CN108250185B

CN108250185B - 6- (2- (o-toluidine amino) acetamido) quinazoline compound, preparation and application thereof

Info

Publication number: CN108250185B
Application number: CN201810069794.5A
Authority: CN
Inventors: 饶国武; 郑泉; 胡成海
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-01-24
Filing date: 2018-01-24
Publication date: 2020-08-21
Anticipated expiration: 2038-01-24
Also published as: CN108250185A

Abstract

The invention discloses a 6- (2- (o-toluidine) acetamido) quinazoline compound, and a preparation method and application thereof. The 6- (2- (o-toluidine) acetamido) quinazoline compound provided by the invention has obvious inhibitory activity on human breast cancer cell strain MCF-7, human lung cancer cell strain A-549, human promyelocytic leukemia cell strain HL-60 and human cervical cancer cell strain Siha, and is expected to be applied to preparation of medicines for preventing or treating human breast cancer, human lung cancer, human leukemia and human cervical cancer. The invention provides a preparation method of the 6- (2- (o-toluidine) acetamido) quinazoline compound, which is simple, easy to operate, easy to obtain raw materials, low in production cost and suitable for practical use.

Description

6- (2- (o-toluidine amino) acetamido) quinazoline compound, preparation and application thereof

(I) technical field

The invention relates to a quinazoline compound and application thereof, in particular to a 6- (2- (o-toluidine) acetamido) quinazoline compound and a preparation method thereof, and application of the compound in preparation of a medicament for preventing or treating tumor diseases.

(II) background of the invention

The quinazoline compounds have a plurality of good biological activities and are widely applied in the field of medicine, particularly, some quinazoline derivatives with special structures have obvious antiviral activity, antibacterial activity, antitumor activity and the like, and the quinazoline compounds are marketed as antitumor drugs. For example, Gefitinib (Gefitinib) and Erlotinib (Erlotinib) are marketed for the treatment of lung cancer, and Lapatinib (Lapatinib) is marketed for the treatment of breast cancer, both of which belong to the quinazoline class of compounds. Novel quinazoline compounds and their biological activities are also commonly reported in the literature (see y. -y. ke, h. -y. shiao, y. c. hsu, c. -y. chu, w. -c. wang, y. -c. lee, w. -h. lin, c. -h. chen, j. t. a. hsu, c. -w. chang, c. -w. lin, t. -k. yeh, y. -s. chao, m.s. coumar, h. -p. hsieh, chemed chem 2013,8, 136-148; a.garofalo, a.farce, s.ravez, a.lemoine, p.six, p.vachatte, l.gos, p.depenux, j.chem. 1204, d. chem. 1189). Of course most quinazoline compounds do not have anti-tumor activity.

Disclosure of the invention

The invention aims to provide a novel quinazoline compound with anticancer activity, namely a 6- (2- (o-toluidine) acetamido) quinazoline compound, and a preparation method and application thereof, wherein the compound has a good inhibition effect on a human lung cancer cell strain A-549, a human breast cancer cell strain MCF-7, a human promyelocytic leukemia cell strain HL-60 or a human cervical cancer cell strain Siha under a certain dosage; and the preparation method of the compound is simple and convenient, easy to operate, easy to obtain raw materials, low in production cost and suitable for industrial application.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a 6- (2- (o-toluidino) acetamido) quinazoline compound shown as a formula (I),

in a second aspect, the present invention provides a method for preparing a 6- (2- (o-toluylamino) acetamido) quinazoline compound represented by formula (i), wherein the method comprises: (1) mixing a compound shown as a formula (II) and a compound shown as a formula (III), reacting at 25-120 ℃ in an organic solvent A under the action of a basic catalyst B (TLC tracking monitoring is carried out, a developing agent is ethyl acetate/petroleum ether which is 1: 3(v/v), and preferably 40-100 ℃ for 0.5-12 h), and after the reaction is completed, separating and purifying a reaction solution to obtain a compound shown as a formula (IV); the organic solvent A is selected from one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the basic catalyst B is selected from one of the following: pyridine, diethylamine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate (preferably pyridine, diethylamine, triethylamine, NN-dimethylaniline or 4-dimethylaminopyridine);

(2) completely reacting a compound shown in a formula (IV) in an organic solvent D under the action of a reducing agent E at 25-100 ℃ (TLC tracking monitoring, a developing agent is ethyl acetate/petroleum ether which is 1: 1(v/v), and preferably reacting for 0.5-12 h at 40-80 ℃), filtering a reaction solution, concentrating a filtrate under reduced pressure, and drying a concentrate (preferably drying at 25 ℃ in vacuum) to obtain a compound shown in a formula (V); the organic solvent D is one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the reducing agent E is one of the following: iron powder/concentrated hydrochloric acid, iron powder/acetic acid, palladium on carbon/ammonium formate or palladium on carbon/hydrazine hydrate; the iron powder/concentrated hydrochloric acid refers to the mixing of iron powder and concentrated hydrochloric acid in any proportion, the iron powder/acetic acid refers to the mixing of iron powder and acetic acid in any proportion, the palladium carbon/ammonium formate refers to the mixing of palladium carbon and ammonium formate in any proportion, and the palladium carbon/hydrazine hydrate refers to the mixing of palladium carbon and hydrazine hydrate in any proportion;

(3) mixing a compound shown as a formula (V) with chloroacetyl chloride or chloroacetic anhydride, completely reacting at-10-50 ℃ in an organic solvent G under the action of an alkaline catalyst F (tracking and monitoring by TLC, wherein a developing agent is ethyl acetate/petroleum ether (1: 1(v/v), preferably reacting at-10-50 ℃ for 3-12 h), and carrying out aftertreatment A on a reaction solution to obtain a compound shown as a formula (VI); the alkaline catalyst F is one of the following: pyridine, diethylamine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate; the organic solvent G is one of the following: tetrahydrofuran, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile, toluene or benzene;

(4) mixing a compound shown as a formula (VI) with o-toluidine, reacting at 25-120 ℃ in an organic solvent J under the action of a basic catalyst K (TLC tracking monitoring is carried out, a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v), preferably 40-100 ℃ for 0.5-36 h), and after the reaction is completed, carrying out aftertreatment B on a reaction liquid to obtain a compound shown as a formula (I); the organic solvent J is selected from one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the basic catalyst K is selected from one of the following: pyridine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate (preferably pyridine, quinoline, triethylamine, N-dimethylaniline or 4-dimethylaminopyridine);

further, in the step (1), the ratio of the amount of the compound represented by the formula (III) to the amount of the compound represented by the formula (II) and the amount of the substance charged as the basic catalyst B is 1.0: 0.8 to 1.2: 1.0 to 8.0.

Further, in the step (1), the amount of the organic solvent A is 10-50 mL/g based on the mass of the compound represented by the formula (III).

Further, the method for separating and purifying the reaction solution in the step (1) of the present invention comprises: after the reaction is completed, evaporating the solvent from the reaction solution, dissolving the concentrate with an organic solvent C to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the organic solvent C in a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component (preferably, ethyl acetate/petroleum ether is 1: 3(v/v) is taken as a developing agent for tracking detection, collecting the target component, preferably, collecting a component with an Rf value of 0.5), concentrating under reduced pressure, and drying (preferably, drying at 50 ℃) to obtain a compound shown in a formula (IV); the organic solvent C is one of the following solvents: ethanol, chloroform, tetrahydrofuran or ethyl acetate. The organic solvent C is used in an amount capable of dissolving the residue.

Further, in the step (2), when the reducing agent E is iron powder/concentrated hydrochloric acid or iron powder/acetic acid, the charging mass ratio of the compound represented by the formula (iv) to the iron powder, concentrated hydrochloric acid or acetic acid in the reducing agent E is 1.0: 1.0 to 3.0: 0.2 to 1.0. In the invention, the mass concentration of the concentrated hydrochloric acid is 36-38%, and the acetic acid is glacial acetic acid.

Further, in the step (2), when the reducing agent E is palladium on carbon/ammonium formate or palladium on carbon/hydrazine hydrate, the feeding mass ratio of the compound represented by the formula (iv) to palladium on carbon, ammonium formate or hydrazine hydrate in the reducing agent E is 1.0: 0.1 to 0.5: 1.0 to 3.0. The mass loading amount of palladium in the palladium-carbon applicable to the invention is 2-10%, preferably 5%, and the mass concentration of hydrazine hydrate is 40-80%, preferably 80%.

Further, in the step (2), the amount of the organic solvent D is 10-50 mL/g based on the mass of the compound represented by the formula (IV).

In the step (3), the ratio of the compound of the formula (v) to the amount of chloroacetyl chloride or chloroacetic anhydride and the basic catalyst F to be fed is 1: 1.0 to 8.0: 1.0 to 3.0.

Further, in the step (3), the amount of the organic solvent G is 11 to 100mL/G based on the mass of the compound represented by the formula (V).

Further, the specific recommended step (3) of the present invention is performed as follows: dropwise adding chloroacetyl chloride or chloroacetic anhydride organic solvent G solution into the compound shown in the formula (V) and the organic solvent G solution of the basic catalyst F at-10 ℃ or the compound shown in the formula (V) and the basic catalyst F, reacting for 3-12 hours at-10-50 ℃, and carrying out aftertreatment on the obtained reaction solution A to obtain the compound shown in the formula (VI); the volume consumption of the organic solvent for dissolving the chloroacetyl chloride or the chloroacetic anhydride does not influence the invention, and the total consumption of the organic solvent G is 11-100 mL/G based on the mass of the compound shown in the formula (V). The total amount of the organic solvent G is the total volume of the organic solvent G in which the basic catalyst F and the compound represented by the formula (V) are dissolved and the organic solvent G in which chloroacetyl chloride or chloroacetic anhydride is dissolved.

Further, the method for post-treating the reaction solution A in the step (3) of the present invention comprises: after the reaction is completed, filtering the reaction solution, evaporating the solvent from the filtrate, dissolving the concentrate with an organic solvent H to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the organic solvent H according to a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component (preferably, ethyl acetate/petroleum ether is 1: 1(v/v) is taken as a developing agent for tracking detection, collecting the target component, preferably, collecting a component with an Rf value of 0.5), concentrating under reduced pressure, and drying (preferably, drying at 50 ℃) to obtain a compound shown in a formula (VI); the organic solvent H is one of the following: ethanol, chloroform, tetrahydrofuran or ethyl acetate. The organic solvent H is used in an amount capable of dissolving the residue.

Further, in the step (4), the ratio of the amount of the compound represented by the formula (vi) to the amounts of the o-toluidine and the basic catalyst K to be charged is 1.0: 0.8 to 8.0: 1.0 to 8.0.

Further, in the step (4), the amount of the organic solvent J is 10-60 mL/g based on the mass of the compound represented by the formula (VI).

Further, the method for post-treating the reaction solution B in the step (4) of the present invention comprises: after the reaction is completed, evaporating the solvent from the reaction solution, dissolving the concentrate with an organic solvent M to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the organic solvent M in a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component (preferably, ethyl acetate/petroleum ether is 1: 1(v/v) is taken as a developing agent for tracking detection, collecting the target component, preferably, collecting a component with an Rf value of 0.5), concentrating under reduced pressure, and drying (preferably, drying at 50 ℃) to obtain the compound shown in the formula (I); the organic solvent M is one of the following: ethanol, chloroform, tetrahydrofuran or ethyl acetate. The organic solvent M is used in an amount capable of dissolving the residue.

In a third aspect, the invention also relates to an application of the 6- (2- (o-toluidine) acetamido) quinazoline compound shown in the formula (I) in preparing a medicament for preventing or treating tumors, in particular to an application in preparing a medicament for preventing or treating human breast cancer.

Preferably, the medicament is a medicament for inhibiting the activity of the human breast cancer cell line MCF-7. The compound (I) provided by the invention has a good inhibition effect on human breast cancer cell strain MCF-7.

The 6- (2- (o-toluidine) acetamido) quinazoline compound shown in the formula (I) also has a remarkable inhibiting effect on a human lung cancer cell strain A-549, a human promyelocytic leukemia cell strain HL-60 or a human cervical cancer cell strain Siha, and can be applied to preparation of medicines for preventing or treating human lung cancer, human leukemia or human cervical cancer.

The organic solvents A, C, D, G, H, J and M are organic solvents, so that the organic solvents used for distinguishing different steps are named for convenience, and letters have no meanings; the catalyst B, the reducing agent E, the catalyst F and the catalyst K are all catalysts, so that the catalysts used in different steps are named for convenience of distinguishing, and letters have no meanings; the post-treatment A and the post-treatment B are both post-treatments, so that the post-treatments used for distinguishing different steps are named for convenience, and the letters have no meanings.

The invention has the following beneficial effects: (1) the 6- (2- (o-toluidino) acetamido) quinazoline compound (I) has good anticancer activity, and is expected to be applied to the preparation of medicaments for preventing or treating tumor diseases, in particular to the application of medicaments for preventing human breast cancer, human lung cancer, human leukemia or human cervical cancer; (2) the preparation method of the 6- (2- (o-toluidine) acetamido) quinazoline compound (I) provided by the invention is simple and easy to operate, has easily available raw materials and lower production cost, and is suitable for practical use.

(IV) detailed description of the preferred embodiments

The invention is further illustrated by reference to specific examples, which are intended to illustrate the invention, but not to limit it in any way.

The compound (II) can be prepared by the method described in Weinstock, J.et al.J.Med.chem.,1986, 29(11), 2315-2325. Preparation of 4-chloro-6-nitroquinazoline (III) according to the method of Fernandes, C.et al bioorg.Med.chem.,2007,15(12), 3974-3980.

The palladium-carbon (Pd/C) model D5H5A used in the embodiment of the invention is purchased from Shaanxi Rui New Material Co., Ltd.

Example 1: preparation of 6-nitroquinazoline (IV)

1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 2.39 g (6.87mmol) of compound (II), 3.62 g (45.76mmol) of pyridine and 12 ml of chloroform were sequentially added to a 50ml reaction flask, heated to 40 ℃ and subjected to TLC follow-up detection (developing solvent ethyl acetate/petroleum ether: 1: 3(v/v)), and the reaction was stopped after stirring for 10 hours, the solvent was distilled off from the reaction solution, and 10 ml of acetic acid was added to the resulting concentrateDissolving the ethyl ester to obtain a dissolved solution, adding 3.0 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating to remove the solvent to obtain a mixture of a dried concentrate and the silica gel, loading the mixture into a column, and then mixing the mixture according to a volume ratio of 1: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 3(v/v)), collecting an eluent containing the compound shown in the formula (IV) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a light yellow solid product shown in the formula (IV), wherein the yield is 85.1%, and the melting point is 164-166 ℃.¹H NMR(500MHz,CDCl₃):3.32-3.38(m,1H),3.63(dt,J＝3.4,15.5Hz,1H),3.75(s,3H),3.82(s,6H),3.91(dd,J＝8.1,14.3Hz,1H),4.03(td,J＝4.1,11.7Hz,1H),4.15(d,J＝11.5Hz,1H),4.72(dd,J＝8.3,14.2Hz,1H),5.14(t,J＝8.9Hz,1H),6.60(s,1H),6.90(d,J＝8.7Hz,2H),7.08(d,J＝8.6Hz,2H),7.93(d,J＝9.1Hz,1H),8.48(dd,J＝2.4,9.2Hz,1H),8.71(s,1H),8.96(d,J＝2.4Hz,1H)。IR(KBr,cm^-1)ν:2917,2848,1616,1580,1510,1463,1355,1327,1249,1038,847。

Example 2: preparation of 6-nitroquinazoline (IV)

Sequentially adding 1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 1.59 g (4.57mmol) of compound (II), 1.67 g (22.83mmol) of diethylamine and 60ml of toluene into a 100ml three-neck flask, heating to 100 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 3(v/v)), stirring for 2 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of ethanol into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 2.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography by using a volume ratio of 1: eluting with a petroleum ether/ethyl acetate mixed solution of 5 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 3(v/v)), collecting an eluent containing the compound shown in the formula (IV) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a light yellow solid product shown in the formula (IV), wherein the yield is 72.6%, and the melting point is 164-166 ℃.¹H NMR and IR were performed simultaneouslyExample 1.

Example 3: preparation of 6-nitroquinazoline (IV)

Sequentially adding 1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 1.99 g (5.72mmol) of compound (II), 0.58 g (5.73mmol) of triethylamine and 60ml of ethanol into a 100ml three-neck flask, heating to 60 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 3(v/v)), stirring for 8 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of chloroform into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 2.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography by using a volume ratio of 10: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 3(v/v)), collecting an eluent containing the compound shown in the formula (IV) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a light yellow solid product shown in the formula (IV), wherein the yield is 77.2%, and the melting point is 164-166 ℃.¹H NMR and IR were the same as in example 1.

Example 4: preparation of 6-nitroquinazoline (IV)

Adding 1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 2.20 g (6.32mmol) of compound (II), 1.40 g (11.46mmol) of 4-dimethylaminopyridine and 60ml of isopropanol into a 100ml three-neck flask, stirring at room temperature and 25 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether ═ 1: 3(v/v)), reacting for 12 hours, closing the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of tetrahydrofuran into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 4.0 g of column chromatography silica gel (300-400 mesh silica gel) into the dissolved solution, mixing uniformly, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography on the mixture in a volume ratio of 5: eluting with petroleum ether/ethyl acetate mixed solution of 1 as eluent, detecting by TLC (developing solvent ethyl acetate/petroleum ether is 1: 3(v/v)), collecting eluate containing compound shown in formula (IV) (Rf value is 0.5) according to TLC detection, concentrating the collected solution, and drying at 50 deg.C to obtain light yellow solid shown in formula (IV)The yield of the product is 80.2%, and the melting point is 164-166 ℃.¹H NMR and IR were the same as in example 1.

Example 5: preparation of 6-nitroquinazoline (IV)

Adding 1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 1.79 g (5.15mmol) of compound (II), 1.04 g (8.58mmol) of N, N-dimethylaniline and 12 ml of N, N-dimethylformamide into a 50ml reaction bottle, heating to 120 ℃, performing TLC tracking detection (ethyl acetate/petroleum ether is 1: 3(v/v)) and stirring for 0.5 hour, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of tetrahydrofuran into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 5.0 g of silica gel (300-400 mesh silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried concentrate and the silica gel, filling the mixture into a column, and then filling the mixture into the column according to the volume ratio of 1: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 3(v/v)), collecting an eluent containing the compound shown in the formula (IV) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a light yellow solid product shown in the formula (IV), wherein the yield is 89.6%, and the melting point is 164-166 ℃.¹H NMR and IR were the same as in example 1.

Example 6: preparation of 6-nitroquinazoline (IV)

Adding 1.20 g (5.73mmol) of 4-chloro-6-nitroquinazoline (III) and 2.39 g (6.87mmol) of compound (II), 3.62 g (45.76mmol) of pyridine and 20 ml of propanol into a 50ml reaction bottle, heating to 40 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 3(v/v)), stirring for 10 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of ethyl acetate into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 3.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography by using a volume ratio of 1: eluting with petroleum ether/ethyl acetate mixed solution of 1 as eluent, detecting by TLC (developing solvent ethyl acetate/petroleum ether is 1: 3(v/v)), and collecting the compound containing formula (IV) according to TLC detectionAnd (3) eluting the eluent (the Rf value is 0.5), concentrating the collected solution, and drying at 50 ℃ to obtain a light yellow solid product shown in the formula (IV), wherein the yield is 78.3%, and the melting point is 164-166 ℃.¹H NMR and IR were the same as in example 1.

Example 7: preparation of 6-aminoquinazolines (V)

0.40 g (0.77mmol) of 6-nitroquinazoline (IV), 0.40 g (6.34mmol) of ammonium formate, 0.04 g of 5% Pd/C and 4.0 ml of chloroform prepared in the method of example 1 are sequentially added into a reaction bottle, stirred at the room temperature of 25 ℃, subjected to TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), reacted for 12 hours, filtered, concentrated, and dried in vacuum at the temperature of 25 ℃ to obtain a light yellow solid product, namely 6-aminoquinazoline (V), with the yield of 98.2% and the melting point of 122-126 ℃.¹H NMR(500MHz,CDCl₃):3.40-3.48(m,2H),3.71(s,3H),3.82(s,3H),3.83(s,3H),3.87-3.98(m,5H),4.45(dd,J＝6.3,13.8Hz,1H),4.95(dd,J＝6.5,9.2Hz,1H),6.47(s,1H),6.90(d,J＝8.7Hz,2H),6.95(d,J＝2.5Hz,1H),7.11(d,J＝8.6Hz,2H),7.15(dd,J＝8.9,2.5Hz,1H),7.69(d,J＝8.9Hz,1H),8.50(s,1H)。IR(KBr,cm^-1)ν:3368,3215,2932,2825,1628,1566,1512,1487,1353,1248,1036,834。

Example 8: preparation of 6-aminoquinazolines (V)

0.40 g (0.77mmol) of 6-nitroquinazoline (IV), 1.20 g (19.18mmol) of 80 wt% hydrazine hydrate, 0.20 g of 5% Pd/C and 20.0 ml of toluene prepared by the method in example 2 are sequentially added into a 50ml reaction bottle, heated to 100 ℃, subjected to TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirred for 0.5 hour, cooled and filtered, the filtrate is concentrated, and vacuum-dried at 25 ℃ to obtain a light yellow solid product, namely 6-aminoquinazoline (V), with the yield of 100.0% and the melting point of 122-126 ℃.¹H NMR and IR were the same as in example 7.

Example 9: preparation of 6-aminoquinazolines (V)

0.40 g (0.77mmol) of 6-nitroquinazoline (IV), 0.08 g of concentrated hydrochloric acid (mass concentration is 36-38%), 0.40 g of iron powder and 20.0 ml of methanol prepared in the method of example 3 are sequentially added into a 50ml reaction bottle, heated to 40 ℃, subjected to TLC tracking detection (ethyl acetate/petroleum ether is used as a developing agent: 1(v/v)), and stirred for reactionCooling and filtering the mixture for 8 hours, concentrating the filtrate, and drying the filtrate in vacuum at 25 ℃ to obtain a light yellow solid product 6-aminoquinazoline (V), wherein the yield is 94.1 percent, and the melting point is 122-126 ℃.¹H NMR and IR were the same as in example 7.

Example 10: preparation of 6-aminoquinazolines (V)

0.40 g (0.77mmol) of 6-nitroquinazoline (IV), 0.40 g of acetic acid, 1.20 g of iron powder and 20.0 ml of isopropanol prepared in the method of example 4 are sequentially added into a 50ml reaction bottle, heated to 80 ℃, subjected to TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirred for reaction for 3 hours, cooled and filtered, and concentrated and dried in vacuum at 25 ℃ to obtain a light yellow solid product, namely 6-aminoquinazoline (V), wherein the yield is 97.5% and the melting point is 122-126 ℃.¹H NMR and IR were the same as in example 7.

Example 11: preparation of chloroacetamidoquinazoline (VI)

Adding 0.27 g (0.55mmol) of 6-aminoquinazoline (V), 0.13 g (1.64mmol) of pyridine and 3 ml of tetrahydrofuran prepared in the method of example 7 into a reaction bottle, dropwise adding 0.497 g (4.40mmol) of chloroacetyl chloride under the condition of stirring at-10 ℃, after dropwise adding, performing TLC tracking detection (the developing agent is ethyl acetate/petroleum ether is 1: 1), reacting for 12 hours at-10 ℃, filtering, evaporating the filtrate to remove the solvent, adding 10 ml of ethyl acetate into the concentrate to dissolve the concentrate to obtain a dissolved solution, adding 0.60 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating to remove the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography on the mixture in a volume ratio of 1: eluting by using a petroleum ether/ethyl acetate mixed solution of 10 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (VI) according to TLC detection (the Rf value is 0.5), concentrating the collected liquid, and drying at 50 ℃ to obtain the chloracetyl amido quinazoline yellow solid shown in the formula (VI), wherein the yield is 95.6%, and the melting point is 255-258 ℃.¹H NMR(500MHz,CDCl₃):3.26-3.33(m,1H),3.54(dt,J＝3.7,15.4Hz,1H),3.74(s,3H),3.81-3.82(m,7H),3.95-4.05(m,2H),4.28(s,2H),4.64(dd,J＝8.2,14.4Hz,1H),5.24(t,J＝8.8Hz,1H).6.64(s,1H),6.88(d,J＝8.8Hz,2H),7.07(d,J＝8.7Hz,2H),7.53(dd,J＝2.3,9.0Hz,1H),7.83(d,J＝9.0Hz,1H),8.54(s,1H),8.60(s,1H),8.69(d,J＝2.2Hz,1H)。IR(KBr,cm^-1)ν:3396,2998,2937,2835,1694,1557,1525,1510,1489,1463,1349,1249,1179,1036,840。

Example 12: preparation of chloroacetamidoquinazoline (VI)

Adding 0.27 g (0.55mmol) of 6-aminoquinazoline (V), 0.04 g (0.55mmol) of diethylamine and 10.0 ml of chloroform prepared in the method of example 8 into a 50ml reaction bottle, dropwise adding a mixed solution of 0.07 g (0.55mmol) of chloroacetyl chloride and 5.0 ml of chloroform under the condition of stirring at 10 ℃, after dropwise adding, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether ═ 1: 1(v/v)), reacting for 8 hours at 10 ℃, filtering, evaporating the solvent from the filtrate, adding 20 ml of ethanol into the concentrate to dissolve the concentrate to obtain a dissolved solution, adding 0.26 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, mixing uniformly, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then filling the mixture into the column at a volume ratio of 1: and (3) eluting by using a petroleum ether/ethyl acetate mixed solution of 5 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent (the Rf value is 0.5) containing the compound shown in the formula (VI) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a chloroacetylamidoquinazoline yellow solid shown in the formula (VI), wherein the yield is 83.4%, and the melting point is 255-258 ℃.¹H NMR and IR were the same as in example 11.

Example 13: preparation of chloroacetamidoquinazoline (VI)

Adding 0.27 g (0.55mmol) of 6-aminoquinazoline (V), 0.111 g (1.10mmol) of triethylamine and 10.0 ml of ethyl acetate prepared in the method of example 9 into a 50ml reaction bottle, dropwise adding 0.14 g (1.09mmol) of chloroacetyl chloride and 5.0 ml of ethyl acetate solution under the condition of stirring at 0 ℃, after dropwise adding, performing TLC tracking detection (the developing agent is ethyl acetate/petroleum ether is 1: 1), reacting for 6 hours at 25 ℃, filtering, evaporating the filtrate to remove the solvent, adding 20 ml of chloroform into the concentrate to dissolve the concentrate to obtain a dissolved solution, adding 0.30 g of silica gel (300-400 mesh silica gel) into the dissolved solution, mixing uniformly, evaporating to remove the solvent to obtain a mixture of dried concentrate and silica gel, and mixing the mixture to obtain a mixture of the dried concentrate and the silica gel, and performing column chromatography on the mixture to obtain a mixtureLoading the column, and then mixing the column with the volume ratio of 10: eluting by using a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (VI) according to TLC detection (the Rf value is 0.5), concentrating the collected liquid, and drying at 50 ℃ to obtain a chloroacetylamidoquinazoline yellow solid shown in the formula (VI), wherein the yield is 70.5%, and the melting point is 255-258 ℃.¹H NMR and IR were the same as in example 11.

Example 14: preparation of chloroacetamidoquinazoline (VI)

0.27 g (0.55mmol) of 6-aminoquinazoline (v), 0.067 g (0.55mmol) of 4-dimethylaminopyridine and 20.0 ml of toluene prepared in example 10 are sequentially added to a 50ml reaction flask, a solution of 0.376 g (2.20mmol) of chloroacetic anhydride and 7.0 ml of toluene is added dropwise under stirring at 5 ℃, the mixture is heated to 50 ℃, TLC tracking detection is carried out (the developing agent is ethyl acetate/petroleum ether is 1: 1), the mixture reacts for 3 hours, the filtrate is filtered, the solvent is distilled off, the concentrate is dissolved by adding 20 ml of tetrahydrofuran to obtain a dissolved solution, 0.40 g of silica gel (300-400 mesh column chromatography silica gel) is added to the dissolved solution, the mixture is uniformly mixed, the solvent is distilled off to obtain a mixture of dried concentrate and silica gel, the mixture is filled into a column, and then the volume ratio of the mixture is 5: eluting by using a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (VI) according to TLC detection (the Rf value is 0.5), concentrating the collected liquid, and drying at 50 ℃ to obtain a chloroacetylamidoquinazoline yellow solid shown in the formula (VI), wherein the yield is 85.3%, and the melting point is 255-258 ℃.¹H NMR and IR were the same as in example 11.

Example 15: preparation of chloroacetamidoquinazoline (VI)

0.27 g (0.55mmol) of 6-aminoquinazoline (V), 0.213 g (1.65mmol) of quinoline and 15.0 ml of benzene prepared in example 7 are added in sequence to a 50ml reaction flask, a solution of 0.28 g (2.19mmol) of chloroacetyl chloride and 5.0 ml of benzene are added dropwise with stirring at-10 ℃ after the dropwise addition, followed by TLC detection (the developing solvent is ethyl acetate/petroleum ether ═ 1: 1), the reaction is carried out at-10 ℃ for 12 hours, the filtrate is filtered, the solvent is distilled off, and concentratedAnd (2) adding 20 ml of tetrahydrofuran into the condensate to dissolve the condensate to obtain a dissolved solution, adding 0.40 g of column chromatography silica gel (300-400-mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture according to a volume ratio of 1: eluting by using a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (VI) according to TLC detection (the Rf value is 0.5), concentrating the collected liquid, and drying at 50 ℃ to obtain a chloroacetylamidoquinazoline yellow solid shown in the formula (VI), wherein the yield is 82.1%, and the melting point is 255-258 ℃.¹H NMR and IR were the same as in example 11.

Example 16: preparation of chloroacetamidoquinazoline (VI)

Adding 0.27 g (0.55mmol) of 6-aminoquinazoline (V), 0.164 g (1.10mmol) of 4-pyrrolidinylpyridine and 15.0 ml of dichloromethane prepared in the method of example 7 into a 50ml reaction bottle, dropwise adding 00.14 g (1.09mmol) of chloroacetyl chloride and 5.0 ml of dichloromethane solution under the condition of stirring at 10 ℃, after dropwise adding, performing TLC tracking detection (ethyl acetate/petroleum ether is used as a developing agent, 1: 1), reacting for 8 hours at 10 ℃, filtering, evaporating the solvent from the filtrate, adding 20 ml of ethanol into the concentrate to dissolve the concentrate to obtain a dissolved solution, adding 0.50 g of silica gel (300-400 mesh silica gel) into the dissolved solution for column chromatography, mixing uniformly, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then filling the mixture into the column at a volume ratio of 10: eluting by using a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (VI) according to TLC detection (the Rf value is 0.5), concentrating the collected liquid, and drying at 50 ℃ to obtain a chloroacetylamidoquinazoline yellow solid shown in the formula (VI), wherein the yield is 90.2%, and the melting point is 255-258 ℃.¹H NMR and IR were the same as in example 11.

Example 17: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

3.25 g (5.73mmol) of the chloroacetamidoquinazoline (VI) prepared in example 11 were combined in succession with 0.736 g (6.87mmol) of o-toluidine, 3.626 gAdding (45.84mmol) pyridine and 32.5 ml methanol into a 50ml reaction bottle, heating to 40 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirring for 10 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 10 ml ethyl acetate into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 1.5 g column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried concentrate and the silica gel, loading the mixture into a column, and then performing column chromatography by using a volume ratio of 1: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 53.4%, and the melting point is 179-181 ℃.¹H NMR(500MHz,CDCl₃):2.32(s,3H),3.26-3.32(m,1H),3.55(dt,J＝3.5,15.3Hz,1H),3.76(s,3H),3.81-3.82(m,7H),4.00-4.06(m,4H),4.33(t,J＝5.3Hz,1H),4.65(dd,J＝8.2,14.3Hz,1H),5.29(t,J＝8.8Hz,1H),6.62(d,J＝8.2Hz,1H),6.68(s,1H),6.84(t,J＝7.3Hz,1H),6.88(d,J＝8.7Hz,2H),7.09(d,J＝8.7Hz,2H),7.15-7.18(m,2H),7.37(dd,J＝2.2,9.0Hz,1H),7.75(d,J＝8.9Hz,1H),8.57(s,1H),8.79(s,2H)。HRMS-ESI m/z:638.2527[M+H]⁺。¹³C NMR(125MHz,CDCl₃):17.6,28.8,49.7,50.0,51.1,54.4,55.3,56.0,60.5,110.9,113.9,114.2,114.4,116.1,120.0,123.0,125.3,127.6,128.0,128.1,128.7,128.8,130.7,133.6,135.0,137.5,144.0,145.0,149.0,152.2,153.3,158.4,163.7,169.1。IR(KBr,cm^-1)ν:2934,2831,1695,1562,1512,1487,1453,1349,1248,1036,837。

Example 18: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

Sequentially adding 3.25 g (5.73mmol) of chloroacetamidoquinazoline (VI) prepared in example 12, 0.490 g (4.57mmol) of o-toluidine, 2.95 g (22.84mmol) of quinoline and 80 ml of toluene into a 100ml three-neck flask, heating to 100 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirring for 2 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of ethanol into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 2.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, loading the mixture into a column, and then mixing in a volume ratio of 1: eluting with a mixed solution of petroleum ether and ethyl acetate as eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating the collected liquid, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 52.8%, and the melting point is 179-181 ℃. The characterization was the same as example 17.

Example 19: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

Sequentially adding 3.25 g (5.73mmol) of chloroacetamidoquinazoline (VI) prepared in example 13, 0.614 g (5.73mmol) of o-toluidine, 0.58 g (5.73mmol) of triethylamine and 80 ml of ethanol into a 100ml three-neck flask, heating to 60 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirring for 8 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of chloroform into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 2.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, loading the mixture into a column, and then performing column chromatography on the mixture according to a volume ratio of 10: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 47.6%, and the melting point is 179-181 ℃. The characterization was the same as example 17.

Example 20: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

3.25 g (5.73mmol) of chloroacetamidoquinazoline (VI) prepared by the method of example 14, 2.456 g (22.92mmol) of o-toluidine, 1.40 g (11.46mmol) of 4-dimethylaminopyridine and 60ml of isopropanol are sequentially added into a 100ml three-neck flask, stirred at room temperature of 25 ℃, subjected to TLC tracing detection (ethyl acetate/petroleum ether ═ 1: 1(v/v)) and reacted for 36 hours, the reaction is stopped, the solvent is distilled off from the reaction solution, 20 ml of tetrahydrofuran is added into the obtained concentrate to be dissolved to obtain a dissolved solution, 3.0 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) is added into the dissolved solution, after uniform mixing, the solvent is distilled off to obtain a mixture of dried concentrate and silica gel, the mixture is packed into a column, and then the volume ratio of the mixture is 5: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 62.2%, and the melting point is 179-181 ℃. The characterization was the same as example 17.

Example 21: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

Sequentially adding 3.25 g (5.73mmol) of chloroacetamidoquinazoline (VI) prepared in example 15, 0.552 g (5.15mmol) of o-toluidine, 1.04 g (8.58mmol) of N, N-xylidine and 33 ml of N, N-dimethylformamide into a 50ml reaction bottle, heating to 120 ℃, performing TLC tracking detection (ethyl acetate/petroleum ether is 1: 1(v/v)) and stirring for reaction for 0.5 hour, stopping the reaction, evaporating the reaction solution to remove the solvent, adding 20 ml of tetrahydrofuran into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 4.0 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, mixing uniformly, evaporating the solvent to obtain a mixture of a dried concentrate and the silica gel, filling the mixture into a column, and then filling the mixture into the column at a volume ratio of 1: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 58.1%, and the melting point is 179-181 ℃. The characterization was the same as example 17.

Example 22: preparation of 6- (2- (o-toluidino) acetamido) quinazoline (I)

Sequentially adding 3.25 g (5.73mmol) of chloroacetamidoquinazoline (VI) prepared in example 16, 4.912 g (45.84mmol) of o-toluidine, 3.626 g (45.84mmol) of pyridine and 195 ml of propanol into a 500 ml reaction bottle, heating to 40 ℃, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1(v/v)), stirring for 10 hours, stopping the reaction, evaporating the reaction liquid to remove the solvent, adding 20 ml of ethyl acetate into the obtained concentrate to dissolve the concentrate to obtain a dissolved solution, adding 6.0 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography by using a volume ratio of 1: eluting with a petroleum ether/ethyl acetate mixed solution of 1 as an eluent, tracking and detecting by TLC (the developing solvent is ethyl acetate/petroleum ether is 1: 1(v/v)), collecting an eluent containing the compound shown in the formula (I) (the Rf value is 0.5) according to TLC detection, concentrating a collected solution, and drying at 50 ℃ to obtain a yellow solid product shown in the formula (I), wherein the yield is 65.3%, and the melting point is 179-181 ℃. The characterization was the same as example 17.

Example 23: in vitro test for anti-cancer Activity

(1) The prepared compound (I) is subjected to a human breast cancer cell line MCF-7 bioactivity test.

The test method comprises the following steps: tetrazolium salt reduction (MTT process).

Cell lines: human breast cancer cell strain MCF-7. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.

The experimental procedure was as follows:

(a) preparation of samples: for soluble samples, each 1mg was dissolved in 40. mu.L DMSO, 2. mu.L was diluted with 1000. mu.L of medium to a concentration of 100. mu.g/mL, and then serially diluted with the culture medium to the use concentration.

(b) Culture of cells

Preparation of culture medium, each 1000mL of DMEM culture medium (Gibco) contains 80 ten thousand units of penicillin, 1.0g of streptomycin and 10% inactivated fetal bovine serum.

② cultivation of cells, inoculating tumor cells into culture medium, standing at 37 deg.C and 5% CO₂Culturing in an incubator, and carrying out passage for 3-5 days.

Measuring the inhibition of the sample on the growth of tumor cells

The 10 th generation cells were digested with EDTA-pancreatin digest and diluted to 1 × 10 with medium⁶Perml, 100. mu.L/well in 96-well cell culture plates, 37 ℃ 5% CO₂Culturing in an incubator. After 24h of inoculation, 100. mu.L of 100. mu.L/well, 10. mu.g/mL and 1. mu.g/mL samples diluted with medium were added to each well at 3 concentrations, and the mixture was incubated at 37 ℃ with 5% CO₂The culture was performed in an incubator, 5mg/mL MTT was added to the cell culture wells after 72h, 10. mu.L per well, incubated at 37 ℃ for 3h, DMSO was added, 150. mu.L per well, shaken with a shaker, and formazan was completely solubilized and colorimetric with a microplate reader at a wavelength of 570 nm. Using cells cultured in the same DMSO concentration medium without the sample under the same conditions as a control, the IC of the sample on tumor cell growth was calculated₅₀。

The results of the test are shown in table 1:

TABLE 1 inhibitory Effect of Compound (I) on the growth of cancer cell line MCF-7

(2) Quinazoline compounds (a), (b) and (c) were synthesized according to example 11 by substituting chloroacetyl chloride with 4-iodobenzoyl chloride, 3-methoxybenzoyl chloride or cinnamoyl chloride, respectively, and following the following structures:

the prepared quinazoline compounds (a), (b) and (c) are subjected to a biological activity test on a human breast cancer cell line MCF-7 according to the method, and the test results show that the quinazoline compounds (a), (b) and (c) have no obvious inhibition effect on the human breast cancer cell line MCF-7, and the compounds (a), (b) and (c) have far lower anticancer activity on the human breast cancer cell line MCF-7 than the compound (I). The specific results are shown in table 2:

TABLE 2 inhibitory Effect of Compounds (a), (b) and (c) on the growth of cancer cell line MCF-7

The anti-cancer activity in vitro test experiment shows that: the other 3 compounds (a), (b) and (c) with similar structures have no obvious inhibition effect on the growth of the human breast cancer cell strain MCF-7. The compound (I) has obvious inhibition effect on the growth of human breast cancer cell strains MCF-7, and is obviously superior to the compounds (a), (b) and (c).

(3) Quinazoline compounds (d), (e) and (f) were synthesized in the same manner as in example 17 except that o-toluidine was replaced with 3, 4-dimethylaniline, 3, 4-dimethoxyaniline or di-n-propylamine, respectively, according to example 17, and the structures thereof were as follows:

the prepared quinazoline compounds (d), (e) and (f) are subjected to a biological activity test of a human breast cancer cell line MCF-7 according to the method, and the results show that the quinazoline compounds (d), (e) and (f) have far lower anticancer activity than the compound (I) on the human breast cancer cell line MCF-7. Specific results are shown in table 3:

TABLE 3 inhibitory Effect of Compounds (d), (e) and (f) on the growth of cancer cell line MCF-7

(4) Referring to the literature (Rao, G. -W.et al. ChemMedChem,2013,8(6),928-933), 4-chloroquinazoline was prepared, 4-chloro-6-nitroquinazoline was substituted with 4-chloroquinazoline according to example 1, and the other operations were the same as in example 1 to synthesize a quinazoline compound (g) having the following structure:

the prepared quinazoline compound (g) is subjected to a biological activity test of a human breast cancer cell line MCF-7 according to the method, and the test result shows that the quinazoline compound (g) has far lower anticancer activity on the human breast cancer cell line MCF-7 than the compound (I). Specific results are shown in table 4:

TABLE 4 inhibitory Effect of Compound (g) on the growth of cancer cell line MCF-7

Example 24: in vitro test for anti-cancer Activity

(1) The prepared compounds (I), (IV) and (VI) are tested for the biological activity of a human lung cancer cell strain A-549.

Cell lines: human lung cancer cell strain A-549. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.

The experimental procedure was as follows:

(b) Culture of cells

Measuring the inhibition of the sample on the growth of tumor cells

The 2 nd generation cells were digested with EDTA-pancreatin digest and diluted to 1 × 10 with medium⁶Perml, 100. mu.L/well in 96-well cell culture plates, 37 ℃ 5% CO₂Culturing in an incubator. After 24h of inoculation, 100. mu.L of 100. mu.L/well, 10. mu.g/mL and 1. mu.g/mL samples diluted with medium were added to each well at 3 concentrations, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in incubator, adding 5mg/mL MTT into cell culture well after 72 hr, placing 10 μ L MTT into each well, and standing at 37 deg.CIncubate for 3h, add DMSO, 150 μ L per well, shake with a shaker, fully solubilize the formazan, and color compare with a microplate reader at a wavelength of 570 nm. Using cells cultured in the same DMSO concentration medium without sample under the same conditions as a control, the IC of the sample on tumor cell growth was calculated₅₀。

The results of the test are shown in table 5:

TABLE 5 inhibitory Effect of Compounds (I), (IV) and (VI) on the growth of cancer cell line A-549

the prepared quinazoline compounds (a), (b) and (c) are subjected to a biological activity test of a human lung cancer cell strain A-549 according to the method, and test results show that the quinazoline compounds (a), (b) and (c) have no obvious inhibition effect on the human lung cancer cell strain A-549, and the compounds (a), (b) and (c) have far lower anti-cancer activity than the compound (I) on the human lung cancer cell strain A-549. Specific results are shown in table 6:

TABLE 6 inhibitory Effect of Compounds (a), (b) and (c) on the growth of cancer cell line A-549

The anti-cancer activity in vitro test experiment shows that: the other 3 compounds (a), (b) and (c) with similar structures have no obvious inhibition effect on the growth of the human lung cancer cell strain A-549. The compound (I) has obvious inhibition effect on the growth of a human lung cancer cell strain A-549, and is obviously superior to the compounds (a), (b) and (c).

the prepared quinazoline compounds (d), (e) and (f) are subjected to a biological activity test of a human lung cancer cell strain A-549 according to the method, and the results show that the anticancer activity of the quinazoline compounds (d), (e) and (f) on the human lung cancer cell strain A-549 is far lower than that of the compound (I). Specific results are shown in table 7:

TABLE 7 inhibitory Effect of Compounds (d), (e) and (f) on the growth of cancer cell line A-549

the prepared quinazoline compound (g) is subjected to a biological activity test of a human lung cancer cell strain A-549 according to the method, and a test result shows that the anticancer activity of the quinazoline compound (g) on the human lung cancer cell strain A-549 is far lower than that of the compound (I). Specific results are shown in table 8:

TABLE 8 inhibitory Effect of Compound (g) on the growth of cancer cell line A-549

Example 25: in vitro test for anti-cancer Activity

(1) The prepared compound (I) is subjected to biological activity test of a human promyelocytic leukemia cell strain HL-60.

Cell lines: human promyelocytic leukemia cell line HL-60. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.

The experimental procedure was as follows:

(b) Culture of cells

Measuring the inhibition of the sample on the growth of tumor cells

The 2 nd generation cells were digested with EDTA-pancreatin digest and diluted to 1 × 10 with medium⁶Perml, 100. mu.L/well in 96-well cell culture plates, 37 ℃ 5% CO₂Culturing in an incubator. After 24h of inoculation, 100. mu.L of 100. mu.L/well, 10. mu.g/mL and 1. mu.g/mL samples diluted with medium were added to each well at 3 concentrations, and the mixture was incubated at 37 ℃ with 5% CO₂The culture was performed in an incubator, 5mg/mL MTT was added to the cell culture wells after 72h, 10. mu.L per well, incubated at 37 ℃ for 3h, DMSO was added, 150. mu.L per well, shaken with a shaker, and formazan was completely solubilized and colorimetric with a microplate reader at a wavelength of 570 nm. Using cells cultured in the same DMSO concentration medium without sample under the same conditions as a control, the IC of the sample on tumor cell growth was calculated₅₀。

The results of the testing are shown in table 9:

TABLE 9 inhibitory Effect of Compound (I) on the growth of cancer cell line HL-60

(2) Quinazoline compounds (b) and (c) were synthesized according to example 11 by substituting chloroacetyl chloride with 3-methoxybenzoyl chloride or cinnamoyl chloride, respectively, and following structures as shown in the following, in the same manner as in example 11:

the prepared quinazoline compounds (b) and (c) are subjected to a biological activity test of a human promyelocytic leukemia cell line HL-60 according to the method, and test results show that the quinazoline compounds (b) and (c) have no obvious inhibition effect on the human promyelocytic leukemia cell line HL-60, and the anticancer activities of the compounds (b) and (c) on the human promyelocytic leukemia cell line HL-60 are far lower than that of the compound (I). Specific results are shown in table 10:

TABLE 10 inhibitory Effect of Compounds (b) and (c) on the growth of cancer cell line HL-60

The anti-cancer activity in vitro test experiment shows that: the other 2 compounds (b) and (c) with similar structures have no obvious inhibition effect on the growth of the human promyelocytic leukemia cell line HL-60. The compound (I) has obvious inhibition effect on the growth of human promyelocytic leukemia cell strain HL-60, and is obviously superior to the compounds (b) and (c).

the biological activity of the quinazoline compounds (d), (e) and (f) prepared by the method in a human promyelocytic leukemia cell line HL-60 test proves that the anticancer activity of the quinazoline compounds (d), (e) and (f) in the human promyelocytic leukemia cell line HL-60 is far lower than that of the compound (I). Specific results are shown in table 11:

TABLE 11 inhibitory Effect of Compounds (d), (e) and (f) on the growth of cancer cell line HL-60

Example 26: in vitro test for anti-cancer Activity

(1) The prepared compound (I) is subjected to a biological activity test of a human cervical cancer cell strain Siha.

Cell lines: human cervical cancer cell line Siha. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.

The experimental procedure was as follows:

(b) Culture of cells

Measuring the inhibition of the sample on the growth of tumor cells

The 2 nd generation cells were digested with EDTA-pancreatin digest and diluted to 1 × 10 with medium⁶Perml, 100. mu.L/well in 96-well cell culture plates, 37 ℃ 5% CO₂Culturing in an incubator. After 24h of inoculation, 100. mu.g of each diluted medium was addedsamples/mL, 10. mu.g/mL and 1. mu.g/mL, 100. mu.L per well, 3 wells per concentration, 37 ℃ 5% CO₂The culture was performed in an incubator, 5mg/mL MTT was added to the cell culture wells after 72h, 10. mu.L per well, incubated at 37 ℃ for 3h, DMSO was added, 150. mu.L per well, shaken with a shaker, and formazan was completely solubilized and colorimetric with a microplate reader at a wavelength of 570 nm. Using cells cultured in the same DMSO concentration medium without sample under the same conditions as a control, the IC of the sample on tumor cell growth was calculated₅₀. The results of the testing are shown in table 12:

TABLE 12 inhibition of growth of cancer cell lines Siha by Compound (I)

the prepared quinazoline compounds (b) and (c) are subjected to a human cervical cancer cell line Siha bioactivity test according to the method, and test results show that the quinazoline compounds (b) and (c) have no obvious inhibition effect on the human cervical cancer cell line Siha, and the anticancer activities of the compounds (b) and (c) on the human cervical cancer cell line Siha are far lower than that of the compound (I). Specific results are shown in table 13:

TABLE 13 inhibitory Effect of Compounds (b) and (c) on growth of cancer cell lines Siha

The anti-cancer activity in vitro test experiment shows that: the other 2 compounds (b) and (c) with similar structures have no obvious inhibition effect on the growth of the human cervical cancer cell strain Siha. The compound (I) has obvious inhibition effect on the growth of human cervical cancer cell strains Siha, and is obviously superior to the compounds (b) and (c).

the prepared quinazoline compounds (d), (e) and (f) are subjected to a biological activity test on human cervical cancer cell lines Siha according to the method, and the results show that the anticancer activity of the quinazoline compounds (d), (e) and (f) on the human cervical cancer cell lines Siha is far lower than that of the compound (I). Specific results are shown in table 14:

TABLE 14 inhibitory Effect of Compounds (d), (e) and (f) on growth of cancer cell line Siha

Claims

1. A6- (2- (o-toluylamino) acetamido) quinazoline compound of the formula (I):

2. a process for the preparation of a 6- (2- (o-toluylamino) acetamido) quinazoline compound of the formula (i) as defined in claim 1, which comprises:

(1) mixing a compound shown as a formula (II) and a compound shown as a formula (III), reacting in an organic solvent A at 25-120 ℃ under the action of a basic catalyst B, and after the reaction is completed, separating and purifying a reaction liquid to obtain a compound shown as a formula (IV); the organic solvent A is selected from one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the basic catalyst B is selected from one of the following: pyridine, diethylamine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate;

(2) completely reacting a compound shown in a formula (IV) in an organic solvent D under the action of a reducing agent E at 25-100 ℃, filtering a reaction solution, and drying a concentrate obtained by concentrating a filtrate under reduced pressure to obtain a compound shown in a formula (V); the organic solvent D is one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the reducing agent E is one of the following: iron powder/concentrated hydrochloric acid, iron powder/acetic acid, palladium on carbon/ammonium formate or palladium on carbon/hydrazine hydrate; the iron powder/concentrated hydrochloric acid refers to the mixing of iron powder and concentrated hydrochloric acid in any proportion, the iron powder/acetic acid refers to the mixing of iron powder and acetic acid in any proportion, the palladium carbon/ammonium formate refers to the mixing of palladium carbon and ammonium formate in any proportion, and the palladium carbon/hydrazine hydrate refers to the mixing of palladium carbon and hydrazine hydrate in any proportion;

(3) mixing a compound shown in a formula (V) with chloroacetyl chloride or chloroacetic anhydride, completely reacting in an organic solvent G at-10-50 ℃ under the action of an alkaline catalyst F, and carrying out post-treatment on a reaction solution A to obtain a compound shown in a formula (VI); the alkaline catalyst F is one of the following: pyridine, diethylamine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate; the organic solvent G is one of the following: tetrahydrofuran, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile, toluene or benzene;

(4) mixing a compound shown as a formula (VI) with o-toluidine, reacting in an organic solvent J at 25-120 ℃ under the action of a basic catalyst K, and after the reaction is completed, carrying out post-treatment B on a reaction liquid to obtain a compound shown as a formula (I); the organic solvent J is selected from one of the following: chloroform, toluene, methanol, ethanol, propanol, isopropanol, acetonitrile or N, N-dimethylformamide; the basic catalyst K is selected from one of the following: pyridine, triethylamine, quinoline, N-dimethylaniline, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or sodium carbonate.

3. The method of claim 2, wherein: the ratio of the compound represented by the formula (III) to the compound represented by the formula (II) and the amount of the charged substance of the basic catalyst B in the step (1) is 1.0: 0.8 to 1.2: 1.0 to 8.0; the dosage of the organic solvent A is 10-50 mL/g based on the mass of the compound shown in the formula (III).

4. The method of claim 2, wherein: the method for separating and purifying the reaction liquid in the step (1) comprises the following steps: after the reaction is completed, evaporating the solvent from the reaction solution, dissolving the concentrate with an organic solvent C to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the organic solvent C in a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component, concentrating under reduced pressure, and drying to obtain a compound shown as a formula (IV); the organic solvent C is one of the following solvents: ethanol, chloroform, tetrahydrofuran or ethyl acetate.

5. The method of claim 2, wherein: in the step (2), when the reducing agent E is iron powder/concentrated hydrochloric acid or iron powder/acetic acid, the feeding mass ratio of the compound shown in the formula (IV) to the iron powder, concentrated hydrochloric acid or acetic acid in the reducing agent E is 1.0: 1.0-3.0: 0.2-1.0; when the reducing agent E is palladium carbon/ammonium formate or palladium carbon/hydrazine hydrate, the feeding mass ratio of the compound shown in the formula (IV) to the palladium carbon, ammonium formate or hydrazine hydrate in the reducing agent E is 1.0: 0.1-0.5: 1.0-3.0.

6. The method of claim 2, wherein: the ratio of the compound of the formula (V) to chloroacetyl chloride or chloroacetic anhydride and the basic catalyst F in the feeding substances in the step (3) is 1: 1.0-8.0: 1.0-3.0; the dosage of the organic solvent G is 11-100 mL/G based on the mass of the compound shown in the formula (V).

7. The method of claim 2, wherein: the method for post-treating the reaction solution A in the step (3) comprises the following steps: filtering the reaction solution, evaporating the solvent from the filtrate, dissolving the concentrate with an organic solvent H to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the silica gel in a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component, concentrating under reduced pressure, and drying to obtain a compound shown in a formula (I); the organic solvent H is one of the following: ethanol, chloroform, tetrahydrofuran or ethyl acetate.

8. The method of claim 2, wherein: in the step (4), the ratio of the compound represented by the formula (VI) to the amounts of the o-toluidine and the basic catalyst K fed is 1.0: 0.8 to 8.0: 1.0 to 8.0; the dosage of the organic solvent J is 10-60 mL/g based on the mass of the compound shown in the formula (VI);

the method for post-treating the reaction solution B comprises the following steps: after the reaction is completed, evaporating the solvent from the reaction solution, dissolving the concentrate with an organic solvent M to obtain a dissolved solution, adding column chromatography silica gel of which the weight is 1.0-2.0 times that of the concentrate into the dissolved solution, uniformly mixing, evaporating the solvent, drying to obtain a mixture of the concentrate and the silica gel, filling the mixture into a column, and then mixing the mixture with the organic solvent M in a volume ratio of 1: taking a mixed solution of petroleum ether and ethyl acetate of 0.1-10 as an eluent, collecting an effluent containing a target component, concentrating under reduced pressure, and drying to obtain a compound shown in a formula (I); the organic solvent M is one of the following: ethanol, chloroform, tetrahydrofuran or ethyl acetate.

9. The use of a 6- (2- (o-toluylamino) acetamido) quinazoline compound of the formula (i) as defined in claim 1 in the manufacture of a medicament for the prophylaxis or treatment of human breast cancer.

10. The use according to claim 9, wherein the medicament is a medicament having the activity of inhibiting the activity of human breast cancer cell line MCF-7.