CN105254615B - Phenylaminopyrimidine derivatives and their use in preparation of drugs for resisting cancers - Google Patents

Abstract

The invention relates to phenylaminopyrimidine derivatives and their use in preparation of drugs for preventing and/or treating cancers. The phenylaminopyrimidine derivatives are ideal high-efficiency non-reversible EGFR kinase inhibitors and can be used for treating or preventing lung cancer, stomach and intestine cancer, breast cancer, pancreas cancer, ovarian cancer, esophagus cancer, head and neck squamous carcinoma, epidermal squamous carcinoma, prostate cancer, glioma and nasopharyngeal carcinoma.

Description

Aniline pyrimidine derivatives and their use in the preparation of anti-malignant tumor drugs

technical field

The invention relates to aniline pyrimidine derivatives and their application in the preparation of drugs for preventing and treating malignant tumors.

Background technique

EGFR (epidermal growth factor receptor, epidermal growth factor receptor) is a transmembrane receptor, EGFR binds to the extracellular domain to form a receptor dimer and activates the intracellular tyrosine kinase domain, triggering kinase autophosphorylation Phosphorylation and phosphorylation of downstream molecules activate a variety of cellular functions including proliferation and survival. EGFR can be detected in nearly 80% to 85% of NSCLC patients, and its expression level varies greatly in a continuous range. The incidence of EGFR mutation in lung adenocarcinoma reaches 50% in the Asian population, and the incidence is higher in never-smokers, females, and non-mucinous tumors.

The most common EGFR mutations are exon 19 deletion (E19del, found in 45% of patients) and exon 21L858R mutation (found in 40% of patients), both of which lead to activation of the tyrosine kinase domain and are associated with tumor It is related to the sensitivity to small molecule TKIs (tyrosine kinase inhibitor, tyrosine kinase inhibitor). These drug-susceptibility mutations are found in nearly 10% of Caucasian NSCLC patients and up to 50% of Asian patients. The EGFR gene is the target with the highest mutation probability in Asian lung adenocarcinoma patients and can benefit the most patients from treatment. Other types of drug susceptibility mutations include exon 21 (L861Q) and exon 18 (G719X) point mutations. T790M mutation can lead to TKI drug resistance, and relevant reports show that this mutation type is found in about 50% of patients with tumor progression.

Contents of the invention

The technical problem to be solved by the present invention is to provide a novel aniline pyrimidine derivative, which is an ideal irreversible EGFR kinase inhibitor and can be used to effectively prevent or treat lung cancer, gastrointestinal cancer, breast cancer, pancreatic cancer, ovarian cancer , Esophageal cancer, head and neck squamous cell carcinoma, epidermal squamous cell carcinoma, prostate cancer, glioma and nasopharyngeal carcinoma and other malignant tumor diseases.

In order to solve the above technical problems, the present invention takes the following technical solutions:

A compound of general formula (I), its pharmaceutically acceptable salts, hydrates, or metabolites formed in any form of metabolism,

in:

R ₁ is CH ₂ F, CHF ₂ , CF ₃ , OCH ₂ F, OCHF ₂ , OCF ₃ , C ₂ ~C ₃ fluorohydrocarbon group, C ₂ ~C ₃ chlorohydrocarbon group; or, R ₁ is OC _k H _{2k +1} , where k is an integer between 1 and 3;

R ₂ and R ₃ are linear or branched hydrocarbon chains with at least one double bond, and one or two other methylene units of R ₂ and R ₃ are optionally and independently modified by -NHC(O)-, - C(O)NH-, -N(H)SO ₂ -or -SO ₂ N(H)-replacement; or, R ₂ and R ₃ are -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , -N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , 4-methylpiperazine-1 -yl, 4-ethylpiperazin-1-yl, wherein R ₂ and R ₃ cannot be the same group;

_R4 is

Wherein, R ₅ is H, CH ₃ , CH ₂ CH ₃ ;

X is N or CH;

In the compound of general formula (I), its pharmaceutically acceptable salt, hydrate, or metabolites formed by any form of metabolism, the non-exchangeable hydrogen is unsubstituted, or partially or completely substituted by deuterium.

According to one aspect of the present invention, in formula (I), R ₁ is one selected from CH ₂ F, CHF ₂ , CF ₃ , OCH ₂ F, OCHF ₂ , OCF ₃ , and OCH ₃ ; R ₂ , R ₃ , R ₄ , R ₅ and X are as defined above.

According to another aspect of the present invention, in formula (I), R ₂ is -NHC(O)CH=CH ₂ , -NHC(O)CH=CHCH ₂ N(CH ₃ ) ₂ , -NHSO ₂ CH=CH ₂ , -NHSO ₂ CH=CHCH ₂ N(CH ₃ ) ₂ ; R ₃ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , -N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , 4-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl; R ₁ , R ₄ , R ₅ and X are as defined above.

According to yet another aspect of the present invention, in formula (I), R ₃ is -NHC(O)CH=CHCH ₂ N(CH ₃ ) ₂ , -NHSO ₂ CH=CH ₂ , -NHSO ₂ CH=CHCH ₂ N( CH ₃ ) ₂ ; R ₂ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , - N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , 4-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl; definitions of R ₁ , R ₄ , R ₅ and X Ditto.

According to a specific and preferred aspect of the present invention, in formula (I), R ₅ is H or CH ₃ , R ₁ , R ₂ , R ₃ , R ₄ and X are as defined above.

According to a specific aspect, X is CH.

According to a preferred aspect of the present invention, in formula (I), R ₁ is one selected from OCH ₂ F, OCHF ₂ , OCF ₃ , OCH ₃ and OCH ₂ CH ₃ ; R ₃ is -NHC(O)CH =CHCH ₂ N(CH ₃ ) ₂ , -NHSO ₂ CH=CH ₂ , -NHSO ₂ CH=CHCH ₂ N(CH ₃ ) ₂ ; R ₂ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , -N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , 4-Methylpiperazine- 1-yl, 4-ethylpiperazin-1-yl; X is CH, R ₄ and R ₅ are as defined above.

According to the present invention, representative compounds are as follows:

According to the present invention, the compound includes not only a single compound form, but also a mixture of compounds whose structures meet the requirements of general formula (I), and different isomer forms of the same compound such as racemic isomers, enantiomers, diastereomers, etc. The pharmaceutically acceptable salts include but not limited to hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, benzenesulfonate, benzoate, toluenesulfonate , succinate, fumarate, fumarate, tartrate, gallate, citrate, etc. The "prodrug of the compound having the general formula (I)" refers to a substance that, when administered by an appropriate method, can undergo metabolism or chemical reactions in the body of a subject to convert into at least one compound of the structural formula (I). compound or its salt.

The compounds of the present invention may be prepared by synthetic routes to methods analogous to those well known in the chemical arts, in particular by synthesizing the compounds of the present invention according to the description contained herein. Reagents are generally obtained from commercial sources or are readily prepared using methods well known to those skilled in the art.

The present invention also relates to an intermediate for preparing the above-mentioned compound with general formula (I), which is shown in general formula (II):

In the general formula (II), the definitions of R ₁ , R ₂ and R ₄ are the same as above.

Further, in the above general formula (II), R ₂ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N( H) _CH3 , -N( _CH3 ) _CH2CH2N ( _CH3 ) ₂ , 4-methylpiperazin- ₁ -yl or 4-ethylpiperazin-1-yl.

Further, in the above general formula (II), R ₁ is OCH ₂ F, OCHF ₂ , OCF ₃ or OCH ₃ .

Representative general formula (II) intermediates are for example:

Using the above intermediates, the corresponding compound of general formula (I) can be obtained through a one-step amidation reaction.

Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:

The compound provided by the present invention is a novel aniline derivative, which is an ideal high-efficiency irreversible EGFR tyrosine kinase inhibitor, and inhibits the activity and phosphorylation of the kinase by acting on the intracellular part of EGFR and competitively combining with ATP. And block the EGFR tyrosine kinase ATP binding site so as to achieve the purpose of specific inhibition of EGFR. Therefore, the compound of the present invention can be used to prepare and treat or prevent various indications related to EGFR kinase function, including but not limited to lung cancer, gastrointestinal cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, head and neck squamous cell carcinoma, epidermis Squamous cell carcinoma, prostate cancer, glioma and nasopharyngeal carcinoma and other malignant tumor diseases. What is more special is that the compound of the present invention can specifically act on EGFR kinase with T790M mutation, but has only weak inhibitory effect on wild-type EGFR kinase. Tumor has therapeutic significance, and less toxic and side effects.

detailed description

The present invention will be described in further detail below in conjunction with specific examples, but the present invention is not limited to the following examples.

Example 1

Formula Ia compound, its chemical structure is as follows:

The compound of formula Ia can be obtained by the following synthetic route:

The preparation method of formula Ia compound specifically comprises the following steps:

(1), Preparation of Intermediate 3: Add ferric chloride (5.5g, 33.9 mmol) and 1-methylindole (5.0 g, 38 mmol). The resulting mixture was stirred overnight at around 60°C. After cooling, the solid was precipitated by adding methanol (50 mL) and water (100 mL). The resulting slurry was stirred for 3 hours. The solid was collected by filtration, rinsed with methanol (100 mL), and dried overnight at 50 °C to afford Intermediate 3 (6 g, 77%) as a purple solid.

(2), preparation of intermediate 5: tosylate hydrate (772.82mg, 4.1mmol) was added to 2-pentanol (10mL) of intermediate 3 (1g, 4.1mmol) and intermediate 4 (763mg, 4.1mmol) ) in the mixture. The resulting mixture was stirred at 105°C for 2.5 hours. Then cooled to room temperature, the resulting precipitate was collected by filtration, rinsed with 2-pentanol (20 mL), and dried under vacuum to afford Intermediate 5 (1.2 g, 75%) as a yellow solid.

(3) Preparation of Intermediate 7: A solution of Intermediate 5 (500 mg, 1.27 mmol) and Intermediate 6 (155.8 mg, 1.53 mmol) in DMA (50 mL) was stirred at 85°C for 5-6 hours, then cooled to room temperature. Water (50 mL) was added, and the mixture was stirred for 3-4 hours. The solid material was collected by filtration, washed with water (30 mL) and dried at 50 °C for 12 hours to give Intermediate 7 (400 mg, 96%) as an orange solid.

(4), preparation of intermediate 8: intermediate 7 (400mg, 0.84mmol), iron (281.84mg, 5.04mmol), ammonium chloride (31.5mg, 0.588mmol), ethanol (30mL) and water (10mL) mixture Heat to reflux for 2 hours. The desired product was combined by eluting the column with 7M methanolic ammonia and concentrated onto silica gel in vacuo. Purification by column chromatography gave intermediate 8 as a beige foam, which was directly used in the next step reaction.

(5), preparation of formula Ia compound: intermediate 9 (222mg, 1.34mmol), HATU (255mg, 0.67mmol) and TEA (135mg, 1.34 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was purified by preparative HPLC to directly afford the crude product (300 mg) as a dark brown oil. The crude product was further purified by TLC chromatography to give compound Ia as a dark yellow semi-solid.

Proton nuclear magnetic resonance H ^- NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product Ia obtained, and the results are as follows:

Absorption peak in ¹ H-NMR spectrum: δ=8.88(s,1H)8.33(m,1H)8.27(m,2H)7.47(m,1H)7.24(m,3H)6.99(m,2H)6.83( d,1H)4.01(s,3H)3.92(s,3H)3.46(t,2H)3.33(m,2H)3.29(m,2H)2.89(s,6H)2.72(m,9H).

m/z[MH] ⁺ :557.3. It is calculated that the product has the molecular formula C ₃₁ H ₄₀ N ₈ O ₂ and the exact molecular mass (exact mass) is 556.33.

Example 2

Formula Ib compound, its chemical structure is as follows:

Formula Ib compound can be obtained by following synthetic route:

The preparation method of formula Ib compound specifically comprises the following steps:

(1), Preparation of Intermediate 12: Add DIPEA (144 mg, 1.12 mmol), Intermediate 11 (117 mg, 1.03 mmol) to a suspension of Intermediate 5 (340 mg, 0.86 mmol) in DMA (10 mL), and heat the mixture to 80°C for 16 hours. The mixture was poured into water (40 mL), stirred for 5 min and filtered to afford Intermediate 12 (290 mg, 69%) as an orange solid with m/z [MH] ⁺ of 488.

(2) Preparation of Intermediate 13: Iron (200 mg, 3.57 mmol) and ammonium chloride (22 mg, 0.41 mmol) were added to the solution of Intermediate 12 (290 mg, 0.59 mmol) in EtOH (12 mL)/water (4 mL). Heat to reflux for 2 hours. The mixture was extracted with ethanol (100 mL) and water (80 mL), filtered through celite, dried and chromatographed to afford Intermediate 13 (250 mg, 93%) as a dark semi-solid.

(3), preparation of formula Ib compound: intermediate 9 (145 mg, 0.88 mmol), HATU (168 mg, 0.44 mmol) and TEA (88 mg, 0.88 mmol). The mixture was stirred at room temperature for 2 hours. Purification by preparative HPLC afforded compound Ib (140 mg, 56%) as a green solid.

Proton nuclear magnetic resonance ¹ H-NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product 1b that obtains, and the results are as follows:

Absorption peak in ¹ H-NMR spectrum: δ=8.65(s,1H)6.58(s,1H)8.29(m,1H)8.05(m,1H)7.52(d,J=8.0Hz,1H)7.34(m ,2H)7.24(m,1H)7.05(s,1H)6.85(m,2H)4.01(m,2H)3.97(s,3H)3.95(s,3H)3.72(m,2H)3.46(m,2H ) 3.37 (m, 2H) 3.33 (m, 4H) 2.94 (s, 6H) 1.44 (m, 3H).

m/s:[MH] ⁺ :569.4. It is calculated that the product has the molecular formula C ₃₂ H ₄₀ N ₈ O ₂ and the exact molecular mass (exact mass) is 568.33.

Example 3

Formula Ic compound, its chemical structure is as follows:

Compounds of formula Ic can be obtained through the following synthetic routes:

The preparation method of formula Ic compound specifically comprises the following steps:

(1) Preparation of Intermediate 15: Sodium carbonate (100 g, 943 mmol) was added to a solution of Intermediate 14 (25 g, 159 mmol) in DMF (500 mL) at room temperature. Then, the reactant was heated to 90° C., and Compound A (71.5 g, 550 mmol) was added to the solution. The reaction was stirred for 2 hours. The mixture was poured into ice water (300 mL), and extracted with MTBE. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude material was purified by silica gel chromatography to afford Intermediate 15 (31 g, 94%) as a yellow oil.

(2) Preparation of Intermediate 16: Pd/C (3 g, 3.0 mmol) was added to a stirred solution of Intermediate 15 (31 g, 150 mmol) in EtOH (80 mL) at room temperature. The reaction mixture was reacted overnight under 40 psi of hydrogen. The mixture was concentrated by filtration under reduced pressure to intermediate 16 (22 g, 82%) as a brown oil.

(3) Preparation of Intermediate 17: Intermediate 16 (11.0 g, 62 mmol) was added dropwise into concentrated sulfuric acid at °C. Then, _KNO3 (6.2 g, 62 mmol) was added in portions. Stir at °C for 30 minutes. The solution was poured into ice water, the pH was adjusted to 7-8, extracted with saturated NaHCO ₃ and ethanol. Washed with brine, dried and concentrated in vacuo to afford Intermediate 17 (9.6 g, 70%) as a yellow solid.

(4), Preparation of Intermediate 18: Add Intermediate 3 (2.2 g, 9.0 mmol) and p-toluenesulfonic acid (2.1 g, 10.8 mmol) to Intermediate 17 (2.0 g, 9.0 mmol) 2-pentanol (15 mL) solution ). The reaction mixture was stirred overnight at 120 °C. The solvent was evaporated under reduced pressure to afford intermediate 18 (2.9 g, 75%) as a brown solid.

(5) Preparation of Intermediate 19: Intermediate 6 (0.8 g, 8.1 mmol) and DIPEA (1.1 g, 8.7 mmol) were added to Intermediate 18 (2.9 g, 6.7 mmol) in DMA (20 mL). The mixture was stirred at 85°C for 2 hours. The mixture was poured into ice water and stirred for half an hour. Then, the mixture was filtered under reduced pressure. The filter cake was washed with ethanol to give intermediate 19 (2.0 g, 65%) as a yellow solid.

(6) Preparation of Intermediate 20: Ammonium chloride (150 mg, 2.7 mmol) and iron (1.3 g, 23.4 mmol) were added to Intermediate 19 (2.0 g, 3.9 mmol) in ethanol (25 mL). The mixture was stirred at 75°C for 2 hours. The mixture was filtered under reduced pressure. The filtrate extract was washed with brine, dried and concentrated in vacuo. The crude material was purified by silica gel chromatography to afford Intermediate 20 (800 mg, 41%) as a gray solid.

(7), preparation of formula Ic compound: Intermediate 9 (165 mg, 1.26 mmol), HATU (237 mg, 0.63 mmol) and TEA (126 mg, 1.26 mmol). The reaction was stirred at room temperature for 2 hours. The mixture was purified by preparative HPLC to give compound Ic (20 mg, 5.3%) as a yellow solid.

Proton nuclear magnetic resonance ¹ H-NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product Ic obtained, and the results are as follows:

Absorption peaks in ¹ H-NMR spectrum: δ=8.88(s, 1H), 8.32(t, J=4.0Hz, 1H), 8.28(s, 1H), 7.48(d, J=8.0Hz, 1H), 7.21(d, J=12.0Hz, 1H), 7.20-7.14(m, 4H), 7.10-6.96(m, 2H), 6.74(t, J=12.0Hz, 1H), 3.94(s, 3H), 3.70 (d,J=4.0Hz,2H),3.44(t,J=8.0Hz,2H),3.28(t,J=8.0Hz,2H),2.89(s,6H),2.75(s,3H),2.70 (s,6H).

m/s: [MH] ⁺ :536.3. It is calculated that the product has the molecular formula C ₂₈ H ₃₁ F ₂ N ₇ O ₂ and the exact molecular mass (exact mass) is 535.25.

Example 4

Formula Id compound, its chemical structure is as follows:

This compound can be obtained by reacting intermediate 20 described in Example 3 with intermediate 9, and the specific preparation process can be referred to Example 3.

Proton nuclear magnetic resonance ¹ H-NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product Id that obtains, and the results are as follows:

Absorption peaks in ¹ H-NMR spectrum: δ=8.97(s, 1H), 8.31(t, J=4.0Hz, 2H), 8.27(d, J=8.0Hz, 1H), 7.47(d, J=8.0 Hz, 1H), 7.28-7.13(m, 4H), 6.94(t, J=76.0Hz, 1H), 6.61-6.51(m, 1H), 6.47(d, J=4.0Hz, 1H), 3.92(s , 3H), 3.05(t, J=4.0Hz, 2H), 3.01-2.88(m, 2H), 2.74(s, 3H), 2.72(s, 6H).

m/s: [MH] ⁺ :593.3. It is calculated that the product has the molecular formula C ₃₁ H ₃₈ F ₂ N ₈ O ₂ and the exact molecular mass (exact mass) is 592.31.

Example 5

Formula Ie compound, its chemical structure is as follows:

The compound can be obtained by reacting the intermediate 8 described in Example 1 with the intermediate 8a [ClS(O ₂ )CH ₂ CH ₂ Cl]. The specific preparation process can be found in Example 1.

Proton nuclear magnetic resonance ¹ H-NMR (400MHz, CDCl ₃ ) and mass spectrometry were carried out to the target product Ie obtained, and the results are as follows:

Absorption peak in ¹ H-NMR spectrum: δ=9.0(s,1H)8.72(s,1H)8.32(m,1H)8.08(m,1H)7.70(s,1H)7.41(m,1H)7.30( m,2H)7.21(m,1H)6.77(s,1H)6.55(m,1H)6.15(m,1H)5.72(d,J＝10Hz,1H)3.96(s,3H)3.89(s,3H) 2.85(m,2H)2.71(s,3H)2.33(s,6H)2.25(m,2H).

m/s: [MH] ⁺ :536.3. It is calculated that the product has the molecular formula C ₂₇ H ₃₃ N ₇ O ₃ S and the exact molecular mass (exact mass) is 535.24.

Example 6

Formula If compound, its chemical structure is as follows:

The compound can be obtained by reacting the intermediate 16 described in Example 3 with the intermediate 8a [ClS(O ₂ )CH ₂ CH ₂ Cl]. The specific preparation process can be found in Example 3.

Proton nuclear magnetic resonance ¹ H-NMR (400MHz, CDCl ₃ ) and mass spectrometry were carried out on the obtained target product If, and the results are as follows:

Absorption peak in ¹ H-NMR spectrum: δ=9.0(s,1H)8.63(s,1H)8.38(d,J=5.2Hz,1H)8.08(d,J=8.0Hz,1H)7.41(m, 2H)7.28(m,3H)7.03(s,1H)6.55(m,1H)6.21(m,1H)5.76(d,J=10Hz,1H)3.93(s,3H)2.81(m,2H)2.70( s,3H)2.35(s,6H)2.33(m,2H).

m/s: [MH] ⁺ :572.1. It is calculated that the product has the molecular formula C ₂₇ H ₃₁ F ₂ N ₇ O ₃ S and the exact molecular mass (exact mass) is 571.22.

Example 7

Formula Ig compound, its chemical structure is as follows:

Formula Ig compound can be obtained by following synthetic route:

The preparation method of formula Ig compound specifically comprises the steps:

(1) Preparation of Intermediate 21: CH ₃ MgBr (35 mL, 105 mol in THF) was added dropwise to a THF solution (45 mL) of indole (11.50 g, 98.16 mmol) at 0° C. within 10 minutes. The solution was then stirred at 0-5°C for 0.5 hours. A solution of 2,4-dichloro-5-methylpyrimidine (8 g, 49.08 mmol) in THF (25 mL) was added dropwise to the solution. The ice bath was then removed and the solution was stirred at room temperature for 1 hour, then at 60°C for 18 hours. A solution of acetic acid (6 mL) was added followed by water (80 mL). The resulting suspension was stirred at 60°C for 0.5 hours. The resulting solid was collected by filtration, washed with water and methanol, and dried in vacuo to afford Intermediate 21 (2.8 g, 23.4%) as a white solid.

(2) Preparation of Intermediate 22: NaH (945 mg, 39.4 mol) was added to a solution of Intermediate 21 (8.7 g, 35.8 mmol) in THF (100 mL). Stir at 0°C for half an hour. MeI (6.1 g, 43 mmol) was then added to the mixture. The reaction was stirred at room temperature for 3 hours. The mixture was filtered under reduced pressure to afford Intermediate 22 (6.0 g, 65%) as a white solid.

(2) Preparation of intermediate 23: Intermediate 4 (2.5 g, 244 mmol) and p-toluenesulfonic acid (2.3 g, 13 mmol) were added to a solution of intermediate 22 (2.9 g, 11 mmol) in 2-pentanol (30 mL). The reaction was stirred overnight at 120 °C. The mixture was filtered under reduced pressure to afford Intermediate 23 (4.3 g, 88%) as a brown solid.

(2) Preparation of Intermediate 24: Intermediate 6 (1.2 g, 12 mmol) and DIPEA (1.6 g, 13 mmol) were added to Intermediate 23 (4.3 g, 9.6 mmol) in DMA (30 mL). The reaction mixture was stirred at 85 °C for 2 hours. The mixture was poured into ice water and stirred for half an hour. Then, the mixture was filtered under reduced pressure. The filter cake was washed with ethanol to afford Intermediate 24 (2.3 g, 45%) as a yellow solid.

(2) Preparation of Intermediate 25: Ammonium chloride (115 mg, 2.1 mmol) and iron (1.2 g, 21.4 mmol) were added to Intermediate 24 (2.3 g, 4.3 mmol) in ethanol (25 mL). The reaction mixture was stirred at 75°C for 2 hours. The mixture was filtered under reduced pressure. The filtrate was extracted, washed with brine, dried and concentrated in vacuo. The crude material was purified by silica gel chromatography to afford Intermediate 25 (1.2 g, 56%) as a gray solid.

(3) Preparation of compound of formula Ig: Add cobalt chloride (1 mL) and a drop of DMF to a solution of intermediate 9 (64 mg, 0.4 mmol) in DCM (5 mL). The reaction was stirred at room temperature for 1 hour. Cobalt chloride was removed by evaporation under reduced pressure, and the residue was dissolved in DCM (5 mL). To this mixture was added compound 5 (100 mg, 0.3 mmol). The reaction was stirred at 0°C for 30 minutes. The mixture was concentrated in vacuo. The crude product was purified by preparative TLC to afford 1 g of compound (26 mg, 10%) as a yellow solid.

Proton nuclear magnetic resonance H ^- NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product Ig that obtains, and the results are as follows:

Absorption peaks in ¹ H-NMR spectrum: δ=8.71(s,1H), 8.33(d,J=8.0Hz,1H),8.15-8.10(m,2H),7.49(d,J=8.0Hz,1H ),7.39(d,J=8.0Hz,1H),7.23(s,1H),7.15-6.74(m,5H),6.32-6.28(m,1H),4.04(d,J=8.0Hz,2H) , 3.95(s, 3H), 3.44(d, J=8.0Hz, 2H), 2.94(s, 12H), 2.77(s, 3H), 2.47(s, 3H).

m/s:[MH] ⁺ :607.2. It is calculated that the product has the molecular formula C ₃₂ H ₄₀ F ₂ N ₈ O ₂ and the exact molecular mass (exact mass) is 606.32.

Example 8

Formula Ih compound, its chemical structure is as follows:

This compound can be obtained by reacting intermediate 21 and intermediate 4 described in Example 7, and following the same reaction steps in the next three steps. For the specific preparation process, please refer to Example 7.

Proton nuclear magnetic resonance H ^- NMR (400MHz, MeOD) and mass spectrometry were carried out to the target product Ih that obtains, and the results are as follows:

Absorption peaks in ¹ H-NMR spectrum: δ=9.02(s, 1H), 8.26-8.23(m, 2H), 7.93(s, 1H), 7.43(d, J=8.0Hz, 1H), 7.17(d ,J=8.0Hz,1H),7.14(s,1H),7.03(d,J=8.0Hz,1H),6.87-6.82(m,1H),6.77(d,J=76Hz,1H),6.43- 6.39(m, 1H), 3.21(d, J=8.0Hz, 2H), 3.05(t, J=4.0Hz, 2H), 2.70(s, 3H), 2.52(t, J=8.0Hz, 2H), 2.44(s,3H), 2.34(s,6H), 2.31(s,6H).

m/s: [MH] ⁺ :593.2. It is calculated that the product has the molecular formula C ₃₁ H ₃₈ F ₂ N ₈ O ₂ and the exact molecular mass (exact mass) is 592.31.

drug efficacy test

1. Compound enzyme activity test:

1. Test method

The half-inhibitory concentration IC ₅₀ (the concentration of the compound required to inhibit the enzyme activity to 50%) of the compound is determined by immobilizing the enzyme mixed with a specific substrate and different concentrations of the test compound. The assay method used is Caliper Mobility Shift Assay, the assayed kinases are EGFR ^WT and EGFR ^790M/L858R , and the applied standard reference compound is staurosporine.

2. Test results

Table 1 summarizes the results of the enzyme activity inhibition experiments of the compounds. The results showed that the target compounds (Ia, Ib, Ic, Id, Ie, If, Ig and Ih) had very strong inhibitory effects on the two EGFR kinases, and at the same time, the results showed that the target compounds (Ia, Ib, Ic, Id, Ie and The selective inhibitory activity of If) is better. This selective inhibition has important therapeutic implications for acquired drug-resistant tumors carrying the T790M mutation.

Table 1 Results of compound enzyme activity inhibition experiments

2. Tumor cell inhibition test:

1. Test method

(1) Compound: In the in vitro study, the test compound was first dissolved in 100% DMSO, and then diluted to the required concentration, and the final concentration of DMSO was 0.1%. 0.1% (v/v) DMSO was added to the culture medium as a solvent control, a total of 9 concentration gradients, and the test was repeated twice.

(2) Tumor cell line: The tested tumor cell line was cultured in RPMI10 medium containing 10% fetal bovine serum in an incubator at 5% CO ₂ at 37°C. The tested tumor cell lines are: A431, Calu-3, H1975, H1650 and HCC827.

(3) MTS method: the cells were seeded in a 96-well plate, with 3000 cells per well, and incubated overnight in a 5% CO ₂ , 37° C. humidified incubator. The next day after addition of the test compound to the wells, incubation was continued for an additional 72 hours. Cell viability was detected using MTS. _IC50 (concentration of drug required to inhibit cell growth by 50% compared to DMSO control, calculated using non-linear regression analysis of GraphPad Prism software) was calculated.

2. Test results

The inhibitory activity of the target compound Id on A431, Calu-3, H1975, H1650 and HCC827 tumor cells is summarized in Table 2.

Table 2 Results of tumor cell inhibition test

It can be seen from Table 2 that the compound Id of the present invention exhibited inhibitory activity on various tested tumor cells.

3. Pharmacokinetic experiment

1. Experimental method:

Experimental animals: nude mice, female, 6-7 weeks; body weight: 20-25g;

Preparation of the test article: the test compound was formulated into 0.2 mg/mL (for intravenous administration) and 1.0 mg/mL (for oral administration), ready for use. Route of administration: oral/intravenous. Dosing volume and frequency: 5mL/kg, single administration.

Sample collection: blood was collected according to the following time points, 3 animals were collected at each time point, and about 0.5-1.0 mL of whole blood was collected. Blood was collected at 5min, 15min, 30min, 1h, 2h, 4h, 8h and 24h after administration. All animals were euthanized after completion of the experiment.

2. Sample analysis and results

Sample analysis: LC-MS/MS method was used to detect the collected samples. The instrument model used is SHIMADZU20A-API4000.

Pharmacokinetic data analysis: use WinNolin to fit and calculate the obtained plasma concentration data according to the non-compartmental model method, and some results are summarized in Table 3.

Table 3 The pharmacokinetic parameters of the target compound calculated according to the non-compartmental model method

The test results in nude mice show that the compound of the present invention has good pharmacokinetic characteristics.

In addition, the toxic and side effects of the compounds of the present invention have also been experimentally tested. Nude mice were given continuous administration once a day for 21 days, and rats were given continuous administration once a day for 14 days. The results showed that the compound of the present invention had less toxic and side effects and was well tolerated in animals. The highest dose tested 100mg/kg/day.

The above examples are representative only. It can be seen from the above examples that the compounds of the present invention are ideal high-efficiency dual irreversible tyrosine kinase inhibitors, and can be expected to be used for the treatment or prevention of lung cancer, gastrointestinal cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, Head and neck squamous cell carcinoma, epidermal squamous cell carcinoma, prostate cancer, glioma, nasopharyngeal carcinoma and other malignant tumor diseases have achieved very good results. It can also be combined with different types of medicinal salts to make oral preparations (tablets or capsules, etc.). Tablets or capsules made with the compounds of this invention may be administered one or more times daily. The compound of the present invention can also be combined with other drugs to make compound preparations.

The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (8)

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof: in: R ₁ is CH ₂ F, CHF ₂ , CF ₃ , OCH ₂ F, OCHF ₂ , OCF ₃ , C ₂ ~C ₃ fluorohydrocarbon group, C ₂ ~C ₃ chlorohydrocarbon group; or, R ₁ is OC _k H _{2k +1} , where k is an integer between 1 and 3; R ₂ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , -N(CH ₃ ) _CH2CH2N ( _CH3 ) _{2 ;} R ₃ is -NHC(O)CH=CHCH ₂ N(CH ₃ ) ₂ , -NHSO ₂ CH=CH ₂ , -NHSO ₂ CH=CHCH ₂ N(CH ₃ ) ₂ ; R ₄ is Wherein, R ₅ is H, CH ₃ , CH ₂ CH ₃ ; X is CH. 2. The compound with general formula (I) or its pharmaceutically acceptable salt according to claim 1, characterized in that: in formula (I), R ₁ is selected from CH ₂ F, CHF ₂ , CF ₃ , OCH One of ₂ F, OCHF ₂ , OCF ₃ , OCH ₃ and OCH ₂ CH ₃ . 3. The compound with general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: in formula (I), R ₅ is H or CH ₃ . 4. The compound with general formula (I) or its pharmaceutically acceptable salt according to claim 1, characterized in that: in formula (I), R ₁ is selected from OCH ₂ F, OCHF ₂ , OCF ₃ , OCH ₃ and one of OCH ₂ CH ₃ ; R ₃ is -NHC(O)CH=CHCH ₂ N(CH ₃ ) ₂ , -NHSO ₂ CH=CH ₂ , -NHSO ₂ CH=CHCH ₂ N(CH ₃ ) ₂ ; R ₂ is -NHCH ₂ CH ₂ N(H)CH ₃ , -NHCH ₂ CH ₂ N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₂ N(H)CH ₃ , -N(CH ₃ ) _CH2CH2N ( _CH3 ) _{2 ;} X is CH. 5. The compound with general formula (I) or its pharmaceutically acceptable salt according to claim 1, characterized in that: the compound is one of the compounds represented by the following structural formula: 6. A compound represented by formula Ic or a pharmaceutically acceptable salt thereof, 7. Use of the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating indications related to EGFR kinase function. 8. The use according to claim 7, characterized in that: the indications related to EGFR kinase function are breast cancer, ovarian cancer, gastrointestinal cancer, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, Epidermal squamous cell carcinoma, prostate cancer, glioma and nasopharyngeal carcinoma.