CN108069913B

CN108069913B - Bis (morpholinylalkoxy) quinazoline derivative and application thereof in anti-tumor aspect

Info

Publication number: CN108069913B
Application number: CN201611024326.3A
Authority: CN
Inventors: 李宝林; 陈丽; 张娅玲; 刘娟; 张喜全; 顾红梅; 徐宏江; 杨玲
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Shaanxi Normal University
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Shaanxi Normal University
Priority date: 2016-11-18
Filing date: 2016-11-18
Publication date: 2022-03-01
Anticipated expiration: 2036-11-18
Also published as: CN108069913A

Abstract

The invention belongs to the field of medicines, discloses a bis (morpholinyl alkoxy) quinazoline derivative and application thereof in the aspect of tumor resistance, and particularly relates to a compound shown as a formula (I) or pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the compound and application thereof in the aspect of tumor resistance. The compound has good inhibition effect on the proliferation of human skin squamous cell carcinoma cell line A431, human non-small cell lung cancer cell line A549, human colon cancer cell line SW480 and human lung cancer cell line NCI-H1975 containing EGFR T790M/L858R double mutation.

Description

Bis (morpholinylalkoxy) quinazoline derivative and application thereof in anti-tumor aspect

Technical Field

The invention relates to a novel bis (morpholinyl alkoxy) quinazoline derivative or pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the compound and an application of the compound in the aspect of tumor resistance.

Background

The traditional anticancer drugs are mostly cytotoxic drugs, the drugs have great toxic and side effects on normal human tissue cells while having a killing effect on cancer cells, and in order to improve the selectivity of the drugs on the cancer cells, the research on a targeted drug treatment method taking key genes, regulatory molecules and specific cell receptors as targets has become a hotspot.

Epidermal growth factor receptor protein (EGFR) tyrosine kinase is one of the important targets for cancer therapy. When EGFR is over-expressed and abnormally activated in a human body, the growth regulation and control of cells are out of control, the death is blocked, and the cells are always in an abnormal proliferation state, thus finally resulting in malignant tumors. The tyrosine kinase inhibitor taking the EGFR as a target plays an important role in treating tumors, and mainly combines with the EGFR competitively through ATP, so that the catalytic activity of the EGFR and the phosphorylation of tyrosine residues of the EGFR are inhibited, thereby blocking a downstream signal transduction path, further inhibiting the proliferation of tumor cells, accelerating the apoptosis of the tumor cells and inhibiting the infiltration and the metastasis of the tumor cells. Some small molecule EGFR inhibitors such as flavonoids, isoflavones, quinazolines, quinolines, pyrimidines, indoles, and indazoles have been discovered. Among them, a class of small molecule EGFR inhibitors with high activity and good selectivity is 4-arylamino quinazoline compounds, such as Erlotinib (Tarceva) and Gefitinib (Gefitinib, Iressa) which are representatives of the first generation quinazoline EGFR inhibitors have been on the market for many years. These drugs play an important role in the treatment of EGFR-dependent non-small cell lung cancer. However, after the medicine is used for a long time, obvious medicine tolerance can be generated, and difficulty is brought to the later treatment of tumors. It is considered that one of the causes of drug resistance is mutation of EGFR gene, resulting in T790M change of EGFR protein. This conversion of threonine residue to methionine residue at position 790 in the EGFR protein results in steric exclusion of binding to first and second generation (Afatibib and Dacomitinib) EGFR inhibitors, creating tolerance to such inhibitors. The third-generation EGFR inhibitors (nazarrtinib, osimertinib, rociletinib and the like) can avoid the spatial interference of Met790 on the binding of a drug molecule and EGFR, so that the drug molecule and EGFR can generate good antitumor activity through the irreversible binding reaction with Cys797 on the ATP binding site of EGFR (Y.Jia et al cancer Res.2016,76, 1591-. C797S is a recently discovered new mutation in EGFR that prevents covalent binding of third generation inhibitors to EGFR and reduces their potency (j.engel et al.angelw.chem.int.ed.2016, 55, 10909-. Therefore, there is still a need to further develop new EGFR inhibitors to overcome the resistance of tumor cells.

Disclosure of Invention

In one aspect, the invention relates to a compound of formula (I):

wherein the content of the first and second substances,

R¹、R²independently selected from hydrogen, halogen, C₂～C₆Alkynyl, C₂～C₆Alkenyl radical, C₁～C₆Alkyl, -OC₁～C₆Alkyl, -SC₁～C₆Alkyl, -OC₂～C₆Alkenyl, -COC₁～C₆Alkyl, -COOC₁～C₆Alkyl, -CONH₂、-CONHC₁～C₆Alkyl, -CON (C)₁～C₆Alkyl radical)₂-CN, amino, -OH, -COOH, -CHO, -NO₂And a benzene ring is optionally substituted with one or more selected from halogen, C₂～C₆Alkynyl, C₂～C₆Alkenyl radical, C₁～C₆Alkyl, -OC₁～C₆Alkyl, -SC₁～C₆Alkyl, -OC₂～C₆Alkenyl, -COC₁～C₆Alkyl, -COOC₁～C₆Alkyl, -CONH₂、-CONHC₁～C₆Alkyl, -CON (C)₁～C₆Alkyl radical)₂、-CH₂O, amino, -CN, -NO₂、-OH、-COOH、C₃～C₉A benzyloxy group substituted with a cycloalkyl group;

R³、R⁴independently selected from hydrogen, halogen, amino, -CN, -NO₂、-SC₁～C₆Alkyl, -OH, -COOH, C₁～C₆Alkyl, -OC₁～C₆Alkyl radical, C₃～C₉A cycloalkyl group;

n, m are independently 1, 2, 3, or 4; p, q are independently 0, 1, 2, 3, or 4.

In some embodiments, R¹、R²Independently selected from halogen, C₂～C₄Alkynyl, C₂～C₄Alkenyl and benzene ring are substituted by one or more selected from halogen, C₂～C₄Alkynyl, C₂～C₄Alkenyl, -CHO, amino, -CN, -NO₂-OH and-COOH.

In some embodiments, R¹、R²Independently selected from F, Cl, ethynyl, propenyl, and phenyl ring substituted with one or more groups selected from F, Cl, Br, ethynyl, propenyl, -CHO, -CN, -NO₂-OH and-COOH.

In some embodiments, R¹、R²Independently selected from the group consisting of propen-1-yl, ethynyl, F, Cl and benzyloxy having a phenyl ring substituted by 1, 2, 3 or 4 identical or different groups selected from F, Cl, Br, propen-1-yl, ethynyl.

In some embodiments, R¹、R²Independently selected from the group consisting of propen-1-yl, ethynyl, F, Cl and benzyloxy substituted in the 3-and/or 4-position of the phenyl ring by the same or different groups selected from the group consisting of F, Cl, Br, propen-1-yl, ethynyl.

In some embodiments, R²Is halogen and R¹Is benzyloxy substituted by same or different groups selected from F, Cl, Br, propylene-1-radical and ethynyl at the 3-position and/or the 4-position of the benzene ring.

In some embodiments, the morpholine ring is substituted with 1, 2, 3 or 4R, which may be the same or different³Substitution; in some embodiments, the morpholine ring is substituted with 1, 2, 3 or 4R, which may be the same or different⁴Substitution; in some embodiments of the present invention, the substrate is,p and q are both 0.

In some embodiments, n, m are independently 2 or 3.

In some embodiments, the present invention relates to any one of compounds a-H and pharmaceutically acceptable salts thereof:

a: 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline

B: 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline

C: 4- (3-ethynylphenylamino) -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline

D: 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline

E: 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline

F: 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline

G: 4- (3-ethynylphenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline

H: 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline.

The synthetic route for the above compounds is exemplified as follows:

wherein R is¹、R²Independently selected from hydrogen, halogen, C₂～C₆Alkynyl, C₂～C₆Alkenyl radical, C₁～C₆Alkyl radical, C₁～C₆Alkoxy, -SC₁～C₆Alkyl, -OC₂～C₆Alkenyl, -COC₁～C₆Alkyl, -COOC₁～C₆Alkyl, -CONH₂、-CONHC₁～C₆Alkyl, -CON (C)₁～C₆Alkyl radical)₂-CN, amino, -OH, -COOH, -CHO, -NO₂And the benzene ring is optionally substituted by one or more selected from halogen, C₂～C₆Alkynyl, C₂～C₆Alkenyl radical, C₁～C₆Alkyl radical, C₁～C₆Alkoxy, -SC₁～C₆Alkyl, -OC₂～C₆Alkenyl, -COC₁～C₆Alkyl, -COOC₁～C₆Alkyl, -CONH₂、-CONHC₁～C₆Alkyl, -CON (C)₁～C₆Alkyl radical)₂、-CH₂O, amino, -CN, -NO₂、-OH、-COOH、C₃～C₉A benzyloxy group substituted with a cycloalkyl group; n, m are independently 1, 2, 3, or 4.

The compound can be synthesized by taking 3, 4-dihydroxy benzonitrile as a raw material, and directly obtaining di (halogenated alkoxy) benzonitrile through nucleophilic substitution reaction with dihaloalkane or halogenated alcohol in the presence of an acid-binding agent; or firstly obtaining the bis (hydroxyl-substituted alkoxy) benzonitrile, and then reacting the bis (halogenated alkoxy) benzonitrile with a halogenating reagent. Further carrying out nitration, reduction, formamidine generation and Dimroth rearrangement reaction between the di (haloalkoxy) benzonitrile and substituted arylamine in sequence to obtain the di (haloalkoxy) quinazoline derivative, and then carrying out nucleophilic substitution reaction on the di (haloalkoxy) quinazoline derivative and morpholine in the presence of KI to obtain the target product, namely the bis (morpholinyl alkoxy) quinazoline derivative.

In another aspect, the invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be prepared by mixing the compound of the present invention with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid preparations, such as tablets, pills, capsules, powders, granules, pastes, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.

The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

Typical routes of administration of the compounds of the present invention or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

In some embodiments, the pharmaceutical composition is administered by oral administration. Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, the mixture obtained is optionally milled, if appropriate with further suitable adjuvants, the mixture is then processed to granules and further tablets are prepared. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, flavoring agents, and the like.

The pharmaceutical compositions may also be administered parenterally, for example, as a sterile solution, suspension or lyophilisate for reconstitution.

The amount of the compound of the present invention to be administered may vary depending on the route of administration, the age, body weight, type, severity and the like of the patient, and the daily dose may be 0.01 to 50mg/Kg, preferably 0.1 to 10 mg/Kg. Generally, administration by parenteral injection is carried out at a lower dose, preferably orally.

The compounds of the present invention and their pharmaceutically acceptable salts may be administered alone or in combination with other therapeutic agents. In particular, in the anti-tumor therapy, combination with other chemotherapeutic agents, hormones or antibody drugs should be considered. Accordingly, the combination therapy of the present invention comprises at least one compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional active agent. The compound of formula (I) or a pharmaceutically acceptable salt thereof and the other active agent may be administered together or separately, and when administered separately, may be administered simultaneously or sequentially in any order.

In a further aspect, the invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of an anti-tumour agent. The tumors comprise skin squamous cell carcinoma, lung cancer (such as EGFR T790M/L858R mutant lung cancer), non-small cell lung cancer, cervical cancer, liver cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, head and neck cancer, ovarian cancer, prostate cancer, genitourinary tract cancer, melanoma, squamous cell carcinoma, astrocytic carcinoma, Kaposi sarcoma, glioblastoma, leukemia and the like.

The compound of the formula (I) or pharmaceutically acceptable salt thereof has good inhibition effect on the proliferation of tumor cells, and experimental results show that the compound has good inhibition effect on human skin squamous cell carcinoma cell line A431, human non-small cell lung cancer cell line A549, human colon cancer cell line SW480, human lung cancer cell line NCI-H1975 containing EGFR T790M/L858R double mutation, and the proliferation of 4 tumor cell lines, and can be used for preparing antitumor drugs.

Definition of

The following terms used in the present invention have the following meanings, unless otherwise specified.

Herein C_m-nIt is the moiety that has an integer number of carbon atoms in the given range. E.g. "C_1-6By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "amino" refers to the group-NH₂A radical which may be further substituted, for example by one or two identical or different radicals selected from C₁～C₆Alkyl and C₁～C₆Alkoxy groups.

The term "alkyl" refers to a group of formula C_nH_2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "C_1-6Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like). Preferably, is C_1-4An alkyl group. The alkyl group may be further substituted, for example substituents including but not limited to-CN, -NH₂、-OH、-COOH、-CH₂O、-NO₂。

The term "alkenyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one double bond, consisting of carbon atoms and hydrogen atoms. C₂～C₆Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1, 3-butadienyl, and the like, preferably C₂～C₄An alkenyl group. The alkenyl group may be further substituted including, but not limited to, -CN, -NH₂、-OH、-COOH、-CHO、-NO₂。

The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one triple bond composed of carbon atoms and hydrogen atoms. C₂～C₆Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C ≡ CH), 1-propynyl (-C ≡ C-CH)₃) 2-propynyl (-CH)₂-C.ident.CH), 1, 3-butadiynyl (-C.ident.C-C.ident.CH), etc., preferably C₂～C₄Alkynyl. The alkynyl group may be further substituted including, but not limited to, -CN, -NH₂、-OH、-COOH、-CHO、-NO₂。

The term "cycloalkyl" refers to a carbon ring that is fully saturated and may exist as a single ring, a bridged ring, or a spiro ring. Unless otherwise indicated, the carbocycle is typically a 3,4, 5, 6,7, 8, 9 or 10 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "benzyloxy" refers to

Wherein the 2, 3,4, 5 and/or 6 position of the phenyl ring may be substituted, preferably the 3 or 4 position is substituted by a substituent.

The term "pharmaceutically acceptable salts" includes, but is not limited to, ammonium salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) inhibiting the disease or disease state, i.e., arresting its development;

(ii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The invention also includes atoms which are the same as those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in natureSubstituted isotopically labeled compounds of the present invention. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine, such as²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、¹⁸F、¹²³I、¹²⁵I and³⁶cl, and the like.

Certain isotopically-labelled compounds of the invention (e.g. by³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation)³H) And carbon-14 (i.e.¹⁴C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as¹⁵O、¹³N、¹¹C and¹⁸f can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

In addition, heavier isotopes are used (such as deuterium (i.e., deuterium)²H) Substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances where deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is substituted with at least one deuterium.

The compounds of the invention may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, cis-trans isomers, enantiomers and conformational isomers.

Detailed Description

The following examples will help to understand the present invention, but do not limit the scope of the present invention.

The reagents used in the examples of the present invention were all analytical grade. Nuclear magnetic resonance data used for determining the structure of the compound are measured by Bruker Avance300, 400 and 600 superconducting nuclear magnetic resonance instruments, and TMS is used as an internal standard; measuring infrared spectrum data by a Nicolet170SXFT-IR infrared spectrometer; the melting point was measured using an X-6 micro melting point apparatus (Beijing Take instruments, Ltd.); mass spectral data were measured with a Bruker Esquire3000plus mass spectrometer.

Example 1: synthesis of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (A)

(1) Preparation of 3, 4-bis (3-chloropropoxy) benzonitrile

Sequentially adding anhydrous K₂CO₃(0.03mol), 0.6mL of PEG-400 and 30mL of acetonitrile, 1, 3-bromochloropropane (0.06mol), heated to 60 ℃, added in six portions with 3, 4-dihydroxybenzonitrile (0.01mol), allowed to react further, followed by TLC [ developer: ethyl acetate petroleum ether (1:4, V/V)]The reaction time is about 3 hours after the reaction is finished. Stopping the reaction and filtering the solution to remove K when the solution is hot₂CO₃Washing the filter cake with hot acetonitrile for three times, distilling the filtrate to recover acetonitrile and 1, 3-bromochloropropane to obtain brown oily residue, and separating by silica gel column chromatography [ ethyl acetate: petroleum ether (1:6, V/V)]White solid 3, 4-bis (3-chloropropyloxy) benzonitrile was obtained (yield 86.4%).

(2) Preparation of 2-nitro-4, 5-di (3-chloropropoxy) benzonitrile

Adding 3, 4-bis (3-chloropropoxy) benzonitrile (0.02mol) and 10mL of acetic acid into a 100mL single-neck flask, stirring uniformly, and adding 7.5mL of H₂SO₄(70%) and 2.5mLHNO₃(68%) the mixture was added dropwise to the reaction system under ice-water bath conditions. After the dropwise addition, the ice water bath was removed, the mixture was stirred at 35 ℃ and the reaction was followed by TLC until completion, and the reaction was carried out for about 1.5 hours. Cooling to room temperature, pouring into ice water, stirring for 30min, vacuum filtering, washing with water to neutrality, and vacuum drying to obtain yellow2-Nitro-4, 5-bis (3-chloropropyloxy) benzonitrile as a solid (yield 90.2%).

(3) Preparation of 2-amino-4, 5-bis (3-chloropropyloxy) benzonitrile

Dissolving 2-nitro-4, 5-di (3-chloropropoxy) benzonitrile (0.01mol) in a mixed solvent of 15mL of water and 15mL of ethanol, heating to 50 ℃, and adding sodium hydrosulfite (0.03mol) in 1h for six times under stirring. TLC tracking [ developing agent: ethyl acetate: petroleum ether (1:2, V/V) ] until the reaction is finished, it takes about 30 min. 8ml of concentrated hydrochloric acid is added dropwise at 50 ℃, and stirring is carried out for 2 hours after the dropwise addition. Cooled to room temperature, adjusted to pH 9-11 with 20% NaOH aqueous solution, and extracted with ethyl acetate. The extract was dried and evaporated to dryness to give 2-amino-4, 5-bis (3-chloropropyloxy) benzonitrile as a yellow-green solid (yield 67.3%).

(4) Preparation of N' - [ 2-cyano-4, 5-bis (3-chloropropyloxy) phenyl ] -N, N-dimethylformamidine

2-amino-4, 5-bis (3-chloropropyloxy) benzonitrile (2.0mmol) and DMF-DMA (4.0mmol) were dissolved in 5mL of toluene, and after warming to 35 ℃, 2 drops of glacial acetic acid were added dropwise, and TLC followed [ developer: ethyl acetate petroleum ether (1:2, V/V)]About 15min is required until the reaction is finished. The toluene is distilled off under reduced pressure, 20mL of water are added, the pH is adjusted to 11 with 20% aqueous NaOH solution and CH is used₂Cl₂Extracting (2X 15mL), combining extract liquid, drying by anhydrous magnesium sulfate, concentrating to obtain oily matter N' - [ 2-cyano-4, 5-di (3-chloropropoxy) phenyl]-N, N-dimethylformamidine (yield 92.1%).

(5) Preparation of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropoxy) quinazoline

N' - [ 2-cyano-4, 5-bis (3-chloropropyloxy) phenyl ] -N, N-dimethylformamidine (1.00mmol) was taken, 2.0mL glacial acetic acid and 4- (E) -aminophenylpropene (1.10mmol) were added, refluxed at 130 ℃ and TLC-tracked [ developing solvent: and (3) ethyl acetate: petroleum ether (1:2, V/V) until the reaction is finished, wherein the reaction time is about 15 min. Stopping heating, distilling off glacial acetic acid under reduced pressure, adding 10mL of water, adjusting pH to 9-10 with ammonia water, stirring for 30min, filtering, washing the filter cake with water for 3 times, and separating by silica gel column chromatography [ ethyl acetate: petroleum ether (1:4, V/V) ] to obtain 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropoxy) quinazoline as a white solid (yield 91.9%).

(6) Preparation of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (A)

4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropoxy) quinazoline (0.11mmol), KI (0.06mmol) in a 10mL single neck flask was added 1mL morpholine, heated in an oil bath to 120 ℃ and followed by TLC [ developer: methanol, ethyl acetate (1:5, V/V) ] for about 30-40min until the reaction is finished. Suction filtration, reduced pressure evaporation to dryness and silica gel column chromatography of the residue [ methanol: ethyl acetate (1:10, V/V) ] gave the title compound as a pale yellow solid a (yield 49.8%).

m.p.:91.3–92.2℃；HRMS(C₃₁H₄₁N₅O₄)m/z[M+H]⁺548.3246 (calculated value: 548.3237).

¹H NMR(300MHz,DMSO-d₆)δ(ppm):9.47(s,1H),8.44(s,1H),7.86(s,1H),7.76(d,J＝8.2Hz,2H),7.39(d,J＝8.2Hz,2H),7.17(s,1H),6.40(d,J＝16.0Hz,1H),6.31–6.12(m,1H),4.25–4.10(m,4H),3.34–3.55(m,12H),2.45–2.31(m,8H),2.05–1.90(m,4H),1.85(d,J＝6.1Hz,3H).

¹³C NMR(75MHz,DMSO-d₆)δ(ppm):156.2,153.7,152.8,148.3,146.9,138.2,132.5,130.5,125.7,124.0,122.2,108.9,107.9,103.2,67.1,66.5,66.1,66.1,54.9,54.8,53.4,53.3,25.8,25.6,18.2.

IRν_max(KBr)cm^-1:3435,3026,2935,2839,1613,1500,1435,1371,1226,1113,984.

Example 2: synthesis of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (B)

Steps (1) to (4) were the same as in example 1,

(5) preparation of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (3-chloropropyloxy) quinazoline

By following the procedure of example 1, step 5, using 3-chloro-4- (3-fluorobenzyloxy) aniline (1.10mmol) instead of 4- (E) -aminophenylpropene used in example 1, step 5, 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (3-chloropropyloxy) quinazoline was prepared as a white solid (yield 93.8%).

(6) Preparation of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (B)

Following the procedure of example 1, step 6, substituting 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (3-chloropropyloxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropyloxy) quinazoline in example 1, step 6, prepared as a light yellow solid B (yield 72.1%).

m.p.:71.5–73.2℃；HRMS(C₃₅H₄₁ClFN₅O₅)m/z[M+H]⁺666.2862 (calculated value: 666.2859).

¹H-NMR(600MHz,DMSO-d₆)δ(ppm):9.47(s,1H),8.45(s,1H),7.98(s,1H),7.83(s,1H),7.72(dd,J＝9.0,2.3Hz,1H),7.47(dd,J＝14.9,7.6Hz,1H),7.37–7.29(m,2H),7.26(d,J＝9.0Hz,1H),7.20–7.15(m,2H),5.25(s,2H),4.19–4.14(m,4H),3.66–3.52(m,12H),2.43–2.30(m,8H),2.00–1.93(m,4H).

¹³C-NMR(151MHz,DMSO-d₆)δ(ppm):163.0(d,¹J_C-F＝243.6Hz),156.2,153.8,152.7,149.3,148.3,146.8,139.7(d,³J_C-F＝7.4Hz),133.6,130.5(d,³J_C-F＝8.4Hz),123.9,123.3,122.0,121.0,114.7(d,²J_C-F＝21.0Hz),114.3(d,⁴J_C-F＝2.3Hz),114.0(d,²J_C-F＝21.9Hz),108.7,107.9,103.1,69.4,67.1,66.5,66.2,66.1,55.0,54.8,53.4,53.3,25.9,25.6.

IRν_max(KBr)cm^-1:3453,3083,2922,2853,1619,1502,1431,1215,1110,1060,853.

Example 3: synthesis of 4- (3-ethynylphenylamino) -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (C)

Steps (1) to (4) were the same as in example 1.

(5) Preparation of 4- (3-ethynylphenylamino) -6, 7-bis (3-chloropropoxy) quinazoline

Following the procedure of example 1, step 5, substituting 3-ethynylaniline (1.10mmol) for 4- (E) -aminophenylpropene of example 1, step 5, prepared 4- (3-ethynylphenylamino) -6, 7-bis (3-chloropropyloxy) quinazoline as a white solid (yield 90.6%).

(6) Preparation of 4- (3-ethynylphenylamino) -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (C)

Following the procedure of example 1, step 6, substituting 4- (3-ethynylphenylamino) -6, 7-bis (3-chloropropoxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropoxy) quinazoline in example 1, step 6, prepared as a light yellow solid C (yield 71.5%).

m.p.:91.5–92.7℃；HRMS(C₃₀H₃₇N₅O₄)m/z[M+H]⁺532.2920 (calculated value: 532.2924).

¹H-NMR(600MHz,DMSO-d₆)δ(ppm):9.51(s,1H),8.49(s,1H),8.02–8.01(m,1H),7.92(dd,J＝8.2,1.2Hz,1H),7.85(s,1H),7.40(t,J＝7.9Hz,1H),7.21(d,J＝7.6Hz,1H),7.17(s,1H),4.19(s,1H),4.18–4.15(m,4H),3.62–3.55(m,12H),2.43–2.28(m,8H),2.00–1.92(m,4H).

¹³C-NMR(151MHz,DMSO-d₆)δ(ppm):156.1,153.9,152.6,148.4,146.9,139.8,128.8,126.3,124.7,122.5,121.7,108.9,107.9,103.1,83.5,80.4,67.1,66.5,66.2,64.4,55.0,54.8,53.4,53.4,25.9,25.6.

IRν_max(KBr)cm^-1:3431,3072,2954,2856,2818,1621,1580,1510,1428,1110,863.

Example 4: synthesis of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [3- (morpholinyl) propoxy ] quinazoline (D)

Steps (1) to (4) were the same as in example 1.

(5) Preparation of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis (3-chloropropoxy) quinazoline

By following the procedure of example 1, step 5, using 3-chloro-4-fluoroaniline (1.10mmol) instead of 4- (E) -aminophenylene in example 1, step 5, 4- (3-chloro-4-fluorophenylamino) -6, 7-bis (3-chloropropyloxy) quinazoline was prepared as a white solid (yield 91.6%).

(6) Preparation of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [3- (4-morpholinyl) propoxy ] quinazoline (D)

Following the procedure of example 1, step 6, substituting 4- (3-chloro-4-fluoroanilino) -6, 7-bis (3-chloropropoxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (3-chloropropoxy) quinazoline in example 1, step 6, prepared as a light yellow solid D (yield 71.4%).

m.p.:86.1–87.2℃；HRMS(C₂₈H₃₅ClFN₅O₄)m/z[M+H]⁺560.2435 (calculated value: 560.2440.).

¹H NMR(600MHz,DMSO-d₆)δ(ppm):9.55(s,1H),8.49(s,1H),8.14(dd,J＝6.8,2.6Hz,1H),7.83–7.78(m,2H),7.42(t,J＝9.1Hz,1H),7.16(s,1H),4.16(dd,J＝15.0,6.2Hz,4H),3.60–3.57(m,8H),3.52–3.30(m,4H),2.42–2.30(m,8H),1.99–1.92(m,4H).

¹³C NMR(151MHz,DMSO-d₆)δ(ppm):155.9,153.9(d,¹J_C-F＝209.1Hz),153.8,152.3,148.4,146.9,136.8(d,⁴J_C-F＝2.3Hz),123.3,122.1(d,³J_C-F＝6.8Hz),118.7(d,²J_C-F＝18.0Hz),116.4(d,²J_C-F＝21.7Hz),108.7,107.9,103.0,67.1,66.5,66.2,66.1,55.0,54.8,53.4,53.3,25.9,25.6.

IRν_max(KBr)cm^-1:3402,3083,2959,2867,1624,1502,1427,1220,1069,956,862.

Example 5: synthesis of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (E)

(1) Preparation of 3, 4-bis (2-chloroethoxy) benzonitrile

Taking 3, 4-dihydroxy benzonitrile (0.01mol) and anhydrous K₂CO₃(0.04mol) and 15mL of DMF were put in a 100mL single-neck flask, stirred at room temperature for 10min, added with chloroethanol (0.03mol), and heated to 90 ℃. TLC tracking [ developing agent: methanol: chloroform (1:5, V/V)]The reaction time is about 5 hours after the reaction is finished. After adding 50mL of water, the pH was adjusted to weak acidity with 2M HCl, followed by extraction with ethyl acetate (3X 30mL), and the combined extracts were dried over anhydrous magnesium sulfate and concentrated to dryness to give 3, 4-bis (2-hydroxyethoxy) benzonitrile as a white solid (yield 80.2%).

Taking 3, 4-di (2-hydroxyethoxy) benzeneNitrile (0.01mol), pyridine (0.03mol) and benzene (10mL) are put in a reaction bottle; stirring the SOCl₂A mixed solution of (0.03mol) and benzene (20mL) was slowly added to the reaction flask, warmed to 85 ℃, and followed by TLC [ developing solvent: ethyl acetate petroleum ether (1:2, V/V)]The reaction time is about 3 hours after the reaction is finished. Cooling to room temperature, evaporating to remove volatile substances, and slowly adding 10% NaOH to adjust pH to alkalescence. Extracting with ethyl acetate (3 × 30ml), mixing extractive solutions, drying with anhydrous magnesium sulfate, concentrating, and separating by silica gel column chromatography [ ethyl acetate: petroleum ether (1:6, V/V)]This gave 3, 4-bis (2-chloroethoxy) benzonitrile as a white solid (yield 78.4%).

(2) Preparation of 2-nitro-4, 5-bis (2-chloroethoxy) benzonitrile

3, 4-bis (2-chloroethoxy) benzonitrile (0.02mol) and 10mL of acetic acid were added to a 100mL single-neck flask and stirred well, 7.5mL of H₂SO₄(70%) and 2.5mL of HNO₃(68%) the mixture was added dropwise to the reaction system with cooling in an ice-water bath. After the dropwise addition, the ice water bath was removed, the mixture was stirred at 35 ℃ and the reaction was completed by TLC, which took about 1.5 hours. Cooling to room temperature, pouring into ice water, stirring for 30min, performing suction filtration, washing with water to neutrality, and performing vacuum drying to obtain yellow solid 2-nitro-4, 5-bis (2-chloroethoxy) benzonitrile (yield 90.3%).

(3) Preparation of 2-amino-4, 5-bis (2-chloroethoxy) benzonitrile

Dissolving 2-nitro-4, 5-bis (2-chloroethoxy) benzonitrile (0.01mol) in a mixed solvent of 15mL of water and 15mL of ethanol, heating to 50 ℃, and adding sodium hydrosulfite (0.03mol) for 1h in six times under stirring. TLC tracking [ developing agent: and (3) ethyl acetate: petroleum ether (1:2, V/V) until the reaction is finished, wherein the reaction time is about 30 min. 8mL of concentrated hydrochloric acid was added dropwise at 50 ℃ and stirred for 2 hours after the addition. Cooled to room temperature, adjusted to pH 9-11 with 20% NaOH aqueous solution, and extracted with ethyl acetate. The extract was dried and evaporated to dryness to give a yellow-green solid, 2-amino-4, 5-bis (2-chloroethoxy) benzonitrile (yield 70.5%).

(4) Preparation of N' - [ 2-cyano-4, 5-bis (2-chloroethoxy) phenyl ] -N, N-dimethylformamidine

2-amino-4, 5-bis (2-chloroethoxy) benzonitrile (2.0mmol) and DMF-DMA (4.0mmol) were dissolved in 5mL of toluene, heated to 35 ℃ and 2 drops of glacial acetic acid were added dropwise, followed by TLC [ developer: ethyl acetate petroleum ether (1:2V/V)]The reaction time is about 15min after the reaction is finished. The toluene is distilled off under reduced pressure, 20mL of water are added, the pH is adjusted to 11 with 20% aqueous NaOH solution and CH is used₂Cl₂Extraction (2X 15mL), combining extracts, drying over anhydrous magnesium sulfate, and concentration to give N' - [ 2-cyano-4, 5-bis (2-chloroethoxy) phenyl ] as a white oil]-N, N-dimethylformamidine (yield 87.8%).

(5) Preparation of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline

N' - [ 2-cyano-4, 5-bis- (2-chloroethoxy) -phenyl ] -N, N-dimethylformamidine (1.00mmol) was added with 2.0mL glacial acetic acid and 4- (E) -aminophenylene (1.10mmol), and the mixture was refluxed at 130 ℃. TLC tracking [ developing agent: and (3) ethyl acetate: petroleum ether (1:2, V/V) until the reaction is finished, wherein the reaction time is about 15 min. Stopping heating, distilling off glacial acetic acid under reduced pressure, adding 10mL of water, adjusting pH to 9-10 with ammonia water, stirring for 30min, filtering, washing the filter cake with water for 3 times, and separating by silica gel column chromatography [ ethyl acetate: petroleum ether (1:4, V/V) ] to obtain 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline as a white solid (yield 86.1%).

(6) Preparation of 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (E)

In a 10mL single-neck flask was taken 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline (1.00mmol), KI (0.60mmol), morpholine (1mL), heated to 120 ℃ with stirring, and TLC tracked [ developing solvent: and (3) adding methanol, ethyl acetate (1:5, V/V) until the reaction is finished, wherein the reaction time is about 30-40 min. After cooling, suction filtration and reduced pressure evaporation to dryness, the residue was subjected to silica gel column chromatography [ methanol: ethyl acetate (1:10, V/V) ] to obtain a pale yellow solid E (yield 73.1%).

m.p.:197.4–198.5℃；HRMS(C₂₉H₃₇N₅O₄)m/z[M+H]⁺520.2926 (calculated value: 520.2924).

¹H-NMR(400MHz,DMSO-d₆)δ(ppm):9.43(s,1H),8.46(s,1H),7.87(s,1H),7.76(d,J＝8.5Hz,2H),7.38(d,J＝8.5Hz,2H),7.20(s,1H),6.39(d,J＝15.8Hz,1H),6.31–6.09(m,1H),4.27–4.23(m,4H),3.71–3.50(m,8H),2.81–2.75(m,4H),2.60–2.53(m,8H),1.85(d,J＝6.2Hz,3H).

¹³C NMR(101MHz,DMSO-d₆)δ(ppm):158.1,155.4,154.8,149.9,148.8,140.1,134.4,132.4,127.6,125.9,124.1,110.8,110.0,105.1,69.1,68.8,68.1,61.6,58.8,58.6,55.7,55.7,20.2.

IRν_max(KBr)cm^-1:3296,3077,2964,1648,1514,1454,1427,1262,1038,969,855.

Example 6: synthesis of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (F)

Steps (1) to (4) were the same as in example 5.

(5) Preparation of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline

By following the procedure of example 5, step 5, using 3-chloro-4- (3-fluorobenzyloxy) aniline (1.10mmol) instead of 4- (E) -aminophenylpropene used in example 5, step 5, 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline was prepared as a white solid (yield 85.7%).

(6) Preparation of 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (F)

Following the procedure of example 5, step 6, substituting 4- [ 3-chloro-4- (3-fluorobenzyloxy) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline in example 5, step 6, prepared F as a white solid (yield 75.2%).

m.p.:173.5–174.4℃；HRMS(C₃₃H₃₇ClFN₅O₅)m/z[M+H]⁺638.2547 (calculated value: 638.2545).

¹H-NMR(600MHz,DMSO-d₆)δ(ppm):9.36(s,1H),8.41(s,1H),7.92(d,J＝2.5Hz,1H),7.77(s,1H),7.66(dd,J＝9.0,2.5Hz,1H),7.42(td,J＝7.9,6.3Hz,1H),7.30–7.25(m,2H),7.20(d,J＝9.0Hz,1H),7.15–7.13(m,2H),5.19(s,2H),4.20(dd,J＝9.4,5.5Hz,4H),3.55(dd,J＝9.4,4.9Hz,6H),3.37(bs,8H),2.75(t,J＝5.8Hz,2H),2.71(t,J＝5.5Hz,2H),2.47–2.46(m,2H).

¹³C NMR(151MHz,DMSO-d₆)δ(ppm):163.0(d,¹J_C-F＝243.6Hz),156.2,153.5,152.8,149.4,148.0,146.8,139.7(d,³J_C-F＝7.5Hz),133.6,130.5(d,³J_C-F＝8.0Hz),123.8,123.3(d,⁴J_C-F＝2.1Hz),122.0,121.0,114.7(d,²J_C-F＝20.9Hz),114.3,114.0(d,²J_C-F＝21.9Hz),108.7,108.1,103.1,69.4,67.2,66.9,66.2,66.1,56.9,56.7,53.8,53.7.

IRν_max(KBr)cm^-1:3449,3077,2924,2853,1623,1507,1343,1125,1002,852.

Example 7: synthesis of 4- (3-ethynylphenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (G)

Steps (1) to (4) were the same as in example 5.

(5) Preparation of 4- (3-ethynylphenylamino) -6, 7-bis (2-chloroethoxy) quinazoline

4- (3-ethynylphenylamino) -6, 7-bis (2-chloroethoxy) quinazoline was prepared as a white solid according to the procedure of example 5, step 5, substituting 3-ethynylaniline (1.10mmol) for 4- (E) -aminophenylpropene in example 5, step 5 (yield 82.0%).

(6) Preparation of 4- (3-ethynylphenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (G)

Prepared according to the method of example 5 step 6, substituting 4- (3-ethynylphenylamino) -6, 7-bis (2-chloroethoxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline in example 5 step 6 to give G as a white solid (yield 71.5%).

m.p.:91.2–92.5℃；HRMS(C₂₈H₃₃N₅O₄)m/z[M+H]⁺504.2609 (calculated value: 504.2611).

¹H NMR(600MHz,DMSO-d₆)δ(ppm):9.48(s,1H),8.53(s,1H),8.04(s,1H),7.94(d,J＝8.3Hz,1H),7.86(s,1H),7.42(t,J＝7.9Hz,1H),7.25(d,J＝7.6Hz,1H),7.22(s,1H),4.29(dd,J＝13.7,5.6Hz,4H),4.21(s,1H),3.68–3.61(m,8H),2.89(t,J＝5.6Hz,2H),2.86(t,J＝5.3Hz,2H),2.64(bs,8H).

¹³C NMR(151MHz,DMSO-d₆)δ(ppm):156.1,153.5,152.7,148.0,146.8,139.7,128.8,126.3,124.8,122.6,121.7,108.9,108.1,103.1,83.5,80.4,66.9,66.6,66.0,65.9,56.8,56.5,53.7,53.6.

IRν_max(KBr)cm^-1:3401,3069,2959,2865,1623,1438,1388,1243,1117,1005,860.

Example 8: synthesis of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (H)

Steps (1) to (4) were the same as in example 5.

(5) Preparation of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis (2-chloroethoxy) quinazoline

By following the procedure of example 5, step 5, using 3-chloro-4-fluoroaniline (1.10mmol) instead of 4- (E) -aminophenylene in example 5, step 5, 4- (3-chloro-4-fluoroanilino) -6, 7-bis (2-chloroethoxy) quinazoline was prepared as a white solid (yield 83.6%).

(6) Preparation of 4- (3-chloro-4-fluorophenylamino) -6, 7-bis [2- (4-morpholinyl) ethoxy ] quinazoline (H)

Following the procedure of example 5, step 6, substituting 4- (3-chloro-4-fluoroanilino) -6, 7-bis (2-chloroethoxy) quinazoline (0.50mmol) for 4- [4- (E) - (propen-1-yl) phenylamino ] -6, 7-bis (2-chloroethoxy) quinazoline in example 5, step 6, prepared as a white solid H (yield 75.2%).

m.p.:176.5–177.4℃；HRMS(C₂₆H₃₁ClFN₅O₄)m/z[M+H]⁺532.2121 (calculated value: 532.2127).

¹H NMR(600MHz,DMSO-d₆)δ(ppm):9.53(s,1H),8.51(s,1H),8.14(dd,J＝6.8,2.6Hz,1H),7.84(s,1H),7.81(ddd,J＝8.9,4.2,2.7Hz,1H),7.44(t,J＝9.1Hz,1H),7.22(s,1H),4.28(t,J＝5.2Hz,4H),3.63(bs,8H),2.88–2.84(m,4H),2.63(bs,8H).

¹³C NMR(151MHz,DMSO-d₆)δ(ppm):156.0,153.9(d,¹J_C-F＝242.7Hz),153.6,152.6,148.1,146.9,136.7(d,⁴J_C-F＝2.2Hz),123.4,122.2(d,³J_C-F＝6.7Hz),118.8(d,²J_C-F＝18.2Hz),116.5(d,²J_C-F＝21.6Hz),108.8,108.1,103.1,67.0,66.6,66.0,65.9,56.8,56.5,53.7,53.6.

IRν_max(KBr)cm^-1:3480,3055,2931,2865,1632,1584,1498,1425,1219,1115,939,863.

Example 9: growth inhibition experiment of compounds A to H on tumor cells

1. Cell line

Human skin squamous cell carcinoma cell line A431, human non-small cell lung cancer cell line A549, human colon cancer cell line SW480 and human lung cancer cell line NCI-H1975 containing EGFR T790M/L858R double mutation are all purchased from Shanghai cell bank of Chinese academy of sciences.

2. Reagents and materials

MTT (MPBIO), 96-well cell culture plate (Costar), fetal bovine serum (Gibco), culture medium (Gibco), and enzyme labeling instrument (ThermoMULTISKANMK 3).

3. Experimental procedure

The growth inhibitory activity of the compounds A to H on tumor cells was measured by the MTT method. Respectively taking human tumor cells SW480, A549, A431 and NCI-H1975 in logarithmic growth phase, digesting with 0.25% trypsin digestion solution, centrifuging, re-suspending, counting, preparing cell suspension, and adjusting the concentration of the cell suspension to be 2 x 10⁴～5×10⁴one/mL. The cell suspension was inoculated into a 96-well cell culture plate (100. mu.L/well), and placed under saturated humidity at 37 ℃ and 5% CO₂Culturing in an incubator for 24 h. Test compounds were diluted with medium to the desired concentration, added to 96-well cell culture plates (100 μ L/well) inoculated with human tumor cells, DMSO at a final concentration of 0.5%, and placed in an incubator for 72 h. MTT was added to 96-well plates (20. mu.L/well) and reacted in an incubator for 4 h. The liquid in the wells was removed by blotting, DMSO (150 μ L/well) was added, and the formazan was shaken on a shaker for 10min to dissolve the formazan completely. Then, the absorbance (OD) at the wavelength of 570nm was measured by a microplate reader₅₇₀) Absorbance (OD) at wavelength of 630nm₆₃₀) As a reference, the cell growth inhibition rate was calculated with the corresponding solvent as a control.

The inhibition rate of the test compounds on the growth of tumor cells was calculated as follows:

tumor cell growth inhibition rate [% 1- (OD)_s-OD_NC)/(OD_PC-OD_NC)]×100％

Wherein: OD_SThe absorbance values (cells + test compound + MTT), OD of the sample wells are expressed_PCAbsorbance values (cell + DMSO + MTT), OD of control wells_NCAbsorbance values (Medium + DMSO + MTT), ODs ═ OD, for the zeroed wells_570s-OD_630s，OD_PC＝OD_570PC-OD_630PC，OD_NC＝OD_570NC-OD_630NC。

Fitting of test Compounds to tumor cell growth inhibition curves and IC₅₀The calculation of (2):

graphpadprism5 was used to fit the inhibition curves of test compounds on tumor cell growth and IC was derived₅₀The value is obtained. Each set was set with 3 replicate wells and the experiment was repeated at least 3 times.

4. Results of the experiment

The results of the experiment are shown in table 1.

TABLE 1 IC inhibition of tumor cell proliferation by test compounds₅₀(μmol/L)

Note: -means no test.

As can be seen from the experimental data in Table 1, the tested compounds have a very good inhibitory effect on the proliferation of tumor cells.

Claims

1. A bis (morpholinylalkoxy) quinazoline derivative is characterized in that the derivative is any one of the following compounds A-D:

A：4-[4-(E) -propenylphenylamino]-6, 7-bis [3- (4-morpholinyl) propoxy group]Quinazoline

。

2. Use of the bis (morpholinoalkoxy) quinazoline derivative of claim 1 and pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating human squamous cell carcinoma of skin, human non-small cell lung cancer, human colon cancer or human lung cancer containing EGFR T790M/L858R double mutation.