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

Phenylaminopyrimidine derivatives and their use in preparation of drugs for resisting cancers Download PDF

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CN105254615B
CN105254615B CN201510405448.6A CN201510405448A CN105254615B CN 105254615 B CN105254615 B CN 105254615B CN 201510405448 A CN201510405448 A CN 201510405448A CN 105254615 B CN105254615 B CN 105254615B
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CN105254615A (en
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殷建明
李邦良
吕裕斌
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Hangzhou Bangshun Pharmaceutical Co ltd
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HANGZHOU HUADONG MEDICINE GROUP NEW MEDICINE RESEARCH INSTITUTE Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

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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

Anilinopyrimidine derivative and application thereof in preparation of anti-malignant tumor drugs
Technical Field
The invention relates to an aniline pyrimidine derivative and application thereof in preparing a medicament for preventing and treating malignant tumors.
Background
EGFR (epidermal growth factor receptor) is a transmembrane receptor, and binding of EGFR to an extracellular domain forms a receptor dimer and activates an intracellular tyrosine kinase domain, triggering kinase autophosphorylation and phosphorylation of downstream molecules, activating various cellular functions including proliferation and survival. EGFR is detectable in nearly 80% to 85% of NSCLC patients, with expression levels that vary widely over a continuous range. The incidence of EGFR mutations in lung adenocarcinoma reaches 50% in asian populations and is higher in non-smokers, women and non-mucinous tumors.
The most common EGFR mutations are an exon 19 deletion (E19del, seen in 45% of patients) and an exon 21L858R mutation (seen in 40% of patients), both of which result in tyrosine kinase domain activation and are associated with tumor sensitivity to small molecule TKIs (tyrosine kinase inhibitors). These drug-sensitive mutations are found in nearly 10% of caucasian NSCLC patients and up to 50% of asian patients. The EGFR gene is the target of the Asian lung adenocarcinoma patients with the largest mutation probability and the most patients with treatment benefit. Other drug-sensitive mutation types include exon 21(L861Q) and exon 18(G719X) point mutations. The T790M mutation may result in TKI class drug resistance, and reports have shown that this type of mutation is found in about 50% of tumor progression patients.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel aniline pyrimidine derivative which is an ideal non-reversible EGFR kinase inhibitor and can be used for effectively preventing or treating various malignant tumor diseases such as 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 the like.
In order to solve the technical problems, the invention adopts the following technical scheme:
a compound having the general formula (I), a pharmaceutically acceptable salt, hydrate, or metabolite formed metabolically in any form thereof,
wherein:
R1is CH2F，CHF2，CF3，OCH2F，OCHF2，OCF3，C2～C3Fluorinated hydrocarbon radical, C2～C3A chlorinated hydrocarbon group; or, R1Is OCkH2k+1Wherein k is an integer between 1 and 3;
R2and R3Is a straight or branched hydrocarbon chain having at least one double bond, and R2And R3Optionally and independently of one or two other methylene units of (a) via-NHC (O) -, -C (O) NH-, -N (H) SO2-or-SO2N (h) -substitution; or, R2And R3is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)24-methylpiperazin-1-yl group, 4-ethylpiperazin-1-yl group, wherein R is2And R3Cannot be the same group;
R4is composed of
Wherein R is5Is H, CH3、CH2CH3；
X is N or CH;
the compound with the general formula (I), the pharmaceutically acceptable salt, the hydrate or the metabolite formed by metabolism in any form has non-exchangeable hydrogen which is not substituted or is partially or completely substituted by deuterium.
According to one aspect of the invention, in formula (I), R1Is selected from CH2F，CHF2，CF3，，OCH2F，OCHF2，OCF3And OCH and3one of (1); r2、R3、R4、R5And X is as defined above.
According to still another aspect of the present invention, in the formula (I), R2is-NHC (O) CH ═ CH2、-NHC(O)CH＝CHCH2N(CH3)2、-NHSO2CH＝CH2、-NHSO2CH＝CHCH2N(CH3)2；R3is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)24-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl; r1、R4、R5And X is as defined above.
According to still another aspect of the present invention, in the formula (I), R3is-NHC (O) CH ═ CHCH2N(CH3)2、-NHSO2CH＝CH2、-NHSO2CH＝CHCH2N(CH3)2；R2is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)24-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl; r1、R4、R5And X is as defined above.
According to a particular and preferred aspect of the invention, in formula (I), R5Is H or CH3，R1、R2、R3、R4And X is as defined above.
According to a particular aspect, X is CH.
According to a preferred aspect of the present invention, in the formula (I), R1Is selected from OCH2F，OCHF2，OCF3，OCH3And OCH2CH3One of (1); r3is-NHC (O) CH ═ CHCH2N(CH3)2、-NHSO2CH＝CH2、-NHSO2CH＝CHCH2N(CH3)2；R2is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)24-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl; x is CH, R4、R5The definition of (A) is as above.
According to the invention, representative compounds are as follows:
according to the invention, the compound not only comprises a single compound form, but also comprises a plurality of compound mixtures with the structure meeting the requirements of the general formula (I), and different isomer forms of the same compound, such as raceme, enantiomer, diastereoisomer and the like. The pharmaceutically acceptable salts include, but are not limited to, hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, benzenesulfonate, benzoate, methylbenzenesulfonate, succinate, fumarate, tartrate, gallate, citrate, and the like. The "prodrug of a compound having the general formula (I)" means a substance which, when administered by an appropriate method, undergoes a metabolic or chemical reaction in the subject to be converted into at least one compound of the formula (I) or a salt thereof.
The compounds of the invention may be prepared by synthetic routes analogous to those well known in the chemical arts, particularly by synthesizing the compounds of the invention according to the description contained herein. Reagents are generally obtained from commercial sources or readily prepared using methods well known to those skilled in the art.
The invention also relates to an intermediate for preparing the compound with the general formula (I), which is shown as the general formula (II):
in the general formula (II), R1，R2，R4The definition of (A) is as above.
Further, in the above general formula (II), R2is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)24-methylpiperazin-1-yl or 4-ethylpiperazin-1-yl.
Further, in the above general formula (II), R1Is OCH2F，OCHF2，OCF3Or OCH3。
Representative intermediates of formula (II) are for example:
the intermediate is utilized to obtain the corresponding compound with the general formula (I) through one-step amidation reaction.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the compound provided by the invention is a novel aniline pyrimidine derivative, is an ideal high-efficiency irreversible EGFR tyrosine kinase inhibitor, inhibits the activity and phosphorylation of kinase by acting on the intracellular part of EGFR and competitively combining with ATP, and blocks the ATP binding site of EGFR tyrosine kinase so as to achieve the aim of specifically inhibiting EGFR. Therefore, the compounds of the invention can be used for preparing medicines for treating or preventing 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, epidermal squamous cell carcinoma, prostatic cancer, glioma, nasopharyngeal carcinoma and other malignant tumor diseases. More particularly, the compound of the invention can specifically act on EGFR kinase with T790M mutation, but has weak inhibition effect on wild type EGFR kinase, and the selective inhibition effect has therapeutic significance on acquired drug-resistant tumor with T790M mutation and has small toxic and side effects.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.
Example 1
A compound of formula Ia, having the chemical structure:
the compounds of formula Ia can be obtained by the following synthetic route:
the process for the preparation of the compound of formula Ia comprises the following steps:
(1) preparation of intermediate 3: to a stirred solution of 2, 4-dichloropyrimidine (4.83g, 32.4mmol) and dimethoxyethane (50mL) at about 60 deg.C was added ferric chloride (5.5g, 33.9mmol) and 1-methylindole (5.0g, 38 mmol). The resulting mixture was stirred at about 60 ℃ overnight. After cooling, the solid precipitated by adding methanol (50mL) and water (100 mL). The resulting slurry was stirred for 3 hours. The solid was collected by filtration, washed with methanol (100mL) and dried overnight at 50 ℃ to give intermediate 3(6g, 77%) as a purple solid.
(2) Preparation of intermediate 5: tosylate hydrate (772.82mg, 4.1mmol) was added to a mixture of intermediate 3(1g, 4.1mmol) and intermediate 4(763mg, 4.1mmol) in 2-pentanol (10 mL). The resulting mixture was stirred at 105 ℃ for 2.5 hours. Then cooled to room temperature and the resulting precipitate was collected by filtration, washed with 2-pentanol (20mL) and dried under vacuum to give intermediate 5(1.2g, 75%) as a yellow solid.
(3) Preparation of intermediate 7: a solution of intermediate 5(500mg, 1.27mmol) and intermediate 6(155.8mg, 1.53mmol) in DMA (50mL) was stirred at 85 deg.C for 5-6 hours and then cooled to room temperature. Water (50mL) was added, and the mixture was stirred for 3-4 hours. The solid material was collected by filtration, washed with water (30mL) and dried at 50 ℃ for 12 hours to give intermediate 7(400mg, 96%) as an orange solid.
(4) Preparation of intermediate 8: a mixture of intermediate 7(400mg, 0.84mmol), iron (281.84mg, 5.04mmol), ammonium chloride (31.5 mg, 0.588mmol), ethanol (30mL) and water (10mL) was heated at reflux for 2 h. The desired product was combined with a 7M methanolic ammonia elution column and concentrated in vacuo onto silica gel. Purification by column chromatography gave intermediate 8 as a beige foam which was used directly in the next reaction step.
(5) Preparing a compound of formula Ia: to a solution of intermediate 8(300mg, 0.67mmol) in DMF (12mL) was added 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 give the crude product directly (300mg) as a dark brown oil. The crude product was further purified by TLC chromatography to give compound Ia as a dark yellow semi-solid.
Performing hydrogen nuclear magnetic resonance on the obtained target product Ia1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 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. The product has molecular formula C31H40N8O2The exact molecular mass (exact) was 556.33.
Example 2
A compound of formula Ib having the chemical structure:
the compounds of formula Ib can be obtained by the following synthetic route:
the preparation method of the compound shown in the formula Ib specifically comprises the following steps:
(1) preparation of intermediate 12: to the suspension of intermediate 5(340mg, 0.86mmol) in DMA (10mL) was added DIPEA (144mg, 1.12mmol), intermediate 11(117mg, 1.03mmol) and the mixture was heated to 80 ℃ for 16 h. The mixture was poured into water (40mL), stirred for 5min and filtered to give intermediate 12(290mg, 69%) as an orange solid, m/z [ MH)]+Is 488.
(2) Preparation of intermediate 13: to a solution of intermediate 12(290mg, 0.59mmol) in EtOH (12 mL)/water (4mL) were added iron (200mg, 3.57mmol) and ammonium chloride (22mg, 0.41 mmol). Heated to reflux for 2 hours. The mixture was extracted with ethanol (100mL) and water (80mL), filtered through celite, dried and chromatographed to give intermediate 13(250mg, 93%) as a dark semi-solid.
(3) Preparing a compound of formula Ib: to a solution of intermediate 13(200mg, 0.44mmol) in DMF (8mL) was added intermediate 9(145mg, 0.88mmol), HATU (168mg, 0.44mmol) and TEA (88mg, 0.88 mmol). The mixture was stirred at room temperature for 2 hours. Purification by preparative HPLC gave compound Ib (140mg, 56%) as a green solid.
Performing hydrogen nuclear magnetic resonance on the obtained target product Ib1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 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. The product has molecular formula C32H40N8O2The exact molecular mass (exact) was 568.33.
Example 3
A compound of formula Ic having the chemical structure:
compounds of formula Ic can be obtained by the following synthetic route:
the process for the preparation of compounds of formula Ic comprises in particular the following steps:
(1) preparation of intermediate 15: to a solution of intermediate 14(25g, 159mmol) in DMF (500 mL) was added sodium carbonate (100g, 943mmol) at room temperature. The reaction was then heated to 90 ℃ and Compound A (71.5g, 550mmol) was added to the solution. The reaction was stirred for 2 hours. The mixture was poured into ice water (300mL) 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(31g, 94%) as a yellow oil.
(2) Preparation of intermediate 16: Pd/C (3g, 3.0mmol) was added to a stirred solution of intermediate 15(31g, 150mmol) in EtOH (80mL) at room temperature. The reaction mixture was reacted under 40psi of hydrogen overnight. The mixture was concentrated by filtration under reduced pressure to intermediate 16(22g, 82%) as a brown oil.
(3) Preparation of intermediate 17: intermediate 16(11.0g, 62mmol) was added dropwise to concentrated sulfuric acid at deg.C. Then, KNO is added3(6.2g, 62mmol) was added in portions. Stirring at deg.C for 30 minutes. The solution was poured into ice water, the pH was adjusted to 7-8, saturated NaHCO3And ethanol extraction. Washed with brine and concentrated by vacuum drying to give intermediate 17(9.6g, 70%) as a yellow solid.
(4) Preparation of intermediate 18: to a solution of intermediate 17(2.0g, 9.0mmol) in 2-pentanol (15mL) was added intermediate 3(2.2g, 9.0mmol) and p-toluenesulfonic acid (2.1 g, 10.8 mmol). The reaction mixture was stirred at 120 ℃ overnight. The solvent was evaporated under reduced pressure to give intermediate 18(2.9g, 75%) as a brown solid.
(5) Preparation of intermediate 19: to a solution of intermediate 18(2.9g, 6.7mmol) in DMA (20mL) was added intermediate 6(0.8g, 8.1mmol) and DIPEA (1.1 g, 8.7 mmol). The mixture was stirred at 85 ℃ 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(2.0g, 65%) as a yellow solid.
(6) Preparation of intermediate 20: to an ethanol solution (25mL) of intermediate 19(2.0g, 3.9mmol) was added ammonium chloride (150mg, 2.7mmol) and iron (1.3g, 23.4 mmol). The mixture was stirred at 75 ℃ 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 give intermediate 20(800mg, 41%) as a grey solid.
(7) Preparation of a compound of formula Ic: to a stirred solution of intermediate 20(300mg, 0.63mmol) in DMF (10mL) was added intermediate 9(165mg, 1.26mmol), HATU (237mg, 0.63mmol) and TEA (126mg, 1.26 mmol). The reaction was stirred at room temperature for 2 hours. The mixture was purified by preparative HPLC to give Ic compound (20mg, 5.3%) as a yellow solid.
The obtained target product Ic is subjected to hydrogen nuclear magnetic resonance1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 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 to 7.14(m,4H),7.10 to 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. The product has molecular formula C28H31F2N7O2Accuracy ofThe molecular mass (exact) was 535.25.
Example 4
A compound of formula Id having the chemical structure:
this compound can be obtained by reacting intermediate 20 with intermediate 9 as described in example 3, and the specific preparation process can be seen in example 3.
Performing hydrogen nuclear magnetic resonance on the obtained target product Id1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 8.97(s,1H),8.31(t, J ═ 4.0Hz,2H),8.27(d, J ═ 8.0Hz,1H),7.47(d, J ═ 8.0Hz,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. The product has molecular formula C31H38F2N8O2The exact molecular mass (exact) was 592.31.
Example 5
A compound of formula Ie having the chemical structure:
the compound can be prepared by reacting intermediate 8 described in example 1 with intermediate 8a [ ClS (O)2)CH2CH2Cl ] is obtained by reaction, and the specific preparation process can be seen in the examples1。
Performing hydrogen nuclear magnetic resonance on the obtained target product Ie1H-NMR(400MHz，CDCl3) And mass spectrometry, the results are as follows:
1absorption peaks in H-NMR spectrum: 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. The product has molecular formula C27H33N7O3S, exact molecular mass (exact) 535.24.
Example 6
A compound of formula If, having the chemical structure:
the compound can be prepared by reacting intermediate 16 described in example 3 with intermediate 8a [ ClS (O)2)CH2CH2Cl ] is obtained by reaction, and the specific preparation process can be seen in example 3.
Performing hydrogen nuclear magnetic resonance on the obtained target product If1H-NMR(400MHz，CDCl3) And mass spectrometry, the results are as follows:
1absorption peaks in H-NMR spectrum: 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. The product has molecular formula C27H31F2N7O3S, exact molecular mass (exact)mass) is 571.22.
Example 7
A compound of formula Ig having the chemical structure:
the compounds of formula Ig can be obtained by the following synthetic routes:
the process for the preparation of the compound of formula Ig comprises the following steps:
(1) preparation of intermediate 21: at 0 ℃ adding CH3MgBr (in THF, 35mL, 105mol) was added dropwise over 10 min to a solution of indole (11.50g, 98.16mmol) in THF (45 mL). The solution was then stirred at 0-5 ℃ for 0.5 h. A solution of 2, 4-dichloro-5-methylpyrimidine (8g, 49.08mmol) in THF (25mL) was added dropwise to the solution. The ice bath was then removed and the solution was stirred at room temperature for 1 hour and then at 60 ℃ for 18 hours. A solution of acetic acid (6mL) was added, followed by water (80 mL). The resulting suspension was stirred at 60 ℃ for 0.5 hour. The resulting solid was collected by filtration, washed with water and methanol, and dried under vacuum to give intermediate 21(2.8 g, 23.4%) as a white solid.
(2) Preparation of intermediate 22: to a solution of intermediate 21(8.7g, 35.8mmol) in THF (100mL) was added NaH (945mg, 39.4 mol). Stirring at 0 deg.C for half an hour. MeI (6.1g, 43mmol) was then added to the mixture. The reaction was stirred at room temperature for 3 hours. The mixture was filtered under reduced pressure to give intermediate 22(6.0g, 65%) as a white solid.
(2) Preparation of intermediate 23: to a solution of intermediate 22(2.9g, 11mmol) in 2-pentanol (30mL) was added intermediate 4(2.5g, 244mmol) and p-toluenesulfonic acid (2.3g, 13 mmol). The reaction was stirred at 120 ℃ overnight. The mixture was filtered under reduced pressure to give intermediate 23(4.3g, 88%) as a brown solid.
(2) Preparation of intermediate 24: to a solution of intermediate 23(4.3g, 9.6mmol) in DMA (30mL) was added intermediate 6(1.2g, 12mmol) and DIPEA (1.6g, 13 mmol). The reaction mixture was stirred at 85 ℃ 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 24(2.3g, 45%) as a yellow solid.
(2) Preparation of intermediate 25: to an ethanol solution (25mL) of intermediate 24(2.3g, 4.3mmol) were added ammonium chloride (115mg, 2.1mmol) and iron (1.2g, 21.4 mmol). The reaction mixture was stirred at 75 ℃ for 2 hours. The mixture was filtered through reduced pressure. The filtrate was extracted, washed with brine, dried and concentrated in vacuo. The crude material was purified by silica gel chromatography to give intermediate 25(1.2g, 56%) as a grey solid.
(3) Preparing a compound of formula Ig: to a solution of intermediate 9(64mg, 0.4mmol) in DCM (5mL) was added 1mL of cobalt chloride and one drop of DMF. The reaction was stirred at room temperature for 1 hour. The cobalt chloride was evaporated under reduced pressure and the residue was dissolved in DCM (5 mL). To the mixture was added compound 5(100mg, 0.3 mmol). The reaction was stirred at 0 ℃ for 30 minutes. The mixture was concentrated in vacuo. The crude product was purified by preparative TLC to give the Ig compound (26mg, 10%) as a yellow solid.
Performing hydrogen nuclear magnetic resonance on the obtained target product Ig1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 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. Calculating to obtain the score of the productSub-formula C32H40F2N8O2The exact molecular mass (exact) was 606.32.
Example 8
A compound of formula Ih having the chemical structure:
this compound can be obtained by reacting intermediate 21 with intermediate 4 as described in example 7, followed by 3 identical reaction steps, the specific preparation of which is described in example 7.
Performing hydrogen nuclear magnetic resonance on the obtained target product Ih1H-NMR (400MHz, MeOD) and mass spectrometry results were as follows:
1absorption peaks in H-NMR spectrum: 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, 31H).
m/s:[MH]+:593.2. The product has molecular formula C31H38F2N8O2The exact molecular mass (exact) was 592.31.
Test of drug efficacy
Firstly, testing the activity of compound enzyme:
1. test method
Half inhibitory concentration of compound IC50(the concentration of the compound required to inhibit the enzyme activity to 50%) was determined by mixing the immobilized enzyme with the specific substrate and the test compound at different concentrations. The assay method usedIs a Caliper Mobility Shift Assay (Caliper Shift Assay) in which the kinase measured is EGFRWTAnd EGFR790M/L858RThe standard reference compound used was staurosporine (staurosporine).
2. Test results
Table 1 summarizes the results of the compound enzyme activity inhibition experiments. The results showed that the target compounds (Ia, Ib, Ic, Id, Ie, If, Ig and Ih) had very strong inhibitory effects on two EGFR kinases, and at the same time, the results showed that the selective inhibitory activity of the target compounds (Ia, Ib, Ic, Id, Ie and If) was better. This selective inhibition has important therapeutic implications for acquired-resistant tumors carrying the T790M mutation.
TABLE 1 results of inhibition experiment of enzyme Activity of Compounds
Secondly, tumor cell inhibition test:
1. test method
(1) And a compound: in vitro studies test compounds were first dissolved in 100% DMSO and then diluted to the desired concentration, with a final DMSO concentration of 0.1%. 0.1% (v/v) DMSO was added to the medium as a solvent control for a total of 9 concentration gradients and the test was repeated twice.
(2) And tumor cell lines: the tumor cell lines tested were cultured in RPMI10 medium containing 10% fetal bovine serum in 5% CO2And culturing in an incubator at 37 ℃. The tumor cell lines tested were: a431, Calu-3, H1975, H1650 and HCC 827.
(3) And MTS method: cells were seeded in 96-well plates at 3000 cells per well at 5% CO2Incubated overnight in a humidified incubator at 37 ℃. The next day test compounds were added to the wells and incubated for a further 72 hours. The activity of the cells was measured using MTS. Computing IC50(fine compared to DMSO control groupThe concentration of drug required for 50% inhibition of cell growth, calculated using non-linear regression analysis of GraphPadPrism software).
2. Test results
The tumor cell inhibitory activity of the target compound Id against A431, Calu-3, H1975, H1650 and HCC827 is summarized in Table 2.
TABLE 2 tumor cell inhibition test results
As can be seen from Table 2, the compound Id of the present invention exhibited inhibitory activity against various tumor cells tested.
Third, pharmacokinetic experiment
1. The experimental method comprises the following steps:
experimental animals: nude mice and females for 6-7 weeks; weight: 20-25 g;
preparing a test article: test compounds were formulated for 0.2mg/mL (for intravenous administration) and 1.0mg/mL (for oral administration) for use. The administration route is as follows: oral/intravenous. Dose volume and frequency: 5mL/kg, single administration.
Collecting samples: blood was collected at the following time points, 3 animals per time point, and whole blood was taken at approximately 0.5-1.0 mL. Blood was taken 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
And (3) sample analysis: the collected samples were tested using LC-MS/MS method. The instrument model number SHIMADZU20A-API4000 was used.
Pharmacokinetic data analysis: the resulting plasma concentration data were fitted and calculated using WinNolin according to a non-compartmental model and part of the results are summarized in table 3.
TABLE 3 pharmacokinetic parameters of target Compounds calculated according to a non-compartmental model
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 compound are tested. The compound is continuously administrated to nude mice once a day for 21 days, and rats are continuously administrated once a day for 14 days, and the results show that the compound has less toxic and side effects and good tolerance in animals, and the tested highest dose is 100 mg/kg/day.
The above embodiments are merely representative. As can be seen from the above examples, the compound of the present invention is an ideal high-efficiency dual irreversible tyrosine kinase inhibitor, and can be expected to be used for treating or preventing various malignant tumor diseases such as lung cancer, gastrointestinal cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, head and neck squamous cancer, epidermal squamous cancer, prostate cancer, glioma and nasopharyngeal carcinoma, and can be combined with different types of pharmaceutically acceptable salts to prepare oral preparations (tablets or capsules, etc.). Tablets or capsules made with the compounds of the present invention may be administered one or more times daily. The compound of the invention can also be combined with other medicines to prepare a compound preparation.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (8)

1. A compound having the general formula (I):
wherein:
R1is CH2F，CHF2，CF3，OCH2F，OCHF2，OCF3，C2～C3Fluorinated hydrocarbon radical, C2～C3A chlorinated hydrocarbon group; or,R1is OCkH2k+1Wherein k is an integer between 1 and 3;
R2is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)2；
R3is-NHC (O) CH ═ CHCH2N(CH3)2、-NHSO2CH＝CH2、-NHSO2CH＝CHCH2N(CH3)2；R4Is composed of
Wherein R is5Is H, CH3、CH2CH3；
X is CH.
2. A compound having the general formula (i) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein: in the formula (I), R1Is selected from CH2F，CHF2，CF3，OCH2F，OCHF2，OCF3，OCH3And OCH2CH3One kind of (1).
3. A compound having the general formula (i) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein: in the formula (I), R5Is H or CH3。
4. A compound having the general formula (i) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein: in the formula (I), R1Is selected from OCH2F，OCHF2，OCF3，OCH3And OCH2CH3One of (1); r3is-NHC (O) CH ═ CHCH2N(CH3)2、-NHSO2CH＝CH2、-NHSO2CH＝CHCH2N(CH3)2；R2is-NHCH2CH2N(H)CH3、-NHCH2CH2N(CH3)2、-N(CH3)CH2CH2N(H)CH3、-N(CH3)CH2CH2N(CH3)2(ii) a X is CH.
5. A compound having the general formula (i) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein: 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 a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of an indication relating to EGFR kinase function.
8. 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 cancer.
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