CN108299419B

CN108299419B - Novel crystal forms of novel EGFR kinase inhibitor and preparation method thereof

Info

Publication number: CN108299419B
Application number: CN201810021796.7A
Authority: CN
Inventors: 赵立文; 符伟; 吴伟杰; 刘建设; 金建淋
Original assignee: Nanjing Sanhome Pharmaceutical Co Ltd
Current assignee: Nanjing Sanhome Pharmaceutical Co Ltd
Priority date: 2017-01-11
Filing date: 2018-01-10
Publication date: 2022-04-26
Anticipated expiration: 2038-01-10
Also published as: CN108299419A

Abstract

The present invention relates to several novel crystalline forms of a novel EGFR kinase inhibitor. In particular, the invention relates to a crystal form and a preparation method of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) methacrylamide, and the crystal form can be used for preparing a medicament for treating and/or preventing drug-resistant tumors.

Description

Novel crystal forms of novel EGFR kinase inhibitor and preparation method thereof

Technical Field

The present invention relates to several novel crystalline forms of a novel EGFR kinase inhibitor. In particular to several new crystal forms of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidine-2-yl) amino) phenyl) allyl amide and preparation methods and applications thereof.

Background

Epidermal Growth Factor Receptor (EGFR) is a member of the erbB receptor family of transmembrane protein tyrosine kinases. Homodimerization and/or heterodimerization of erbB receptors results in phosphorylation of key tyrosine residues in the intracellular domain, stimulating many intracellular signaling pathways involved in cell proliferation and survival. Dysregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and tumor cell survival, and has been described in many cancers, such as lung, head and neck, and breast cancers, among others. The small molecule EGFR tyrosine kinase inhibitor and ATP compete to bind to the intracellular region phosphorylation site of EGFR, so that the autophosphorylation process of EGFR is inhibited, and a downstream signal path is blocked, thereby achieving the purpose of inhibiting tumor cells.

Gefitinib and erlotinib are the first-generation reversible small-molecule inhibitors of EGFR, mainly used for treating non-small cell lung cancer. However, clinical studies have shown that many patients develop resistance to these small molecule EGFR inhibitors soon (12-14 months). The research shows that the mutation of the gatekeeper residue T790M is a mutation point of the No. 20 exon of the EGFR gene and is one of the main mechanisms for causing the generation of drug resistance. Second generation irreversible inhibitors such as afatinib have strong inhibitory effects on L858R and T790M mutated EGFR, and have significant therapeutic effects on patients who have developed resistance to gefitinib or erlotinib. However, the second generation EGFR mutant inhibitors also have strong inhibitory effects on wild-type EGFR, resulting in toxic side effects such as skin rash and diarrhea in most patients during clinical treatment.

Therefore, third generation EGFR inhibitors should first remain on EGFR^L858RThe activation mutant, the Exon19 deletion activation mutant and the T790M resistance mutant have stronger inhibition effect, and simultaneously, the toxic and side effects of a second generation inhibitor are overcome, namely, the inhibition effect on wild type EGFR is reduced. AZD9291 (also known as N- (2- { 2-dimethylaminoethyl-methylamino } -4-methoxy-5- { [4- (1-methylindol-3-yl) pyrimidin-2-yl) developed by AstraZeneca]Amino } phenyl) prop-2-enamide) is an oral, irreversible EGFR inhibitor with superior therapeutic efficacy for patients with EGFR-T790M mutation-positive non-small cell lung cancer. But its metabolite AZ5104 also has strong inhibitory effect on wild type EGFR. Therefore, there is still a need to develop a novel EGFR inhibitor having better drug efficacy, and the inventors of the present invention have studied and found that a compound represented by the formula (I) [ hereinafter referred to as "compound of the formula (I)"]Is an irreversible novel EGFR kinase inhibitor.

The chemical name of the compound of formula (I) is N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1, 2-a)]Indol-10-yl) -pyrimidin-2-yl) amino) phenyl) acrylamide. In vitro kinase Activity assays for mutant EGFR kinases, e.g., EGFR^L858R/T790MThe kinase has good propertyGood inhibitory activity, IC₅₀The value is less than 1nM, the influence on wild type EGFR kinase is small, and the selectivity is good. In vitro cell experiment results show that the compound of the formula (I) has better inhibition effect on double mutant cells, small inhibition effect on EGFR wild type cells and good selectivity. This will help to reduce adverse clinical effects.

As known to those skilled in the art, different recrystallization methods not only have large purity difference and yield, but also have large influence on the stability, water solubility and other relevant properties. The crystal form structure of the medicinal active compound has great significance to the synthesis process and the preparation process development and research process, and the crystal form suitable for medicinal use needs to be deeply researched and searched.

The invention content is as follows:

in a first aspect, the present invention provides a crystal form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide, named as form I, having good stability and suitable for formulation development, as shown in formula (I) below, and a preparation method thereof:

in the crystal form research of the compound shown in the formula (I), the inventor of the invention discovers more than ten crystal forms, wherein the crystal form I has good stability, no crystal transformation phenomenon, less impurities and high purity, and is suitable for preparation development.

The X-ray powder diffraction pattern of the I-type crystal form provided by the invention uses Cu-Ka radiation and represents an X-ray powder diffraction pattern at an angle of 2 theta, wherein characteristic peaks exist at about 7.75 degrees +/-0.2 degrees and 10.26 degrees +/-0.2 degrees.

Further, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.66 degrees +/-0.2 degrees, 7.75 degrees +/-0.2 degrees, 10.26 degrees +/-0.2 degrees, 11.77 degrees +/-0.2 degrees and 12.39 degrees +/-0.2 degrees.

Still further, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.66 degrees +/-0.2 degrees, 7.75 degrees +/-0.2 degrees, 10.26 degrees +/-0.2 degrees, 11.77 degrees +/-0.2 degrees, 12.39 degrees +/-0.2 degrees, 15.47 degrees +/-0.2 degrees and 16.37 degrees +/-0.2 degrees.

Further, the crystal form has an X-ray powder diffraction pattern as shown in figure 1.

Without limitation, the DSC profile of this form (see figure 6) shows an absorption peak around 130.6 ℃ (onset temperature).

Without limitation, the thermogravimetric analysis (TGA) profile of this crystalline form (see fig. 7) shows no significant weight loss before 200 ℃ and a decomposition temperature of about 300 ℃.

The crystal form has good stability, and the crystal form is heated to 80 ℃ without crystal transformation. The purity is not obviously reduced after the product is stored for 6 months at room temperature.

The invention provides a preparation method of a compound I crystal form of a formula (I), which comprises the following steps:

(1) dissolving N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide in a crystallization solvent, and separating out crystals; and

(2) filtering, washing and drying.

In the above reaction step (1), the form of the raw material N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide is not particularly limited, and any crystalline or amorphous solid can be used.

The crystallization solvent in the reaction step (1) is a lower organic solvent or a mixed solution thereof, the lower organic solvent is an alcohol having less than 6 carbon atoms or the like, the lower organic solvent is preferably methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol or the like, and more preferably ethanol.

In a second aspect, the present invention provides a crystalline form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide, named form II, as shown in formula (I) below, and a method for preparing the same.

The X-ray powder diffraction pattern of the II type crystal form provided by the invention uses Cu-Ka radiation and represents an X-ray powder diffraction pattern by a 2 theta angle, wherein characteristic peaks exist at about 6.68 degrees +/-0.2 degrees, 7.79 degrees +/-0.2 degrees, 8.41 degrees +/-0.2 degrees, 9.79 degrees +/-0.2 degrees and 10.29 degrees +/-0.2 degrees.

Further, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.33 degrees +/-0.2 degrees, 6.68 degrees +/-0.2 degrees, 7.79 degrees +/-0.2 degrees, 8.41 degrees +/-0.2 degrees, 9.79 degrees +/-0.2 degrees and 10.29 degrees +/-0.2 degrees.

Still further, an X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.33 degrees +/-0.2 degrees, 6.68 degrees +/-0.2 degrees, 7.79 degrees +/-0.2 degrees, 8.41 degrees +/-0.2 degrees, 9.79 degrees +/-0.2 degrees, 10.29 degrees +/-0.2 degrees, 11.81 degrees +/-0.2 degrees, 13.37 degrees +/-0.2 degrees, 13.86 degrees +/-0.2 degrees, 15.47 degrees +/-0.2 degrees and 16.41 degrees +/-0.2 degrees.

Further, the crystal form has an X-ray powder diffraction pattern as shown in figure 2.

The present invention provides a process for the preparation of form II crystal form of the compound of formula (I) comprising the steps of:

(2) filtering, washing and drying.

The crystallization solvent in the reaction step (1) is preferably a mixed solution of ethanol and water.

In a third aspect, the invention provides a crystal form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allyl amide shown in formula (I) below, named as type III crystal form, and a preparation method thereof.

The X-ray powder diffraction pattern of the III type crystal form provided by the invention uses Cu-Ka radiation and represents an X-ray powder diffraction pattern by a 2 theta angle, wherein characteristic peaks exist at 5.72 degrees +/-0.2 degrees, 9.03 degrees +/-0.2 degrees and 9.40 degrees +/-0.2 degrees.

Furthermore, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 5.72 degrees +/-0.2 degrees, 9.03 degrees +/-0.2 degrees, 9.40 degrees +/-0.2 degrees, 18.19 degrees +/-0.2 degrees and 18.92 degrees +/-0.2 degrees.

Still further, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 5.72 degrees +/-0.2 degrees, 9.03 degrees +/-0.2 degrees, 9.40 degrees +/-0.2 degrees, 18.19 degrees +/-0.2 degrees, 18.92 degrees +/-0.2 degrees, 24.10 degrees +/-0.2 degrees and 24.34 degrees +/-0.2 degrees.

Further, the crystal form has an X-ray powder diffraction pattern as shown in figure 3.

The present invention provides a process for preparing form III crystal form of the compound of formula (I) comprising the steps of:

(2) filtering, washing and drying.

The crystallization solvent in the reaction step (1) is preferably a mixed solution of acetone and water.

In a fourth aspect, the present invention provides a crystalline form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide, named form IV, as shown in formula (I) below, and a preparation method thereof.

The X-ray powder diffraction pattern of the IV crystal form provided by the invention uses Cu-Ka radiation and represents an X-ray powder diffraction pattern at an angle of 2 theta, wherein characteristic peaks exist at 6.67 degrees +/-0.2 degrees and 6.94 degrees +/-0.2 degrees.

Furthermore, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.67 degrees +/-0.2 degrees, 6.94 degrees +/-0.2 degrees, 9.68 degrees +/-0.2 degrees, 10.92 degrees +/-0.2 degrees and 11.20 degrees +/-0.2 degrees.

Still further, an X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 6.67 DEG + -0.2 DEG, 6.94 DEG + -0.2 DEG, 8.49 DEG + -0.2 DEG, 8.79 DEG + -0.2 DEG, 9.68 DEG + -0.2 DEG, 10.92 DEG + -0.2 DEG, 11.20 DEG + -0.2 DEG, 11.72 DEG + -0.2 DEG, 13.30 DEG + -0.2 DEG, 13.96 DEG + -0.2 DEG, 15.04 DEG + -0.2 DEG, 18.23 DEG + -0.2 DEG, 19.03 DEG + -0.2 DEG, 19.62 DEG + -0.2 DEG, 20.17 DEG + -0.2 DEG, 20.99 DEG + -0.2 DEG, 23.96 DEG + -0.2 DEG, 27.29 DEG + -0.2 DEG, and 28.47 DEG + -0.2 deg.

Further, the crystal form has an X-ray powder diffraction pattern as shown in figure 4.

The present invention provides a process for the preparation of form IV crystal form of the compound of formula (I) comprising the steps of:

(2) filtering, washing and drying.

The crystallization solvent in the reaction step (1) is preferably isopropyl alcohol.

In a fifth aspect, the present invention provides a crystalline form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide, named form V, as shown in formula (I) below, and a preparation method thereof.

The X-ray powder diffraction pattern of the V-type crystal form provided by the invention uses Cu-Ka radiation and represents an X-ray powder diffraction pattern by a 2 theta angle, wherein characteristic peaks exist at about 7.70 degrees +/-0.2 degrees, 8.33 degrees +/-0.2 degrees and 10.21 degrees +/-0.2 degrees.

Furthermore, the X-ray powder diffraction pattern of the crystal form has characteristic peaks at about 7.70 degrees +/-0.2 degrees, 8.33 degrees +/-0.2 degrees, 10.21 degrees +/-0.2 degrees and 15.47 degrees +/-0.2 degrees.

Still further, the crystal form has an X-ray powder diffraction pattern as shown in figure 5.

The present invention provides a process for the preparation of form V crystal form of the compound of formula (I) comprising the steps of:

(2) filtering, washing and drying.

The crystallization solvent in the reaction step (1) is preferably a nitrile solvent, and the nitrile solvent is more preferably acetonitrile.

In the present invention, the method of recrystallization is not particularly limited, and conventional recrystallization procedures can be employed. For example, N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide may be dissolved in a crystallization solvent, crystallized, filtered, and dried to obtain form I, form II, form III, form IV, or form V of the present invention. Or heating and dissolving the compound of the formula (I) with any crystal form or amorphous form in a crystallization solvent, cooling, crystallizing, or stirring and crystallizing, and then filtering and drying to obtain the crystal form of the invention. The crystals obtained by filtration are usually dried under vacuum at about 20 ℃ to 100 ℃, preferably 25 ℃ to 60 ℃ to remove the recrystallization solvent.

The crystal form prepared by the method does not contain or contains low-content residual solvent, meets the limit requirement of related medicinal product residual solvent specified by national formulary, and can be better used as a medicinal active ingredient.

The purity of the crystal forms I, II, III, IV and V obtained by the preparation method is improved, the crystal forms accord with pharmacopoeia standards, the yield is high, and the preparation method is suitable for large-scale purification and synthesis.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the crystalline form of the present invention and at least one pharmaceutically acceptable carrier. The crystal form is selected from one or more of I crystal form, II crystal form, III crystal form, IV crystal form and V crystal form.

The invention provides a pharmaceutical composition comprising the crystalline form of the invention and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition consisting of a crystalline form of the compound of formula (I) and a pharmaceutically acceptable carrier, wherein the crystalline form of the compound of formula (I) is mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical formulation suitable for oral or parenteral administration. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulations may be administered by any route, for example by oral administration, by infusion or bolus injection, by a route of absorption through epithelial or cutaneous mucosa (e.g. oral mucosa or rectum, etc.). Administration may be systemic or local. Examples of the formulation for oral administration include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and the like. The formulations may be prepared by methods known in the art and include carriers conventionally used in the art of pharmaceutical formulation.

In a seventh aspect, the invention provides a method for treating and/or preventing tumors by using the crystal form of the invention or a pharmaceutical composition comprising the crystal form and at least one pharmaceutically acceptable carrier, and an application of the crystal form or the pharmaceutical composition in preparation of drugs for preventing and/or treating tumors. In particular to the application in the preparation of the medicine for treating and/or preventing the tumor with drug resistance. The drug resistant tumor may be a tumor that is resistant to multiple drugs, preferably to EGFR inhibitors, such as first, second, and third generation EGFR inhibitors, such as gefitinib, erlotinib, and lapatinib. The tumor includes but is not limited to solid tumor, preferably lung cancer, head and neck tumor, colorectal cancer, bladder cancer, pancreatic cancer, breast cancer, prostate cancer, stomach cancer, oral cancer, liver cancer, ovarian cancer. More preferably, the tumor is non-small cell lung cancer. In some embodiments, the present invention provides methods of treating a drug resistant tumor with a crystalline form of the compound of formula (I), wherein the tumor carries an EGFR mutant gene. In one embodiment, the EGFR mutant gene carried by the tumor is the T790M mutation in exon 20. In another embodiment, the EGFR mutant gene carried by the tumor is a L858R mutation and/or a deletion/insertion mutation in exon 21. In another embodiment, the EGFR mutant gene carried by the tumor is a T790M and L858R double mutation. In other embodiments, the present invention provides a crystalline form of a compound of formula (I) of the present invention or a pharmaceutical composition of the present invention for use in the treatment of tumors, wherein the tumor treatment effect is manifested by an outstanding therapeutic effect, a high degree of selectivity and/or fewer side effects. In still other embodiments, the present invention provides a method of treating tumors by administering to a patient in need thereof a therapeutically effective amount of a crystalline form of a compound of formula (I) of the present invention or a pharmaceutical composition of the present invention, resulting in effects in treating tumors that are manifested by superior therapeutic efficacy, high selectivity and/or fewer side effects.

It is specifically stated herein that the X-ray powder diffraction pattern is characteristic for a particular crystalline form. To determine if it is the same as the known crystal type, care should be taken with respect to the relative positions of the peaks (i.e., 2 θ) rather than their relative intensities. This is because the relative intensities of the spectra (especially at low angles) vary due to the dominant orientation effects resulting from differences in crystal conditions, particle size or other measurement conditions, and the relative intensities of the diffraction peaks are not characteristic for the determination of the crystalline form. In addition, the 2 theta value of the same crystal form may have slight error, which is about +/-0.2 degrees. Therefore, this error should be taken into account when determining each crystalline structure. Peak positions are typically expressed in XRPD patterns in terms of 2 θ angles or crystal plane distances d, with a simple conversion between the two: d ═ λ/2sin θ, where the value of d represents interplanar spacing, λ represents the wavelength of the X-rays, and θ is the diffraction angle. It should also be noted that in the identification of mixtures, where partial loss of diffraction lines is caused by, for example, a reduction in the amount of the compound, one band may be characteristic of a given crystal without relying on all bands observed in a high purity sample.

DSC measures the transition temperature when a crystal absorbs or releases heat due to a change in its crystal structure or melting of the crystal. For the same crystal form of the same compound, the thermal transition temperature and melting point errors are typically within about 5 ℃ in a continuous analysis. When we say that a compound has a given DSC peak or melting point, this means that the DSC peak or melting point ± 5 ℃. It is noted that the DSC peak or melting point for the mixture may vary over a larger range. Furthermore, the melting temperature is related to the rate of temperature rise due to decomposition that accompanies the process of melting the substance.

Description of the drawings:

FIG. 1: an X-ray powder diffraction pattern of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt form I crystal form.

FIG. 2: an X-ray powder diffraction pattern of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt form II crystal form.

FIG. 3: an X-ray powder diffraction pattern of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide form III crystalline form.

FIG. 4: an X-ray powder diffraction pattern of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide form IV crystalline form.

FIG. 5: an X-ray powder diffraction pattern of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide form V crystalline form.

FIG. 6: a DSC diagram of form I crystal form of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt. .

FIG. 7: a TGA profile of form I of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt.

FIG. 8: an X-ray powder diffraction pattern of the N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt form I after heating to 80 ℃.

FIG. 9: DSC diagram after heating form I of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt to 80 ℃.

FIG. 10: a TGA diagram of form I of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide salt after heating to 80 ℃.

Detailed Description

Test instrument for experiments

Powder X-ray diffraction

The instrument model is as follows: d8Advance X-ray Diffract

Ray: monochromatic copper radiation with a wavelength of 1.54nm

The scanning mode is as follows: theta-2 theta

Scanning range: 3-40 degrees 2 theta

Step length: 0.02 degree

Voltage: 40Kv

Current: 40Ma

Slit 1.0/1.0/Ni/0.2

2.DSC

The instrument model is as follows: NETZSCH DSC 204 type differential thermal analyzer

The heating rate is as follows: 10 ℃/min

Temperature range: 40-250 deg.C

3.TGA

The instrument model is as follows: NETZSCH TG 209 type thermogravimetric analyzer

Temperature range: 30-350 deg.C

The heating rate is as follows: 10 ℃/min

Example 1: n- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide

Step a Synthesis of 1- (4-bromobutyl) -1H-indole

Sequentially adding NaH (60% content, 1.23g,30.73mmol) and DMF (10mL) into a 100mL reaction bottle, stirring at room temperature for 5min, cooling to 0-4 ℃, slowly adding 10mL of DMF solution dissolved with indole (3g,25.61mmol), heating to room temperature after addition, and reacting for 20min to obtain an indole activation solution.

Another 250mL reaction flask was charged with 1, 4-dibromobutane (16.59g,76.82mmol), DMF (50 mL). Slowly dripping the prepared indole activating solution at 0-4 ℃, and reacting for 0.5h at room temperature after dripping. After the reaction, water (100mL) is added for quenching, ethyl acetate is used for extraction, ethyl acetate layers are combined, anhydrous sodium sulfate is dried, and the mixture is filtered, concentrated and purified by column chromatography to obtain the title compound.

ESI-Ms m/z:252.1[M+H]⁺。

Step b Synthesis of 1- (4-iodobutyl) -1H-indole

In a 250mL reaction flask, the product of step a, 1- (4-bromobutyl) -1H-indole (5g,19.83mmol), sodium iodide (13.39g,89.93mmol), and acetonitrile (100mL) were added in that order and refluxed overnight. After the reaction, water was added, ethyl acetate was extracted, and the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated and dried to give the title compound which was used in the next step.

ESI-Ms m/z:300.0[M+H]⁺。

Step c Synthesis of 6,7,8, 9-tetrahydropyrido [1,2-a ] indole

1- (4-iodobutyl) -1H-indole (5.93g, 19.83mmol), potassium phosphate (8.4g, 39.67mmol), palladium tetratriphenylphosphine (2.3g,1.98mmol), 1, 4-dioxane (80mL), argon blanket, reflux overnight, obtained in step b, were added to a 250mL three-necked flask. After the reaction is finished, water is added to quench the reaction, ethyl acetate is used for extraction, an ethyl acetate layer is combined, anhydrous sodium sulfate is used for drying, filtering, concentrating and column chromatography purification are carried out, and the title compound is obtained.

ESI-Ms m/z:172.1[M+H]⁺。

Step d Synthesis of 10- (2-Chloropyrimidin-4-yl) -6,7,8, 9-tetrahydropyrido [1,2-a ] indole

In a 100mL reaction flask, aluminum trichloride (2.18g,16.35mmol), ethylene glycol dimethyl ether (50mL), 2, 4-dichloropyrimidine (2.44g,16.35mmol) and the product of step c, 6,7,8, 9-tetrahydropyrido [1,2-a ] indole (2.8g,16.35mmol) were added in this order, and the reaction was refluxed for 2 hours. After the reaction is finished, the reaction solution is cooled to room temperature, filtered, washed by filter cake water and dried to obtain the subject.

ESI-Ms m/z:284.1[M+H]⁺。

Step e N Synthesis of- (4-fluoro-2-methoxy-5-nitrophenyl) -4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-amine

In a 100mL reaction flask, the product of step d, 10- (2-chloropyrimidin-4-yl) -6,7,8, 9-tetrahydropyrido [1,2-a ] indole (1.05g,3.52mmol), 4-fluoro-2-methoxy-5-nitroaniline (655mg,3.52mmol) and p-toluenesulfonic acid (605mg,3.52mmol) were added, dissolved in 15mL sec-butanol, reacted at 110 ℃ for 5h, after the reaction was complete, cooled to room temperature, filtered and dried to give the title compound.

ESI-Ms m/z:434.2[M+H]⁺。

Step f N Synthesis of- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) -4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-amine

In a 50mL single-neck flask, the product obtained in step e, namely N- (4-fluoro-2-methoxy-5-nitrophenyl) -4- (6,7,8, 9-tetrahydropyrido [1, 2-a)]Indol-9-yl) -pyrimidin-2-amine (1.65g,3.70mmol), N, N, N' -trimethylethylenediamine (373 mg,3.70mmol), diisopropylethylamine (1.41g,11.1mmol) and 30mL of 1, 4-dioxane were dissolved, reacted at 110 ℃ for 3h, stopped, concentrated, and purified by column chromatography to give the title compound. ESI-Ms M/z 516.3[ M + H ]]⁺。

Step g N Synthesis of- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-aminophenyl) -4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-amine

The product of step f, N- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) -4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-amine (1.7g,3.20mmol), 10% palladium on carbon (50mg) and 30mL of methanol were added sequentially in a 50mL single-necked flask and reduced with hydrogen at 1 atm for 1h to stop the reaction, filtered and concentrated to give the title compound, which was used directly in the next step.

ESI-Ms m/z：486.3[M+H]⁺。

Step h N Synthesis of- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) pyrimidin-2-yl) amino) phenyl) allylamide

In a 100mL single-necked flask, the product obtained in step g, N- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-aminophenyl) -4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-amine (1.14g,2.27mmol), diisopropylethylamine (878mg,6.8mmol) and 30mL of anhydrous dichloromethane were added, and a solution of allyl chloride (204mg, 2.27mmol) in dichloromethane (5mL) was dissolved and added dropwise to the mixture to react for 30min, followed by concentration and purification by column chromatography to obtain the title compound with a purity of 98.5%.

¹H NMR(300MHz,DMSO-d₆)δ10.20(s,1H),8.65(s,1H),8.34(d,1H),8.11(s,1H),8.06(d,1H),7.43(d,1H),7.19-7.03(m,3H),6.98(s,1H),6.57-6.41(m,1H),6.28-6.15(m,1H),5.82-5.71(m,1H),4.09(t,2H),3.84(s,3H),3.18(t,2H),3.06-2.92(m,2H),2.66(s,3H),2.47-2.40(m,2H),2.27(s,6H),2.08-1.96(m,2H),1.87-1.74(m,2H)。

ESI-Ms m/z:540.3[M+H]⁺。

Example 2: preparation of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide form I crystal form

Weighing 50mg of the compound shown as the formula (I) and dissolving the compound in 30mL of absolute ethyl alcohol, heating to dissolve the compound clearly, cooling, standing for crystallization, performing suction filtration, washing, and drying at 60 ℃ in vacuum to obtain the I-type crystal form of the compound shown as the formula (I), wherein the yield is 86.7%, and the purity is 99.5%.

The X-ray diffraction pattern of the I-type crystal form is shown in figure 1, Cu-Ka radiation is used, an X-ray powder diffraction pattern is expressed by 2 theta angles, and characteristic peaks are arranged at about 3.25, 6.66, 7.75, 10.26, 11.77, 12.39, 13.31, 14.43, 15.47, 16.37, 16.91, 17.67, 18.23, 19.59, 20.46, 21.01, 21.78, 22.05, 23.67, 24.39, 25.60, 26.05 and 30.83. The values of 2 θ, the interplanar spacings d, and the relative intensities of the peaks in FIG. 1 are shown in Table 1. The peak intensity depends on the sample morphology and particle size and varies, with low intensity peaks (intensity less than 20%) possibly not being present in some cases.

Table 1: details of XRD pattern of crystal form I

Example 3 preparation of crystalline form II of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide

Weighing 50mg of the compound shown in the formula (I) and dissolving the compound in 50mL of ethanol water solution with the volume ratio of 1:1, heating to dissolve the compound, cooling, standing for crystallization, performing suction filtration, washing, and drying at 60 ℃ in vacuum to obtain the II type crystal form of the compound shown in the formula (I), wherein the yield is 63.0%, and the purity is 99.1%. .

An X-ray diffraction pattern of the II type crystal form is shown in figure 2, Cu-Ka radiation is used, an X-ray powder diffraction pattern is expressed by 2 theta angles, and characteristic peaks exist at about 6.33, 6.68, 7.79, 8.41, 9.79, 10.29, 11.41, 11.81, 12.43, 12.68, 13.37, 13.86, 14.48, 15.47, 16.41, 16.89, 17.73, 18.84, 19.15, 19.69, 20.51, 21.05, 21.50, 22.10, 22.68, 23.72, 25.72 and 30.84. The values of 2 θ, d-plane spacing, and relative intensities of peaks in FIG. 2 are shown in Table 2. The peak intensity depends on the sample morphology and particle size and varies, with low intensity peaks (intensity less than 20%) possibly not being present in some cases.

Table 2: XRPD pattern of type II crystal form

Example 4 preparation of crystalline form III of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide

Weighing 50mg of the compound shown in the formula (I) and dissolving the compound in 8mL of acetone aqueous solution with the volume ratio of 1:1, heating and dissolving the mixture, cooling, standing and crystallizing, filtering, washing, and drying at 60 ℃ in vacuum to obtain the III crystal form of the compound shown in the formula (I), wherein the yield is 62%, and the purity is 99.1%.

The X-ray diffraction pattern of the III type crystal form is shown in figure 3, and the X-ray powder diffraction pattern is expressed by 2 theta angles by using Cu-Ka radiation, and has characteristic peaks at about 5.72, 9.03, 9.40, 9.95, 10.24, 11.05, 11.48, 12.06, 12.95, 15.61, 16.06, 17.74, 18.19, 18.92, 19.91, 20.65, 23.58, 24.10, 24.34, 26.00, 26.37 and 33.32. The values of 2 θ, the interplanar spacings d and the relative intensities of the peaks in FIG. 3 are shown in Table 3. The peak intensity depends on the sample morphology and particle size and varies, with low intensity peaks (intensity less than 20%) possibly not being present in some cases.

Table 3: XRPD pattern of type III crystal form

Example 5 preparation of form IV of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide

Weighing 50mg of the compound of the formula (I) and dissolving the compound in 10mL of isopropanol, heating to dissolve the mixture clearly, cooling, standing for crystallization, performing suction filtration, washing, and drying at 60 ℃ in vacuum to obtain the IV crystal form of the compound of the formula (I), wherein the yield is 60%, and the purity is 98.5%.

The X-ray diffraction pattern of the IV type crystal form is shown in figure 4, Cu-Ka radiation is used, an X-ray powder diffraction pattern is expressed by 2 theta angles, and characteristic peaks are arranged at about 6.67, 6.94, 8.49, 8.79, 9.68, 10.92, 11.20, 11.72, 12.39, 13.30, 13.96, 15.04, 18.23, 19.03, 19.62, 20.17, 20.99, 23.96, 27.29 and 28.47. The values of 2 θ, the interplanar spacings d and the relative intensities of the peaks in FIG. 4 are shown in Table 4. The peak intensity depends on the sample morphology and particle size and varies, with low intensity peaks (intensity less than 20%) possibly not being present in some cases.

Table 4: XRPD pattern of IV crystal form

Peak number	2θ	d value	Counting	Relative strength
						1	6.67	13.25	653.0	100.0
2	6.94	12.73	648.0	99.4
					3	8.49	10.41	130.0	19.9
4	8.79	10.05	161.0	24.7
					5	9.68	9.13	273.0	41.8
6	10.92	8.09	264.0	40.5
					7	11.20	7.90	260.0	39.8
8	11.72	7.54	149.0	22.8
					9	12.39	7.14	105.0	16.1
10	13.30	6.65	196.0	30.0
					11	13.96	6.34	200.0	30.7
12	15.04	5.89	266.0	40.7
					13	18.23	4.86	202.0	30.9
14	19.03	4.66	420.0	64.3
					15	19.62	4.52	329.0	50.4
16	20.17	4.40	217.0	33.2
					17	20.99	4.23	277.0	42.5
18	23.96	3.71	227.0	34.8
					19	27.29	3.27	126.0	19.4
20	28.47	3.13	175.0	26.8

Example 6 preparation of crystalline form V of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (6,7,8, 9-tetrahydropyrido [1,2-a ] indol-10-yl) -pyrimidin-2-yl) amino) phenyl) allylamide

Weighing 50mg of the compound shown as the formula (I) and dissolving the compound in 10mL of acetonitrile, heating to dissolve the acetonitrile, cooling, standing for crystallization, performing suction filtration, washing, and drying at 60 ℃ in vacuum to obtain the V-type crystal form of the compound shown as the formula (I), wherein the yield is 63%, and the purity is 98.5%.

An X-ray diffraction pattern of the V-shaped crystal form is shown in figure 5, and a Cu-Ka radiation is used, an X-ray powder diffraction pattern is expressed by 2 theta angles, and characteristic peaks exist at about 6.26, 6.60, 6.94, 7.70, 8.33, 9.72, 10.21, 11.31, 11.73, 12.41, 12.80, 13.29, 13.80, 14.39, 15.47, 16.34, 16.83, 17.68, 18.25, 19.07, 19.59, 20.45, 21.04, 21.41, 21.72, 22.04, 22.66, 22.93, 23.67, 24.35, 24.75, 25.36, 25.63, 26.15, 28.68, 30.85 and 39.30. The values of 2 θ, the interplanar spacings d and the relative intensities of the peaks in FIG. 5 are shown in Table 5. The peak intensity depends on the sample morphology and particle size and varies, with low intensity peaks (intensity less than 20%) possibly not being present in some cases.

Table 5: XRPD pattern of V crystal form

Experimental example 1: in vitro kinase Activity evaluation of Compounds of formula (I)

1 materials of the experiment

1.1 enzymes

EGFR^WTKinases available from Carna corporation;

EGFR^T790M/L858Rkinase, purchased fromInvitrogen corporation.

1.2 reagents

Adenosine Triphosphate (ATP), purchased from Sigma;

peptide (Peptide FAM-P22) available from GL Biochem;

ethylenediaminetetraacetic acid (EDTA), available from Sigma.

1.3 instruments

Caliper EZ reader microfluidic chip instrument, available from Caliper Life Sciences, Inc.

2 method of experiment

2.1 preparation of 1 Xkinase base buffer and stop buffer

1 × kinase base buffer (for EGFR)^WT)：50mM HEPES，pH7.5，0.0015％Brij-35，10mM MgCl₂，10mM MnCl₂，2mM DTT；

1 × kinase base buffer (for EGFR)^T790M/L858R)：50mM HEPES，pH7.5，0.0015％Brij-35，10mM MgCl₂，2mM DTT；

Stop buffer: 100mM HEPES, pH7.5, 0.0015% Brij-35, 0.2% Coating Reagent #3, 50mM EDTA.

2.2 preparation of Compounds

Compounds of formula (I) were dissolved in 100% DMSO to 10mM each, diluted to 50. mu.M in complete medium, then diluted to 5. mu.M in complete medium containing 0.1% DMSO, and then diluted 3-fold in sequence for 10 concentrations (for EGFR)^WT)；

Compounds of formula (I) were dissolved in 100% DMSO to 10mM each, diluted to 50. mu.M in complete medium, diluted to 1. mu.M in complete medium containing 0.1% DMSO, and then diluted 3-fold in sequence for 10 concentrations (for EGFR)^T790M/L858R)；

Add 100. mu.l 100% DMSO to empty wells for preparing kinase no compound control and kinase no compound control;

the 96-well plate used above was labeled as the source plate.

2.3 preparation of the intermediate plate

Transfer 10. mu.l of the solution from the source plate to a new 96-well plate as an intermediate plate, add 90. mu.l of 1 Xkinase buffer to each well of the intermediate plate, and mix by shaking for 10 min.

2.4 preparation of the test plate

From a 96-well intermediate plate, 5. mu.l of the solution was transferred per well to a 384-well plate.

2.5 kinase reaction

2.5.1. Preparation of 2.5 × kinase solution: EGFR (epidermal growth factor receptor)^WTKinase and EGFR^T790M/L858RAdding the kinase stock solution into 1 × basic buffer solution respectively to prepare 2.5 × kinase solution;

2.5.2. preparation of a 2.5 Xpeptide solution: adding FAM labeled peptide and ATP into 1 × basic buffer solution to prepare 2.5 × peptide solution;

2.5.3. transfer 10. mu.l of 2.5 Xkinase solution to 384 well assay plates and incubate for 10min at room temperature;

2.5.4. transfer 10. mu.l of 2.5 Xpeptide solution to 384 well plates, incubate at 28 ℃ for a period of time, and stop the reaction by adding 25. mu.l of stop buffer.

A kinase-free no compound control group (containing DMSO, 1 Xbase buffer and 2.5 Xpeptide solution) and a kinase-free no compound control group (containing DMSO, 2.5 Xkinase solution and 2.5 Xpeptide solution) were set at the same time.

Reading and fitting a curve of the Caliper instrument, and calculating the inhibition rate

Reading data on a Caliper instrument, obtaining conversion data from a Caliper program, and calculating the inhibition rate according to the following formula:

inhibition rate [% ], [% ] represents [% ], [% ] represents "[% ], [% ] represents". its inhibition rate is calculated by (max-com)/(max-min) × 100.

2.5.6. Computing IC Using Graphpad 5.0 data processing software₅₀The value is obtained. The results are shown in Table 6.

TABLE 6

As can be seen from the above results,the compounds of formula (I) against mutant EGFR kinases, e.g. EGFR^L858R/T790MThe kinase has good inhibitory activity, IC₅₀Values were less than 1 nM. Therefore, the compound shown in the formula (I) has good inhibition effect on mutant EGFR kinase and better selectivity relative to EGFR wild-type kinase.

Experimental example 2: evaluation of the in vitro cellular Activity of Compounds of formula (I)

1 materials of the experiment

1.1 cells

Experimental cell line NCI-H1975(EGFR double mutant cell, with L858R and T790M mutations)

And a431(EGFR wild type cells) purchased from ATCC.

1.2 reagents

Cell Titer-Glo luminescence Cell viability assay, available from Promega corporation;

RPMI1640medium, available from Invitrogen;

DMEM medium, available from Invitrogen corporation;

fetal bovine serum, purchased from Invitrogen;

DMSO, available from Sigma company;

NCI-H1975 cells were cultured in RPMI1640 containing 10% inactivated fetal bovine serum (GIBCO)

The nutrient medium contains 100IU/mL of penicillin and 100 mu g/mL of streptomycin;

a431 cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum (GIBCO),

containing 100IU/mL of penicillin and 100 mu g/mL of streptomycin.

2 method of experiment

2.1 Experimental Process (CTG assay)

NCI-H1975 cells and A431 cells in logarithmic growth phase were digested, blown into single cell suspension, inoculated into 96-well culture plates with 100. mu.L of culture medium per well, and plated in 3-well plates for each cell line, wherein NCI-H1975 cells were plated in 3X 10-well plates per well³Individual cells, a431 cells were seeded at 4X 10 per well³And (4) cells. Inoculating NCI-H1975 cells and A431 cells in 5% CO₂Culturing in an incubator for 16-24 hours, after the cells adhere to the wall,the compounds were added as required (highest tested concentration of compound on NCI-H1975 cells was 4. mu.M, 3-fold dilution for 9 concentrations; highest tested concentration on A431 cells was 10. mu.M, 3-fold dilution for 9 concentrations) and incubated in the incubator for a further 72 hours. Both a blank control (medium only, no cells and DMSO solution) and a DMSO control (medium with cells and 0.5% DMSO solution) were set. Add 100. mu.L of CTG solution, shake for 2min in the dark, incubate for 10 min.

2.2 reading, calculating IC₅₀Value of

Placing the culture plate in

Reading the plate by a multi-mode microplate detector, recording the luminescence reading result, and calculating the inhibition rate according to the following formula:

inhibitor (%) - (1- (RLU)_com-RLU_blank)/(RLU_DMSO–RLU_blank))×100％，

Wherein RLU_comDenotes the absorbance, RLU, of the test Compound group_blankAbsorbance values, RLU, of blank control group_DMSOThe absorbance of the DMSO control was expressed,

the drug effect inhibition rate curve is drawn by using XLFit curve fitting software and the IC is calculated₅₀The values, results are shown in Table 7.

TABLE 7

It has been shown that one of the major side effects of EGFR inhibitors on the market is skin rash, diarrhea, etc., which are associated with the inhibition of wild-type EGFR. The results of the above experiments show that the compound of formula (I) has good inhibitory effect on double mutant cells (NCI-H1975), and has little inhibition and good selectivity on EGFR wild type cells (A431). Is expected to become a drug with specific curative effect and less side effect for resisting the drug-resistant tumor caused by EGFR mutation.

Experimental example 3: the stability of the crystal forms I, II, III, IV and V of the compound shown in the formula (I) is researched.

The forms I, II, III, IV and V obtained in examples 2, 3, 4, 5 and 6 were vacuum-dried at 80 ℃ for 24 hours, respectively, and then the obtained samples were subjected to X-ray powder diffraction test, DSC test and TGA test.

The experimental result shows that the XPRD pattern (see figure 8), the DSC pattern (see figure 9) and the TGA pattern (see figure 10) of the I-type crystal form after high-temperature treatment are consistent with those of the initial sample, which indicates that the I-type crystal form after high-temperature treatment does not generate crystal transformation phenomenon, has high stability and is suitable for the research of process preparations.

The samples of crystal forms I, II, III, IV and V obtained in examples 2, 3, 4, 5 and 6 were placed at high temperature (60 ℃) and high humidity (RH 75%) for 1 month, 3 months and 6 months, respectively, and purity was checked by HPLC.

The results show that the purity of the I crystal form of the compound shown in the formula (I) is not obviously changed after the compound is placed under the conditions of high temperature and high humidity for 1 month, 3 months and 6 months, and the influence of the high temperature and high humidity on the compound is small.

Claims

1. The I-type crystal form of the compound shown as the formula (I) is characterized in that an X-ray powder diffraction spectrum has characteristic peaks at 6.66 degrees +/-0.2 degrees, 7.75 degrees +/-0.2 degrees, 10.26 degrees +/-0.2 degrees, 11.77 degrees +/-0.2 degrees and 12.39 degrees +/-0.2 degrees, which are expressed by 2 theta angles:

2. a crystalline form according to claim 1, characterized by an X-ray powder diffraction spectrum having characteristic peaks, expressed in degrees 2 Θ, at 6.66 ° ± 0.2 °, 7.75 ° ± 0.2 °, 10.26 ° ± 0.2 °, 11.77 ° ± 0.2 °, 12.39 ° ± 0.2 °, 15.47 ° ± 0.2 °, 16.37 ° ± 0.2 °.

3. A process for preparing the form I crystalline form of claim 1 or 2, comprising the steps of:

(1) dissolving the compound of formula (I) in a crystallization solvent, cooling to precipitate crystals, and

(2) filtering, washing and drying the mixture,

the crystallization solvent is absolute ethyl alcohol.

4. A pharmaceutical composition comprising the crystalline form of claim 1 or 2 and a pharmaceutically acceptable carrier.

5. Use of the crystalline form according to claim 1 or 2 or the pharmaceutical composition according to claim 4 for the preparation of a medicament for the treatment and/or prevention of tumors.

6. The use of claim 5, wherein the tumor is a drug resistant tumor.

7. The use of claim 6, wherein the tumor is a tumor resistant to an EGFR inhibitor.