Quinazoline derivatives substituted by aniline, preparation method and use thereof

Abstract

The invention relates to quinazoline derivatives substituted by aniline which are represented by the below formula (I), pharmaceutical acceptable salts and stereoisomer thereof, wherein these groups of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, L and n have the meanings given in the specification. The invention also relates to preparation methods, pharmaceutical compositions, pharmaceutical preparation and the use for preparation of medicine of treating excessive hyperplasia and chronic obstructive pulmonary disease and uses for treating excessive hyperplasia and chronic obstructive pulmonary disease thereof. ##STR00001##

Claims

1. A compound represented by a general formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof: ##STR00251## wherein R.sup.1 is selected from the group consisting of ##STR00252## ##STR00253## ##STR00254## R.sup.2 is selected from the group consisting of hydrogen and a C.sub.1-4alkyl group that is unsubstituted or substituted by 1-2 Q.sub.2 substituents, Q.sub.2 is selected from the group consisting of a di(C.sub.1-4alkyl)amino group and a saturated 5-8 membered heterocyclyl group, R.sup.3 is selected from the group consisting of fluoro, chloro, and bromo; R.sup.4, R.sup.5 and R.sup.6 are each hydrogen; L is O; n is 1, 2 or 3.

2. A compound according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein R.sup.1 is selected from the group consisting of: ##STR00255## ##STR00256## R.sup.2 is selected from the group consisting of hydrogen, methyl that is unsubstituted or substituted by 1-2 Q.sub.2 substituents, and ethyl that is unsubstituted or substituted by 1-2 Q.sub.2 substituents, Q.sub.2 is selected from the group consisting of: (1) a di(C.sub.1-4alkyl)amino group, (2) piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl; R.sup.3 is selected from the group consisting of fluoro and chloro; R.sup.4, R.sup.5 and R.sup.6 are hydrogen; L is O; and n is 2.

3. A compound according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein R.sup.1 is selected from the group consisting of: ##STR00257## ##STR00258## R.sup.2 is hydrogen; R.sup.3 is selected from the group consisting of fluoro and chloro; R.sup.4, R.sup.5 and R.sup.6 are hydrogen; L is O; and n is 2.

4. A compound, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the compound is selected from the group consisting of: (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide, (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((8-methyl-1-oxa-8-azaspiro[4,5]decan-3-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4,5]decan-2-ylmethoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-6-yl]-acrylamide, (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-butenamide, (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide, N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-acrylamide, N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide, (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino-2-butenamide, (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((2-(3-methyl-3-aza-bicyclo[3.1.0]-hexan-6-yl)-ethoxy)quinazolin-6-yl)]-4-dimethylamino-crotonamide, (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(((spiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide, and (E)-N-(7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide.

5. A process for preparing a compound of general formula (I) according to claim 1, comprising the steps of: Reaction Procedure: ##STR00259## wherein, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, L and n are as defined in claim 1; the starting material 2=R.sup.1-LH; the starting material 3=R.sup.2CH═CH—C(O)Cl or R.sup.2CH═CH—COOH, (1) Dissolving the starting material 2 in a non-protonic polar solvent, and reacting with the starting material 1 in the presence of a base to produce the Intermediate 1; (2) Reacting the Intermediate 1 with a reducing agent optionally in the presence of an acid to produce the Intermediate 2; and (3) Dissolving the Intermediate 2 in an organic solvent, and reacting with the starting material 3 in the presence of an organic base to produce the compound of formula (I).

6. A pharmaceutical composition, which contains a compound according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

7. A pharmaceutical composition according to claim 6, which further contains a second therapeutical agent selected from the group consisting of an antimetabolite, a growth factor inhibitor, an antibody, a mitotic inhibitor, an antineoplastic hormone, an alkylating agent, carboplatin, cisplatin, and oxaliplatin; a topoismerase inhibitor, and an immunosuppressant.

8. A pharmaceutical formulation containing a compound according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof and one or more pharmaceutically acceptable carriers, which formulation is in a form of any pharmaceutically acceptable dosage form.

9. The pharmaceutical composition according to claim 7, wherein the second therapeutical agent is selected from the group consisting of capecitabine, gemcitabine, pazopanib, imatinib, Herceptin, bevacizumab, paclitaxel, vinorelbine, docetaxel, doxorubicin, letrozole, tamoxifen, fulvestrant, cyclophosphamide, carmustine topotecan, and everolimus.

Description

BEST MODE OF CARRYING OUT THE INVENTION

(1) The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. All of the technical solutions that can be accomplished based on the above disclosure fall in the scope of the present invention.

(2) In the examples, the used starting materials are commercially available, for example, from Jingyan Chemicals (Shanghai); Titan chemical (Shanghai); Darui (Shanghai); Ouhechem (Beijing); Tetranov Biopharm (Zhengzhou); Guanghan Bio-Tech (Sichuan); Accela ChemBio (Shanghai); Alfa Aesar (Tianjin); TCI (Shanghai), J&K (Beijing); and Bepharm (Shanghai).

(3) For convenience, the following well-known abbreviations are used hereinafter to describe the compounds.

(4) DMF: dimethylformamide

(5) THF: tetrahydrofuran

(6) DIPEA/DIEA: diisopropylethylamine

(7) EA: ethyl acetate

(8) EtOH: ethanol

(9) DCM: dichloromethane

(10) MeOH: methanol

(11) HATU: 2-(7-azobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

(12) DCC: N,N-dicyclohexylcarbodiimide

(13) EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

(14) DMAP: 4-dimethylaminopyridine

I. PREPARATION EXAMPLES FOR THE PRESENT COMPOUND

(15) N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6nitroquinazolin-4-amine, as the starting material for the present compound, was prepared according to US 2005/0250761 A1:

(16) ##STR00040##

(17) The steps were as follows:

(18) Reaction Procedures:

(19) ##STR00041##

(20) 2-amino-4-fluorobenzoic acid, acetic acid and formamidine were reacted under heating to reflux in the presence of 2-methoxyethanol to produce 7-fluoro-3H-quinazolin-4-one. The resulting product was nitrified to produce 7-fluoro-6-nitro-3H-quinazolin-4-one, which was treated with thionyl chloride to produce 4-chloro-6-nitro-7-fluoro-3H-quinazoline. The resulting product was dissolved in isopropanol, 4-fluoro-3-chlorophenylamine was added to produce N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6nitroquinazolin-4-amine.

Example 1 Preparation of (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide (Compound 1)

(21) ##STR00042##

(1) Preparation of 7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine

(22) ##STR00043##

(23) To a round-bottom flask containing NaH (468 mg, 12 mmol) was added DMF (20 mL) under an ice bath, and then was added dropwise a solution of 8-oxabicyclo[3.2.1]octan-3-ol (1.0 g, 7.8 mmol) in DMF (2 mL). The mixture was stirred for 30 min. Then N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.75 g, 5.2 mmol) was added in batch thereto. The mixture was warmed up spontaneously to room temperature and reacted overnight. Water (60 mL-80 mL) was added. The precipitate was formed and filtered by suction to produce a solid, which was dried in vacuum to produce 7-(8-oxabicyclo[3,2,1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine (2.3 g) in a yield of 100%.

(2) Preparation of 7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine

(24) ##STR00044##

(25) 7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine (2.3 g, 5.2 mmol) was dissolved in a mixed solution (120 mL) of glacial acetic acid and ethanol (glacial acetic acid/ethanol=1/3). Then Fe powder (2.04 g, 36.4 mmol) was added. The mixture was warmed up spontaneously to room temperature and reacted overnight. The reaction was filtered by suction, and ethanol was removed in vacuum. An appropriate amount of water was added. The mixture was neutralized with a saturated sodium bicarbonate solution until the mixture became neutral. The mixture was extracted with ethyl acetate. The organic layer was concentrated to produce a crude product, which was purified by a silica gel column chromatography (eluted with DCM/methanol=10/1) to produce 7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine 500 mg) in a yield of 23%.

(3) Preparation of (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-bromo-2-butenamide

(26) ##STR00045##

(27) 7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine (500 mg, 1.2 mmol) was dissolved in dichloromethane (20 mL). To the mixture were successively added triethylamine (976 mg) and 4-bromo-2-butenoyl chloride (275 mg, 1.5 mmol). The mixture was stirred at room temperature for 12 h. An appropriate amount of water was added to the reaction. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate and concentrated to produce a crude product, which was directly used in the next step without purification.

(4) Preparation of (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide

(28) ##STR00046##

(29) The product from the previous step, (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-bromo-2-butenamide was dissolved in acetonitrile (20 mL). Piperidine (205 mg, 2.4 mmol) and cesium carbonate (787 mg, 2.4 mmol) were added. The reaction was conducted at 40° C. for 12 h under stirring. An appropriate amount of water was added to the reaction. The reaction was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate, concentrated, and purified by a silica gel column chromatography (eluted with dichloromethane/methanol=5/1) to produce (E)-N-[7-(8-oxabicyclo[3.2.1]octan-3-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide 20 mg) in a yield of 3%.

(30) Molecular formula: C.sub.30H.sub.33ClFN.sub.5O.sub.3

(31) Mass spectrum (m/e): 566.3 (M+1) 283.6 (M/2)

(32) .sup.1HNMR: (400 MHz, CDCl.sub.3) δ9.13 (s, 1H), 8.67 (s, 1H), 8.10 (s, 1H), 8.00 (d, 1H), 7.54 (m, 2H), 7.18 (t, 1H), 7.07 (m, 1H), 6.22 (d, 1H), 4.98 (m, 1H), 4.60 (m, 2H), 3.25 (m, 2H), 2.51 (m, 4H), 2.23 (m, 4H), 1.88-2.03 (m, 10H).

Example 2 Preparation of (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide (Compound 2)

(33) ##STR00047##

(1) Preparation of 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine

(34) ##STR00048##

(35) To a round-bottom flask containing NaH (531 mg, 22 mmol) was added DMF (20 mL) under an ice bath, and then was added dropwise a solution of 7-oxabicyclo[2.2.1]heptan-2-ol (1.0 g, 8.8 mmol) in DMF (2 mL). The mixture was stirred for 30 min. Then N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.98 g, 5.9 mmol) was added in batch. The mixture was warmed up spontaneously to room temperature and reacted overnight. Water (60 mL-80 mL) was added. The precipitate was formed and filtered by suction to produce a filtered cake, which was dried in vacuum to produce 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine (2.5 g) in a yield of 100%.

(2) Preparation of 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine

(36) ##STR00049##

(37) 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine (2.5 g, 5.8 mmol) was dissolved in a mixed solution (120 mL) of glacial acetic acid and ethanol (glacial acetic acid/ethanol=1/3). Then Fe powder (2.28 g, 40.7 mmol) was added. The mixture was warmed up spontaneously to room temperature and reacted overnight. The reaction was filtered by suction, and ethanol was removed in vacuum. An appropriate amount of water was added. The mixture was neutralized with a saturated sodium bicarbonate solution until the mixture became neutral. The mixture was extracted with ethyl acetate. The organic layer was concentrated to produce a crude product, which was purified by a silica gel column chromatography (eluted with DCM/methanol=10/1) to produce 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine 600 mg) in a yield of 25%.

(3) Preparation of (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-bromo-2-butenamide

(38) ##STR00050##

(39) 7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine (600 mg, 1.5 mmol) was dissolved in dichloromethane (20 mL). To the mixture were successively added triethylamine (1.21 g) and 4-bromo-2-butenoyl chloride (366 mg, 2.0 mmol). The mixture was stirred at room temperature for 12 h. An appropriate amount of water was added to the reaction. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate and concentrated to produce a crude product, which was directly used in the next step without purification.

(4) Preparation of (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide

(40) ##STR00051##

(41) The product from the previous step, (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-bromo-2-butenamide was dissolved in acetonitrile (20 mL). Piperidine (255 mg, 3.0 mmol) and cesium carbonate (978 mg, 3.0 mmol) were added. The reaction was conducted at 40° C. for 12 h under stirring. An appropriate amount of water was added to the reaction. The reaction was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate, concentrated, and purified by a silica gel column chromatography (eluted with dichloromethane/methanol=5/1) to produce (E)-N-[7-(7-oxabicyclo[2.2.1]heptan-2-yloxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide (25 mg) in a yield of 3%.

(42) Molecular formula: C.sub.29H.sub.31ClFN.sub.5O.sub.3

(43) Mass spectrum (m/e): 552.2 (M+1)

(44) .sup.1HNMR: (400 MHz, CDCl.sub.3) δ9.13 (s, 1H), 8.65 (s, 1H), 8.20 (s, 1H), 7.97 (d, 1H), 7.72 (s, 1H), 7.54 (m, 1H), 7.18 (m, 2H), 6.23 (d, 1H), 4.80 (m, 2H), 4.64 (m, 1H), 3.23 (m, 2H), 2.49 (m, 4H), 2.20 (m, 2H), 1.88-2.03 (m, 10H).

Example 3 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide (Compound 3)

(45) ##STR00052##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)-6-nitroquinazolin-4-amine

(46) ##STR00053##

(47) 2-methyl-2,7-diazaspiro[4.5]decane (500 mg, 3.2 mmol), potassium carbonate (1.0 g, 7.2 mmol) and N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (560 mg, 1.7 mmol) were dissolved in acetonitrile (20 mL). The mixture was added to 82° C. and reacted for 4 h. The reaction was cooled to room temperature. Water (30 mL) was added. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated. The concentrate was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=40/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)-6-nitroquinazolin-4-amine (600 mg) in a yield of 75%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-4,6-diamine

(48) ##STR00054##

(49) N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)-6-nitroquinazolin-4-amine (600 mg, 1.3 mmol) was dissolved in ethanol (9 mL) and acetic acid (3 mL). The mixture was added to 80° C. and reacted for 2 h. After the completion of reaction, the solvent was evaporated off, and the residual material was extracted with ethyl acetate. The extracted liquid was dried over anhydrous sodium sulphate and concentrated to dryness to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4,5]decan-7-yl)quinazolin-4,6-diamine (500 mg) in a yield of 87%.

(3) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-bromo-2-butenamide

(50) ##STR00055##

(51) N-(4-(3-chloro-4-fluorophenyl))-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-4,6-diamine (500 mg, 1.1 mmol) and (E)-4-bromo-2-butenoyl chloride (1.1 g, 6 mmol) was dissolved in THF (20 mL). To the mixture was successively added DIPEA (2 mL). The mixture was stirred at room temperature for 1 h. To the solution was added water (30 mL). The solution was extracted with ethyl acetate. The organic layers were combined and dried over anhydrous sodium sulphate, and evaporated to dryness. The resulting solid was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=60/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-bromo-2-butenamide (230 mg) in a yield of 36%.

(4) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide

(52) ##STR00056##

(53) (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-bromo-2-butenamide (240 mg, 0.4 mmol), piperidine (70 mg, 0.8 mmol) and potassium carbonate (110 mg, 0.8 mmol) were dissolved in acetonitrile (20 mL). The mixture was reacted at 50° C. for 8 h. After the completion of reaction, to the reaction mixture was added an appropriate amount of water. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated. The concentrate was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methyl-2,7-diazaspiro[4.5]decan-7-yl)quinazolin-6-yl]-4-(piperidin-1-yl)-2-butenamide (18 mg) in a yield of 8%.

(54) Molecular formula: C.sub.32H.sub.39ClFN.sub.7O

(55) Mass spectrum (m/e): 592.3 (M+1), 296.6 (M/2)

(56) .sup.1HNMR: (400 MHz, CDCl.sub.3) δ9.00 (s, 1H), 8.74 (s, 1H), 8.65 (s, 1H), 8.06 (s, 1H), 7.99 (d, 1H), 7.58 (s, 1H), 7.58 (s, 1H), 7.08 (m, 2H), 6.16 (d, 1H), 3.21 (d, 2H), 2.90-3.20 (m, 7H), 2.46-2.78 (m, 5H), 2.38 (s, 3H), 1.28-1.79 (m, 12H).

Example 4 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-6-yl]-acrylamide (Compound 4)

(57) ##STR00057##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)-6-nitroquinazolin-4-amine

(58) ##STR00058##

(59) 8-methyl-8-azabicyclo[3.2.1]octan-3-ol (0.7 g, 5 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (0.4 g, 10 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.12 g, 3.3 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)-6-nitroquinazolin-4-amine (560 mg) in a yield of 37%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-4,6-diamine

(60) ##STR00059##

(61) N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)-6-nitroquinazolin-4-amine (560 mg, 1.22 mmol) was dissolved in a mixed solvent (20 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). Then Fe powder (343 mg, 6.12 mmol) was added. The mixture was warmed up to 70° C. and stirred for 1 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was extracted with EA, and adjusted with 1 mol/L NaOH solution until the mixture became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-4,6-diamine (360 mg) in a yield of 69%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-6-yl]-acrylamide

(62) ##STR00060##

(63) Acrylic acid (243 mg, 3.37 mmol) was dissolved in DMF (10 mL). To the resulting mixture was added DMAP (162 mg, 1.35 mmol), N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-4,6-diamine (360 mg, 0.84 mmol) and EDC (193 mg, 1.01 mmol) under an ice bath. The mixture was stirred at room temperature overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The residue was washed with diethyl ether to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-8-azabicyclo[3.2.1]octan-3-yloxy)quinazolin-6-yl]-acrylamide (54 mg) in a yield of 13%.

(64) Molecular formula: C.sub.25H.sub.25ClFN.sub.5O.sub.2

(65) Mass spectrum (m/e): 482.2 (M+1), 241.6 (M/2)

(66) .sup.1HNMR: (400 MHz, DMSO-d.sub.6) δ9.78 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.52 (s, 1H), 8.14 (d, 1H), 7.80 (d, 1H), 7.41 (t, 1H), 7.11 (s, 1H), 6.58 (m, 1H), 6.30 (d, 1H), 5.80 (d, 1H), 4.83 (m, 1H), 3.03 (m, 2H), 2.12 (s, 3H), 2.10 (m, 2H), 1.88 (m, 6H).

Example 5 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-6-yl]-acrylamide (Compound 5)

(67) ##STR00061##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)-6-nitroquinazolin-4-amine

(68) ##STR00062##

(69) 8-methyl-1-oxa-8-azaspiro[4.5]decan-3-ol (0.4 g, 2.5 mmol) was dissolved in DMF (60 mL). 60% sodium hydride (0.4 g, 10 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.12 g, 3.3 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)-6-nitroquinazolin-4-amine (1.0 g) in a yield of 82%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-4,6-diamine

(70) ##STR00063##

(71) N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)-6-nitroquinazolin-4-amine (1.0 g, 2.05 mmol) was dissolved in a mixed solvent (80 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (1.5 g, 26 mmol). The mixture was warm up to 70° C. and stirred for 1 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-4,6-diamine (400 mg) in a yield of 43%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-6-yl]-acrylamide

(72) ##STR00064##

(73) N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-4,6-diamine (400 mg, 0.9 mmol) was dissolved in DCM (20 mL). To the resulting mixture were added triethylamine (0.3 mL) and acryloyl chloride (81 mg, 0.9 mmol). The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=5/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yloxy)quinazolin-6-yl]-acrylamide (100 mg) in a yield of 22%.

(74) Molecular formula: C.sub.26H.sub.27ClFN.sub.5O.sub.3

(75) Mass spectrum (m/e): 512.2 (M+1), 256.6 (M/2)

(76) .sup.1HNMR: (400 MHz, CDCl.sub.3) δ9.15 (s, 1H), 8.64 (s, 1H), 7.98 (s, 1H), 7.96 (d, 1H), 7.55 (m, 2H), 7.16 (t, 1H), 7.12 (s, 1H), 6.50 (d, 1H), 6.35 (m, 1H), 5.92 (d, 1H), 5.20 (m, 1H), 4.23 (m, 2H), 3.07 (m, 1H), 2.96 (m, 2H), 2.64 (s, 3H), 2.38 (m, 4H), 2.04 (d, 1H), 1.84 (d, 1H), 1.44 (t, 1H).

Example 6 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)meth oxy)quinazolin-6-yl]-acrylamide (Compound 6)

(77) ##STR00065##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)-6-nitroquinazolin-4-amine

(78) ##STR00066##

(79) (8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methanol (300 mg, 1.08 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (97 mg, 2.43 mmol) was added in batch in an ice bath under an atmosphere of N2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (362 mg, 1.08 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, a large quantity of water was added. The mixture was filtered, and the filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)-6-nitroquinazolin-4-amine (516 mg) in a yield of 95%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)quinazolin-4,6-diamine

(80) ##STR00067##

(81) N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)-6-nitroquinazolin-4-amine (516 mg, 1.03 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (346 mg, 6.18 mmol). The mixture was stirred at room temperature for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was concentrated, and the resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)quinazolin-4,6-diamine (100 mg) in a yield of 21%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)meth oxy)quinazolin-6-yl]-acrylamide

(82) ##STR00068##

(83) In a reaction flask, N-(4-(3-chloro-4-fluorophenyl))-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)methoxy)quinazolin-4,6-diamine (100 mg, 0.21 mmol) was dissolved in dichloromethane (10 mL). The mixture was cooled down to 0° C. Triethylamine (42 mg, 0.42 mmol) was added to the reaction flask. Acryloyl chloride (17 mg, 0.19 mmol) was dissolved in DCM (1 mL). The resulting solution was slowly added to the reaction flask. The mixture was reacted at room temperature for 30 min. The reaction was washed with distilled water (10 mL) triple, and distillated at a reduced pressure to remove dichloromethane to produce a crude yellow powdery product, which was purified by a silica gel column chromatography (eluted with DCM/MeOH=15/1) to produce a pale-yellow powdery solid N-[4-(3-chloro-4-fluorophenylamino)-7-((8-methyl-1-oxa-8-azaspiro[4.5]decan-3-yl)meth oxy)quinazolin-6-yl]-acrylamide (15 mg) in a yield of 14%.

(84) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.3

(85) Mass spectrum (m/e): 526.2 (M+1), 263.6 (M/2)

(86) .sup.1HNMR: (400 MHz, CDCl.sub.3) δ9.10 (s, 1H), 8.63 (s, 1H), 8.32 (s, 1H), 7.86 (d, 1H), 7.74 (s, 1H), 7.48 (d, 1H), 7.15 (s, 1H), 7.10 (m, 1H), 6.48 (d, 1H), 6.38 (m, 1H), 5.86 (d, 1H), 4.18 (t, 2H), 4.08 (t, 1H), 3.87 (t, 1H), 2.91 (s, H), 2.60 (m, 4H), 2.36 (m, 3H), 2.13 (t, 1H), 1.80-2.11 (m, 4H), 1.60 (m, 1H).

Example 7 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-6-yl]-acrylamide (Compound 7)

(87) ##STR00069##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)-6-nitroquinazolin-4-amine

(88) ##STR00070##

(89) 8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethanol (280 mg, 1.5 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (1.1 g, 27 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (508 mg, 1.5 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)-6-nitroquinazolin-4-amine (380 mg) in a yield of 50%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-4,6-diamine

(90) ##STR00071##

(91) N-(4-(3-chloro-4-fluorophenyl))-7-8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)-6-nitroquinazolin-4-amine (380 mg, 0.76 mmol) was dissolved in a mixed solvent (8 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (343 mg, 6.12 mmol). The mixture was warm up to 70° C. and stirred for 1 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-4,6-diamine (180 mg) in a yield of 50%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-6-yl]-acrylamide

(92) ##STR00072##

(93) N-(4-(3-chloro-4-fluorophenyl))-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-4,6-diamine (175 mg, 0.38 mmol) was dissolved in dichloromethane (30 mL). Triethylamine (77 mg) was added. Acryloyl chloride (31 mg, 0.34 mmol) was added dropwise under an ice bath. The mixture was stirred at room temperature for 0.5 h. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=20/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(8-methyl-1-oxa-8-azaspiro[4.5]decan-2-ylmethoxy)quinazolin-6-yl]-acrylamide (14 mg) in a yield of 8%.

(94) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.3

(95) Mass spectrum (m/e): 526.2 (M+1), 263.7 (M/2)

(96) .sup.1HNMR (400 MHz, CDCl.sub.3) δ8.92 (s, 1H), 8.55 (s, 1H), 7.98 (d, 1H), 7.62 (d, 1H), 7.20 (s, 1H), 7.13 (t, 1H), 6.47 (d, 2H), 5.86 (d, 1H), 4.47 (m, 1H), 4.24 (d, 1H), 4.10 (t, 1H), 3.05 (m, 4H), 2.49 (s, 3H), 1.78-1.97 (m, 8H).

Example 8 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-6-yl]-acrylamide (Compound 8)

(97) ##STR00073##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)-6-nitroquinazolin-4-amine

(98) ##STR00074##

(99) 2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethanol (0.2 g, 1.4 mmol) was dissolved DMF (10 mL). 60% sodium hydride (1.12 g, 2.8 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 30 min. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (470 mg, 1.4 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl ethoxy)-6-nitroquinazolin-4-amine (500 mg) in a yield of 78%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-4,6-diamine

(100) ##STR00075##

(101) N-(4-(3-chloro-4-fluorophenyl))-7-2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl ethoxy)-6-nitroquinazolin-4-amine (500 mg, 1.1 mmol) was dissolved in a mixed solvent of EtOH (10 mL) and CH.sub.3COOH (3 mL). To the mixture was added Fe powder (343 mg, 6.12 mmol). The reaction was conducted at room temperature 12 h under stirring. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-4,6-diamine (200 mg) in a yield of 43%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-6-yl]-acrylamide

(102) ##STR00076##

(103) N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-4,6-diamine (200 mg, 0.47 mmol) was dissolved in dichloromethane (20 mL). Triethylamine (200 mg) and acryloyl chloride (43 mg, 0.47 mmol) were added under an ice bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=5/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-ylethoxy)quinazolin-6-yl]-acrylamide 40 mg) in a yield of 18%.

(104) Molecular formula: C.sub.25H.sub.25ClFN.sub.5O.sub.2

(105) Mass spectrum (m/e): 482.2 (M+1), 241.6 (M/2)

(106) .sup.1HNMR (400 MHz, CDCl.sub.3) δ9.15 (s, 1H), 8.94 (s, 1H), 8.60 (s, 1H), 7.89 (s, 1H), 7.83 (d, 1H), 7.58 (d, 1H), 7.22 (t, 1H), 7.05 (m, 1H), 6.49 (d, 1H), 5.86 (d, 1H), 4.38 (t, 2H), 3.78 (d, 2H), 3.10 (d, 2H), 2.80 (s, 3H), 2.39 (m, 1H), 1.83 (m, 2H), 1.74 (m, 2H).

Example 9 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-6-yl]-acrylamide (Compound 9)

(107) ##STR00077##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)-6-nitroquinazolin-4-amine

(108) ##STR00078##

(109) (2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methanol (380 mg, 2.45 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (1.6 g, 40 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.08 g, 3.2 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)-6-nitroquinazolin-4-amine (318 mg) in a yield of 28%.

(2) N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-4,6-diamine

(110) ##STR00079##

(111) N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)-6-nitroquinazolin-4-amine (318 mg, 0.68 mmol) was dissolved in a mixed solvent (8 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (200 mg, 3.57 mmol). The mixture was warmed up to 70° C. and stirred for 1.5 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-4,6-diamine (90 mg) in a yield of 30%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-6-yl]-acrylamide

(112) ##STR00080##

(113) N-(4-(3-chloro-4-fluorophenyl))-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)quinazolin-4,6-diamine (80 mg, 0.18 mmol) was dissolved in dichloromethane (30 mL). Triethylamine (40 mg) was added. Acryloyl chloride (16 mg, 0.18 mmol) was added dropwise under an ice bath. The mixture was stirred at room temperature for 0.5 h. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=20/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-((2-methyloctahydrocyclopenta[c]pyrrol-4-yl)methoxy)-acrylamide 20 mg) in a yield of 22%.

(114) Molecular formula: C.sub.26H.sub.27ClFN.sub.5O.sub.2

(115) Mass spectrum (m/e): 496.3 (M+1), 248.7 (M/2)

(116) .sup.1HNMR (400 MHz, CDCl.sub.3) δ8.79 (s, 1H), 8.56 (s, 1H), 7.96 (d, 1H), 7.61 (d, 1H), 7.29 (s, 1H), 7.15 (m, 1H), 6.70 (m, 1H), 6.51 (d, 1H), 5.85 (d, 1H), 4.18 (m, 1H), 4.10 (m, 1H), 3.41-3.95 (m, 2H), 3.38 (d, 1H), 3.03-3.12 (m, 4H), 2.82 (s, 3H), 2.15 (s, 3H), 1.22 (m, 3H).

Example 10 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-6-yl]-acrylamide (Compound 10)

(117) ##STR00081##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)-6-nitroquinazolin-4-amine

(118) ##STR00082##

(119) (7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methanol (283 mg, 2.0 mmol) was dissolved in DMF (10 mL). 60% sodium hydride (160 g, 4.0 mmol) was added in batch in an ice bath under an atmosphere of N2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.13 g, 3.0 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=8/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)-6-nitroquinazolin-4-amine (427 mg) in a yield of 47%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-4,6-diamine

(120) ##STR00083##

(121) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)-6-nitroquinazolin-4-amine (427 mg, 0.93 mmol) was dissolved in a mixed solvent (28 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (312 mg, 5.58 mmol). The mixture was warm up to 70° C. and stirred for 1 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-4,6-diamine (157 mg) in a yield of 40%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-6-yl]-acrylamide

(122) ##STR00084##

(123) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-4,6-diamine (157 mg, 0.37 mmol) was dissolved in dichloromethane (10 mL). Triethylamine (111 mg, 1.10 mmol) and acryloyl chloride (33 mg, 0.37 mmol) were added under an ice bath. The mixture was stirred at room temperature overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azabicyclo[2.2.1]heptan-2-yl)methoxy)quinazolin-6-yl]-acrylamide 20 mg) in a yield of 11%.

(124) Molecular formula: C.sub.25H.sub.25ClFN.sub.5O.sub.2

(125) Mass spectrum (m/e): 482.2 (M+1), 241.7 (M/2)

(126) .sup.1HNMR (400 MHz, CDCl.sub.3) δ9.08 (s, 1H), 8.64 (s, 1H), 8.16 (s, 1H), 7.88 (m, 2H), 7.51 (d, 1H), 7.24 (s, 1H), 7.10 (t, 1H), 6.48 (d, 1H), 6.38 (m, 1H), 5.88 (d, 1H), 4.22 (m, 1H), 4.10 (t, 1H), 3.56 (s, 1H), 3.44 (s, 1H), 2.94 (m, 1H), 2.46 (s, 3H), 2.25 (m, 1H), 1.99 (m, 1H), 1.70 (m, 1H), 1.68 (m, 1H), 1.41 (m, 1H), 1.05 (m, 1H).

Example 11 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-6-yl]-acrylamide (Compound 11)

(127) ##STR00085##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)-6-nitroquinazolin-4-amine

(128) ##STR00086##

(129) 2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethanol (338 mg, 2 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (0.4 g, 10 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (1.12 g, 3.3 mmol) was added. The mixture was stirred at 50° C. overnight. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)-6-nitroquinazolin-4-amine (560 mg) in a yield of 58%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-4,6-diamine

(130) ##STR00087##

(131) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)-6-nitroquinazolin-4-amine (560 mg, 1.15 mmol) was dissolved in a mixed solvent (20 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (343 mg, 6.12 mmol). The mixture was warmed up to 70° C. and stirred for 1 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-4,6-diamine (360 mg) in a yield of 69%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-6-yl]-acrylamide

(132) ##STR00088##

(133) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-4,6-diamine (360 mg, 0.79 mmol) and triethylamine (112 mg) were dissolved in dichloromethane (20 mL). Acryloyl chloride (71 mg, 0.79 mmol) was added dropwise. The mixture was stirred at room temperature for 1 h. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA, and rotary-vaporated to dryness under a reduced pressure. The residue was washed with diethyl ether to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-azabicyclo[3.2.1]octan-8-yl)ethoxy)quinazolin-6-yl]-acrylamide (34 mg) in a yield of 8%.

(134) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.2

(135) Mass spectrum (m/e): 510.3 (M+1), 255.8 (M/2)

(136) .sup.1HNMR (400 MHz, CDCl.sub.3) δ9.13 (s, 1H), 8.67 (s, 1H), 8.15 (s, 1H), 7.94 (d, 1H), 7.61 (s, 1H), 7.53 (d, 1H), 7.15 (t, 1H), 6.50 (d, 1H), 6.34 (m, 1H), 5.90 (d, 1H), 4.29 (m, 2H), 2.69 (t, 1H), 2.66 (d, 1H), 2.50 (s, 3H), 1.27-2.35 (m, 11H).

Example 12 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-6-yl]-acrylamide (Compound 12)

(137) ##STR00089##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)-6-nitroquinazolin-4-amine

(138) ##STR00090##

(139) (5-methyl-5-azaspiro[2.4]heptan-1-yl)methanol (370 mg, 2.62 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (157 mg, 3.93 mmol) was added in batch in an ice bath under an atmosphere of N2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (588 mg, 1.75 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)-6-nitroquinazolin-4-amine (690 mg) in a yield of 86%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-4,6-diamine

(140) ##STR00091##

(141) N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)-6-nitroquinazolin-4-amine (69 mg, 1.51 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (507 mg, 9.06 mmol). The mixture was warm up to 30° C. and stirred for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-4,6-diamine (100 mg) in a yield of 15%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-6-yl]-acrylamide

(142) ##STR00092##

(143) N-(4-(3-chloro-4-fluorophenyl))-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-4,6-diamine (100 mg, 0.23 mmol) was dissolved in DCM (10 mL). Triethylamine (46 mg, 0.46 mmol) was added. Acryloyl chloride (19 mg, 0.21 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA, and rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-((5-methyl-5-azaspiro[2.4]heptan-1-yl)methoxy)quinazolin-6-yl]-acrylamide (14 mg) in a yield of 14%.

(144) Molecular formula: C.sub.25H.sub.25ClFN.sub.5O.sub.2

(145) Mass spectrum (m/e): 482.3 (M+1), 241.6 (M/2)

(146) .sup.1HNMR (400 MHz, CDCl.sub.3) δ9.35 (s, 1H), 9.00 (s, 1H), 8.60 (s, 1H), 7.98 (s, 1H), 7.78 (d, 1H), 7.54 (d, 1H), 6.95 (m, 2H), 6.52 (m, 2H), 5.83 (m, 1H), 4.57 (m, 1H), 3.65 (m, 1H), 0.68-3.23 (m, 12H).

Example 13 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-6-yl]-acrylamide (Compound 13)

(147) ##STR00093##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)-6-nitro quinazolin-4-amine

(148) ##STR00094##

(149) (6-methyl-6-azaspiro[2.5]octan-1-yl)methanol (400 mg, 2.58 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (230 mg, 3.87 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (954 mg, 2.83 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)-6-nitro quinazolin-4-amine (300 mg) in a yield of 25%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-4,6-diamine

(150) ##STR00095##

(151) N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)-6-nitro quinazolin-4-amine (300 mg, 0.64 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (177 mg, 3.18 mmol). The mixture was warmed up to 30° C. and stirred for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure to produce N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-4,6-diamine (200 mg) in a yield of 71%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-6-yl]-acrylamide

(152) ##STR00096##

(153) N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-4,6-diamine (200 mg, 0.45 mmol) was dissolved in DCM (10 mL). Triethylamine (46 mg, 0.46 mmol) was added. Acryloyl chloride (39 mg, 0.43 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)quinazolin-6-yl]-acrylamide 25 mg) in a yield of 12%.

(154) Molecular formula: C.sub.26H.sub.27ClFN.sub.5O.sub.2

(155) Mass spectrum (m/e): 496.2 (M+1), 248.6 (M/2)

(156) .sup.1HNMR (400 MHz, CDCl.sub.3) δ9.13 (s, 1H), 8.66 (s, 1H), 8.32 (s, 1H), 7.97 (d, 1H), 7.66 (s, 1H), 7.55 (d, 1H), 7.25 (s, 1H), 7.17 (t, 1H), 6.52 (d, 1H), 6.42 (m, 1H), 5.90 (d, 1H), 4.39 (m, 1H), 4.07 (t, 1H), 2.69 (m, 2H), 2.55 (m, 2H), 2.40 (s, 3H), 2.04 (s, 1H), 1.80 (m, 2H), 1.56 (m, 2H), 1.30 (m, 2H).

Example 14 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-6-yl]-acrylamide (Compound 14)

(157) ##STR00097##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)-6-nitroquinazolin-4-amine

(158) ##STR00098##

(159) 2-(6-methyl-6-azaspiro[2,5]octan-1-yl)ethanol (9 g, 53 mmol) was dissolved in DMF (200 mL). 60% sodium hydride (10 g, 0.25 mol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (18 g, 53 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=20/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)-6-nitroquinazolin-4-amine (17.0 g) in a yield of 66%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-4,6-diamine

(160) ##STR00099##

(161) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)-6-nitroquinazolin-4-amine (17 g, 35 mmol) was dissolved in a mixed solvent (300 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (13.6 g, 243 mmol). The mixture was stirred at room temperature for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=20/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-4,6-diamine (8.2 g) in a yield of 51%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-6-yl]-acrylamide

(162) ##STR00100##

(163) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-4,6-diamine (300 mg, 6.6 mmol) was dissolved in DCM (50 mL). Triethylamine (2.0 g, 145 mmol) was added. Acryloyl chloride (600 mg, 6.7 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=30/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(6-methyl-6-azaspiro[2.5]octan-1-yl)ethoxy)quinazolin-6-yl]-acrylamide 0.5 g) in a yield of 15%.

(164) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.2

(165) Mass spectrum (m/e): 510.2 (M+1), 255.9 (M/2)

(166) .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.14 (s, 1H), 8.69 (s, 1H), 8.24 (s, 1H), 7.99 (d, 1H), 7.55 (m, 1H), 7.53 (m, 1H), 7.27 (s, 1H), 7.19 (t, 1H), 6.52 (d, 1H), 6.41 (m, 1H), 5.90 (d, 1H), 4.34 (m, 2H), 2.82 (m, 2H), 2.80 (m, 2H), 2.38 (s, 3H), 0.62-2.19 (m, 9H).

Example 15 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl)ethoxy)quinazolin-6-yl]-2-butenamide (Compound 15)

(167) ##STR00101##

(168) N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl)ethoxy)quinazolin-4,6-diamine was prepared according to steps (1) and (2) of Example 8.

(169) Trans-2-butenoic acid (0.12 g, 1.2 mmol) was dissolved in DMF (5 mL). Then HATU (0.05 g, 1.32 mmol), triethylamine (0.5 mL) and N-(4-(3-chloro-4-fluorophenyl))-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl)ethoxy)quinazolin-4,6-diamine (500 mg, 1.2 mmol) were added. The mixture was stirred at room temperature for 12 h. After the completion of reaction, water (50 mL) was added. The reaction was extracted with dichloromethane. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1-5/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl)ethoxy)quinazolin-6-yl]-acrylamide (30 mg) in a yield of 5%.

(170) Molecular formula: C.sub.26H.sub.27ClFN.sub.5O.sub.2

(171) Mass spectrum (m/e): 496.2 (M+1), 248.6 (M/2)

(172) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.45 (br s, 1H), 9.40 (br s, 1H), 8.97 (s, 1H), 8.69 (s, 1H), 8.05 (s, 1H), 7.72 (m, 1H), 7.46 (t, 1H), 7.29 (m, 1H), 6.98 (m, 1H), 6.47 (d, 1H), 4.26 (m, 2H), 3.54 (m, 2H), 3.34 (m, 3H), 2.76 (s, 3H), 1.74-1.91 (m, 6H), 1.23 (m, 1H).

Example 16 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-butenamide (Compound 16)

(173) ##STR00102##

(174) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine was prepared according to steps (1) and (2) of Example 18.

(175) Trans-2-butenoic acid (98 mg, 1.1 mmol) was dissolved in DMF (10 mL). Then HATU (563 g, 1.32 mmol), DIEA (441 mg, 3.4 mmol) and N-(4-(3-chloro-4-fluorophenyl)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl))methoxy)quinazolin-4,6-diamine (400 mg, 0.88 mmol) were added. The mixture was stirred at room temperature for 12 h. After the completion of reaction, water (50 mL) was added. The reaction was extracted with dichloromethane. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1-5/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl))methoxy)quinazolin-6-yl]-2-butenamide 30 mg) in a yield of 7%.

(176) Molecular formula: C.sub.28H.sub.31ClFN.sub.5O.sub.2 MW: 524

(177) Mass spectrum (m/e): 524.2 (M+1), 262.6 (M/2)

(178) .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.08 (s, 1H), 8.65 (s, 1H), 8.03 (s, 1H), 7.96 (d, 1H), 7.52 (d, 1H), 7.23 (s, 1H), 7.15 (t, 1H), 7.06 (m, 1H), 5.99 (d, 1H), 4.17 (d, 2H), 2.84 (m, 1H), 2.27 (m, 4H), 2.26 (s, 3H), 2.00 (m, 2H), 1.97 (d, 3H), 1.62-1.82 (m, 6H).

Example 17 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide (Compound 17) and its hydrochloride

(179) ##STR00103##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine

(180) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine was prepared according to steps (1) and (2) of Example 18.

(2) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide

(181) Trans-2-pentenoic acid (86 mg, 0.86 mmol) was dissolved in DMF (10 mL). Then HATU (425 g, 1.12 mmol), DIEA (333 mg, 2.6 mmol) and N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazoline-4,6-diamine (300 mg, 0.66 mmol) were added. The mixture was stirred at room temperature for 12 h. After the completion of reaction, water (50 mL) was added. The reaction was extracted with dichloromethane. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1-5/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide 30 mg) in a yield of 8%.

(3) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide (Compound 17) hydrochloride

(182) (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide (58 mg, 0.11 mmol) was dissolved in methanol (10 mL). HCl was added dropwise at room temperature. The reaction was conducted for 2 h under stirring, and then the solvent was evaporated off to produce a yellow solid (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-2-pentenamide hydrochloride 61 mg) in a yield of 100%.

(183) Molecular formula: C.sub.29H.sub.34O.sub.2FN.sub.5O.sub.2

(184) Mass spectrum (m/e): 538.1 (M+1), 269.6 (M/2)

(185) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ11.0 (br s, 1H), 9.41 (s, 1H), 9.01 (s, 1H), 8.94 (s, 1H), 8.82 (s, 1H), 7.98 (d, 1H), 7.65 (d, 1H), 7.52 (t, 1H), 7.26 (s, 1H), 5.67 (m, 2H), 4.24 (d, 2H), 3.28 (d, 2H), 2.80 (m, 2H), 2.74 (s, 3H), 1.63-1.98 (m, 14H).

Example 18 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-acrylamide (Compound 18)

(186) ##STR00104##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)-6-nitroquinazolin-4-amine

(187) ##STR00105##

(188) (7-methyl-7-azaspiro[3.5]nonan-2-yl)methanol (9 g, 53 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (10 g, 0.25 mol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (18 g, 53 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)-6-nitroquinazolin-4-amine (17 g) in a yield of 66%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine

(189) ##STR00106##

(190) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)-6-nitroquinazolin-4-amine (17 g, 35 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (13.6 g, 243 mmol). The mixture was stirred at room temperature for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine 10 g) in a yield of 63%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-acrylamide

(191) ##STR00107##

(192) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine (3 g, 6.6 mmol) was dissolved in DCM (10 mL). Triethylamine (2 g, 19.8 mmol) was added. Acryloyl chloride (600 mg, 6.6 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA, and rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl]-acrylamide (1.26 g) in a yield of 37%.

(193) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.2

(194) Mass spectrum (m/e): 510.2 (M+1), 255.8 (M/2)

(195) .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.12 (s, 1H), 8.67 (s, 1H), 8.18 (s, 1H), 7.98 (d, 1H), 7.55 (m, 1H), 7.52 (s, 1H), 7.26 (s, 1H), 7.17 (t, 1H), 6.50 (d, 1H), 6.34 (m, 1H), 5.88 (d, 1H), 4.21 (d, 2H), 2.86 (m, 1H), 2.21-2.50 (m, 7H), 2.07 (t, 2H), 1.82 (m, 2H), 1.63-1.71 (m, 4H).

Example 19 Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide (Compound 19) and its hydrochloride

(196) ##STR00108##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)-6-nitroquinazolin-4-amine

(197) ##STR00109##

(198) 2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethanol (2.7 g, 14.8 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (1.78 g, 44.5 mmol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (4.95 g, 14.7 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)-6-nitroquinazolin-4-amine (5.0 g) in a yield of 68%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-4,6-diamine

(199) ##STR00110##

(200) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)-6-nitroquinazolin-4-amine (5 g, 10 mmol) was dissolved in a mixed solvent (250 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (1.96 g, 35 mmol). The mixture was warmed up to 30° C. and stirred for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-4,6-diamine 2.5 g) in a yield of 53%.

(3) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide

(201) ##STR00111##

(202) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-4,6-diamine (300 mg, 0.64 mmol) was dissolved in DCM (10 mL). Triethylamine (194 mg, 1.92 mmol) was added. Acryloyl chloride (60 mg, 0.67 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide 100 mg) in a yield of 30%.

(4) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide (Compound 19) hydrochloride

(203) N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide (100 mg, 0.19 mmol) was dissolved in methanol (10 mL). A HCl gas was introduced under an ice-water bath. The reaction was conducted for 30 min under stirring, and then the solvent was evaporated off to produce a white solid N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(7-methyl-7-azaspiro[3.5]nonan-2-yl)ethoxy)quinazolin-6-yl]-acrylamide hydrochloride (105 mg) in a yield of 97%.

(204) Molecular formula: C.sub.28H.sub.32Cl.sub.2FN.sub.5O.sub.2

(205) Mass spectrum (m/e): 524.0 (M+1), 262.5 (M/2)

(206) .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ10.72 (br s, 1H), 9.77 (br s, 1H), 9.73 (s, 1H), 9.00 (s, 1H), 8.75 (s, 1H), 8.03 (d, 1H), 7.52 (d, 1H), 7.49 (t, 1H), 7.36 (s, 1H), 6.73 (m, 1H), 6.30 (d, 1H), 5.83 (m, 1H), 4.18 (t, 2H), 2.81 (m, 1H), 2.77 (m, 1H), 2.68 (s, 3H), 1.52-2.08 (m, 13H).

Example 20 Preparation of (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino-2-butenamide (Compound 20)

(207) ##STR00112##

(1) Preparation of (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-bromo-2-butenamide

(208) ##STR00113##

(209) N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine was prepared according to steps (1) and (2) of Example 18.

(210) 4-bromocrotonic acid (900 mg, 5.5 mmol) was dissolved in THF (10 mL) under nitrogen. DCC (1130 mg, 5.5 mmol) was added under an ice bath. The mixture was stirred for 0.5 h. Then a solution of N-(4-(3-chloro-4-fluorophenyl))-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-4,6-diamine (500 mg, 1.1 mmol) in DMF (10 mL) was added. The mixture was stirred for 40 min, and then a crude product of (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-bromo-2-butenamide was obtained. This crude product was directly used in the next step with purification.

(2) Preparation of (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino-2-butenamide

(211) ##STR00114##

(212) To the product in the previous step, i.e. (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-bromo-2-butenamide, were successively added dimethylamine hydrochloride (1.25 g, 15.3 mmol) and DIEA (2.68 mL, 15.4 mmol). The mixture was continuously stirred for 2 h under an ice bath. The mixture was moved to an atmosphere of room temperature and stirred overnight. To the reaction was added a saturated sodium bicarbonate solution. The reaction was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate and concentrated. Then, the concentrate was separated by a reverse-phase preparative column (C.sub.18, ODS-AQ 40-60 um, mobile phase: methanol/water=50/50) to produce (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-((7-methyl-7-azaspiro[3.5]nonan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino-2-butenamide (120 mg) in a yield of 19.2%.

(213) Molecular formula: C.sub.30H.sub.36ClFN.sub.6O.sub.2

(214) Mass spectrum (m/e): 567 (M+1)

(215) .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ9.79 (s, 1H), 9.46 (s, 1H), 8.80 (s, 1H), 8.54 (s, 1H), 8.13-8.17 (m, 1H), 7.79-7.83 (m, 1H), 7.39-7.46 (m, 1H), 7.27 (s, 1H), 6.77 (dd, 1H), 6.51 (d, 1H), 4.17 (d, 2H), 3.08 (d, 2H), 2.70-2.82 (m, 1H), 2.21 (m, 10H), 2.11 (s, 3H), 1.88 (t, 2H), 1.71 (t, 2H), 1.22-1.51 (m, 4H).

Example 21 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide (Compound 21)

(216) ##STR00115##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-azabicyclo[3.1.0]-6-hexyl)-ethoxy)-6-nitroquinazolin-4-amine

(217) ##STR00116##

(218) 2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethanol (7.5 g, 53 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (10 g, 0.25 mol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (18 g, 53 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 mL) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)-6-nitroquinazolin-4-amine (14.8 g) in a yield of 61%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)quinazolin-4,6-diamine

(219) ##STR00117##

(220) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy))-6-nitroquinazolin-4-amine (14.8 g, 32 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (13.6 g, 243 mmol). The mixture was stirred at room temperature for 12 h. After the completion of reaction, the rotary-evaporation was conducted to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=10/1) to produce N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy))quinazolin-4,6-diamine (8.3 g) in a yield of 62%.

(3) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)-quinazolin-6-yl-4-chloro)]-crotonamide

(221) ##STR00118##

(222) N-(4-(3-chloro-4-fluorophenyl))-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy))quinazolin-4,6-diamine (2.73 g, 6.6 mmol) was dissolved in DCM (10 mL). Triethylamine (2 g, 19.8 mmol) was added. (E)-4-chloro-crotonyl chloride (600 mg, 6.6 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)-quinazolin-6-yl-4-chloro)]-crotonamide (2.81 g) in a yield of 81%.

(4) Preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)-quinazolin-6-((E)-4-dimethylamino)]-crotonamide

(223) ##STR00119##

(224) N-[4-(3-chloro-4-fluorophenylamino)-7-(2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)-quinazolin-6-((E)-4-chloro)]-crotonamide (0.23 g, 0.4 mmol) was dissolved in acetonitrile (30 mL). Methylamine hydrochloride (0.32 g, 4 mmol) and cesium carbonate (2.6 g, 8 mmol) were added under the nitrogen gas protection. The mixture was heated to reflux and filtered. The filtrate was rotary-evaporated to dryness under a reduce pressure. Then the resulting residue was directly separated by a reverse phase preparative column (C18, ODS-AQ 40-60 um, mobile phase: methanol/water=50/50) to produce a compound named (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-((2-(3-methyl-3-aza-bicyclo[3.1.0]-6-hexyl)-ethoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide (0.14 g) in a yield of 37%.

(225) Molecular formula: C.sub.28H.sub.32ClFN.sub.6O.sub.2

(226) Mass spectrum (m/e): 539.2 (M+1), 270 (M/2)

(227) .sup.1HNMR (400 MHz, CD.sub.3OD) δ 9.20 (s, 1H), 8.76 (s, 1H), 7.94 (d, 1H), 7.68-7.64 (m, 1H), 7.40-7.35 (m, 2H), 7.11-7.00 (m, 2H), 4.44-4.41 (m, 2H), 4.08 (d, 2H), 3.73 (d, 2H), 3.36 (s, 4H), 3.00-2.67 (m, 8H), 1.96-1.92 (m, 2H), 1.82-1.75 (m, 3H).

Example 22 Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(((spiro[3.5]octan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide (Compound 22)

(228) ##STR00120##

(1) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)-6-nitroquinazolin-4-amine

(229) ##STR00121##

(230) (Spiro[3.5]octan-2-yl)methanol (8.16 g, 53 mmol) was dissolved in DMF (20 mL). 60% sodium hydride (10 g, 0.25 mol) was added in batch in an ice bath under an atmosphere of N.sub.2. The mixture was moved to an atmosphere of room temperature and stirred for 1 h. N-(4-(3-chloro-4-fluorophenyl))-7-fluoro-6-nitroquinazolin-4-amine (18 g, 53 mmol) was added. The mixture was stirred at room temperature overnight. After the completion of reaction, water (20 ml) was added. The mixture was filtered. The filtered cake was dried in vacuum to produce N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)-6-nitroquinazolin-4-amine (18.68 g) in a yield of 75%.

(2) Preparation of N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)-quinazolin-4,6-diamine

(231) ##STR00122##

(232) N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)-6-nitroquinazolin-4-amine (16.45 g, 35 mmol) was dissolved in a mixed solvent (60 mL) of acetic acid and ethanol (CH.sub.3COOH/EtOH=1/3). To the mixture was added Fe powder (13.6 g, 243 mmol). The mixture was stirred at room temperature for 12 h. After the completion of reaction, the mixture was rotary-evaporated under a reduced pressure to remove EtOH. Water (30 mL) was added. The mixture was adjusted with 1 mol/L NaOH solution until it became basic. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduce pressure. The resulting residue was purified by a silica gel column chromatography (dichloromethane/methanol=10/1, V/V) to produce N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)quinazolin-4,6-diamine (10 g) in a yield of 63%.

(3) Preparation of ((E)-4-dimethylamino)-crotonyl chloride hydrochloride

(233) ##STR00123##

(234) ((E)-4-dimethylamino)-crotonic acid hydrochloride (1.65 g, 10 mmol) was dissolved in THF (50 ml). DMF (0.1 mL) was added. The mixture was cooled to 0° C., and SOCl.sub.2 (5 mL) was slowly added dropwise. The reaction was warmed up to room temperature. After 0.5 h, the mixture was heated to reflux, stirred for 3 h under reflux, then cooled down to room temperature, and evaporated off the excess of SOCl.sub.2 under the nitrogen gas protection. The resulting product was directly used in the next step.

(4) Preparation of (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(((spiro[3.5]octan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide

(235) ##STR00124##

(236) N-(4-(3-chloro-4-fluorophenyl))-7-((spiro[3.5]octan-2-yl)methoxy)quinazolin-4,6-diamine (3 g, 6.6 mmol) was dissolved in DCM (10 mL). Triethylamine (2 g, 19.8 mmol) was added. 6-((E)-4-dimethylamino)]-crotonyl chloride (600 mg, 6.6 mmol) was added dropwise in an ice-water bath. The mixture was stirred at room temperature for 30 min. After the completion of reaction, water (50 mL) was added. The mixture was extracted with EA. The organic layer was rotary-evaporated to dryness under a reduced pressure. The resulting residue was purified by a silica gel column chromatography (eluted with dichloromethane/methanol=15/1) to produce (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(((spiro[3.5]octan-2-yl)methoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide (1.26 g) in a yield of 38%.

(237) Molecular formula: C.sub.30H.sub.35ClFN.sub.5O.sub.2

(238) Mass spectrum (m/e): 552.2 (M+1), 256.2 (M/2)

(239) .sup.1HNMR (400 MHz, CD.sub.3OD) δ 9.21 (s, 1H), 8.79 (s, 1H), 7.96 (d, 1H), 7.69-7.68 (m, 1H), 7.42 (d, 1H), 7.34 (s, 1H), 7.04 (t, 1H), 6.84 (d, 1H), 4.37 (d, 2H), 4.09 (d, 2H), 3.0-2.96 (m, 7H), 2.10-2.05 (m, 5H), 1.77-1.63 (m, 7H), 1.37-1.33 (m, 3H).

Example 23 Preparation of (E)-N-(7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (Compound 23)

(240) ##STR00125##

(1) Preparation of 7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine

(241) ##STR00126##

(242) To dioxane (50 ml) were added bicyclo[3.1.0]hexan-6-ylmethanol (3.36 g, 30 mmol), potassium carbonate (4.14 g, 30 mmol) and 7-fluoro-4-(3-chloro-4-fluorophenylamine)-6-nitroquinazoline (3.36 g, 10 mmol). The mixture was stirred at room temperature for 24 hr. After the completion of reaction, water was added. The mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate. The resulting residue was purified with a silica gel column chromatography (petroleum ether/ethyl acetate=1/1) to produce a product (7.57 g) in a yield of 59%.

(2) Preparation of 7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-N-(4-(3-chloro-4-fluorophenyl))quinazolin-4,6-diamine

(243) ##STR00127##

(244) 7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-N-(4-(3-chloro-4-fluorophenyl))-6-nitroquinazolin-4-amine (1.71 g, 4 mmol) and Pd/C (0.2 g) were added to tetrahydrofuran (30 mL). The mixture was stirred at room temperature overnight. After the completion of reaction, water was added. The mixture was extracted with ethyl acetate. The organic layer was evaporated to dryness to produce a product (1.40 g) in a yield of 88%.

(3) Preparation of (E)-N-(7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide

(245) ##STR00128##

(246) 4-(dimethylamino)but-2-enoic acid (0.52 g, 4 mmol) was added to dichloroethane (20 mL). Thionyl chloride (0.95 g, 8 mmol) was added dropwise under an ice bath. The mixture was heated to reflux for 2 hr. After the completion of reaction, the reaction was evaporated to dryness. The resulting residue was dissolved in acetonitrile (50 mL). A solution of triethylamine (0.3 g, 3 mmol) and 7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-N-(4-(3-chloro-4-fluorophenyl)quinazolin-4,6-diamine (1.19 g, 3 mmol) in tetrahydrofuran (100 mL) was added dropwise. The mixture was stirred for 12 hr, and water was added. The reaction was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate. The resulting residue was purified by a preparative liquid chromatography (C.sub.18, ODS-AQ 40-60 um, mobile phase: methanol/water=50/50) to produce (E)-N-(7-(bicyclo[3.1.0]hexan-6-ylmethoxy)-4-(3-chloro-4-fluorophenylamino)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (0.168 g) in a yield of 11%.

(247) Molecular formula: C.sub.27H.sub.29ClFN.sub.5O.sub.2

(248) Mass spectrum (m/e): 510 (M+1), 255.7 (M/2)

(249) .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ 9.78 (s, 1H), 951-9.54 (m, 1H), 8.86 (s, 1H), 8.51-8.52 (m, 1H), 8.11-8.13 (m, 1H), 7.78-7.80 (m, 1H), 7.41 (t, 1H), 7.23 (s, 1H), 6.77-6.81 (m, 1H), 6.54-6.57 (m, 1H), 4.21-4.23 (m, 1H), 4.05-4.07 (m, 1H), 3.08-3.09 (m, 2H), 2.19 (s, 6H), 1.95-2.01 (m, 1H), 1.83-1.91 (m, 2H), 1.88 (m, 2H), 1.71-1.73 (m, 2H), 1.36 (m, 1H), 1.04 (m, 1H).

(250) Compounds 1-16, 18 and 20-23 can be prepared into salts according to the salt-formation methods described for Compound 17 and Compound 19.

(251) The following compounds can also be prepared according to the above-mentioned methods.

(252) TABLE-US-00002 No. R.sup.1 R.sup.2 (R.sup.3)n L 24 0 H 3′-Cl, 4′-F O 25 H 3′-Cl, 4′-F O 26 H 3′-Cl, 4′-F O 27 H 3′-Cl, 4′-F O 28 H 3′-Cl, 4′-F O 29 H 3′-Cl, 4′-F O 30 H 3′-Cl, 4′-F O 31 —CH.sub.2CH.sub.3 3′-Cl, 4′-F O 32 —CH.sub.2CH.sub.3 3′-Cl, 4′-F O 33 0 3′-Cl, 4′-F O 34 3′-Cl, 4′-F O 35 —CH.sub.3 3′-Cl, 4′-F O 36 —CH.sub.3 3′-Cl, 4′-F O 37 3′-Cl, 4′-F O 38 3′-Cl, 4′-F O 39 0 3′-Cl, 4′-F O 40 3′-Cl, 4′-F O 41 3′-Cl, 4′-F O 42 3′-Cl, 4′-F O 43 3′-Cl, 4′-F O 44 0 3′-Cl, 4′-F O 45 3′-Cl, 4′-F O 46 3′-Cl, 4′-F O 47 3′-Cl, 4′-F O 48 3′-Cl, 4′-F O 49 0 3′-Cl, 4′-F O 50 3′-Cl, 4′-F O 51 3′-Cl, 4′-F O 52 3′-Cl, 4′-F O 53 3′-Cl, 4′-F O 54 0 3′-Cl, 4′-F O 55 3′-Cl, 4′-F O 56 3′-Cl, 4′-F O 57 3′-Cl, 4′-F S 58 3′-Cl, 4′-F O 59 0 3′-Cl, 4′-F S 60 3′-Cl, 4′-F O 61 3′-Cl, 4′-F O 62 3′-Cl, 4′-F NH 63 3′-Cl, 4′-F NH 64 00 3′-Cl, 4′-F O 65 01 02 3′-Cl, 4′-F O 66 03 04 3′-Cl, 4′-F O 67 05 06 3′-Cl, 4′-F O 68 07 08 3′-Cl, 4′-F O 69 09 0 3′-Cl, 4′-F O 70 3′-Cl, 4′-F O 71 3′-Cl, 4′-F O 72 3′-Cl, 4′-F O 73 3′-Cl, 4′-F S 74 0 3′-Cl, 4′-F O 75 3′-Cl, 4′-F O 76 3′-Cl, 4′-F O 77 3′-Cl, 4′-F O 78 3′-Cl, 4′-F O 79 0 3′-Cl, 4′-F O 80 3′-Cl, 4′-F NH 81 3′-Cl, 4′-F S 82 3′-Cl, 4′-F S 83 3′-Cl, 4′-F O 84 0 3′-Cl, 4′-F O 85 3′-Cl, 4′-F O 86 3′-Cl, 4′-F O 87 3′-Cl, 4′-F O 88 3′-Cl, 4′-F O 89 0 3′-Cl, 4′-F O

II. IN VITRO ASSAYS FOR THE ANTINEOPLASTIC ACTIVITIES OF THE PRESENT COMPOUNDS

(253) Hereinafter, the beneficial effects of the present compounds will be illustrated by in vitro enzyme inhibitory activity and in vitro cellular inhibitory activity. However, it should be noted that the beneficial effects of the present compounds are not limited to the effects as illustrated below.

(254) Assay 1

(255) In Vitro Enzyme Inhibitory Activity of the Present Compounds

(256) Samples:

(257) Controls: Gefitinib, erlotinib hydrochloride, purchased from Anqing worldchem Co., LTD.; lapatinib ditosylate, purchased from Taizhou Xingcheng Chempharm Co., Ltd.; CI-1033 hydrochloride, purchased from Shanghai hanxiangchem, Co., Ltd.; and

(258) The present compounds: lab-made, their chemical names and structural formulae are shown in the preparation examples.

(259) Assay Procedures:

(260) The abbreviations used in the following assay have the following meanings:

(261) HEPES: hydroxyethyl piperazine ethanesulfonic acid;

(262) Brij-35: polyoxyethylene lauryl ether;

(263) DTT: dithiothreitol;

(264) Coating Reagent #3: #3 coating agent;

(265) EDTA: ethylene diamine tetraacetic acid, purchased from Sigma Co. Ltd.;

(266) FAM labeled peptide: fluorescein labeled peptide 22 (GL Biochem);

(267) ATP: adenosine triphosphate (Sigma);

(268) DMSO: dimethyl sulfoxide;

(269) EGFR: human epidermal growth factor receptor (Carna);

(270) HER2: human epidermal growth factor receptor 2 (Carna);

(271) HER4: human epidermal growth factor receptor 4 (Carna).

(272) 1. Formulating the agents to be used in the assay

(273) (1) 1.25-fold MnCl.sub.2-free kinase buffer (62.5 mM HEPES, PH 7.5, 0.001875% Brij-35, 12.5 mM MgCl.sub.2, 2.5 mM DTT);

(274) (2) 1.25-fold MnCl.sub.2-containing kinase buffer (62.5 mM HEPES, pH 7.5, 0.001875% Brij-35, 12.5 mM MgCl.sub.2, 12.5 mM MnCl.sub.2, 2.5 mM DTT);

(275) (3) Stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA);

(276) (4) 2.5-fold kinase solutions (to the 1.25-fold kinase buffers were added the corresponding kinases to formulate 2.5-fold EGFR, HER2, HER4 kinase solutions);

(277) (5) 2.5-fold peptide solutions (to the 1.25-fold kinase buffers were added FAM labeled peptide and ATP to formulate the peptide solutions);

(278) (6) 5-fold compound solutions (using 100% DMSO to formulate 50-fold compound solutions having different concentration gradients, and diluting with water by 10 times to obtain 5-fold compound solutions having different concentration gradients);

(279) 2. Adding 5 μL of a 5-fold compound solution to a 384-well plate;

(280) 3. Adding 10 μL of a 2.5-fold kinase solution to incubate for 10 min;

(281) 4. Then adding 10 μL of a 2.5-fold peptide solution, and reacting at 28° C. for 1 h; and

(282) 5. Finally, adding 25 μL of stop buffer to terminate the reaction, and reading the data with Caliper.

(283) 6. Curve fitting to obtain an IC.sub.50 value.
The calculated inhibition ratio (%)=(the maximum conversion rate−the conversion rate)/(the maximum conversion rate−the minimum conversion rate)×100

(284) The curve fitting was conducted with the Xlfit software to obtain IC.sub.50 values.

(285) The results are shown below.

(286) TABLE-US-00003 TABLE 1 In vitro enzyme inhibitory activity Enzyme inhibitory activity IC.sub.50 (nM) Compound EGFR HER2 HER4 Gefitinib 1.6 318 7.6 Erlotinib hydrochloride 1.3 454 49 Lapatinib ditosylate 16 4.0 250 CI-1033 hydrochloride 0.46 4 2.2 Compound 6 1 7.1 1.4 Compound 7 0.93 4.3 1.7 Compound 8 0.66 6.5 3.4 Compound 11 0.8 12 8.3 Compound 14 0.39 2.6 1.2 Compound 18 1 6.5 1.9 Compound 19 hydrochloride 0.56 3.1 3.7
Conclusion:

(287) It can be seen from table 1 that the present compounds have stronger inhibitory activities on EGFR, HER2, HER4 kinases, and are comparable with CI-1033 hydrochloride in activity; the present compounds have a remarkably better inhibitory activity on the HER2 kinase than gefitinib and erlotinib hydrochloride; and the present compounds have a remarkably better inhibitory activity on the HER4 kinase than erlotinib hydrochloride and lapatinib ditosylate.

(288) Assay 2

(289) In Vitro Cellular Inhibitory Activity of the Present Invention

(290) Samples:

(291) Controls: Gefitinib, erlotinib hydrochloride, purchased from Anqing worldchem Co., LTD. Anqing worldchem Co., LTD.; lapatinib ditosylate, purchased from Taizhou Xingcheng Chempharm Co., Ltd.; CI-1033 hydrochloride, purchased from Shanghai hanxiangchem, Co., Ltd.; and
The present compounds: lab-made, their chemical names and structural formulae are shown in the preparation examples.
Assay Procedures:

(292) The abbreviations used in the following assay have the following meanings:

(293) XTT: 3,3′-Sodium [1-(carbaniloyl)-3,4-tetrazolium]-di(4-methoxy-6-nitro)benzene-sulfonate/2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide salt, purchased from Amresco Ltd.;

(294) RPMI1640: a medium designed by Roswell Park Memorial Institute; purchased from Hyclone Company;

(295) FBS: fetal calf serum, purchased from Hyclone Company;

(296) PBS: phosphate buffer, purchased from Homemade Company.

(297) 1. Formulating the Agents and the Compounds

(298) 1) Formulating PBS:

(299) NaCl (8 g), KCl (0.2 g), Na.sub.2HPO.sub.4 (1.44 g), and KH.sub.2PO.sub.4 (0.24 g) were added to ultrapure water (800 mL). After adjusting the pH to 7.4, ultrapure water was further added until the volume reached 1 L. The mixture was autoclaved for 20 min.

(300) 2) Formulating the XTT Working Liquor:

(301) XTT powder (100 mg) was taken and, while being kept in darkness, dissolved into 300 ml of the serum-free RPMI1640 culture medium that was warmed to 50° C. and did not contain phenol red. The mixture was filtered, packaged separately, and used immediately or within one week. It is necessary for all of the processes to be kept in darkness.

(302) 3) Formulating Test Compounds

(303) Formulating a Stock Solution of Test Compound:

(304) The compound powder was dissolved into DMSO until a concentration of 10 mM reached.

(305) Formulating Gradient Dilute Solutions of Test Compound:

(306) First, the 10 mM stock solution of test compound was diluted with DMSO in a 4-fold successive gradient for 10 concentrations. 2 μL DMSO-diluted compound was added to 998 μL of the culture medium containing 10% FBS. Therefore, the maximum concentration of the compound is 20 μM, the concentration of DMSO is 0.2%, and there are 10 concentration gradients in total.

(307) 2. Culturing Cells

(308) 1) Thawing Cells:

(309) A cell-freezing tube was removed from liquid nitrogen, and placed in a water bath of 37° C.-39° C. to thaw the cells quickly. A freezing-preserving solution was transferred to 15 ml sterile centrifuge tube, to which was added a culture medium in a volume 10 times larger than that of the freezing-preserving solution. The mixture was centrifuged at 1000 rpm at 4° C. for 5 min. The culture medium in the centrifuge tube was discarded, and then a culture medium containing 10% FBS was added. The cells were resuspended and transferred to the culture bottle. On the next day, the solution was changed.
2) Passing Cells For the logarithmic growth phase cells, the culture medium was discarded and an appropriate volume of PBS was added to wash the cells once. Then an appropriate volume of a digestive juice containing 0.25% pancreatic enzyme and 0.02% EDTA was added. The solution was placed on stand at 37° C. for 2-5 min, and then washed once with PBS after the digestive juice was discarded. An appropriate volume of a culture medium containing 10% FBS was added to terminate the digestion. The pipette was blown and hit slightly, and the cells were digested down to produce a cell suspension for cell passage and further experiment.
3) Freezing and Preserving Cells For the logarithmic growth phase cells, a digestive juice containing 0.25% pancreatic enzyme and 0.02% EDTA was used to digest cells to produce a cell suspension. The suspension was centrifuged at 1000 rpm at 4° C. for 5 min. The culture medium was discarded and a freezing-preserving solution containing 10% DMSO and 90% FBS was added to resuspend the cells. The cells were packaged separately in the cell-freezing tubes in 2×10.sup.6 cells/tube. The cell-freezing tubes were placed in a programmed cooling cassette, kept at −80° C. for 24 hours, and then transferred to liquid nitrogen for freezing and preserving.
3. Plating Cells
1) Preparing the Cell Suspension The culture medium was removed from the culture bottle. The cells were rinsed twice with PBS. The pancreatic enzyme was added to digest cells. The digested cells were collected by centrifuge. The cells were resuspended with a culture medium containing 10% fetal calf serum, counted and adjusted to an appropriate concentration (the cell viability should be over 90%). The cell concentration was 5×10.sup.4/ml.
2) The cell suspension was added to the 96-well plate, 100 μL per well.
3) The plate was placed in the incubator and incubated at 37° C. under 5% CO.sub.2 overnight.
4. Treating with Drugs

(310) Drugs were added to the cell culture plate. The plate was placed in the incubator and incubated at 37° C. under 5% CO.sub.2 for 72 hours.

(311) 5. Testing the Cell Viability with the XTT Method

(312) The XTT working solution was added to the plate. The plate was placed in the incubator and incubated at 37° C. under 5% CO.sub.2 for 2 hr. Then the plate was placed in a microplate reader to read the absorbance at 450 nm.

(313) 6. Data Processing

(314) 1) The percent inhibition was calculated by the following calculation.
% inhibitor=(Absorbance(medium)−Absorbance(Compound))/(Absorbance(medium)−Absorbance(positive control)×100%;

(315) 2) Data were input into GraphPad Prism 5.0 to plot a curve and obtain IC.sub.50.

(316) Result:

(317) TABLE-US-00004 TABLE 2 in vitro cellular inhibitory activities on H1975 (NSCLC, nonsmall-cell lung cancer) H1975 Cells Compound IC.sub.50 (nM) erlotinib hydrochloride 3985.0 lapatinib ditosylate 4534.0 CI-1033 hydrochloride 157.3 Compound 8 305.6 Compound 18 92.3 Compound 19 hydrochloride 104.5

(318) TABLE-US-00005 TABLE 3 in vitro cellular inhibitory activities on Calu-3(NSCLC, nonsmall-cell lung cancer) Calu-3 Cells Compound IC.sub.50 (nM) erlotinib hydrochloride 1319.0 lapatinib ditosylate 94.3 CI-1033 hydrochloride 685.6 Compound 18 38.2

(319) TABLE-US-00006 TABLE 4 in vitro cellular inhibitory activities on A431(Epidermoid carcinoma) A431 cells Compounds IC.sub.50 (nM) erlotinib hydrochloride 1269.0 lapatinib ditosylate 3282.0 CI-1033 hydrochloride 402.4 Compound 19 hydrochloride 114.0 Note: The cells H1975, Calu-3 and A431 used in the above assay were available from Chinese Vendor.
Conclusions:

(320) It can be seen from Table 2 that the cellular proliferation inhibition effect of the present compounds on H1975 (NSCLC, nonsmall-cell lung cancer) is remarkably superior to erlotinib hydrochloride and lapatinib ditosylate.

(321) It can be seen from Table 3 that the cellular proliferation inhibition effect of the present compounds on Calu-3 (NSCLC, nonsmall-cell lung cancer) is superior to lapatinib ditosylate, and remarkably superior to erlotinib hydrochloride and CI-1033 hydrochloride.

(322) It can be seen from Table 4 that the cellular proliferation inhibition effect of the present compounds on A431 (Epidermoid carcinoma) is superior to CI-1033 hydrochloride, and remarkably superior to erlotinib hydrochloride and lapatinib ditosylate.