nitriles, lower alcohols containing 2 or more carbon atoms, oxygen-containing heterocyclics, or any mixed solvent of the above organic solvents;

   More preferably, the organic solvent is selected from acetonitrile, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, 1,4-dioxane, or any mixed solvent of the above organic solvents.
9. The method for preparing crystalline form I of tartrate according to claim 4, characterized in that the compound of formula (I) is added to a single solvent or a mixed solvent, after heating to a certain temperature or under normal temperature slurry washing conditions, a certain equivalent of L is added -(+)-tartaric acid, maintain the temperature and continue the reaction to the end of the reaction, lower the temperature or crystallize at room temperature, separate, and dry to obtain L-(+)-tartrate crystal form I of the compound of formula (I);

   Preferably, the single solvent is selected from lower alcohols, lower ketones, lower esters, oxygenated heterocyclics, lower nitriles, lower alkanes or lower halogenated alkanes, and the mixed solvent is selected from alcohols Mixed solvent with water, mixed solvent with nitriles and water, mixed solvent with ketones and water, or mixed solvent with oxygenated heterocyclics and water.
10. The preparation method according to claim 9, wherein the single solvent is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, acetone, butanone, methyl Ethyl ketone, acetonitrile, ethyl formate, ethyl acetate, methyl acetate, tetrahydrofuran, 1,4-dioxane, n-pentane, isopentane, n-hexane, isohexane, cyclohexane, cycloheptane , Toluene or methylene chloride; the mixed solvent is selected from ethanol/water, propanol/water, isopropanol/water, butanol/water, sec-butanol/water, tert-butanol/water, acetonitrile/water, 1 ,4-dioxane/water or acetone/water;

    Preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1-20:1, further preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 1:1-10:1 More preferably, in the mixed solvent, the ratio of organic solvent to water is selected from 2:1-10:1; the certain temperature is selected from 40°C-80°C, preferably 50°C-70°C.
11. The preparation method according to claim 9, wherein the certain equivalent is selected from 0.9-1.2 equivalent, preferably 1.0-1.1 equivalent, more preferably 1.0 equivalent.
12. L-(+)-tartrate crystal form II of a compound of formula (I),

    

    It is characterized in that the molar ratio of the compound of formula (I) to tartaric acid is 1:1, and the Cu-Kα radiation is used to express the X-ray powder diffraction pattern at an angle of 2θ at 9.0±0.2°, 11.3±0.2°, 12.0 There are characteristic peaks at ±0.2°, 12.7±0.2°, 13.9±0.2°, 17.9±0.2°, 19.1±0.2°, 20.3±0.2°, 21.2±0.2°.
13. The crystalline form II according to claim 12, wherein the X-ray powder diffraction pattern expressed at an angle of 2θ using Cu-Kα radiation is also 16.3±0.2°, 17.3±0.2°, 23.5±0.2°, There are characteristic peaks at 24.4±0.2°, 25.5±0.2°, and 25.9±0.2°; preferably, the X-ray powder diffraction pattern of the crystal form II is basically shown in FIG. 5.
14. A pharmaceutical composition containing a tartrate salt of a compound represented by formula (I) according to any one of claims 1-3, and a compound represented by formula (I) according to any one of claims 4-7 The tartrate crystal form I or the tartrate crystal form II of the compound represented by formula (I) according to any one of claims 12-13 and one or more pharmaceutically acceptable carriers, the pharmaceutical composition can be prepared into a pharmaceutical Any dosage form that is acceptable.
15. A tartrate salt of the compound represented by formula (I) according to any one of claims 1-3, or a tartrate crystal form I of the compound represented by formula (I) according to any one of claims 4-7, or the right The use of the tartrate crystal form II of the compound represented by formula (I) according to any one of claims 12 to 13 in the preparation of a medicament for the treatment and/or prevention of EGFR-mediated hyperproliferative diseases, said The hyperproliferative diseases are selected from cancer.
16. The use according to claim 15, wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, liver cancer, gastric cancer, prostate cancer, gum Glioblastoma and epithelial cell carcinoma.
17. The use according to claim 15 or 16, wherein the EGFR is selected from wild type EGFR and mutant EGFR, preferably, mutant EGFR includes EGFR 19 exon mutation, EGFR 20 exon mutation and EGFR One or two or more mutations among the 21 exon mutations, more preferably, the EGFR 20 exon mutation is selected from NPG, ASV, or T790M, and the EGFR 19 exon mutation is selected from insertion Mutation, the EGFR 21 exon mutation is selected from point mutations.

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