CN111410651B

CN111410651B - Salts of tyrosine kinase inhibitors and crystalline forms thereof

Info

Publication number: CN111410651B
Application number: CN201910009711.8A
Authority: CN
Inventors: 王海波; 徐艳君; 吴笃信
Original assignee: Shandong Xuanzhu Pharma Co Ltd
Current assignee: Shandong Xuanzhu Pharma Co Ltd
Priority date: 2019-01-05
Filing date: 2019-01-05
Publication date: 2021-06-04
Anticipated expiration: 2039-01-05
Also published as: CN111410651A

Abstract

The invention relates to a pharmaceutically acceptable salt of N- (5- ((4- (7-cyano-1, 3-dimethyl-1H-indol-5-yl) pyrimidine-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide shown in the following formula (I), in particular to a mesylate and a crystal form of the mesylate of a compound shown in the formula (I), a preparation method thereof, a pharmaceutical preparation containing the salt and the crystal form, and an application thereof in preparing a medicament for treating and/or preventing diseases mediated by EGFR.

Description

Salts of tyrosine kinase inhibitors and crystalline forms thereof

1. Field of the invention

The invention relates to salts of selective inhibitors of EGFR tyrosine kinase and mutants thereof, crystal forms thereof, a preparation method, pharmaceutical compositions containing the salts and the crystal forms of the inhibitors, and applications of the salts and the crystal forms in preparation of medicines for treating and/or preventing abnormal cell proliferation diseases.

2. Background of the invention

Protein Kinases (PKs) are very important signaling entities in intracellular communication, where they modify many proteins by catalyzing the transport of phosphate groups from ATP, which acts as a phosphorus donor, to phenolic hydroxyl groups on protein tyrosine side chains. Sometimes, tyrosine kinases are incorporated into the intracellular domain of very large transmembrane proteins with homologous ligand binding domains in the extracellular domain, whereby ligand binding activates the tyrosine kinase intracellularly. Such molecules are Receptor Tyrosine Kinases (RTKs).

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

When following responders to gefitinib and erlotinib, it was found that the onset of resistance may be associated with several different genetic changes. Although in rare cases tumors seem to pick a completely different signaling system to drive them, resistance usually involves a distortion of the original system. EGFR is a member of the erbB (type I) subfamily of RTKs, along with erbB-2, erbB-3 and erbB-4. These receptors are activated by ligands that induce their dimerization, and while EGFR-EGFR homodimers are commonly used for signaling, the more common process in this family is ligand-induced heterodimerization, such that the signaling entities will be, for example, EGFR: erbB-2 or erb-B2: erbB-3 and the appropriate ligands. The simplest way to reactivate the system is to increase the expression of one of the other erbB, 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. One somewhat related mechanism involves the RTK HGFR, which although not an erbB family member, has been shown to form tumorigenic heterodimers with erbB family members, particularly erbB-3, upon overexpression, and overexpression of HGFR is a common mechanism for resistance to EGFR inhibitors. Addition of HGFR inhibitor to these cells will restore sensitivity to EGFR inhibitors, at least in a laboratory setting. The third and most common mode of resistance is further mutation of EGFR, resulting in a double mutant receptor (dm-EGFR) that reduces its sensitivity to EGFR inhibitors. The most common of these is the so-called "gatekeeper" mutation, T790M, and NSCLC with a double mutant of, for example, L858R/T790M is common in initial responders who subsequently develop resistance to EGFR inhibitors. Although it is not known whether such subclones are consistently present or only present after treatment, it appears most likely that mutations are already present in short-term responders and may appear as re-mutations in later long-term responders developing resistance.

Novel compounds that selectively modulate the activity of certain mutant forms of protein kinases, especially the type I Receptor Tyrosine Kinase (RTK) family or the erbB family, more especially the EGFR receptor, that are resistant to current EGFR-based inhibition therapies, and that are useful for inhibiting cell proliferation and cell invasion, inhibiting metastasis, inducing apoptosis, or inhibiting angiogenesis, are 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 described by formula (I) below.

The research on the crystal form plays an important role in the research and development process of the medicine, the compounds with different forms have different physical and chemical properties such as bioavailability, solubility, stability and the like, and in order to meet the requirements of the conditions such as preparation, production, transportation and the like, the salt of the compound shown in the formula (I) and the crystal form thereof are researched so as to discover the crystal form with good properties.

3. Summary of the invention

The invention provides a mesylate of an EGFR inhibitor N- (5- ((4- (7-cyano-1, 3-dimethyl-1H-indol-5-yl) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) methylamino) -4-methoxyphenyl) acrylamide shown as a formula (I).

The invention also provides a mesylate crystal form I (hereinafter referred to as crystal form I) of an EGFR inhibitor N- (5- ((4- (7-cyano-1, 3-dimethyl-1H-indol-5-yl) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) methylamino) -4-methoxyphenyl) acrylamide shown in the formula (I).

The invention also provides a preparation method of the mesylate crystal form I of the compound shown in the formula (I), a pharmaceutical composition containing the mesylate of the compound shown in the formula (I) and the crystal form I of the mesylate, and an application of the mesylate in preparing a medicament for preventing and/or treating abnormal cell proliferation diseases.

The present invention provides a mesylate salt of a compound of formula (I),

in some embodiments, the mesylate salt of the compound of formula (I) has a molar ratio of the compound of formula (I) to methanesulfonic acid of from 1:3 to 1:1, preferably 1: 1.

The invention also provides a mesylate crystal form I of the compound shown as the formula (I),

wherein the molar ratio of the compound of the formula (I) to methanesulfonic acid is 1:1, and an X-ray powder diffraction pattern expressed by 2 theta angles using Cu-Kalpha radiation has characteristic peaks at 10.0 + -0.2 DEG, 13.2 + -0.2 DEG, 17.6 + -0.2 DEG, 22.0 + -0.2 DEG, and 23.3 + -0.2 deg.

In some embodiments, said form I further has characteristic peaks at 14.8 ± 0.2 °, 20.2 ± 0.2 °, 26.1 ± 0.2 °, 29.3 ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-ka radiation, based on the inclusion of the above characteristic peaks.

In some preferred embodiments, said form I, on the basis of comprising the above characteristic peaks, is further at 11.6 ± 0.2 °, 11.8 ± 0.2 °, 18.1 ± 0.2 °, 18.3 ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-ka radiation.

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

In some embodiments, the form I has a differential scanning calorimetry (DSC profile) with an endothermic peak in the range of 220 ℃ to 250 ℃. In some embodiments, the DSC profile of form I has a maximum endothermic transition temperature of 238.1 ± 5 ℃. In some embodiments, the crystalline form I has a differential scanning calorimetry curve substantially as shown in figure 2.

In some embodiments, the thermogravimetric analysis (TGA) profile of form I shows no significant weight loss at 0 ℃ to 220 ℃. In some embodiments, the crystalline form I has a TGA profile substantially as shown in figure 3.

On the other hand, the invention also provides a preparation method of the mesylate crystal form I of the compound shown in the formula (I), which comprises the steps of adding the compound shown in the formula (I) into a single solvent or a mixed solvent, heating to a certain temperature, adding a certain equivalent of methanesulfonic acid or a solution containing a certain equivalent of methanesulfonic acid, maintaining the temperature, continuing to react until the reaction is finished, cooling, crystallizing, separating, and drying to obtain the mesylate crystal form I of the compound shown in the formula (I).

In some preferred embodiments, the single solvent described in the above preparation method is selected from the group consisting of lower nitriles; the mixed solvent is selected from nitrile and water mixed solvents.

In some preferred embodiments, the solution of methanesulfonic acid in the above-described production method is selected from solutions of methanesulfonic acid and lower nitriles.

In some preferred embodiments, the single solvent in the above preparation method is selected from acetonitrile; the mixed solvent is selected from acetonitrile/water.

In some preferred embodiments, the volume of water in the mixed solvent is not more than 5% of the total volume of the solvent; preferably, the volume of water in the mixed solvent is not more than 3% of the total volume of the solvent; more preferably, the volume of water in the mixed solvent is not more than 1% of the total volume of the solvent.

In some preferred embodiments, the certain temperature described in the above preparation method is selected from the range of 40 ℃ to 80 ℃, preferably 50 ℃ to 60 ℃.

In some preferred embodiments, the certain temperature described in the above preparation method is selected from the temperatures required for heating reflux. In some preferred embodiments, the certain equivalent weight described in the above preparation method is selected from 0.9 to 1.2 equivalent, preferably 1.0 to 1.1 equivalent, more preferably 1.0 equivalent.

The invention also provides a pharmaceutical composition of the mesylate of the compound of the formula (I) or the crystal form I thereof and one or more pharmaceutical carriers and/or diluents, wherein the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form and is administered to a patient in need thereof by oral, parenteral, rectal or pulmonary administration and the like. For oral administration, it can be made into conventional solid preparations such as tablet, capsule, pill, granule, etc.; it can also be made into oral liquid, such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, appropriate filler, binder, disintegrating agent, lubricant, etc. can be added. For parenteral administration, it can be made into injection, including injection solution, sterile powder for injection and concentrated solution for injection. The injection can be prepared by conventional method in the existing pharmaceutical field, and can be prepared without adding additives or adding appropriate additives according to the properties of the medicine. For rectal administration, it can be made into suppository, etc. For pulmonary administration, it can be made into inhalant or spray.

The invention also provides the use of the mesylate salt of the compound of formula (I) or the crystalline form I thereof 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 the treatment and/or prevention of a hyperproliferative disease mediated by EGFR comprising the step of administering to a subject in need thereof an effective amount of the mesylate salt of the compound of formula (I) or crystalline form I thereof.

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 EGFR mutant comprises any combination of one or more of an EGFR 19 exon mutation, an EGFR20 exon mutation and an EGFR 21 exon mutation.

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

In some embodiments, the cancer 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, and epithelial cell cancer.

In some preferred embodiments, the cancer is caused by an exon 20 mutation of EGFR.

In some preferred embodiments, the cancer is caused by a T790M mutation in an EGFR exon 20 mutation, together with an EGFR exon 19 insertion mutation or an EGFR exon 21 point mutation.

The present disclosure also provides the use of the mesylate salt of the compound of formula (I) or crystalline form I thereof in the preparation of a reagent useful for inhibiting the level of EGFR enzyme in a cell. In some preferred embodiments, the agents are used in vivo or in vitro methods.

The present disclosure also provides a method of inhibiting EGFR levels in a cell comprising the step of administering to the cell an effective amount of a mesylate salt of a compound of formula (I) or crystalline form I thereof. In some preferred embodiments, the method is performed in vivo or in vitro.

Unless defined otherwise in the present invention, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill 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 nitriles" refers to nitrile compounds containing 1 to 6 carbon atoms, and examples thereof include, but are not limited to: acetonitrile, propionitrile, and adiponitrile.

In the present invention, the term "separation" means separation of solids by a conventional method such as filtration, centrifugation and discarding of supernatant, etc.

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

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. The drying temperature is room temperature or 30-80 ℃, preferably 40-80 ℃, preferably 50-80 ℃, preferably 60-80 ℃. Room temperature refers to the ambient room temperature and typically refers to 10-30 c, e.g., 20-25 c.

In the present invention, the term "temperature reduction and crystallization" means that the temperature of the reaction system is reduced to 0 to 35 ℃, preferably to 10 to 30 ℃, more preferably to 15 to 25 ℃, and even more preferably to a temperature at which most of crystals are precipitated, by means of cooling with an ice water bath, self-heating cooling, cooling with a refrigeration apparatus, or the like.

The compounds may exist in two or more crystalline states, molecules of the same structure, crystallized in different solid forms, referred to as polymorphs or polymorphs, and the like. When referring to a particular crystalline form, it is often referred to as "crystal form", that is, the term "crystalline form" as used herein.

In the present disclosure, the position of the absorption peak in each crystalline form X-ray powder diffraction pattern may be within a range of ± 0.2 °, for example, within a range of ± 0.1 ° of the specific value of the above invention. .

It should be understood that slightly different XRPD patterns and peaks may be given with different types of equipment or with different test conditions. The spectra, peak values and relative intensities of the various diffraction peaks of the different crystal forms will be affected by the purity of the compound, the pre-treatment of the sample, the scanning speed, the particle size and the calibration and maintenance of the test equipment. The values provided cannot be taken as absolute values.

It will be appreciated that somewhat different DSC profiles and endothermic transition temperature readings may be given with different types of equipment or with different test conditions. This number will be affected by compound purity, sample weight, heating rate, particle size, and check-up and maintenance of the test equipment. The endothermic transition temperature as determined by differential scanning calorimetry can be within a range of + -5.0 deg.C (e.g. + -3.0 deg.C or + -2.0 deg.C) of the specific values disclosed above.

The present disclosure also analyzes the relationship between the degree of decomposition or sublimation of the crystalline form, evaporation (weight loss) and temperature using thermogravimetric analysis (TGA). It should be understood that the same crystal form is affected by the purity of the sample, the particle size, different types of equipment, different testing methods, etc., and that there is a certain error in the obtained numerical values. The temperature at which the crystalline form decomposes or sublimes, evaporates may be within a range of + -3.0 deg.C, such as + -2.0 deg.C, of the specific values disclosed above.

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 prophylactic effect, e.g., an amount that achieves alleviation of symptoms associated with the disease being treated.

In the present invention, the term "treatment" is intended to reduce or eliminate the disease state or condition for which it is directed. A subject is successfully "treated" if the subject, following the methods described herein, receives a therapeutic amount of the crystalline form or pharmaceutical composition thereof and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms. It is also understood that treatment of the disease state or condition described includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.

The main advantages of the compounds of formula (I), their mesylate salts and their mesylate salt forms of the invention include:

(1) the compound of the formula (I), the mesylate and the mesylate crystal form thereof have excellent EGFR (epidermal growth factor receptor) inhibition activity, particularly have high selectivity and inhibition activity on certain mutation forms of an EGFR receptor, and can effectively inhibit cell proliferation and cell invasion, inhibit metastasis, induce apoptosis or inhibit angiogenesis.

(2) The compound of the formula (I), the mesylate and the mesylate crystal form thereof have small toxic and side effects, and particularly have small side effects on aspects of increasing blood sugar, cardiotoxicity and the like.

(3) The compound of the formula (I), the mesylate and the mesylate crystal form thereof have good pharmacokinetic properties, excellent half-life, higher exposure and other properties, and good pharmacy.

(4) The mesylate of the compound shown in the formula (I) has good crystal form stability, good properties, fluidity and compressibility, and is convenient for detection, preparation, transportation and storage;

(5) the mesylate crystal form of the compound shown in the formula (I) has high purity, less residual solvent, higher solubility, good dissolution rate and easily controlled quality;

drawings

Figure 1 is an X-ray powder diffraction pattern of the mesylate salt form I of the compound of formula (I) with diffraction intensity (intensity) on the ordinate and diffraction angle (2 θ) on the abscissa.

FIG. 2 is a Differential Scanning Calorimetry (DSC) thermogram of the mesylate salt form I of the compound of formula (I) with heat flow (W/g) on the ordinate and temperature (. degree. C.) on the abscissa.

Figure 3 is a thermogravimetric analysis (TGA) curve of the mesylate salt form I of the compound of formula (I) with weight percent (%) on the left ordinate, the derivative of weight (%/deg.c) on the right ordinate and temperature (deg.c) on the abscissa.

FIG. 4 is a hydrogen spectrum of the compound of formula (I).

FIG. 5 is an X-ray powder diffraction pattern of the mesylate amorphous form of the compound of formula (I) with diffraction intensity (intensity) on the ordinate and diffraction angle (2 θ) on the abscissa.

4. Detailed description of the preferred embodiments

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

EXAMPLE 1 preparation of the Compound of formula (I)

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

5-bromo-3-methyl-1H-indole-7-carbonitrile (1.17g,5mmol) was dissolved in tetrahydrofuran (20mL), cooled to 0 deg.C, and NaH (260mg,6.5mmol) was added portionwise. After addition, stirred for 30 min at 0 ℃ and then MeI (781mg,5.5mmol) was slowly added dropwise. After dropping, the reaction mixture was naturally warmed to room temperature and stirred for two hours, quenched with water carefully, extracted with ethyl acetate (20 mL. times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a crude product, which was purified by column chromatography to give the objective compound (694mg, yield: 56%).¹HNMR(300MHz,CDCl₃):δ7.87(d,J＝1.5Hz,1H),7.60(d,J＝1.5Hz,1H),6.88(s,1H),4.06(s,3H),2.28(s,3H).

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

The compound 5-bromo-1, 3-dimethyl-1H-indole-7-carbonitrile (523mg, 2.1mmol, 1.0eq) was dissolved in dioxane (5mL), pinacolato diboron (592mg,2.3mmol,1.1eq), potassium acetate (125mg,6.3mol,3eq), Pd (dppf) Cl was added₂(124mg, 0.168mmol,0.08eq) was substituted with nitrogen for 3 times, and then heated to 85 ℃ for 6 hours. TLC, LCMS detection, after the reaction is completed, suction filtration, mother liquor concentration, column chromatography purification to obtain the target compound (350mg, yield: 56%).¹HNMR(300MHz,CDCl₃):δ8.23(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 the protection of nitrogen, the compound 1, 3-dimethyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-indole-7-carbonitrile (296mg, 1mmol, 1.0eq) was dissolved in dioxane and water (5/1mL), and 2, 4-dichloropyrimidine (162mg, 1.1mmol, 1.1eq), Pd (PPh) and the like were added thereto, respectively₃)₄(115mg, 0.1mmol,0.1eq), potassium carbonate (411mg,3mmol,3eq) and then heated to 100 ℃ for reaction for 3 hours. TLC, LCMS detection, after the reaction is completed, suction filtration, concentration of filtrate and column chromatography purification are carried out to obtain the target compound (164mg, yield: 58%).

¹HNMR(300MHz,DMSO-d₆):δ8.65(d,J＝5.1Hz,1H),8.55(s,1H),8.29(s,1H),7.70(d,J＝5.1Hz,1H),6.95(s,1H),4.13(s,3H),2.35(s,3H).

(4) Preparation of Compounds of formula (I)

Dissolving 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) in 2-pentanol (4mL), adding p-toluenesulfonic acid monohydrate (123mg,0.64mmol,1.1eq) in portions at room temperature, heating to 120 ℃ for 5 hours after completion of reaction, detecting by TLC and LCMS, cooling to room temperature after completion of reaction, adding water (10mL), dichloromethane/methanol ═ 10:1(10 mL. times.3) for extraction, washing the organic phase with brine (10mL), drying, concentrating, purifying by column chromatography to obtain compound (48 mg) of formula (I), yield: 15%).

¹HNMR(300MHz,DMSO-d₆):δ10.19(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 the mesylate salt of the Compound of formula (I)

Adding a compound (300mg,0.56mmol) of the formula (I), acetone (1.8mL) and water (1.2mL) into a 10mL single-neck flask, heating to 45-55 ℃ under stirring, dropwise adding an acetone solution (0.6mL) of methanesulfonic acid (53.5mg,0.56mmol), reacting at 54 ℃ for 2.5 hours, cooling, concentrating, adding acetone into the concentrate, filtering, leaching and drying in vacuum at 60 ℃ to obtain yellow solid powder.

The solid was amorphous as determined by XRPD and the pattern is shown in figure 5.

¹HNMR(400MHz,DMSO-d6)：δ9.54(s,1H),8.74(s,1H),8.68(d,1H),8.50(s,1H),8.48(s,1H),8.24(s,1H),7.60(d,1H),7.32(s,1H),7.01(s,1H),6.7(m,1H),6.33(m,1H),6.29(m,1H),5.79(m,1H),4.04(s,3H),3.28(m,4H),2.81(s,3H),2.81(s,3H),2.61(s,3H),2.25(s,3H),2.33(s,3H),2.30(s,3H).

EXAMPLE 3 preparation of the mesylate salt form I of the Compound of formula (I)

The preparation method comprises the following steps:

placing a compound (100mg and 0.186mmol) of the formula (I) into a 10mL single-neck flask, adding acetonitrile (1.82mL) and water (18.2uL), heating to 50-60 ℃, dropwise adding an acetonitrile solution (91uL) containing methanesulfonic acid (17.8mg and 0.186mmol), maintaining the temperature to continue reacting until the reaction is finished, cooling, filtering, and vacuum-drying at 60 ℃, wherein the obtained solid (58.4mg and the yield is 49.5%) is tested as a crystal form I by XRPD.

Preparation method II

Placing the compound (1g,1.86mmol) of the formula (I) in a 20mL single-neck flask, adding acetonitrile (9.1mL), stirring, heating to 50-60 ℃, dropwise adding an acetonitrile solution (0.91mL) containing methanesulfonic acid (0.178g,1.86mmol), keeping the temperature and stirring until the reaction is finished, cooling, filtering, and vacuum drying at 80 ℃. The resulting solid (1.05g, yield 89.1%) was tested by XRPD as form I.

The crystal form I prepared by the above method was tested:

x-ray powder diffraction measurement

The crystal structure of the present invention is not limited to a crystal structure providing an X-ray powder diffraction pattern identical to that depicted in the drawings disclosed in the present application, and any crystal structure providing an X-ray powder diffraction pattern substantially identical to those disclosed in the drawings is included in the scope of the present invention.

Conditions for X-ray powder diffraction measurement: a Cu-Pd alloy containing a Cu-Pd alloy,

1.54060, step size 0.0203, steps 0.3 seconds each.

The crystal form of X-ray powder diffraction has characteristic peak in 2 theta angle (degree) by Cu-Kalpha radiation.

An X-ray powder diffraction pattern of the mesylate salt form I of the compound of formula (I) having peaks at the following diffraction angles 2 Θ (°) is shown in fig. 1:

10.0±0.2°、13.2±0.2°、17.6±0.2°、22.0±0.2°、23.3±0.2°、14.8±0.2°、20.2±0.2°、26.1±0.2°、29.3±0.2°、11.6±0.2°、11.8±0.2°、18.1±0.2°、18.3±0.2°。

differential scanning calorimetry

The solid state thermal properties of the mesylate salt form I of the compound of formula (I) were studied by Differential Scanning Calorimetry (DSC). The DSC curve for form I is shown in figure 2.

The measurement conditions were as follows: the nitrogen purge was used at 50 ml/min, the temperature was equilibrated at 25 ℃ and then raised to 250 ℃ at a heating rate of 10 ℃/min, and the data was collected and plotted with the endothermic peak facing downward.

In DSC measurement, there is a degree of variability in the actual measured starting and maximum temperatures, depending on the measured parameters and the heating rate.

Thermogravimetric analysis

And (3) testing conditions are as follows: the nitrogen was purged at 60 ml/min and data was collected at a 10 c/min heating rate between room temperature and 300 c.

The TGA profile of the mesylate salt form I of the compound of formula (I) is shown in figure 3.

EXAMPLE 4 examination of EGFR and tumor inhibitory Activity of Compounds of formula (I)

Abbreviations

DMSO dimethyl sulfoxide

DTT dithiothreitol

ATP adenosine triphosphate

EDTA ethylene diamine tetraacetic acid

Ki enzyme inhibition constant

DMEM Darber's modified eagle's medium

NCS newborn calf serum

PBS phosphate buffered saline

PMSF phenyl methane sulfonyl fluoride

ELISA enzyme-linked immunosorbent assay

IgG immunoglobulin G

FBS fetal bovine serum

BDNF brain-derived neurotrophic factor

Example 4.1 EGFR kinase inhibition assay

Inhibition of kinases by the 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 useful for measuring inhibition of a variety of 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 the compounds of formula (I-VIII) was measured by means of the following assay.

Purification of epidermal growth factor receptor tyrosine kinase

Human EGF receptor tyrosine kinase was isolated from a431 human squamous cell carcinoma cells overexpressing the EGF receptor by the following method. Cells were grown in 50% Darber modified eagle and 50% HAM F-12 nutrient medium (Gibco) in roller bottles containing 10% fetal bovine serum. Approximately 109 cells were lysed in two volumes of buffer containing: 20mM2- (4N- [ 2-hydroxyethyl ] piperazin-1-yl) ethanesulfonic acid (hepes) pH 7.4, 5mM ethylene glycol bis (2-aminoethyl ether) N, N, N ', N' -tetraacetic acid, 1% Triton X-lOO, 10% glycerol, 0.1mM sodium orthovanadate, 5mM sodium fluoride, 4mM pyrophosphate, 4mM benzamide, 1mM dithiothreitol, 80. mu.g/mL aprotinin, 40. mu.g/mL leupeptin, and 1mM phenylmethylsulfonyl fluoride. After centrifugation at 25,000 Xg for 10 minutes, the supernatant was equilibrated at 40 ℃ with 10mL of wheat embryo agglutinated Soranose previously equilibrated with 50mM Hepes, 10% glycerol, 0.1% Triton X-100, and 150mM NaCl, pH 7.5 (equilibration buffer), for 2 hours. The contaminating proteins were washed from the resin with equilibration buffer containing 1M NaCl, and the enzyme was eluted with equilibration buffer containing 0.5M N-acetyl-1-D-glucosamine followed by 1mM urea. The enzyme was eluted with 0.1mg/ml EGF. The receptor appeared to be homogeneous as assessed by coomassie blue stained polyacrylamide electrophoresis gel.

Using the same techniques as described in the previous paragraph, various mutant forms of the epidermal growth factor receptor can be isolated from an appropriate cell line containing the epidermal growth factor receptor. For example, the EGFR del746-750 mutein can be extracted from PC-9 cells, and the L858R/T790M double mutant EGFR protein can be isolated from H1975 cells.

IC of Single mutant EGFR _ d746-750₅₀Determination of value

For determining IC₅₀The enzyme assay of (4) was performed in a total volume of 25. mu.L. All compounds were diluted in 100% DMSO to 500 μ M stock solution and serially diluted 4-fold to achieve 10 doses. The "Max" and "Min" controls contained 100% DMSO. "Max" represents DMSO control without enzyme, and "Min" represents low pair without compoundAnd (6) irradiating. Mu.l of compound was transferred to 90. mu.l of 1 Xkinase base buffer for intermediate dilution. Mu.l of the intermediate diluted compound was transferred to a 384 well assay plate, followed by the addition of 10. mu.l of 2.5 Xenzyme buffer containing (12.5nM EGFR _ d746-750, 5mMDTT, 1 Xkinase base buffer) to the assay plate. Incubate at room temperature for 10 min and add 10. mu.l of a buffer containing (7.5. mu.M peptide, 35. mu.M ATP, 25mM MgCl)₂1x kinase base buffer) to initiate the reaction. The reaction was terminated by incubation at room temperature for 1 hour and addition of 25. mu.l of stop buffer. Translation data is collected from the Caliper and translation data is collected from the Caliper program. Fitting the data in XLFit to obtain IC₅₀The value is obtained.

IC of double mutant EGFR (EGFR _ T790M/L858R)₅₀Determination of value

For determining IC₅₀The enzyme assay of (4) was performed in a total volume of 25. mu.L. All compounds were diluted in 100% DMSO to 500 μ M stock solutions and serially diluted 4-fold to achieve 10 doses. The "Max" and "Min" controls contained 100% DMSO. "Max" represents DMSO control without enzyme and "Min" represents low control without compound. Mu.l of compound was transferred to 90. mu.l of 1 Xkinase base buffer for intermediate dilution. Mu.l of the intermediate diluted compound was transferred to a 384 well assay plate, followed by the addition of 10. mu.l of 2.5 Xenzyme buffer containing (25nM EGFR _ T790M/L858R, 5mM DTT, 1 Xkinase base buffer) to the assay plate. Incubate at room temperature for 10 min and add 10. mu.l of a buffer containing (7.5. mu.M peptide, 47.5. mu.M ATP, 25mM MgCl)₂1x kinase base buffer) to initiate the reaction. The reaction was terminated by incubation at room temperature for 1 hour and addition of 25. mu.l of stop buffer. Translation data is collected from the Caliper and translation data is collected from the Caliper program. Fitting the data in XLFit to obtain IC₅₀The value is obtained.

IC of wt EGFR₅₀Determination of value

For determining IC₅₀The enzyme assay of (4) was performed in a total volume of 25. mu.L. All compounds were diluted in 100% DMSO to 500 μ M stock solutions and serially diluted 4-fold to achieve 10 doses. The "Max" and "Min" controls contained 100% DMSO. "Max" represents DMSO control without enzyme, "Min" represents no compoundLow control of (2). Mu.l of compound was transferred to 90. mu.l of 1 Xkinase base buffer for intermediate dilution. Mu.l of the intermediate diluted compound was transferred to a 384 well assay plate, followed by the addition of 10. mu.l of 2.5 Xenzyme buffer containing (20nM EGFR, 5mM DTT, 1 Xkinase base buffer) to the assay plate. Incubate at room temperature for 10 min and add 10. mu.l of a solution containing (7.5. mu.M peptide, 5.75. mu.M ATP, 25mM MgCl2, 25mM MnCl)₂1x kinase base buffer) to initiate the reaction. The reaction was terminated by incubation at room temperature for 1 hour and addition of 25. mu.l of stop buffer. Translation data is collected from the Caliper and translation data is collected from the Caliper program. Fitting the data in XLFit to obtain IC₅₀The value is obtained.

Example 4.2 EGFR cell anti-proliferation assay

Test article

Compounds of formula (I), prepared according to the methods in the examples; the compound AZD9291 has the following structure and is prepared according to prior art methods.

Test procedure

H1975 inhibition assay (cell proliferation).

H1975 cells were cryopreserved in liquid nitrogen. Before cell thawing, 15mL of cell culture medium (RPMI 1640 medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin) was placed in a T75 flask and humidified at 37 ℃/5% CO₂The flasks were pre-incubated in the incubator for 15 minutes to allow the medium to equilibrate to the appropriate pH and temperature. The vials were removed from the liquid nitrogen and quickly thawed by placing in a water bath at 37 ℃ for 1-2 minutes with gentle agitation, then decontaminated by wiping with 70% ethanol, followed by opening in a class II biosafety cabinet. The vial contents were transferred drop-wise to 10mL of cell culture medium in a sterile 15mL conical tube. The tube was then centrifuged at 200 Xg for 5 minutes and the supernatant aspirated. The cell pellet was resuspended in 1mL of fresh cell culture medium and transferred to a T75 flask containing cell culture medium.

To passage H1975 cells, adherent cells were first washed with trypsin/EDTA. trypsin/EDTA (3 mL in T75 flask) was then added to the flask and swirled to ensure that the cells were evenly coated with trypsin. The flasks were then incubated at 37 ℃ until the cells detached. Equal volumes of cell culture medium were added to stop the reaction. Detached cells were collected and centrifuged at 200 Xg for 5 minutes, followed by resuspension in fresh medium. Next, the cells were transferred to a new T75 flask containing cell culture medium. Cells were subcultured three times per week in medium at a ratio of 1:2 or 1: 4.

Test compounds were dissolved at 30mM in DMSO. mu.L of compound was transferred to 384-well compound source plates (LABCYTE catalog number P-05525) and serially diluted in a 1:3 ratio, resulting in 13-point dilutions. The same volume of DMSO was taken as a high control. 20nL of DMSO dilutions of these compounds (10 spots, from 1.11mM to 0.056. mu.M) were dispensed through Echo 550 into new 384 well assay plates.

Cells were harvested from flasks to cell culture medium as described above and used an automated cell counter (Thermo Fisher Scientific, Countess)^TM) The number of cells was counted. Cells were diluted with medium to 25,000 cells/ml and 40 μ L of cell suspension was added to each well of a 384-well cell culture plate as indicated. The final concentration was 1,000 cells/well. Medium alone was added as a low control. The plate was covered with a lid and placed at 37 deg.C/5% CO₂The incubator was 72 hours.

After 72 hours of incubation, the plates were removed from the incubator and allowed to equilibrate at room temperature for 15 minutes. CellTiter Glo reagent (Promega, G9243) was incubated at 37 ℃ prior to the experiment. The buffer was equilibrated to room temperature and used to dissolve the matrix. To determine cell viability, 40. mu.L of CellTiter-Glo reagent was added to each well (1: 1 to medium) to be assayed. The plates were then left at room temperature for 30 minutes and then read on an EnSpire (PerkinElmer).

To estimate IC₅₀The luminescence reading is converted to percent inhibition: percent by applying the following equation

Connecting deviceIC was calculated by fitting a four parameter logarithmic curve to XLFit₅₀。

PC-9 growth inhibition assay (cell proliferation).

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

A431 inhibition assay (cell proliferation).

The medium for A431 cells was Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. DMSO dilutions for a431 assays were from 30nM to 10 points of 1.52 uM. The remainder of the procedure was performed in the same manner as described for H1975 cells.

Test results

Table 1: results of cell proliferation assay

PC9 cells contained EGFR d746-750 Single Mutant (SM). H1975 cells contained the EGFR L858R/T790M Double Mutant (DM). A431 cells contain unmutated EGFR Wild Type (WT).

Conclusion of the experiment

The compound of formula (I) has excellent EGFR inhibitory activity and anti-tumor proliferation activity, and particularly has higher inhibitory activity and selectivity on EGFR mutants.

Example 4.3 cellular EGFR autophosphorylation assay

Test article

Compounds of formula (I), prepared according to the methods in the examples; compound AZD9291, prepared according to prior art methods.

Test procedure

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

H1975 cells were cryopreserved in liquid nitrogen. Before cell thawing, 15mL of cell culture medium (RPMI 1640 medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin) was placed in a T75 flask and humidified at 37 ℃/5% CO₂IncubatorThe flasks were pre-incubated for 15 minutes to allow the medium to equilibrate to the appropriate pH and temperature. The vials were removed from the liquid nitrogen and quickly thawed by placing in a water bath at 37 ℃ for 1-2 minutes with gentle agitation, then decontaminated by wiping with 70% ethanol, followed by opening in a class II biosafety cabinet. The vial contents were transferred drop-wise to 10mL of cell culture medium in a sterile 15mL conical tube. The tube was then centrifuged at 200 Xg for 5 minutes and the supernatant aspirated. The cell pellet was resuspended in 1mL of fresh cell culture medium and transferred to a T75 flask containing cell culture medium.

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

Cells were harvested from flasks to cell culture medium and used with an automated cell counter (Thermo Fisher Scientific, Countess)^TM) The number of cells was counted. Cells were diluted with medium to 250,000 cells/ml and 40 μ Ι _ of cell suspension was added to each well of a 384-well cell culture plate as indicated. The final concentration was 10,000 cells/well. The plate was covered with a lid and placed in a 37 ℃/5% CO2 incubator overnight to adhere the cells.

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

The incubation plate was returned to 37 deg.C/5% CO₂The incubator was 2 hours. The medium of each well was replaced with ice-cold HBSS. Then HBSS was removed and 30. mu.L of cell lysate was addedBuffer to each well and shake the plate on a plate shaker for 30 minutes. Centrifuge at 1,000rpm for 5 minutes to remove air bubbles and transfer 25uL lysate supernatant to use commercial ELISA (R)&D, DYC1095B-5) was performed for p-EGFR assay.

To estimate IC50, the absorption readings were converted to relative activity%: by applying the following equation

IC was then calculated by fitting a four-parameter logarithmic curve in XLFit (IDBS, Guildford, Surrey)₅₀。

Wild-type EGFR a431 autophosphorylation inhibition assay (ELISA).

The medium for A431 cells was Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The DMSO dilutions used for the A431 assay were from 10mM to 0.17uM at 11 points. After 2 hours of treatment with test compound, 4.5 μ L EGF (1 μ g/mL) was added to each well and stimulated for 10 minutes. The remainder of the procedure was performed in the same manner as described for H1975 cells.

Exonl9 deletion EGFR (activated Single mutant) PC-9 cell autophosphorylation assay.

The human lung cell line PC9(Exon 19 deficient in EGFR) was obtained from the american type culture collection. PC9 cells were maintained in RPMI 1640 containing 10% fetal bovine serum and 2mM glutamine. Cells were incubated at 37 ℃ with 5% CO in a humidified incubator₂And (4) growing. The assay to measure cellular phosphorylation of endogenous p-EGFR in cell lysates is based on R&D Systems DuoSet IC human phosphorylation-EGF R ELISA (R)&D Systems catalog # DYCI 095). mu.L of cells were seeded (10000 cells/well) in growth medium in Coming black clear bottom 384-well plates at 37 ℃ with 5% CO₂Incubate overnight. Cells were acoustically administered using Echo 555, with compounds serially diluted in 100% DMSO. The plates were incubated for an additional 2 hours, then after aspirating the medium, 40 μ L 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, 1mu.L of lysate was transferred to Greiner black high binding 384 well plates and incubated for 2 hours. After aspiration and washing of the plate with PBS, 20 μ L of detection antibody was added and incubated for 2 hours. After aspiration and washing of the plates with PBS, 20. mu.L of QuantaBlu fluorescent peroxidase substrate (Thermo Fisher Scientific Cat No. 15169) was added and incubated for 1 hour. mu.L of QuantaBlu stop solution was added to the plate and fluorescence was read on an Envision plate reader using excitation 352nm wavelength and emission 460nm wavelength. The data obtained from each compound was entered into a suitable software package (e.g., Origin) to perform curve fitting analysis. From this data, IC was determined by calculating the concentration of compound required to obtain 50% effect₅₀The value is obtained.

Test results

TABLE 2 cellular EGFR autophosphorylation assay results

Conclusion of the experiment

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

Example 5 stability study of the mesylate salt form I of the Compound of formula (I)

Test article

Compound of formula (I) mesylate form I, prepared according to the methods in the examples.

Examination conditions

The examination condition one is as follows: the sample is placed under the conditions of 60 ℃ opening, RH 92.5% closing and illumination-opening respectively, and is sampled on the 5 th day and the 10 th day respectively, and related substances, content and water content are measured and compared with the sample on the 0 th day.

And (3) related substance determination: the determination is carried out according to the high performance liquid chromatography 0512 of the general rules of four departments in 2015 version of Chinese pharmacopoeia.

And (3) moisture determination: measured according to the first method 1 volumetric titration method of 0832 in the four general rules of the chapter of the book of Chinese pharmacopoeia 2015.

Content determination: the determination is carried out according to the high performance liquid chromatography 0512 of the general rules of four departments in 2015 version of Chinese pharmacopoeia.

Test results

Table 3 stability test results for form I

*: total illumination is greater than or equal to 1.2 multiplied by 10⁶Lux.h, near ultraviolet energy not less than 200 w.hr/m²

Conclusion of the experiment

The mesylate crystal form I of the compound shown in the formula (I) is placed under the high-temperature and high-humidity condition for 10 days, and related substances, content and water content of a sample are not obviously changed. Under the illumination condition, related substances and water content of the crystal form I are slightly increased, and the content of the crystal form I is not obviously changed. In conclusion, the crystal form I has better stability, is beneficial to the preparation, transportation and storage of medicines, and basically meets the requirement of druggability.

EXAMPLE 6 stability assay of the mesylate salt of the Compound of formula (I) in buffered saline solution

Test article

Examination conditions

And dissolving the crystal form I in buffer salt solution with pH 1.2 and pH 4.5, sampling at 0h, 3h, 6h, 9h and 25h respectively, and determining the conditions of related substances.

The preparation method of the pH 1.2 buffer salt comprises the following steps: 6.8g of monopotassium phosphate is weighed, dissolved by adding water (1000mL), and the pH value is adjusted to 1.2 by hydrochloric acid.

The preparation method of the pH 4.5 buffer salt comprises the following steps: 6.8g of monopotassium phosphate was weighed, dissolved in water (1000mL), and the pH was adjusted to 4.5 with phosphoric acid.

Test results

Table 4 stability test results in buffer

Conclusion of the experiment

The mesylate of the compound of the formula (I) is placed for 25 hours under the condition of potassium dihydrogen phosphate buffer salt with the pH value of 1.2, and the total related substances are basically unchanged, so that the stability is good; under the condition of potassium dihydrogen phosphate buffer salt with pH value of 4.5, after standing for 9h, the total related substances are not changed greatly, and after standing for 25h, the total related substances are increased by about 0.2%. The overall stability of the mesylate salt of the compound of formula (I) is good.

Claims

1. A mesylate salt of a compound of formula (I),

2. the mesylate salt of the compound of claim 1, wherein the molar ratio of the compound of formula (I) to the methanesulfonic acid is from 1:3 to 1: 1.

3. The mesylate salt of the compound of claim 1, wherein the molar ratio of the compound of formula (I) to the methanesulfonic acid is 1: 1.

4. A mesylate salt form I of a compound of formula (I),

the compound is characterized in that the molar ratio of the compound of the formula (I) to methanesulfonic acid is 1:1, and an X-ray powder diffraction pattern expressed by 2 theta angles by using Cu-Kalpha radiation has characteristic peaks at 10.0 +/-0.2 degrees, 13.2 +/-0.2 degrees, 17.6 +/-0.2 degrees, 22.0 +/-0.2 degrees and 23.3 +/-0.2 degrees.

5. Crystalline mesylate form I according to claim 4, which has an X-ray powder diffraction pattern, expressed in terms of 2 θ, using Cu-Ka radiation, and further has characteristic peaks at 14.8 ± 0.2 °, 20.2 ± 0.2 °, 26.1 ± 0.2 °, 29.3 ± 0.2 °.

6. The crystalline mesylate salt form I of claim 5, having an X-ray powder diffraction pattern substantially as shown in figure 1.

7. The mesylate salt form I of claim 4, having a differential scanning calorimetry trace with an endothermic peak in the range of 220 ℃ to 250 ℃ and a maximum endothermic transition temperature of 238.1 ± 5 ℃.

8. The crystalline mesylate form I of claim 4, having a differential scanning calorimetry curve substantially as shown in figure 2.

9. The crystalline mesylate form I of claim 4, which has a thermogravimetric analysis profile without significant weight loss at 0 ℃ to 220 ℃ having a thermogravimetric analysis curve substantially as shown in figure 3.

10. The preparation method of the mesylate crystal form I according to claim 4, wherein the compound of the formula (I) is added into a single solvent or a mixed solvent, heated to a certain temperature, added with a certain equivalent of methanesulfonic acid or added with a solution containing a certain equivalent of methanesulfonic acid, kept at the temperature for continuous reaction until the reaction is finished, cooled for crystallization, separated and dried to obtain the mesylate crystal form I of the compound of the formula (I);

the single solvent is selected from acetonitrile, the mixed solvent is selected from a mixed solvent of nitrile and water, and the solution of methanesulfonic acid is selected from a solution of methanesulfonic acid and acetonitrile.

11. The method of claim 10, wherein the solvent mixture is selected from the group consisting of acetonitrile/water, and the volume of water in the solvent mixture is not more than 5% of the total volume of the solvent.

12. The method of claim 11, wherein the volume of water in the solvent mixture is not more than 3% of the total volume of the solvent.

13. The method of claim 10, wherein the certain temperature is selected from the range of 40 ℃ to 80 ℃.

14. The method of claim 10, wherein the certain temperature is selected from the range of 50 ℃ to 60 ℃.

15. The method according to claim 10, wherein the specific temperature is selected from the group consisting of a temperature required for heating and refluxing the reaction system.

16. The method of any one of claims 10-15, wherein the certain equivalent weight is selected from 0.9 to 1.2 equivalent weight.

17. The method of any one of claims 10-15, wherein the certain equivalent weight is selected from 1.0 to 1.1 equivalent weight.

18. The method of any one of claims 10-15, wherein the certain equivalent weight is 1.0 equivalent weight.

19. A pharmaceutical composition comprising the mesylate salt of the compound of formula (I) according to any one of claims 1 to 3 or the crystalline form I of the mesylate salt of the compound of formula (I) according to any one of claims 4 to 9, and one or more pharmaceutically acceptable carriers, which can be prepared in any pharmaceutically acceptable dosage form.

20. Use of the mesylate salt of the compound of formula (I) according to any one of claims 1 to 3 or the crystalline form I of the mesylate salt of the compound of formula (I) according to any one of claims 4 to 9 for the preparation of a medicament for the treatment and/or prevention of a hyperproliferative disease mediated by EGFR, selected from cancer.

21. The use of claim 20, wherein the cancer is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, liver cancer, stomach cancer, prostate cancer, glioblastoma, and epithelial cell cancer.

22. The use of claim 20, wherein the cancer is non-small cell lung cancer.

23. The use of claim 20, wherein the EGFR is selected from the group consisting of wild-type EGFR and mutant EGFR.

24. The use of claim 23, wherein the mutant EGFR comprises one or more of an EGFR 19 exon mutation, an EGFR20 exon mutation, and an EGFR 21 exon mutation.

25. The use according to claim 24, wherein the EGFR exon 20 mutation is selected from the group consisting of NPG, ASV and T790M, wherein the EGFR exon 19 mutation is selected from the group consisting of an insertion mutation, and wherein the EGFR exon 21 mutation is selected from the group consisting of a point mutation.