CN110092781A

CN110092781A - A kind of novel quinazoline quinoline class EGFR inhibitor and its preparation and application

Info

Publication number: CN110092781A
Application number: CN201810096251.2A
Authority: CN
Inventors: 邹敏; 郑立运; 陈慧平; 张长征; 赵志鸿; 张小俊; 马方; 王桂芳
Original assignee: Henan Academy of Medical and Pharmaceutical Sciences
Current assignee: Henan Academy of Medical and Pharmaceutical Sciences
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2019-08-06

Abstract

A kind of novel quinazoline quinoline class EGFR inhibitor and its preparation and application, belong to pharmaceutical synthesis field, general formula is as follows:, R is selected from one of H, F, Cl, Br, I in formula；R₁、R₂Identical or different, they are respectively selected from H, nitro, amino, hydroxyl, 2- methoxy ethoxy, C₁‑C₄Alkoxy, C₁‑C₄One of alkyl.The invention has the advantages that: be different from the existing 4 quinazoline inhibitor replaced by various aniline, the present invention is replaced at 4, quinazoline skeleton using the thiophene methyl amine with extensive bioactivity, simultaneously 6,7 by rational modification but do not include electrophilic property acrylamide key, covalent bond is formed to avoid with EGFR, design has synthesized Novel series EGFR inhibitor.Compared with existing anti-tumor drug, such compound can significantly inhibit EGFR phosphorylation, and being overexpressed tumour cell (such as A431 and MCF-7) to EGFR has good antiproliferative activity.

Description

A kind of novel quinazoline quinoline class EGFR inhibitor and its preparation and application

Technical field

The invention belongs to technical field of medicine synthesis, and in particular to a kind of to form the novel of covalent bond independent of with target enzyme Quinazoline ditosylate salt inhibitor, i.e. N- ((5- substituted thiophene -2- base) methyl) -6,7- substituted quinazoline -4- aminated compounds or its medicine With salt and its preparation and application.

Background technique

EGF-R ELISA EGFR (epidermal growth factor receptor) is a kind of transmembrane After tyrosine kinases receptors, with ligand effect, signal path is activated, including Ras-Raf-MAP kinase pathway and PI3- Kinase/AKT access, therefore various cell functions are affected, such as cell cycle progress, proliferation, transfer, apoptosis.EGFR It is overexpressed or mutation is found in lung, mammary gland, colon, ovary and head and neck neoplasm, EGF-R ELISA EGFR has become For the important target spot for studying treating cancer.

Currently, there are many RGFR inhibitor to be used for clinical treatment, wherein 4 4- amido quinazolines (be shown in by basic structure Fig 1) analog derivative such as gefitinib, erlotinib, lapatinib, and afatinib by FDA approval be used for non-small cell Lung cancer, cancer of pancreas, breast cancer.

Fig 1

In non-small cell lung cancer, 90% patient is to cause [Clin Cancer by the mutation (L858R) of EGFR Leu858 Res, 2010,16 (12): 3164], although with these mutation non-small cell lung cancer patient to first generation EGFR inhibitor, such as Gefitinib, erlotinib reaction are sensitive, but the state of an illness recurs again after the medication several months, and 50% patient is due to T790M mutation Produce acquired resistance [Ann.Onco, 2013,24,2080-2087].Drug resistance caused by order to overcome T790M to be mutated occurs Second generation EGFR inhibitor, using 4- amido quinazoline as parent, unlike first generation EGFR inhibitor quinazoline parent nucleus Side chain include one have electrophilic property acryloyl amine key, i.e. Michael Acceptor, with EGFR cysteine residues The irreversible formation covalent bond of cys797, but these irreversible covalent bond inhibitor with other albumen are also irreversible is bonded, cause Additional toxic side effect, clinically fails in dose-limiting toxicity.

Third generation Osimertinib (AZD9291), the compound of an Aminopyrimidines are both comprising Michael The irreversible inhibitor 2015 non-small cell lung carninomatosis by FDA approval for being clinically mutated with T790M of Acceptor People, however bonding position cys797 mono- new mutation (C797S) result in drug resistance [Nat Med, 2015,21,560- 564], due to crossing high toxicity and binding site mutation, these irreversible inhibitors illustrate only limited clinical effectiveness.

The mutation three times that EGFR T790M/C797S/L858R is generated affects patient to the sensitivity of current all inhibitor Property [Clin Cancer Res, 2015,21,3924-3933].The studies above shows EGFR mutation or is overexpressed related cancer Treat the new inhibitor for needing to be formed independent of covalent bond.

In recent years, 2-thenylaminine causes very big concern in organic chemistry and drug design, and 2-thenylaminine is drawn Enter in a class medicine molecular skeleton, compound can be generated intentionally with the physico-chemical property of the degree of bonding of target enzyme, selectivity and molecule The influence of justice, for example, due to rotatable C-N singly-bound (- CH₂- NH -) as connection abutment, the hardness of molecular structure is reduced, Flexibility is improved, is conducive to molecule in conjunction with target enzyme.Khai Huynh Q in 2011 et al. reports N- (thiophene 2- ylmethyl)- 3- (3,4,5 trimethoxyphenyl) imidazo [1,2 β] pyridine piperazine 6- amine is as ALK5 inhibitor, IC₅₀Value is 0.7 μM [Journal of Biomolecular Screening,2011,16(7),724-733]；2015, Pontiki Eleni etc. It is very strong anti-to report that 2- (4- methylpiperazine-1-yl)-N- (thiophene -2- base) pteridine -4- amine has as lipoxygenase inhibitors Scorching activity [Future Med.Chem, 2015,7 (14), 1937-1951]；2017, Kaplan ' s Alan P reported a system Arrange 5- (1- methyl -5- (trifluoromethyl) -1H- pyrazole-3-yl) novel Monoamine Oxidase B of thiophene-2-carboxamide derivatives Inhibitor has selectivity [Chem.Neurosci, 2017,8,2746-2758] well.

The studies above shows, targeted therapy needs further structural modification, design synthesizing efficient low toxicity, independent of with The new inhibitor that target enzyme forms covalent bond has significant clinical medicine meaning as antitumoral compounds.

Summary of the invention

Replaced the technical problem to be solved in the present invention is to provide new 4 by 5- substituted thiophene methylamine while 6,7 are closed N- ((5- substituted thiophene -2- base) methyl) -6, the 7- substituted quinazoline -4- aminated compounds or its pharmaceutical salts of modification are managed, and is mentioned For its preparation and the application in terms of preparation treatment with EGFR overexpression related disease such as breast cancer, epidermis cancer drug.

The technical solution adopted by the present invention is as follows:

General formula be following structural formula N- ((5- substituted thiophene -2- base) methyl) -6,7- substituted quinazoline -4- aminated compounds or Its pharmaceutical salts:

R is selected from one of H, F, Cl, Br, I in formula；R₁、R₂Identical or different, they are respectively selected from H, nitro, amino, hydroxyl, 2- methoxy ethoxy, C₁-C₄Alkoxy, C₁-C₄One of alkyl.

The compounds of this invention has enough alkalinity, therefore can form officinal salt with many organic or inorganic acids.Herein The term " officinal salt " used refers to the salt of the compounds of this invention, substantially nontoxic to organism living.Typical officinal salt The salt that compound and inorganic acid including those through the invention are formed, these salt are referred to as acid-addition salts.

The acid for being used to form acid-addition salts generally includes inorganic acid, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid Deng, organic acid, for example, p-methyl benzenesulfonic acid, oxalic acid, to bromo-benzene sulfonic acid, carbonic acid, benzoic acid, acetic acid etc..Preferred pharmaceutically acceptable acid adds It is the salt and those salt formed with organic acid such as p-methyl benzenesulfonic acid that those are formed with inorganic acid such as hydrochloric acid and hydrobromic acid at salt.

Part preferred compound of the present invention are as follows:

A.N- ((thiophene -2- base) methyl) quinazoline -4- amine；

B.N- ((- 2 base of 5- chlorothiophene) methyl) quinazoline -4- amine；

C.N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine；

D.6,7- two (2- methoxyl group)-N- ((thiophene -2- base) methyl) quinazoline -4- amine；

E.N- ((5- chlorothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

F.N- ((5- bromothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

G.6,7- two (2- methoxy ethoxy)-N- ((thiophene -2- base) methyl) quinazoline -4- amine；

H.6,7- two (2- methoxy ethoxy)-N- ((5- chlorothiophene -2- base) methyl) quinazoline -4- amine；

I.6,7- two (2- methoxy ethoxy)-N- ((5- bromothiophene -2- base) methyl) quinazoline -4- amine；

J.N- ((- 2 base of 5- fluorine thiophene) methyl) quinazoline -4- amine；

K.N- ((5- fluorine thiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

L.6,7- two (2- methoxy ethoxy)-N- ((5- fluorine thiophene -2- base) methyl) quinazoline -4- amine.

Preparation method, steps are as follows:

(1) it using 2- thiophene formonitrile HCN as raw material, is reacted through reduction, Boc and intermediate compound I, i.e. (thiophene -2- base) methyl carbamic acid is made The tert-butyl ester；

(2) intermediate II is made through halogenating reaction in intermediate compound I, i.e. (5- substituted thiophene -2- base) methyl carbamic acid tert-butyl ester；

(3) intermediate III, i.e. (5- substituted thiophene -2- base) methylamine are made after intermediate II is gone Boc to be deprotected；

(4) intermediate compound IV, i.e. 4- chlorine is made through chlorination in (3H) -one of quinazoline -4 or 6, (3H) -one of 7- substituted quinazoline -4 Quinazoline or the chloro- 6,7- substituted quinazoline of 4-；

(5) target product V, i.e. N- ((5- substituted thiophene -2- base) methyl)-is made in intermediate III and IV nucleo philic substitution reaction 6,7- substituted quinazoline -4- amine.

Aforementioned intermediate compound I, intermediate II, intermediate III, intermediate compound IV general structure successively are as follows:

R is selected from one of H, F, Cl, Br, I in formula；R₁、R₂Identical or different, they are respectively selected from H, nitro, amino, hydroxyl, 2- methoxy Base oxethyl, C₁-C₄Alkoxy, C₁-C₄One of alkyl.

Synthetic route is as follows, and group representated by each substituent group is as previously described in reaction equation:

Specifically, detailed process is as follows for step (1): solvent 1 is added to dissolution in 2- thiophene formonitrile HCN and reducing agent 1, and 10-60 DEG C anti- 1-10h is answered, is filtered, by filtrate and di-tert-butyl dicarbonate after 10-60 DEG C is stirred to react 1-10h, twice with saturated salt washing, Desiccant dryness filters, and decompression removes solvent, obtains intermediate compound I；The reducing agent 1 is one of hydrogen or lithium aluminium hydride reduction；It is described Solvent 1 is one of ether, tetrahydrofuran, butyl ether or isopropyl ether or their mixture.

Specifically, detailed process is as follows for step (2): solvent 2 is added to dissolution in intermediate compound I, and halogenating agent, 10- is added 60 DEG C of reaction 10-20h are added solvent 3 and extract, obtain intermediate II after reaction；The solvent 2 is dimethyl sulfoxide, N, N- One kind or their mixture of dimethylformamide or tetrahydrofuran；Solvent 3 is in ethyl acetate, chloroform or methylene chloride A kind of or their mixture.

Specifically, detailed process is as follows for step (3): the 1-3 times of acid measured is added in intermediate II addition solvent 4 to dissolution, 10-60 DEG C of reaction 10-20h, after reaction, decompression remove solvent, obtain intermediate III；The solvent 4 be methylene chloride, chloroform, One of ether or ethyl acetate or their mixture；The acid 1 be one of trifluoroacetic acid or hydrochloric acid or they Mixture.

Specifically, detailed process is as follows for step (4): (3H) -one of quinazoline -4 or 6,7- substituted quinazoline -4 (3H) -one And catalyst 1 is added in solvent 5,30-70 DEG C of reaction 3-10h, and it is after reaction, cooling, PH 6-8 is adjusted, stratification is done Dry organic phase, decompression removal organic phase solvent, obtains intermediate compound IV；The solvent 5 is in methylene chloride, chloroform or thionyl chloride A kind of or their mixture；The catalyst 1 is N,N-dimethylformamide.

Specifically, detailed process is as follows for step (5): intermediate compound IV is added in solvent 6, adds intermediate III and 1-3 The Hunig's base of amount again, 30-70 DEG C of reaction 3-10h, fully reacting is cooling, filters, and filter cake recrystallizes obtained target product V；The solvent 6 is one of methanol, ethyl alcohol, propyl alcohol or isopropanol or their mixture.

In the present invention, the usage amount of solvent 1-6, which can guarantee, dissolves respective reaction raw materials.

Compound of the present invention is in terms of preparation treatment with EGFR overexpression related disease such as breast cancer, epidermis cancer drug Application.

Beneficial effects of the present invention: the quinazoline inhibitor replaced different from existing 4 by various aniline, the present invention exist 4, quinazoline skeleton using having the thiophene methyl amine of extensive bioactivity to be replaced, while 6,7 by rational modification but are not wrapped Acryloyl amine key containing electrophilic property forms irreversible covalent bond to avoid with EGFR, and design has synthesized the EGFR of Novel series Inhibitor.Compared with existing anti-tumor drug, such compound has the strain of EGFR overexpressing cell such as A431 and MCF-7 anti-swollen Tumor activity and apparent selectivity；Expression cell strain A549 non-to EGFR does not have activity, and measurement result is shown: the present invention can be very Good inhibition EGFR autophosphorylation, compared with positive control Erlotinib, part of compounds shows excellent antitumor work Property, to A431 and MCF-7 anti-tumor activity generally close to control drug Erlotinib, there is good application prospect；System of the present invention Synthetic method during standby is at low cost, step is simple, reaction condition is mild, yield is high, is easy to post-process.

Detailed description of the invention

Fig. 1 is result figure of the compound e made from embodiment 5 to p-EGFR in cell (Y1086) protein expression.

Specific embodiment

Specific embodiment provided by the present invention, feature are described in detail below in conjunction with specific embodiment, but the present invention It is not limited in the range of the following example.

Embodiment 1

The synthesis of N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine (V-1)

(1) synthesis of (thiophene -2- base) methyl carbamic acid tert-butyl ester (I)

2- thiophene formonitrile HCN (16g, 0.15mol) and tetrahydrofuran are added in the three-necked bottle equipped with thermometer, electromagnetic agitation (600mL), 0 DEG C of addition lithium aluminium hydride reduction (12.54g, 0.33mol) then rise to 20 DEG C and stir 1 hour, be down to 0 DEG C, add 20mL Water quenching reaction is added 15wt%NaOH solution (40mL), water (60mL), filters, by filtrate and di-tert-butyl dicarbonate (39.24g, 0.18mol) be added another equipped with thermometer, electromagnetic agitation 1000mL three-necked bottle in, 20 DEG C are stirred to react 1 Hour, twice with the washing of 500mL saturated salt, anhydrous magnesium sulfate is dry, filters, and decompression removes solvent, obtains light yellow oil (I) 25.56g yield 80.0%.

(2) synthesis of (5- bromothiophene -2- base) methyl carbamic acid tert-butyl ester (II-1)

I (31.95g, 0.15mol) is added in the 1000mL three-necked bottle equipped with thermometer, electromagnetic agitation, DMF (150mL), NBS (32.4g, 0.18mol), 20 DEG C are stirred 12 hours, fully reacting, and ethyl acetate 250mL is added in reaction solution, and organic phase is used The washing of 150mL 3 times, anhydrous magnesium sulfate is dry, filters, and decompression removes solvent, obtains (5- bromothiophene -2- base) methyl carbamic acid The tert-butyl ester (II-1) 29.32g, yield 67.4%.

(3) synthesis of 5- bromothiophene -2- methylamine (III-1)

In the three-necked bottle equipped with thermometer, electromagnetic agitation, compound (II-1) (5.8g, 0.02mol) is dissolved in CH₂Cl₂ In (200mL), TFA (trifluoroacetic acid) 50mL is added, 20 DEG C are stirred 2 hours, fully reacting, and removal solvent obtains white solid (III-1) 3.05g, yield 80.4%,¹H NMR (400MHz, DMSO) δ: 3.99 (s, 2H), 6.68 (d, J=3.6Hz, 1H), 6.89 (d, J=3.6Hz, 1H).

(4) synthesis of 4- chloro-quinazoline (IV-1)

Equipped with thermometer, electromagnetic agitation and reflux condenser three-necked bottle in be added quinazoline -4 (3H) -one (7.30g, 0.05mol), thionyl chloride (200mL) and DMF (0.4mL), are heated to reflux, and react 3 hours, and solution is concentrated under reduced pressure, cooling, add Enter 500mL methylene chloride, respectively with saturated sodium bicarbonate aqueous solution, distillation washing 2 times, uses anhydrous Na₂SO₄Dry organic phase, takes out Filter is concentrated under reduced pressure filtrate, obtains white solid, and ethyl alcohol recrystallization obtains 4- chloro-quinazoline (IV-1) (6.15g, 75%),¹H NMR (400MHz, DMSO) δ: 7.74-7.78 (m, 1H), 7.97-8.01 (m, 1H), 8.10 (d, J=8.4Hz, 1H), 8.30 (d, J= 8.4Hz,1H),9.07(s,1H)。

(5) synthesis of N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine (V-1)

Equipped with thermometer, electromagnetic agitation and reflux condenser three-necked bottle be added 4- chloro-quinazoline (IV-1) (0.82g, 5mmol), ethyl alcohol (50mL), 5- bromothiophene -2- methylamine (III-1) (7mmol, 1.33g), Hunig's base is (in the present invention Hunig's base refers to n,N-diisopropylethylamine, similarly hereinafter, 1.29g, 10mmol), it is heated to reflux 6h, fully reacting is cooling, Have a white solid precipitation, ethyl alcohol recrystallization obtain N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine (V-1) (1.16g, 73%),¹HNMR(400MHz,CDCl₃): δ 4.97 (d, J=5.6Hz, 2H), 5.98 (br, 1H), 6.86 (d, J=4.0Hz, 1H), 6.92 (d, J=4.0Hz, 1H), 7.46-7.50 (m, 1H), 7.69 (d, J=7.6Hz, 1H), 7.74-7.78 (m, 1H), 7.88 (d, J=8.4Hz, 1H), 8.74 (s, 1H)；¹³CNMR(100MHz,CDCl₃)δ40.25,112.17,114.74, 120.35,126.27,126.80,128.82,129.43,132.84,142.51,149.60,155.13,158.82；ESI-MS: 319.9857(C₁₃H₁₀BrN₃S,[M+H]⁺)。

Embodiment 2

The synthesis of N- ((- 2 base of 5- chlorothiophene) methyl) quinazoline -4- amine (V-2)

(1) synthesis of (5- chlorothiophene -2- base) methyl carbamic acid tert-butyl ester (II-2)

According to the method for step (2) in embodiment 1, chemical combination is added in the 1000mL three-necked bottle equipped with thermometer, electromagnetic agitation Object I (31.95g, 0.15mol), DMF (150mL), NCS (23.9g, 0.18mol), 20 DEG C are stirred 12 hours, fully reacting, instead Addition ethyl acetate (250mL) in liquid is answered, organic phase is with washing 3 times of 150mL, and anhydrous magnesium sulfate is dry, filters, and decompression is except molten Agent obtains compound (II-2) 26.70g, yield 72.7%.

(2) synthesis of 5- chlorothiophene -2- methylamine (III-2)

According to the method for step (3) in embodiment 1, compound (II-2) (4.94g, 0.02mol) is dissolved in CH₂Cl₂(200mL) In, it is added TFA (50mL), 20 DEG C are stirred 2 hours, and removal solvent obtains white solid (III-2) 2.41g, yield 81.9%,¹HNMR (400MHz, DMSO) δ: 3.99 (s, 2H), 7.09 (d, J=3.6Hz, 2H), 8.37 (s, 3H).

(3) synthesis of N- ((- 2 base of 5- chlorothiophene) methyl) quinazoline -4- amine (V-2)

Equipped with thermometer, electromagnetic agitation and reflux condenser 500mL three-necked bottle be added 4- chloro-quinazoline (IV-1) (5mmol, 0.82g), ethyl alcohol (50mL), 5- chlorothiophene -2- methylamine (III-2) (7mmol, 1.02g), Hunig's base (1.29g, 10mmol), it is heated to reflux 6h, fully reacting is cooling, has white solid precipitation, ethyl alcohol recrystallization obtains N- ((- 2 base of 5- bromothiophene) Methyl) quinazoline -4- amine (1.17g, 84.7%),¹HNMR(400MHz,CDCl₃): δ 4.95 (d, J=5.2Hz, 2H), 5.98 (br, 1H), 6.78 (d, J=3.6Hz, 1H), 6.87 (d, J=3.6Hz, 1H), 7.46-7.50 (m, 1H), 7.69 (d, J= 8Hz, 1H), 7.74-7.78 (m, 1H), 7.88 (d, J=8.0Hz, 1H), 8.74 (s, 1H)；¹³CNMR(100MHz,CDCl₃):δ 40.34,114.74,120.35,125.63,125.81,126.27,128.82,129.91,132.84,139.58,149.60, 155.12,158.83；ESI-MS:276.0361(C₁₃H₁₀Cl₃S,[M+H]⁺)。

Embodiment 3

The synthesis of N- ((- 2 base of thiophene) methyl) quinazoline -4- amine (V-3)

(1) synthesis of (thiophene -2- base) methyl methylamine (II-3)

According to the method for step (3) in embodiment 1, compound I (2.26g, g, 0.02mol) is dissolved in CH₂Cl₂In (200mL), add Enter TFA (50mL), 20 DEG C are stirred 2 hours, fully reacting, and removal solvent obtains (III-3) 1.84g, yield 81.5%,¹H NMR (400MHz, CDCl₃) δ: 4.26 (s, 2H), 7.08 (dd, J₁=5.2Hz, J₂=3.6Hz, 1H), 7.23 (d, J=2.8Hz, 1H), 7.59 (d, J=1.2Hz, 1H).

(2) synthesis of N- ((- 2 base of thiophene) methyl) quinazoline -4- amine (V-2)

Equipped with thermometer, electromagnetic agitation and reflux condenser 500mL three-necked bottle be added 4- chloro-quinazoline (IV-1) (5mmol, 0.82g), ethyl alcohol (50m), thiophene -2- methylamine (7mmol, 0.79g), Hunig's base (1.29g, 10mmol), are heated to reflux 6h, fully reacting is cooling, has white solid precipitation, ethyl alcohol recrystallization obtains N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine (0.98g, 81%),¹HNMR(400MHz,CDCl₃): δ 5.05 (d, J=5.2Hz, 2H), 5.99 (b, 1H), 6.99-7.01 (m, 1H), 7.11 (d, J=2.8Hz, 1H), 7.27 (d, J=5.2Hz, 1H), 7.45-7.49 (m, 1H), 7.69 (d, J=8.0Hz, 1H), 7.73-7.77 (m, 1H), 7.87 (d, J=8.0Hz, 1H), 8.74 (s, 1H)；¹³CNMR(100MHz,CDCl₃):δ 40.08,114.81,120.46,125.59,126.16,126.57,126.99,128.74,132.74,140.58,149.58, 155.28,158.88；ESI-MS:242.0751(C₁₃H₁₁N₃S,[M+H]⁺)。

Embodiment 4

The synthesis of N- ((5- bromothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (V-4)

(1) synthesis of the chloro- 6,7- dimethoxyquinazoline (IV-2) of 4-

By 6,7- dimethoxyquinazoline -4 (3H) -one in the three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser (10.3g, 50mmol) is dissolved in thionyl chloride (200mL), and DMF (0.4ml) is added dropwise, is heated to reflux, and is reacted 6 hours, cold But, decompression steams excessive thionyl chloride, and residue is heated azeotropic with toluene (50mL) with cleared thionyl chloride, adds two Chloromethanes (500mL), respectively with saturated sodium bicarbonate aqueous solution, washing 2 times, organic phase is dry with magnesium sulfate, is evaporated in vacuo molten Agent obtains white solid IV-2, (7.84g, 70%),¹HNMR (400MHz, CDCl3) δ 4.08 (d, J=1.6Hz, 6H), 7.35 (s,1H),7.40(s,1H),8.88(s,1H)。

(2) N- ((5- bromothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (V-4)

The chloro- 6,7- dimethoxyquinazoline of 4- is added in the 500mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser (IV-2) (5mmol, 1.12g), ethyl alcohol (50mL), 5- bromothiophene -2- methylamine III-1 (7mmol, 1.33g), Hunig's base (1.29g, 10mmol) is heated to reflux 7h, and fully reacting is cooling, has white solid precipitation, ethyl alcohol recrystallization obtains N- ((5- bromine thiophene Pheno -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (V-4) (1.62g, 85.7%),¹HNMR(400MHz,CDCl₃) δ: 3.94 (s, 3H), 3.95 (s, 3H), 4.90 (d, J=5.6Hz, 2H), 6.84 (d ,=3.6Hz, 1H), 6.90 (d ,= 3.6Hz,1H),7.01(s,1H),7.15(s,1H),8.46(s,1H),9.89(br,1H)；¹³CNMR(100MHz,CDCl₃)δ 40.18,56.19,56.31,100.18,105.25,107.91,112.09,126.96,129.36,142.47,143.38, 149.34,152.14,154.91,158.00,ESI-MS:380.0066(C₁₅H₁₄BrN₃O₂S,[M+H]⁺)。

Embodiment 5

The synthesis of N- ((5- chlorothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (V-5)

The chloro- 6,7- dimethoxyquinazoline of 4- is added in the 500mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser (IV-2) (5mmol, 1.12g), ethyl alcohol (50mL), 5- chlorothiophene -2- methylamine (III-2) (7mmol, 1.02g), Hunig's Base (1.29g, 10mmol) is heated to reflux 7h, and fully reacting is cooling, has white solid precipitation, ethyl alcohol recrystallization obtains N- ((5- Bromothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (1.35g, 80.3%),¹HNMR(400MHz,CDCl₃) δ: 3.95 (s, 3H), 3.98 (s, 3H), 4.93 (d, J=5.6Hz, 2H), 5.94 (br, 1H), 6.76 (d ,=3.6Hz, 1H), 6.84 (d ,=4.0Hz, 1H), 6.89 (s, 1H), 7.21 (s, 1H), 8.63 (s, 1H)；¹³CNMR(100MHz,CDCl₃) δ: 40.36, 56.23,99.26,107.77,108.54,125.56,125.66,129.41,129.76,140.16,146.74,149.23, 153.79,154.58,157.70,ESI-MS:336.0573(C₁₅H₁₄ClN₃O₂S,[M+H]⁺)。

Embodiment 6

The synthesis of 6,7- bis- (2- methoxyl group)-N- ((thiophene -2- base) methyl) quinazoline -4- amine (V-6)

The chloro- 6,7- dimethoxyquinazoline of 4- is added in the 500mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser (IV-2) (5mmol, 1.12g), ethyl alcohol (40mL), thiophene -2- methylamine (III-3) (7mmol, 0.79g), Hunig's base (1.29g, 10mmol) is heated to reflux 7h, and fully reacting is cooling, has white solid precipitation, ethyl alcohol recrystallization obtains N- ((5- bromine thiophene Pheno -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (1.27g, 84.6%),¹HNMR(400MHz,CDCl₃) δ: 3.95 (s, 3H), 3.99 (s, 3H), 5.05 (d, J=5.2Hz, 2H), 5.78 (br, 1H), 6.87 (s, 1H), 7.00 (dd, J₁= 5.2Hz,J₂=3.6Hz, 1H), 7.11 (d, J=2.8Hz, 1H), 7.22 (s, 1H), 7.27 (d, J=2.8Hz, 1H), 8.63 (s,1H)；¹³CNMR(100MHz,CDCl₃) δ: 40.09,56.23,56.27,99.29,107.78,108.53,125.50, 126.48,126.94,141.13,146.66,149.15,153.90,154.50,157.75,ESI-MS:302.0963 (C₁₅H₁₅N₃O₂S,[M+H]⁺)。

Embodiment 7

The synthesis of 6,7- bis- (2- methoxy ethoxy)-N- ((5- bromothiophene -2- base) methyl) quinazoline -4- amine (V-7)

(1) synthesis of chloro- bis- (2- methoxy ethoxy) quinazoline (IV-3) of 6,7- of 4-

In the 1000mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser, 6,7- (2- methoxy ethoxy) quinolines Oxazoline -4 (3H) -one (14.7g, 0.05mol) is dissolved in methylene chloride (150mL), is added DMF (1mL), 20 DEG C are added dropwise chlorine Change sulfoxide (40mL), temperature rising reflux 6h, it is cooling, PH to 7 is adjusted with NaOH, is stirred for reaction 30min, places until layering, receives Collect organic phase, decompression removal solvent obtains white solid, ethyl alcohol recrystallization, chloro- 6,7- bis- (2- methoxy ethoxy) the quinoline azoles of 4- Quinoline IV-3 (12.48g, 80%),¹HNMR(400MHz,CDCl₃)δ3.49(s,3H),3.50(s,3H),3.88-3.90(m,4H), 3.32-4.35(m,4H),7.33(s,1H),7.44(s,1H),8.86(s,1H)。

(2) conjunction of 6,7- bis- (2- methoxy ethoxy)-N- ((5- bromothiophene -2- base) methyl) quinazoline -4- amine (V-7) At

In ethyl alcohol (50mL) solution of chloro- 6,7- bis- (2- methoxy ethoxy) quinazoline (IV-3) (5mmol, 1.56g) of 4- It is added 5- bromothiophene -2- methylamine (7mmol, 1.33g), Hunig's base (1.29g, 10mmol) is heated to reflux 6h, has reacted Entirely, cooling, there is white solid precipitation, ethyl alcohol recrystallization obtains 6,7- bis- (2- methoxy ethoxy)-N- ((5- bromothiophene -2- base) Methyl) quinazoline -4- amine (V-7) (1.84g, 78.8%),¹HNMR(400MHz,CDCl₃)δ3.42(s,3H),3.45(s,3H), 3.75-3.83 (m, 4H), 4.14-4.22 (m, 4H), 4.89 (d, J=4.4Hz, 2H), 6.69 (br, 1H), 6.81 (d, J= 3.6Hz, 1H), 6.88 (d, J=3.6Hz, 1H), 7.08 (s, 1H), 7.18 (s, 1H), 8.52 (s, 1H)；¹³CNMR(100MHz, CDCl₃)40.18,59.26,68.33,68.96,70.47,70.81,102.6,107.2,108.2,111.9,126.7, 129.3,142.7,144.9,148.6,152.9,154.5,157.9,ESI-MS:468.0586(C₁₉H₂₂N₃O₄SBr,[M+H ]⁺)。

Embodiment 8

The synthesis of 6,7- bis- (2- methoxy ethoxy)-N- ((5- chlorothiophene -2- base) methyl) quinazoline -4- amine (V-8)

Chloro- bis- (the 2- methoxyl group second of 6,7- of 4- is added in the 500mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser Oxygroup) quinazoline (IV-3) (5mmol, 1.56g), ethyl alcohol (45mL), 5- chlorothiophene -2- methylamine (III-2) (7mmol, 1.02g), Hunig's base (1.29g, 10mmol) is heated to reflux 7h, and fully reacting is cooling, there is light tan solid precipitation, Ethyl alcohol recrystallization obtains N- ((5- chlorothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (1.69g, 80.3%),¹HNMR(400MHz,CDCl₃) δ: 3.42 (s, 3H), 3.44 (s, 3H), 3.75 (t J=4.8Hz, 2H), 3.81 (t J= 4.8Hz, 2H), 4.15 (t J=4.8Hz, 2H), 4.21 (t J=4.8Hz, 2H), 4.89 (d, J=5.6Hz, 2H), 6.19 (br, 1H), 6.74 (d, J=4.0Hz, 1H), 6.81 (d, J=4.0Hz, 1H), 7.06 (s, 1H), 7.17 (s, 1H), 8.61 (s, 1H)；¹³CNMR(100MHz,CDCl₃) δ: 40.29,59.28,68.28,69.13,70.48,70.87,102.63,108.67, 108.70,125.54,125.61,129.63,140.19,146.76,148.57,153.91,154.40,157.85,ESI-MS: 424.1098(C₁₉H₂₂ClN₃O₄S,[M+H]⁺)。

Embodiment 9

The synthesis of 6,7- bis- (2- methoxy ethoxy)-N- ((thiophene -2- base) methyl) quinazoline -4- amine (V-9)

Chloro- bis- (the 2- methoxyl group second of 6,7- of 4- is added in the 500mL three-necked bottle equipped with thermometer, electromagnetic agitation and reflux condenser Oxygroup) quinazoline (IV-3) (5mmol, 1.56g), ethyl alcohol (45mL), thiophene -2- methylamine (7mmol, 0.79g), (III-3) (7mmol, 1.02g), Hunig's base (1.29g, 10mmol) are heated to reflux 7h, and fully reacting is cooling, has light brown solid Body be precipitated, ethyl alcohol recrystallization obtain N- ((5- chlorothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine (1.49g, 76.9%),¹HNMR(400MHz,CDCl₃) δ: 3.40 (s, 3H), 3.43 (s, 3H), 3.72-3.82 (m, 4H), 4.12-4.21 (m, 4H), 5.00 (d, J=5.2Hz, 2H), 6.26 (t J=4.4Hz, 1H), 6.95-6.97 (m, 1H), 7.07 (s, 1H), 7.15 (s, 1H), 7.16 (s, 1H), 7.23 (d, J=4.8Hz, 1H), 8.59 (s, 1H)；¹³CNMR(100MHz,CDCl₃) δ: 39.97,59.20,68.15,68.94,70.44,70.82,102.63,108.56,108.74,125.35,126.39, 126.88,141.16,146.64,148.37,154.03,154.16,157.93；ESI-MS:390.1487(C₁₉H₂₃N₃O₄S,[M +H]⁺)。

Anti tumor activity in vitro test

Principle: the succinate dehydrogenase in living cells mitochondria can make thiazolyl blue (MTT) be reduced to bluish violet not soluble in water Acicular crystal first a ceremonial jade-ladle, used in libation (Formazan) is simultaneously deposited in cell, and dead cell is then without the function.Dimethyl sulfoxide (DMSO) energy The bluish violet crystallization in cell is dissolved, and has maximum absorption band at 490nm wavelength.Therefore it is being measured at 490nm wavelength Absorbance can reflect living cells quantity.Within the scope of certain cell number, the amount that first a ceremonial jade-ladle, used in libation is formed is directly proportional to cell number.

For trying cell

EGFR overexpressing cell: breast cancer cell MCF-7, people's epidermis cancer cell A431；The non-expression cell of EGFR: human lung carcinoma cell A549,3 kinds of tumor cell lines are purchased from Chinese Academy of Sciences's Shanghai school of life and health sciences cell bank

Key instrument and consumptive material

Biohazard Safety Equipment, model HR60-II42, company, China's Haier

CO₂Incubator, model IL-161HT, Chinese Shanghai

Inverted biologic microscope, model C KX41SF, Japanese Olympus company

Air bath constant-temperature table, Guo Hua enterprise, ZD-85 type

Full-automatic multi-functional microplate reader, the production of Bio-Rad company, the U.S.

Accurate adjustable micropipettor, Eppendorf (Germany) company；

96 porocyte culture plates, Corning (U.S.) company；

Tissue Culture Flask, Corning (U.S.) company.

Main agents

Positive reference substance Erlotinib, selleck company；

Top grade fetal calf serum, Hangzhou Sijiqing Biological Engineering Material Co., Ltd.；

Trypsase, Gibco company；

RPMI1640 culture solution (Beijing Solarbio Science&Technology Co., Ltd.), DMEM culture medium (Hyclone, USA), DMSO and the U.S. Methyl thiazoly tetrazolium assay (methylthiazolyl tetrazolium, MTT) Solarbio company.

Experimental procedure is as follows:

The preparation of MTT solution: weighing MTT50mg, is dissolved in the phosphate buffer (PBS) of 10mL, with 0.22 μm of millipore filter mistake Bacterium is filtered out, is dispensed, 4 DEG C are kept in dark place, in two weeks effectively.

For the configuration of reagent object: compound prepared by Example 1-9, i.e. compound a-i, DMSO dissolution are made into 50mmol/L mother liquor, before use, precision draw appropriate above-mentioned sample mother liquor and are diluted to various concentration gradient with corresponding culture medium Working solution, such as 50 μm of ol/L, 25 μm of ol/L, 12.5 μm of ol/L, 6.25 μm of ol/L, 3.125 μm of ol/L.Erlotinib is dissolved in Corresponding culture medium is configured to mother liquor 50mmol/L, is diluted to 5 various concentrations with corresponding culture medium again for different tumour cells The working solution of gradient, such as 50 μm of ol/L, 25 μm of ol/L, 12.5 μm of ol/L, 6.25 μm of ol/L, 3.125 μm of ol/L.

Cell culture

People epidermis cancer cell A431 uses the culture of DMEM culture medium, and breast cancer cell MCF-7 and human umbilical vein endothelial cell use RPMI1640 culture solution, 37 DEG C of saturated humidities, 5%CO₂Routine culture in incubator；Every 2-3d is changed liquid 1 time, when cell is grown to When logarithmic phase, secondary culture is carried out.Cell activity is detected with trypan blue staining in experimentation.

Measuring method

1. taking logarithmic phase A431, MCF-7, A549 cell, digest, be centrifuged and be resuspended, adjusting separately cell density is 8 × 10⁴A/ mL、5×10⁴A/mL, 5 × 10⁴A/mL is inoculated in 96 porocyte culture plates, wherein 100 microlitres of cells prepared are added in every hole Suspension.

2. wait overnight, i.e., cell it is adherent after, discard culture supernatants.It is 50 μm of ol/ that test group, which is separately added into concentration, L, 100 microlitres of the compound (a-i, Erlotinib) of 25 μm of ol/L, 12.5 μm of ol/L, 6.25 μm of ol/L, 3.125 μm of ol/L, often A concentration sets 3 multiple holes, and sets negative control group (isometric cell suspension is added in hole) and blank control group (is added in hole Isometric culture medium).

3. 20 μ L MTT solution (concentration 5mg/ml) are added in every hole after 96 orifice plates are cultivated 72h respectively, in the incubator Continue culture 4 hours.

4. 96 orifice plates take out from incubator, supernatant is carefully sopped up with micropipettor, and every hole is added 150 microlitre two Methyl sulfoxide, shaking on horizontal oscillator tube is completely dissolved crystallization, then using microplate reader in 490nm wavelength condition Lower measurement absorbance value.

5. test continuously repeats three times, and calculates cell inhibitory rate: cell inhibitory rate (%)=[1- according to following formula (test group OD value-blank control group OD)/(negative control hole OD value-blank control group OD] × 100%, and calculate sample inhibition The half-inhibitory concentration IC of cell growth₅₀Value.

Test result is shown, impact cell can be ignored lower than 0.1v%DMSO solvent, IC₅₀Value statistical software Spss13.0 is calculated, and experimental result is shown in Table 1.

1 target compound anti tumor activity in vitro IC of table₅₀(μm ol/L) data

Measurement result is shown: above compound, which is overexpressed tumor cell line A431 and MCF-7 to EGFR, significant antitumor work Property, and change along with the difference of substituent group.Wherein, compound e, f, h, i shows A431 and MCF-7 anti-swollen very well Tumor activity especially shows excellent anti-tumor activity to A431.Wherein compound e grows activity to A431 and MCF-7 antiproliferative and connects Nearly control drug Erlotinib, IC₅₀Respectively 3.4 μm of ol/L and 7.3 μm of ol/L.

Influence of the preferred compound e to p-EGFR in cell (Y1086) protein expression

Western blot, that is, immunoblotting is to be transferred to the cell or tissue gross protein after electrophoretic separation from gel On solid support NC film or pvdf membrane, a kind of protein detection techniques of detection of specific antibody specific antigen are then used, Specific protein expression in the cell or tissue analyzed is obtained by the position and color depth of analysis coloring.

For trying cell

EGFR overexpressing cell people epidermis cancer cell A431 is purchased from Chinese Academy of Sciences Shanghai school of life and health sciences cell bank.

Key instrument

Electrophoresis apparatus (Beijing 6 1), ((Beijing 6 1) transfers in electrophoresis apparatus (Beijing 6 1), horizontal shaker (Beijing Vertial electrophorestic tank 6 one), hand pressure seal mouth machine (Xingye Mechanical Equipment Co., Ltd., Wenzhou City), ice machine (ningbo of china Lactel Co., Ltd, XB- 70), Amersham Imager 600 (GE company, the U.S.)

Main agents

Anti-EGFR antibody [E235] (abcam company), Anti-EGFR (phospho Y1086) antibody [Y39] (abcam company), pre-dyed albumen Marker (U.S. Genview), rabbit-anti GAPDH resist grand IgG antibody (virtuous to the biological section in Hangzhou more Skill Co., Ltd), HRP marks sample anti-rabbit IGg (Beijing ancient cooking vessel state), Human EGF (connection section biology), the examination of BCA determination of protein concentration Agent box (Beijing ancient cooking vessel state), RIPA lysate (strong) (Beijing ancient cooking vessel state), SDS protein electrophoresis sample-loading buffer (Beijing ancient cooking vessel state), Tris- Glycine transferring film buffer (pH8.3) (health is century), TBST buffer (Beijing ancient cooking vessel state), PAGE gel reagent preparation Box (health is century), inhibitors of phosphatases (50) (Leagene company), ECL luminescent solution (Thermo company), pvdf membrane (Millipore company), skimmed milk power (Amresco company)

Experimental method

One, prepared by protein sample

1, culture A431 cell is to logarithmic phase, bed board after digestion.Be arranged negative control group, blank control group, positive controls and Administration group.The setting of compound e administration group concentration is respectively 0.8 μm of ol/L, 4 μm of ol/L, 20 μm of ol/L, and Erlotinib administration is dense Degree is 1.0 μm of ol/L.1h is acted on respectively, then stimulates 10min with the EGF of 100ng/ml, then terminates stimulation.

2, total protein of cell extracts

(1) cell supernatant liquid is sopped up, PBS is added, lays flat and gently shakes, wash 2 times, pancreatin is added into culture dish, by cell Disappear in the centrifuge tube for getting off and being put into 1.5ml from culture dish bottom surface, discarded supernatant after centrifugation, PBS is washed twice, and RIPA is then added and splits Solve liquid；

(2) it is blown and beaten for several times in 30min on ice, waggle or with rifle, comes into full contact with lysate with cell, cracked on ice 30min shakes every 5min；

(3) after having cracked, lower cell is blown and beaten with head is robbed, cell fragment and lysate are then transferred to 1.5ml centrifuge tube with rifle, All operations carry out on ice；

(4) 12000rpm is centrifuged 5min under refrigerated centrifuge；

(5) supernatant after centrifugation is determined into its concentration with BCA albuminimetry, the protein sample diluted and SDS loading is delayed 4:1 is added fliud flushing by volume, then 5min is boiled in boiling water is denaturalized protein completely, and -70 DEG C save for use.

Two, the preliminary quantitative and processing of protein sample

BCA albuminimetry is a kind of method of rapid sensitive, reliable quantification of protein.It is tried according to BCA determination of protein concentration Agent box (Beijing ancient cooking vessel state) specification prepares BCA working reagent, and (i.e. other reagents such as BCA and the copper sulphate containing bivalent cupric ion form Reagent, be mixed as BCA working reagent).Under alkaline condition, by Cu when BCA reagent is in conjunction with protein²⁺It is reduced into Cu⁺, a Cu⁺Two BCA molecules are chelated, working reagent is had at 562nm by original apple green formation purple compound Maximum absorbance can calculate protein concentration by standard curve comparison.

The specific method is as follows:

1, according to standard items and sample size, appropriate BCA working solution is prepared by bought reagent specification.

2, protein standard substance is added in the protein standard sample wells of 96 orifice plates by 0,1,2,4,6,8,10ul, adds sterilizing ultrapure Water supplies 10ul；10ul sample to be tested is taken to be added in 96 orifice plates, 3 secondary orifices are arranged in each concentration.

3,200ulBCA working solution is added into sample to be tested hole and protein standard sample wells to mix.It is put in 37 DEG C of incubators 30min is set, is then cooled to room temperature.

4, its absorbance is then detected under 562nm wavelength.It is made by the concentration of ordinate, protein standard substance of absorbance Standard curve, so as to find out the concentration of sample.

Three, albumen is separated by electrophoresis in SDS-PAGE

3.1 preparation concentration glue and separation gel

1, the preparation of separation gel

(1) clean fixed device: the two sides of the sponge cleaning glass baffle plate of abluent is rinsed with tap water, until on glass plate Face does not have abluent, and not at stock water flow.Then it is rinsed one time, is directly dried up with hair dryer stand-by with pure water.Large and small glass It is put into clamping in folder after glass plate alignment, then vertical to block on the top of the shelf, encapsulating after leak detection.

(2) SDS-PAGE the and 4.17ml pure water of 30% acrylamide of 3.33ml, 2.5ml are mixed in small beaker, Configure the separation gel of 10ml；

(3) TEMED of the 10%APS and 0.004ml of 0.1ml is added, being gently mixed makes its mixing, avoids generating bubble；

(4) it draws 1ml glue with 1ml liquid-transfering gun when encapsulating to be put into along glass, solution is slowly added into the plate assembled to gel Height is 4.5cm or so, reserves the height of about 1.5cm to configure concentration glue.Then add one layer of water on glue, coagulated more after fluid-tight Fastly.Paying attention to adding will slowly be deformed to prevent glue by punching when water, to keep gel surface holding smooth；

(5) 30-60min is stood, has one between Dang Shui and glue clearly behind interface, shows that gel has polymerize.Then it removes photoresist Upper water is simultaneously blotted water with blotting paper.

2, preparation concentration glue

(1) bought reagent specification is pressed, after removing the water layer being covered on separation gel, by the 30%Acr-Bis of 1.02ml The TEMED of the 10%APS and 0.006ml of 0.06ml is added, one in the pure water of (29:1), the SDS-PAGE of 1.5ml and 3.42ml It is mixed in a small beaker, is careful not to generate bubble, configures the concentration glue of 6ml.

(2) concentration sol solution is added into the upper surface of separation gel, until gel solution reaches the top of glass plate, when encapsulating It flows down glue along glass plate, then comb is inserted into gel and (pays attention to not generating bubble when operating).

(3) 10-20min glue polymerization to be concentrated is stood.Carefully comb is pulled out after concentration gelling is poly-, avoids damage to be loaded Hole.

3.2 loadings and electrophoretic procedures

The each albumen extracted is added in well with liquid-transfering gun, each sample adds 12ug, albumen marker to add 5ul. Then electrophoresis apparatus is adjusted to 120V when going to compression glue near separation gel boundary and continues to run by tune electrophoresis apparatus to 80V, sample strip Glue, fastly to bottom 1cm when stop running glue.After electrophoresis, that block glue of required pillar location is cut out according to marker instruction.

3.3 transferring film

1, enough transfering buffering liquids (the transferring film liquid+200ml methanol+700ml ultrapure water of 100ml) are prepared with full of transfer groove, In addition for 200ml for balanced gel and film and wetting filter paper.

2, gel is removed from glass plate, takes out all concentration glue.Concentration glue is gently scraped off, and avoids scraping separation gel It is broken, while marking in the upper right corner of glue.Gel is transferred to 15-30min in transfering buffering liquid；Filter paper is in transfering buffering liquid Impregnate at least 30s；The glue for cutting and cutting pvdf membrane of the same size, and with 5min is impregnated in proper amount of methanol, it is then placed in and turns It moves in buffer and balances 2min.

3, it makes sandwich structure: turning in liquid in electricity, clip is opened, be horizontally arranged, one layer of sponge is padded on black clip, And bubble is driven with glass rolling；Then thick filter paper is put, drives bubble away with glass bar after fixing, puts gel later, then will Pvdf membrane covers on gel, upwards successively three layers of filter paper and sponge, finally closes white clip.

4, clip is put into transfer groove, glue is in cathode, and film is in anode.

5, deposition condition: (meeting heat production, will be placed in ice bath, put ice when necessary constant current 200mA, 1h 40min when electricity turns Bag).

6, it is immunoreacted

(1) milk is closed: pvdf membrane being taken out and is put into containing 5ml, in shaking in the incubation box of the skim milk of 5% (mass fraction) On bed, it is incubated for 4h at room temperature.

(2) primary antibody is incubated for: P-EGFR, Total-EGFR is diluted with TBST with 1 ︰ 3000 of volume ratio, with 1 ︰ 1000 dilution GAPDH takes out pvdf membrane from confining liquid, and the pvdf membrane taken out in the primary antibody dilution prepared and confining liquid is placed in small plastics In bag, 4 DEG C overnight, and next day takes out, and is rinsed 3 times with TBST, each 10min.

(3) secondary antibody is incubated for: being diluted secondary antibody with TBST with 1 ︰ 1000 of volume ratio, is then placed in secondary antibody diluent and pvdf membrane It in small plastic bag, is placed on shaking table, is incubated at room temperature 2h, rinsed 3 times with TBST later, each 10min.

7, ECL, which shines, detects

Four, strip analysis

Each band is observed, picture is handled using Photoshop CS6 image analysis software, measures the gray value of each band, calculate P-EGFR/GAPDH, experiment have statistical significance in triplicate, with P < 0.05.

Experimental result

Image is analyzed with Photoshop CS6, the result is shown in Figure 1.

As shown in Figure 1, compound e is used after handling 1h to A431 cell in 0.8 μm of ol/L, 4 μm of ol/L, 20 μm of ol/L EGF stimulation, western Blot experimental result show that compound e significantly inhibits work to EGFR autophosphorylation in 4 μm of ol/L With.

Above-mentioned reference embodiment is to N- ((5- substituted thiophene -2- base) methyl) -6,7- substituted quinazoline -4- aminated compounds Or the detailed description that its pharmaceutical salts and its preparation method and application carry out, it is illustrative without being restrictive, therefore not The change and modification being detached under present general inventive concept, should belong within protection scope of the present invention.

Claims

1. a kind of general formula be following Formula V shown in structural formula N- ((thiophene -2- base) methyl) quinazoline -4- amine derivant or its Pharmaceutical salts:

, in Formula V, R is selected from one of H, F, Cl, Br, I；R₁、R₂It is identical or different, their each choosings From H, nitro, amino, hydroxyl, 2- methoxy ethoxy, C₁-C₄Alkoxy, C₁-C₄One of alkyl.

2. compound as described in claim 1, which is characterized in that selected from one of following:

A. N- ((thiophene -2- base) methyl) quinazoline -4- amine；

B. N- ((- 2 base of 5- chlorothiophene) methyl) quinazoline -4- amine；

C. N- ((- 2 base of 5- bromothiophene) methyl) quinazoline -4- amine；

D. 6,7- bis- (2- methoxyl group)-N- ((thiophene -2- base) methyl) quinazoline -4- amine；

E. N- ((5- chlorothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

F. N- ((5- bromothiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

G. 6,7- bis- (2- methoxy ethoxy)-N- ((thiophene -2- base) methyl) quinazoline -4- amine；

H. 6,7- bis- (2- methoxy ethoxy)-N- ((5- chlorothiophene -2- base) methyl) quinazoline -4- amine；

I. 6,7- bis- (2- methoxy ethoxy)-N- ((5- bromothiophene -2- base) methyl) quinazoline -4- amine；

J. N- ((- 2 base of 5- fluorine thiophene) methyl) quinazoline -4- amine；

K. N- ((5- fluorine thiophene -2- base) methyl) -6,7- Dimethoxy-quinazolin -4- amine；

L. 6,7- bis- (2- methoxy ethoxy)-N- ((5- fluorine thiophene -2- base) methyl) quinazoline -4- amine.

3. the method for preparing compound as described in claim 1, which is characterized in that steps are as follows:

(5) target product V, i.e. N- ((5- substituted thiophene -2- base) methyl)-is made in intermediate III and IV nucleo philic substitution reaction 6,7- substituted quinazoline -4- amine；

Formula Middle R is selected from one of H, F, Cl, Br, I；R₁、R₂Identical or different, they are respectively selected from H, nitro, amino, hydroxyl, 2- methoxyl group Ethyoxyl, C₁-C₄Alkoxy, C₁-C₄One of alkyl.

4. preparation method as claimed in claim 3, which is characterized in that detailed process is as follows for step (1): 2- thiophene formonitrile HCN with Solvent 1 is added to dissolution in reducing agent 1, and 10-60 DEG C of reaction 1-10h is filtered, by filtrate and di-tert-butyl dicarbonate at 10-60 DEG C After being stirred to react 1-10h, twice with saturated salt washing, desiccant dryness is filtered, and decompression removes solvent, obtains intermediate compound I；It is described to go back Former agent 1 is one of hydrogen or lithium aluminium hydride reduction；The solvent 1 be one of ether, tetrahydrofuran, butyl ether or isopropyl ether or Their mixture.

5. preparation method as claimed in claim 3, which is characterized in that detailed process is as follows for step (2): intermediate compound I is added To dissolution halogenating agent is added, 10-60 DEG C of reaction 10-20h is added solvent 3 and extracts after reaction, dry, decompression in solvent 2 It is concentrated to give intermediate II；The solvent 2 be dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran one kind or they Mixture；Solvent 3 is one of ethyl acetate, chloroform or methylene chloride or their mixture.

6. preparation method as claimed in claim 3, which is characterized in that detailed process is as follows for step (3): intermediate II is added To dissolution 1-3 times of 1, the 10-60 DEG C of reaction 10-20h of acid measured is added, after reaction, decompression removes solvent, obtains intermediate in solvent 4 III；The solvent 4 is one of methylene chloride, chloroform, ether or ethyl acetate or their mixture；The acid 1 is One of trifluoroacetic acid or hydrochloric acid or their mixture.

7. preparation method as claimed in claim 3, which is characterized in that detailed process is as follows: quinazoline -4 for step (4) (3H) -one or 6, (3H) -one of 7- substituted quinazoline -4 and catalyst 1 are added in solvent 5,30-70 DEG C of reaction 3-10h, reaction knot Shu Hou, it is cooling, pH 6-8, stratification are adjusted, dry organic phase depressurizes removal organic phase solvent, obtains intermediate compound IV；It is described molten Agent 5 is one of methylene chloride, chloroform or thionyl chloride or their mixture；The catalyst 1 is N, N- dimethyl methyl Amide.

8. preparation method as claimed in claim 3, which is characterized in that detailed process is as follows for step (5): intermediate compound IV is added In solvent 6, intermediate III is added and 1-3 times is measured Hunig's base, 30-70 DEG C of reaction 3-10h, fully reacting is cold But, it filters, filter cake, which recrystallizes, is made target product V；The solvent 6 be one of methanol, ethyl alcohol, propyl alcohol or isopropanol or it Mixture.

9. application of the compound in terms of preparing the drug with EGFR related disease as described in claims 1 or 2.

10. application as claimed in claim 9, which is characterized in that the compound is in anti-breast cancer processed, anti-human epidermis cancer drug In application.