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 method of treating ovarian cancer, colorectal cancer, mammary cancer, glioma, non-small cell lung cancer, bladder carcinoma, gastric cancer, adenocarcinoma of the esophagus, esophageal squamous cell cancer, head and neck cancer, or pancreatic cancer, which comprises a step of administering a compound represented by a general formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof to a mammal in need thereof, ##STR00252## wherein R.sup.1 is selected from the group consisting of: ##STR00253## ##STR00254## R.sup.2 is selected from the group consisting of hydrogen, methyl that is unsubstituted or substituted by 1-2 Q.sub.2 substituent(s) and ethyl that is unsubstituted or substituted by 1-2 Q.sub.2 substituent(s), Q.sub.2 is selected from the group consisting of: a di(C.sub.1-4alkyl)amino group, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, furyl, cyclopropyl, cyclopentyl, pyrrolyl, pyridyl, pyrimidinyl, and thiazolyl; 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.

2. A method according to claim 1, 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 substituent(s) and ethyl that is unsubstituted or substituted by 1-2 Q.sub.2 substituent(s), Q.sub.2 is selected from the group consisting of dimethylamino, diethylamino, piperidinyl, piperazinyl and morpholinyl; 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 method according to claim 1, 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, (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, 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-(2-((1R,5S,6S)-3-methyl-3-azabicyclo[3.1.0]hexan-6-yl)ethoxy)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-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]-6-hexyl)-ethoxy)quinazolin-6-yl)-4-dimethylamino]-crotonamide, (E)-N-[4-(3-chloro-4-fluorophenylamino)-7-(((spiro[3.5]octan-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.

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

(15) I. Preparation Examples for the Present Compound

(16) 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:

(17) ##STR00039##

(18) The steps were as follows:

(19) Reaction Procedures:

(20) ##STR00040##

(21) 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-6nitro-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)

(22) ##STR00041##

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

(23) ##STR00042##

(24) 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

(25) ##STR00043##

(26) 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

(27) ##STR00044##

(28) 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

(29) ##STR00045##

(30) The product from the previous step, (E)-N-[7-(8-oxabicyclo[3.2.11]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%.

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

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

(33) .sup.1HNMR: (400 MHz, CDCl.sub.3) 9.13 (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)

(34) ##STR00046##

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

(35) ##STR00047##

(36) 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

(37) ##STR00048##

(38) 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

(39) ##STR00049##

(40) 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

(41) ##STR00050##

(42) 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%.

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

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

(45) .sup.1HNMR: (400 MHz, CDCl.sub.3) 9.13 (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)

(46) ##STR00051##

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

(47) ##STR00052##

(48) 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

(49) ##STR00053##

(50) 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

(51) ##STR00054##

(52) 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

(53) ##STR00055##

(54) (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%.

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

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

(57) .sup.1HNMR: (400 MHz, CDCl.sub.3) 9.00 (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)

(58) ##STR00056##

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

(59) ##STR00057##

(60) 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

(61) ##STR00058##

(62) 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

(63) ##STR00059##

(64) 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%.

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

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

(67) .sup.1HNMR: (400 MHz, DMSO-d.sub.6) 9.78 (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)

(68) ##STR00060##

(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

(69) ##STR00061##

(70) 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

(71) ##STR00062##

(72) 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

(73) ##STR00063##

(74) 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%.

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

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

(77) .sup.1HNMR: (400 MHz, CDCl.sub.3) 9.15 (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)methoxy)quinazolin-6-yl]-acrylamide (Compound 6)

(78) ##STR00064##

(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

(79) ##STR00065##

(80) (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

(81) ##STR00066##

(82) 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)methoxy)quinazolin-6-yl]-acrylamide

(83) ##STR00067##

(84) 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)methoxy)quinazolin-6-yl]-acrylamide (15 mg) in a yield of 14%.

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

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

(87) .sup.1HNMR: (400 MHz, CDCl.sub.3) 9.10 (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)

(88) ##STR00068##

(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

(89) ##STR00069##

(90) 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

(91) ##STR00070##

(92) 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=I/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

(93) ##STR00071##

(94) 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%.

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

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

(97) .sup.1HNMR (400 MHz, CDCl.sub.3) 8.92 (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)

(98) ##STR00072##

(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

(99) ##STR00073##

(100) 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-ylethoxy)-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

(101) ##STR00074##

(102) 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 (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

(103) ##STR00075##

(104) 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%.

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

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

(107) .sup.1HNMR (400 MHz, CDCl.sub.3) 9.15 (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)

(108) ##STR00076##

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

(109) ##STR00077##

(110) (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

(111) ##STR00078##

(112) 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

(113) ##STR00079##

(114) 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%.

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

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

(117) .sup.1HNMR (400 MHz, CDCl.sub.3) 8.79 (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)

(118) ##STR00080##

(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

(119) ##STR00081##

(120) (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

(121) ##STR00082##

(122) 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

(123) ##STR00083##

(124) 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%.

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

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

(127) .sup.1HNMR (400 MHz, CDCl.sub.3) 9.08 (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)

(128) ##STR00084##

(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

(129) ##STR00085##

(130) 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

(131) ##STR00086##

(132) 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

(133) ##STR00087##

(134) 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-evaporated 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%.

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

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

(137) .sup.1HNMR (400 MHz, CDCl.sub.3) 9.13 (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)

(138) ##STR00088##

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

(139) ##STR00089##

(140) (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

(141) ##STR00090##

(142) 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

(143) ##STR00091##

(144) 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%.

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

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

(147) .sup.1HNMR (400 MHz, CDCl.sub.3) 9.35 (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)

(148) ##STR00092##

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

(149) ##STR00093##

(150) (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-nitroquinazolin-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

(151) ##STR00094##

(152) N-(4-(3-chloro-4-fluorophenyl))-7-((6-methyl-6-azaspiro[2.5]octan-1-yl)methoxy)-6-nitroquinazolin-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

(153) ##STR00095##

(154) 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%.

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

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

(157) .sup.1HNMR (400 MHz, CDCl.sub.3) 9.13 (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)

(158) ##STR00096##

(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

(159) ##STR00097##

(160) 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

(161) ##STR00098##

(162) 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

(163) ##STR00099##

(164) 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%.

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

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

(167) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.14 (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)

(168) ##STR00100##

(169) 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. 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) 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) ##STR00101##

(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. 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%.

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

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

(177) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.08 (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

(178) ##STR00102##

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

(179) 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

(180) 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)quinazoline4,6-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

(181) (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%.

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

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

(184) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.0 (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)

(185) ##STR00103##

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

(186) ##STR00104##

(187) (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

(188) ##STR00105##

(189) 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

(190) ##STR00106##

(191) 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%.

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

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

(194) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.12 (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

(195) ##STR00107##

(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

(196) ##STR00108##

(197) 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

(198) ##STR00109##

(199) 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

(200) ##STR00110##

(201) 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

(202) 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%.

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

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

(205) .sup.1HNMR (400 MHz, DMSO-d.sub.6) 10.72 (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)

(206) ##STR00111##

(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

(207) ##STR00112##

(208) 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. 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

(209) ##STR00113##

(210) 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%.

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

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

(213) .sup.1HNMR (400 MHz, DMSO-d.sub.6) 9.79 (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)

(214) ##STR00114##

(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

(215) ##STR00115##

(216) 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

(217) ##STR00116##

(218) 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

(219) ##STR00117##

(220) 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

(221) ##STR00118##

(222) 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%.

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

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

(225) .sup.1HNMR (400 MHz, CD.sub.3OD) 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)

(226) ##STR00119##

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

(227) ##STR00120##

(228) (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

(229) ##STR00121##

(230) 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

(231) ##STR00122##

(232) ((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

(233) ##STR00123##

(234) 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%.

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

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

(237) .sup.1HNMR (400 MHz, CD.sub.3OD) 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)

(238) ##STR00124##

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

(239) ##STR00125##

(240) 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

(241) ##STR00126##

(242) 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

(243) ##STR00127##

(244) 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%.

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

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

(247) .sup.1HNMR (400 MHz, DMSO-d.sub.6) 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).

(248) 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.

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

(250) TABLE-US-00002 No. R.sup.1 R.sup.2 (R.sup.3)n L 24 H 3-Cl, 4-F O 25 0 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 3-Cl, 4-F O 34 0 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 3-Cl, 4-F O 40 0 3-Cl, 4-F O 41 3-Cl, 4-F O 42 3-Cl, 4-F O 43 3-Cl, 4-F O 44 3-Cl, 4-F O 45 0 3-Cl, 4-F O 46 3-Cl, 4-F O 47 3-Cl, 4-F O 48 3-Cl, 4-F O 49 3-Cl, 4-F O 50 0 3-Cl, 4-F O 51 3-Cl, 4-F O 52 3-Cl, 4-F O 53 3-Cl, 4-F O 54 3-Cl, 4-F O 55 0 3-Cl, 4-F O 56 3-Cl, 4-F O 57 3-Cl, 4-F S 58 3-Cl, 4-F O 59 3-Cl, 4-F S 60 0 3-Cl, 4-F O 61 3-Cl, 4-F O 62 3-Cl, 4-F NH 63 3-Cl, 4-F NH 64 3-Cl, 4-F O 65 00 01 3-Cl, 4-F O 66 02 03 3-Cl, 4-F O 67 04 05 3-Cl, 4-F O 68 06 07 3-Cl, 4-F O 69 08 09 3-Cl, 4-F O 70 0 3-Cl, 4-F O 71 3-Cl, 4-F O 72 3-Cl, 4-F O 73 3-Cl, 4-F S 74 3-Cl, 4-F O 75 0 3-Cl, 4-F O 76 3-Cl, 4-F O 77 3-Cl, 4-F O 78 3-Cl, 4-F O 79 3-Cl, 4-F O 80 0 3-Cl, 4-F NH 81 3-Cl, 4-F S 82 3-Cl, 4-F S 83 3-Cl, 4-F O 84 3-Cl, 4-F O 85 0 3-Cl, 4-F O 86 3-Cl, 4-F O 87 3-Cl, 4-F O 88 3-Cl, 4-F O 89 3-Cl, 4-F O
II. In Vitro Assays for the Antineoplastic Activities of the Present Compounds

(251) 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.

(252) Assay 1

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

(254) Samples:

(255) 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

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

(257) Assay Procedures:

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

(259) HEPES: hydroxyethyl piperazine ethanesulfonic acid;

(260) Brij-35: polyoxyethylene lauryl ether;

(261) DTT: dithiothreitol;

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

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

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

(265) ATP: adenosine triphosphate (Sigma);

(266) DMSO: dimethyl sulfoxide;

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

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

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

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

(271) (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);

(272) (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);

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

(274) (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);

(275) (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);

(276) (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);

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

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

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

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

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

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

(283) The results are shown below.

(284) 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:

(285) 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.

(286) Assay 2

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

(288) Samples:

(289) 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

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

(291) 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)benzenesulfonate/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 L DMSO-diluted compound was added to 998 L of the culture medium containing 10% FBS. Therefore, the maximum concentration of the compound is 20 M, 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.

(310) 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.

(311) 2) Passing Cells

(312) 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.

(313) 3) Freezing and Preserving Cells

(314) 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 210.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.

(315) 3. Plating Cells

(316) 1) Preparing the cell suspension

(317) 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 510.sup.4/ml.

(318) 2) The cell suspension was added to the 96-well plate, 100 L per well.

(319) 3) The plate was placed in the incubator and incubated at 37 C. under 5% CO.sub.2 overnight.

(320) 4. Treating with Drugs

(321) 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.

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

(323) 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.

(324) 6. Data Processing

(325) 1) The percent inhibition was calculated by the following calculation.
% inhibitor=(Absorbance(medium)Absorbance(Compound))/(Absorbance(medium)Absorbance(positive control)100%;
2) Data were input into GraphPad Prism 5.0 to plot a curve and obtain IC.sub.50.
Result:

(326) Table 2 in vitro cellular inhibitory activities on H1975 (NSCLC, nonsmall-cell lung cancer)

(327) TABLE-US-00004 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

(328) Table 3 in vitro cellular inhibitory activities on Calu-3 (NSCLC nonsmall-cell lung cancer)

(329) TABLE-US-00005 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

(330) Table 4 in vitro cellular inhibitory activities on A431 (Epidermoid carcinoma)

(331) TABLE-US-00006 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

(332) Note: The cells H1975, Calu-3 and A431 used in the above assay were available from Chinese Vendor.

(333) Conclusions:

(334) 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.

(335) 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.

(336) 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.

(337) Assay 3-1 In Vitro Cellular Inhibitory Activities on Bladder Carcinoma of the Compound of the Present Invention

(338) Material

(339) Samples:

(340) Compound 18, lab-made. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(341) Control Drug:

(342) AZD9291 is purchased or lab-made according to the existing technique and method with the following structural formula.

(343) ##STR00250##
Method

(344) The effect of the compound to be tested on 5637 (bladder carcinoma) cell proliferation was tested using CTG kit.

(345) 1. Formulating the Compounds

(346) 535 l DMSO was added into an EP tube filled with 3.32 mg of Compound 18 and evenly shaken (10 mM);

(347) 590 l DMSO was added into an EP tube filled with 3.16 mg of Compound AZD9291 and evenly shaken (10 mM);

(348) Formulating a stock solution of a 1000-fold compound:

(349) The compound was diluted with DMSO by 4-fold from 10 mM to 2.5 mM, 625 M, 156 M, 39 M, 9.8 M, 2.4 M, 0.61 M, 0.15 M, 0.04 M in turn.

(350) Formulating a stock solution of a 10-fold compound: 2 l 1000-fold Compound 18 or AZD9291 was respectively added into a 198 l culture medium and evenly shaken.

(351) 2. Method for Culturing Cells

(352) The specific cell culture method is the same as Assay 2.

(353) 3. Plating Cells

(354) 1) Preparing cell suspension

(355) a The culture medium was removed from the culture bottle;

(356) b The cells were rinsed twice with PBS;

(357) c The pancreatic enzyme was added to digest cells. The digested cells were collected by centrifuge;

(358) d The cells were resuspended with a culture medium containing 10% fetal bovine serum,

(359) counted and adjusted to an appropriate concentration;

(360) 2) The cell suspension was added to the 384-well plate, with 22.5 l per well.

(361) 3) The plate was placed in the incubator and incubated at 37 C. under 5% CO.sub.2 overnight.

(362) 4. Treating with Drugs

(363) 1) Plating in the 384-well plate with 250 cells/well/22.5 ul;

(364) 2) After 24 hours, each well was added with 2.5 ul 10-fold drug which was well diluted to make the final concentration of 10 M, 2.5 M, 625 nM, 156 nM, 39 nM, 9.8 nM, 2.5 nM, 0.610 nM, 0.153 nM, 0.04 nM.

(365) 3) The plate was placed in the CO.sub.2 incubator and incubated at 37 C. for 72 hours

(366) 5. Testing the Cell Viability with the CTG Method

(367) 1) Formulating CellTiter-Glo agent.

(368) 2) Each well was added with 25 ul CellTiter-Glo agent, mixed for 2 minutes and placed at room temperature while being kept in darkness for 10 minutes.

(369) 3) Reading the absorbance using EnVision 2104 Multilabel Reader.

(370) 6. Data Processing

(371) 1) The percent inhibition was calculated by the following calculation:
% inhibition=(Absorbance(medium)Absorbance(Compound))/(Absorbance(medium)Absorbance(blank control)100%;

(372) wherein the medium group was free of the compound to be tested; the blank control group was free of cells (replaced with a pure culture medium) and the compound to be tested. The other test conditions for the medium group and the blank control group were the same as the compound group.

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

(374) Result

(375) TABLE-US-00007 TABLE 5 Inhibitory activities on 5637 cells of the present compound 5637 Cells Compound IC.sub.50 (nM) Compound 18 38.6 AZD9291 282.5
Conclusion

(376) The cellular proliferation inhibitory effect of Compound 18 of the present application on 5637 (bladder carcinoma) is superior to control AZD9291.

(377) Assay 3-2 In Vitro Cellular Inhibitory Activity on Mammary Cancer of the Compound of the present Invention

(378) Material

(379) Samples:

(380) Compound 18, lab-made, its chemical name, structural formula and the preparation method are shown in the preparation examples.

(381) Controls:

(382) Erlotinib is purchased or lab-made according to the existing technique and method with the following structural formula shown in the background art.

(383) BT474 cells (human breast ductal carcinoma cell, purchased from ATCC).

(384) Method

(385) 1. Formulating the Agents and the Compounds

(386) 1) Formulating XTT Solution

(387) The formulating method is the same as in Assay 2.

(388) 2) Formulating the Compound

(389) a Formulating a stock solution of test compound: the compound powder was dissolved into DMSO until a concentration of 10 mM reached.

(390) b Formulating gradient dilute solutions of test compound: First, the 10 mM stock solution of test compound was diluted with DMSO in a 2.5-fold gradient, i.e., the achieved concentration is 4 mM. Then it was diluted with DMSO in a 4-fold successive gradient. There were 10 concentration gradients in total. 2 L DMSO-diluted compound each was added to 198 L of the culture medium containing 10% FBS respectively. Therefore, the maximum concentration of the compound to be tested is 40 M and the concentration of DMSO is 1%.

(391) 2. Method for Culturing Cells

(392) The specific method for culturing cells is the same as Assay 2.

(393) 3. Plating Cells

(394) 1) Preparing the cell suspension

(395) a The culture medium was removed from the culture bottle;

(396) b The cells were rinsed twice with PBS;

(397) c The pancreatic enzyme was added to digest cells. The digested cells were collected by centrifuge;

(398) d The cells were resuspended with a culture medium containing 10% fetal bovine serum, counted and adjusted to an appropriate concentration;

(399) 2) The cell suspension was added to the 96-well plate with 150 l per well.

(400) 3) The plate was placed in the incubator and incubated at 37 C. under 5% CO.sub.2 overnight.

(401) 4. Treating with Drugs

(402) 1) Each well was added 50 l growth medium containing 10% FBS; and 50 l diluted compound was added into the well. The maximum concentration of the test compound was 10 M, and there were 10 concentration gradients in total. For each compound, the operation was duplicated. The final concentration of DMSO was 0.25%.

(403) 2) The plate was placed in the CO.sub.2 cell incubator and incubated at 37 C. for 72 hours.

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

(405) 1) Formulating XTT test working solution

(406) 2) The culture medium was removed; each well was added with 150 l XTT working solution.

(407) 3) The plate was placed in the incubator and incubated at 37 C. under 5% CO.sub.2 for 1-5 hours.

(408) 4) Slightly and evenly shaking before reading the absorbance. Then the plate was placed in a microplate reader to read the absorbance at 450 nm.

(409) 6. Data Processing

(410) 1) The percent inhibition was calculated by the following calculation:
% inhibition=(Absorbance(medium)Absorbance(Compound))/(Absorbance(medium)Absorbance(blank control)100%;

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

(412) Result

(413) TABLE-US-00008 TABLE 6 Inhibitory activities on BT474 cell of the compound of the present invention BT474 Compound IC.sub.50 (nM) Compound 18 9.7 Erlotinib 5430.0
Conclusion

(414) The compound of the present invention plays a good role in cellular proliferation activity inhibition on BT474 human breast ductal carcinoma cell and the inhibition effect is remarkably superior to control Erlotinib.

(415) Assay 3-3 In Vitro Cellular Inhibitory Activities on NCI-N87 (Gastric Carcinoma) of the Compound of the Present Invention

(416) The Material and Method

(417) Samples:

(418) Compound 18, lab-made, its chemical name, structural formula and preparation method are shown in the preparation examples.

(419) Controls:

(420) Erlotinib and Lapatinib are purchased or lab-made according to the existing technique and method with the following structural formula shown in the background art.

(421) Method

(422) 1. Formulating the Agents

(423) 1) Formulating the XTT Solution

(424) The formulating method is the same as Assay 2.

(425) 2) Formulating the Compound

(426) a Formulating a stock solution of test compound: the compound powder was dissolved into DMSO until a concentration of 10 mM reached.

(427) b Formulating gradient dilute solutions of test compound: first, the 10 mM stock solution of test compound was diluted with DMSO in a 4-fold successive gradient, and there are 10 concentration gradients in total. 2 L DMSO-diluted compound each was added to 198 L of the culture medium containing 10% FBS respectively. Therefore, the maximum concentration of the compound to be tested is 40 M, the concentration of DMSO is 1%.

(428) 2. Method for Culturing Cells

(429) The method for culturing cells is the same as Assay 2.

(430) 3. Plating Cells

(431) The method for plating cells is the same as Assay 3-2.

(432) 4. Treating with Drugs

(433) 1) 50 l of the above diluted compound was added into each well of the cell culture plate. The maximum concentration of the test compound was finally made 10 m and there are 10 concentration gradients in total. For each compound, the above operation was triplicated. The final concentration of DMSO was 0.25%.

(434) 2) The plate was placed in the CO.sub.2 incubator and incubated at 37 C. for 72 hours.

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

(436) 1) Formulating the XTT test working solution

(437) 2) The culture medium was removed and each well was added with 150 l XTT working solution.

(438) 3) The plate was placed in the incubator and incubated at 37 C. under 5% CO.sub.2 for 1-5 hours.

(439) 4) Slightly and evenly shaking before reading the absorbance. Then the plate was placed in a microplate reader to read the absorbance at 450 nm.

(440) 6. Data Processing

(441) 1) The percent inhibition was calculated by the following calculation:
% inhibition=(Absorbance(medium)Absorbance(Compound))/(Absorbance(medium)Absorbance(blank control)100%

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

(443) Result

(444) TABLE-US-00009 TABLE 7 Inhibitory activities on NCI-N87 (gastric carcinoma) of the compound of the present invention NCI-N87 cell Compound IC.sub.50 (nM) Compound 18 7.1 Erlotinib 2108.0 Lapatinib 11.9
Conclusion

(445) It can be seen from Table 7 that the compound of the present invention is remarkably superior to control Erlotinib and slightly superior to control Lapatinib in terms of inhibitory effect on NCI-N87 (gastric carcinoma) cellular proliferation. It exhibits superior cellular proliferation inhibitory activity.

(446) Assay 4 In Vivo Antitumor Activity of the Compound of the Present Invention

(447) Assay 4-1 In Vivo Pharmacodynamic Evaluation on Bladder Carcinoma of the Compound of the Present Invention

(448) Material

(449) Samples:

(450) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(451) Control Drugs:

(452) Neratinib and Lapatinib are purchased or lab-made according to the existing technique and method; the structural formula of Lapatinib is as mentioned in the background art; the structural formula of Neratinib is as follows:

(453) ##STR00251##

(454) Experimental animal: BALB/c nude mice purchased from Beijing Huafukang Bioscience Co. Ltd.

(455) Method

(456) Animal Modeling and Grouping:

(457) 5637 (bladder carcinoma) cells were cultured in RPMI1640 culture medium containing 10% FBS. 5637 cells in exponential phase of growth were collected and were resuspended to an appropriate concentration with PBS. The tumor was inoculated subcutaneously on the right side of the nude mice's back.

(458) The growth of the tumor was observed regularly. When the tumor grew to an average of about 100 mm.sup.3, the mice were randomly grouped and administrated according to the size of the tumor and the weight of the mice. The experiment was divided into treatment group of sample compound 18 (30 mg/kg, 20 mg/kg and 10 mg/kg), treatment group of positive control Neratinib and treatment group of positive control Lapatinib and medium control group (sterile water for injection). There were 8 animals in each group which were orally intragastrical administered once a day. They were administered totally 32 times. The tumor volume (TV) was measured with an ernier caliper. In the whole process of the experiment, the weight and tumor size of the mice were measured twice a week. The efficacy was evaluated according to the relative tumor growth inhibition (TGI) value.

(459) Experimental Observation Index and Calculation:

(460) tumor volume TV=0.5 ab.sup.2, wherein a is the long diameter and b is the short diameter of the tumor.

(461) Relative Tumor Growth Inhibition TGI (%)=(1T/C)100%. T and C are respectively the relative tumor volume (RTV) at a certain point-in-time of the treatment group and control group.

(462) T/C % is the relative tumor proliferation rate, i.e., the percentage ratio of relative tumor volume of the treatment group to that of control group at a certain point-in-time. The calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV100% (T.sub.RTV: the mean RTV of the treatment group; C.sub.RTV: the mean RTV of the medium control group; R.sub.TV=V.sub.t/V.sub.0, V.sub.0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

(463) Statistical Method:

(464) All the experiment results were represented by the average tumor volumeSE (Standard Error) and analyzed by means of the test method of one way ANOVA. All the data were analyzed using SPSS 18.0. p<0.05 represents significant difference.

(465) Result:

(466) TABLE-US-00010 TABLE 8 The TGI and T/C value of each group in 5637 human bladder carcinoma model 32 days after the adminstration P Value Experimental Tumor Volume Relative TGI T/C (Comparison with Groups Dosage (mm.sup.3) Tumor Volume %) (%) the control group) Medium control 319 38 3.21 0.44 Compound 18 30 mg/kg 45 9 0.43 0.08 87 13 0.005 20 mg/kg 49 7 0.51 0.08 84 16 0.006 10 mg/kg 64 21 0.68 0.22 79 21 0.007 Neratinib 80 mg/kg 61 6 0.60 0.05 81 19 0.008 Lapatinib 200 mg/kg 41 7 0.41 0.07 87 13 0.005
Conclusion

(467) Compound 18 has significant antitumor effect on 5637 human bladder carcinoma model under all the conditions set by the present experiment and is remarkably superior to the control drugs. The mice with tumor can tolerate Compound 18 in each tested dosage.

(468) Assay 4-2 In Vivo Pharmacodynamic Evaluation on Esophageal Carcinoma of the Compound of the Present Invention

(469) Material

(470) Samples:

(471) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(472) Experimental Animals:

(473) BALB/c nude mice, 6-8 weeks (weeks of age of the mice upon tumor cell inoculation) purchased from Beijing Huafukang Bioscience Co. Ltd.

(474) Method

(475) Animal Modeling and Grouping:

(476) KYSE270 cells were cultured in RPMI1640 culture medium containing 10% FBS. KYSE270 (esophageal carcinoma) cells in exponential phase of growth were collected. After the cells were resuspended to an appropriate concentration with PBS, 110.sup.7 KYSE270 cells were inoculated subcutaneously on the right side of the female mice's back.

(477) When the tumor grew to an average of about 123 mm.sup.3, the mice were randomly grouped according to the size of the tumor. The experiment was divided into treatment group of sample compound 18 (10 mg/kg and 20 mg/kg) and the medium control group (sterile water for injection). There were 8 animals in each group which were orally intragastrical administered once a day. They were administered totally 21 times. The tumor volume (TV) was measured with an ernier caliper. The weight and tumor size of the mice were measured twice a week. The efficacy was evaluated according to the relative tumor growth inhibition (TGI).

(478) Experimental Observation Index and Calculation:

(479) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(480) Statistical Method:

(481) All the experiment results were represented by the average tumor volumeSE (Standard Error) and analyzed by means of the test method of one way ANOVA. All the data were analyzed using Games-Howell (heterogeneity of variance) for comparing the significant differences between the tumor volume and tumor weight of the control group and each treatment group. p<0.05 represents significant difference.

(482) Result:

(483) TABLE-US-00011 TABLE 9 The TGI and T/C value of each group in KYSE270 human esophageal carcinoma model 21 days after the adminstration P Value Experimental Tumor Volume Relative TGI T/C (Comparison with Groups Dosage (mm.sup.3) Tumor Volume (%) (%) the control group) Medium control 1974 116 16.66 1.54 Compound 18 10 mg/kg 40 6 0.33 0.05 98 2 <0.001 Compound 18 20 mg/kg 14 3 0.11 0.02 99 1 <0.001
Conclusion

(484) Compound 18 has significant antitumor effect on KYSE270 human esophageal carcinoma model under both the conditions of 10 mg/kg and 20 mg/kg. The mice with tumor can tolerate the tested drugs well.

(485) Assay 4-3 In Vivo Pharmacodynamic Evaluation on Head and Neck Cancer of the Compound of the Present Invention

(486) Material

(487) Samples:

(488) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(489) Control Drugs:

(490) Neratinib is purchased or lab-made according to the existing technique and method; the structural formula is mentioned in Assay 4-1.

(491) HN2170, head and neck cancer from a 63-year-old Asian male patient.

(492) Animals

(493) BALB/c nude mice, 6-8 weeks purchased from Beijing Huafukang Bioscience Co. Ltd.

(494) Agents

(495) Methyl Cellulose, MC (Cat. No.: 9004-67-5, SIGMA);

(496) Tween 80 (Cat. No.: 9005-64-5, Amresco).

(497) Method

(498) Animal Modeling and Grouping:

(499) Tumor tissues were collected from mice with tumor of human-derived xenograft model HN2170 and cut into tumor blocks of a diameter of 2-4 mm which were then inoculated subcutaneously on the right anterior portion of the mice. When the average tumor volume reached about 100-150 mm.sup.3, the mice were grouped randomly and the day for grouping was recorded as Day 0. The mice were divided into three experimental groups including the 20 mg/kg group of test Compound 18, 80 mg/kg group of Neratinib and vehicle medium group (0.5% MC/0.4% Tween 80 sterile aqueous solution for injection). In each group there were 4 animals which were orally intragastrical administered once a day. They were administered totally 25 days. The tumor volume (TV) was measured with an ernier caliper twice a week.

(500) Experimental Observation and Calculation

(501) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(502) Result

(503) TABLE-US-00012 TABLE 10 Inhibitory effect on treating xenograft model HN2170 of the compound of the present invention Tumor Volume 25 days after the adminstration Experimental (mm.sup.3).sup.a Tumor Volume T/C TGI Groups Dosage (Day 0) (mm.sup.3).sup.a (%) (%) Medium control 142.7 22.4 783.6 262.4 Compound 18 20 mg/kg 143.1 22.9 46.3 10.6*** 6.5 93.5 Neratinib 80 mg/kg 142.4 23.4 53.7 10.3*** 7.2 92.8 Notes: .sup.aMean standard error; ***P < 0.001, Comparison with the control group
Conclusion

(504) Test compound 18 of the present experiment has significant tumor growth inhibitory effect on human-derived xenograft model HN2170 and the inhibitory effect is superior to the Neratinib control.

(505) Assay 4-4 In Vivo Pharmacodynamic Evaluation on Mammary Cancer of the Compound of the Present Invention

(506) Material

(507) Samples:

(508) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(509) Controls:

(510) Lapatinib is purchased or lab-made according to the existing technique and method; the structural formula is mentioned in the background art.

(511) Experimental Animals:

(512) BALB/c-nude mice of 6-7 weeks purchased from Shanghai SLAC Laboratory Animal Co., Ltd.

(513) Method

(514) Human breast cancer BT-474 purchased from American Type Culture Collection (ATCC) was cultured with DMEM culture medium containing 10% FBS.

(515) After the cells were collected by centrifuge, the cells were inoculated subcutaneously on the nude mice (910.sup.6 cells/mouse). When the tumor grew to 100-200 mm.sup.3, the animals were divided into four groups randomly including 20 mg/kg group and 10 mg/kg group of Compound 18, 200 mg/kg group of Lapatinib and vehicle medium group (10% Hydroxypropyl beta cyclodextrin). There were 8 animals in each group which were orally intragastrical administered once a day. The day for grouping was recorded as Day 0 (DO). They were administered totally 21 times. The tumor volume was measured with an ernier caliper twice a week and the data were recorded.

(516) Experimental Observation Index and Calculation:

(517) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(518) Statistical Method:

(519) The significant differences in RTV between the treatment groups and the control groups were tested using t test. In case of heterogeneity of variance between the samples, t test was used for testing. P<0.05 indicates significant differences.

(520) Result:

(521) TABLE-US-00013 TABLE 11 Efficacy on human breast cancer BT-474 nude mice transplanted tumor of the compound of the present invention 21 days after the adminstration Mean Tumor Mean Tumor Relative P Value Experimental Volume (mm.sup.3) Volume (mm.sup.3) Tumor Volume TGI T/C (Comparison with Groups Dosage D0(X S) D21(X S) D21(X S) (%) (%) the control group) Medium control 141.9 6.4 1924.6 165.4 13.6 1.0 100 Compound 18 10 mg/kg 140.5 8.0 447.0 91.4 3.1 0.6 77 23 0.000 Compound 18 20 mg/kg 136.7 8.8 171.6 52.9 1.2 0.3 91 9 0.000 Lapatinib 200 mg/kg 136.6 7.0 245.8 52.2 1.8 0.4 87 13 0.000
Conclusion

(522) Compound 18 significantly inhibits the growth of HER2-high expression human breast cancer BT-474 nude mice transplanted tumor in each tested dosage. The inhibitory effect is significantly superior to the Lapatinib control. The mice with tumor can tolerate Compound 18 well.

(523) Assay 4-5 In Vivo Pharmacodynamic Evaluation on Gastric Cancer of the Compound of the Present Invention

(524) Material

(525) Samples:

(526) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(527) Experimental Animal:

(528) BALB/c nude mice of 6-8 weeks purchased from Beijing Anikeeper Biosciece Co. Ltd.

(529) Method

(530) Animal Modeling and Grouping:

(531) NCI-N87 tumor cells (purchase from ATCC) was cultured with RPMI-1640 culture medium containing inactivated 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM glutamine. After the tumor cells in logarithmic growth phase were resuspended with RPMI1640 free of serum, the cells were inoculated subcutaneously in the right coastal region of the experimental animals in an appropriate concentration.

(532) When the tumor grew to about 150 mm.sup.3, the mice were grouped and administrated. There were 2 groups in total, each with 8 mice, including solvent control group (sterile water for injection) and treatment group of Compound 18. The dosage was 20 mg/kg and the day for grouping was recorded as Day 0 (DO). The mice were orally intragastrical administered once a day and totally administered for 28 days. The size of the tumor was measured twice a week. The relationship between the mice tumor volume and the administration time was recorded. The tumor volume ratio (T/C) and the tumor growth inhibition (TGI) of the treatment group and solvent control group were calculated for statistical analysis.

(533) Measurement of the Tumor and Experimental Index

(534) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(535) Statistical Method:

(536) One-Way ANOVA test was carried out using statistical software SPSS 17.0. Inter group statistical analysis of the tumor volume was performed. p<0.05 represents significant difference.

(537) Result:

(538) TABLE-US-00014 TABLE 12 The antitumor effect on nude mice with tumor of NCI-N87 human-derived gastric cancer xenograft of the compound of the present invention 28 days after the adminstration Experimental Tumor Volume Tumor Relative T/C TGI Groups Dosage (mm.sup.3).sup.a Volume(%).sup.a (%) (%) P Solvent control group 2,359 177 1,474 70 Compound 18 20 mg/kg 358 32 .sup.228 18 15.4 84.6 <0.001 Notes: .sup.amean standard error;
Conclusion

(539) Test Compound 18 treatment group plays significant inhibitory effect on the growth of tumor in the NCI-N87 human-derived gastric cancer xenograft tumor model. In the treatment, experimental animals of each group tolerated well without obvious adverse reaction.

(540) Assay 4-6 In Vivo Pharmacodynamic Evaluation on Malignant Glioma of the Compound of the Present Invention

(541) Material

(542) Samples:

(543) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(544) Control Drugs:

(545) Neratinib and Lapatinib are purchased or lab-made according to the existing technique and method; the structural formula of Lapatinib is as mentioned in the background art; the structural formula of Neratinib is mentioned in Assay 4-1.

(546) Experimental Animal:

(547) BALB/c nude of 6-8 weeks purchased from Beijing Anikeeper Biosciece Co. Ltd.

(548) Method

(549) Culturing Cells

(550) LN-229 cancer cells were cultured with DMEM culture medium containing inactivated 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM glutamine. After the tumor cells in logarithmic growth phase were resuspended with DMEM culture medium free of serum, the cells were inoculated subcutaneously in the right coastal region of the experimental animals in an appropriate concentration.

(551) When the tumor grew to about 100 mm.sup.3, the mice were grouped and administrated. There were 6 groups in total, each with 8 mice, including solvent control group (sterile water for injection); administration groups of Compound 18 with dosages of 30 mg/kg, 20 mg/kg, and 10 mg/kg; Neratinib administration group with the dosage of 80 mg/kg; Lapatinib administration group with the dosage of 200 mg/kg. The mice were orally intragastrical administered once a day and totally administered for 28 days. The size of the tumor was measured twice a week.

(552) Measurement of the Tumor and Experimental Index

(553) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(554) Statistical Method:

(555) One-Way ANOVA test was carried out using statistical software SPSS17.0. Inter group statistical analysis of the tumor volume was performed. p<0.05 represents significant difference.

(556) Result:

(557) TABLE-US-00015 TABLE 13 The antitumor effect on nude mice with tumor of human-derived LN-229 glioblastoma xenograft of the compound of the present invention 28 days after the adminstration Tumor Volume T/C TGI P Groups Dosage (mm.sup.3).sup.a (%) (%) Value.sup.b Solvent control 570 39 Compound 8 30 mg/kg 121 17 21.3 78.7 <0.001 Compound 18 20 mg/kg 191 11 34.0 66.0 <0.001 Compound 18 10 mg/kg 260 27 45.6 54.4 0.001 Neratinib 80 mg/kg 197 20 34.3 65.7 <0.001 Lapatinib 200 mg/kg 261 42 45.6 54.4 0.002 Notes: .sup.amean standard error; .sup.bComparison with the control group.
Conclusion

(558) Test Compound 18 has inhibitory effect on the tumor growth of mice with LN-229 in three dosages including low dosage (10 mg/kg), medium dosage (20 mg/kg) and high dosage (30 mg/kg) in the LN-229 human-derived glioblastoma xenograft model. The inhibitory effect exhibits obvious dosage-response relationship in the present administration solution.

(559) Assay 4-7 In Vivo Pharmacodynamic Evaluation on Ovarian Cancer of the Compound of the Present Invention

(560) Material

(561) Samples:

(562) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(563) Control Drugs:

(564) Erlotinib and Lapatinib are purchased or lab-made according to the existing technique and method; the structural formulae are as mentioned in the background art.

(565) Experimental Animals:

(566) BALB/c nude of 6-8 weeks purchased from Beijing Huafukang Bioscience Co. Ltd.

(567) Method

(568) Animal Modeling and Grouping:

(569) SKOV-3 cells were cultured in McCoy's 5a culture medium containing 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM glutamine. After the tumor cells in logarithmic growth phase were resuspended with PBS to an appropriate concentration, the cells were inoculated subcutaneously in the right coastal region of the experimental animals. When the tumor grew to about 1000 mm.sup.3, the mice with the tumor were killed. The tumor was then stripped under sterile conditions to remove the necrotic tissues and select the well developed tumor tissues which were inoculated subcutaneously into an animal again. In this way the tumor tissues were subjected to passage for twice in the body of the mice. The tumor tissues after the second passage were cut into tissue blocks of 222 mm using scalpel blade. The tumor blocks were inoculated subcutaneously to the right coastal region of the mice with an inoculation needle.

(570) When the tumor grew to about 100 mm.sup.3, the mice were grouped and administrated. There were 5 groups in total, each with 8 mice, including solvent control group (the 10% Hydroxypropyl beta cyclodextrin formulated with water for injection); group of Compound 18 with a dosage of 20 mg/kg and group of Compound 18 with a dosage of 10 mg/kg; group of Erlotinib with a dosage of 50 mg/kg; and group of Lapatinib with a dosage of 80 mg/kg. The mice were orally intragastrical administered once a day for 14 days in total. The tumor size was measured twice a week with an ernier caliper.

(571) Measurement of Tumor and Experimental Index:

(572) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(573) Statistical Method:

(574) One-Way ANOVA test was carried out using statistical software SPSS 17.0. Inter group statistical analysis of the tumor volume was performed. p<0.05 represents significant difference.

(575) Result:

(576) TABLE-US-00016 TABLE 14 The inhibitory effect on the tumor growth of SKOV-3 human-derived xenografts model in ovarian cancer of the compound of the present invention 14 days after the adminstration Tumor Volume T/C TGI Groups Dosage (mm.sup.3).sup.a (%) (%) P.sup.b Solvent control 1603 160 Compound 18 20 mg/kg 540 86 32.1 67.9 0.003 Compound 18 10 mg/kg 798 88 49.1 50.9 0.023 Erlotinib 50 mg/kg 1349 142 83.0 17.0 0.994 Lapatinib 80 mg/kg 1339 167 83.4 16.6 0.997 Notes: .sup.amean standard error; .sup.bComparison with the control group.
Conclusion

(577) Compound 18 of the present invention has significant inhibitory effect on the tumor growth of mice with SKOV-3 in dosage of 20 mg/kg. However, Erlotinib and Lapatinib controls do not produce significant antitumor effect in the present model.

(578) Assay 4-8 In Vivo Pharmacodynamic Evaluation on Colon Cancer of the Compound of the Present Invention

(579) Material

(580) Samples:

(581) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(582) Control Drug:

(583) Neratinib is purchased or lab-made according to the existing technique and method; the structural formulae are as mentioned in Assay 4-1.

(584) Tumor Cell:

(585) CR0205, colorectal cancer from a 53-year-old Asian male patient.

(586) Animals:

(587) BALB/c nude mice of 6-8 weeks purchased from Beijing Huafukang Bioscience Co. Ltd.

(588) Agents:

(589) Methyl Cellulose (MC) (Cat. No.: 9004-67-5, SIGMA); Tween 80 (Cat. No.: 9005-64-5, Amresco).

(590) Method

(591) Tumor tissues were collected from mice with tumor of human-derived xenografts model CR0205 and cut into tumor blocks of a diameter of 2-4 mm which were then inoculated subcutaneously in the right anterior portion of the mice. When the average tumor volume reached 100-150 mm.sup.3, the mice were randomly grouped, the day for grouping being recorded as Day 0. The mice were randomly divided into three experimental groups according to the tumor volume, including group of Compound 18 with a dosage of 20 mg/kg, group of Neratinib with a dosage of 80 mg/kg, and medium control group (0.5% MC/0.4% Tween 80 sterile aqueous solution for injection). There were five animals in each group which were orally intragastrical administered once a day for 23 days in total. The tumor volume (TV) was measured with an ernier caliper twice a week.

(592) Experimental Observation and Calculation:

(593) The calculation formulae for The tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(594) Statistical Method:

(595) The present experiment was analyzed with the method of One-Way ANOVA. All the data were analyzed using SPSS 17.0. p<0.05 represents significant difference.

(596) Result:

(597) TABLE-US-00017 TABLE 15 The inhibitory effect on CR0205 of Huprime xenografts model of the compound of the present invention Tumor Volume 23 days after the administration Experimental (mm.sup.3).sup.a Tumor Volume T/C TGI groups Dosage (Day 0) (mm.sup.3).sup.a (%) (%) Medium group 129.2 14.1 772.3 100.7 Compound 18 20 mg/kg 129.5 11.1 278.3 56.1** 34.3 65.7 Neratinib 80 mg/kg 129.2 11.7 386.8 91.8* 47.5 52.5 Notes: .sup.amean standard error; *P < 0.05, **P < 0.01 and ***P < 0.001 Comparison with the control group.
Conclusion

(598) Tested Compound 18 of the present invention has significant inhibitory effect on the tumor growth of human-derived xenografts model CR0205. The antitumor effect is superior to the positive control group of Neratinib. Moreover, within the 23 days of time of administration, CR0205 mice with tumors tolerated Compound 18 well.

(599) Assay 4-9 In Vivo Pharmacodynamic Evaluation on Gallbladder Carcinoma of the Compound of the Present Invention

(600) Material

(601) Samples:

(602) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(603) GL6899, gallbladder carcinoma from a 74-year-old Asian female patient

(604) Animal:

(605) BALB/c nude mice of 5-7 weeks purchased from SPF Biotech (Beijing) Co. Ltd.

(606) Method

(607) Tumor Inoculation and Grouping

(608) Tumor tissues were collected from mice with tumor of human-derived xenograft gallbladder carcinoma model GL6899 and cut into tumor blocks of a diameter of 2-4 mm which were then inoculated subcutaneously in the right anterior portion of the mice. When the average tumor volume reached 100-150 mm.sup.3, the mice were randomly grouped, the day for grouping being recorded as Day 0. The mice were randomly divided into two experimental groups according to the tumor volume, including group of Compound 18 with a dosage of 20 mg/kg and Vehicle medium control group (sterile water). There were five animals in each group which were orally intragastrical administered (p.o.) once a day from the grouping day for 21 days in total. The tumor volume (TV) was measured with an ernier caliper twice a week. The efficacy was evaluated according to the relative tumor growth inhibition (TGI).

(609) Experimental Observation Index and Calculation:

(610) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(611) Statistical Analysis:

(612) Comparison between the two groups of data was tested by independent sample t test. All the data were analyzed using SPSS 17.0. p<0.05 represents significant difference.

(613) Result:

(614) TABLE-US-00018 TABLE 16 The antitumor effect in HuPrime gallbladder carcinoma xenografts model GL6899 of the compound of the present invention Tumor Volume Tumor Volume on day 0 after adminstration T/C TGI Groups Dosage (mm.sup.3).sup.a of 21 days (mm.sup.3).sup.a (%) (%) P .sup.b Medium group 135.10 13.20 1015.05 78.52 Compound 18 20 mg/kg 135.53 9.60 26.63 2.37*** 2.6 97.4 0.000 Notes: .sup.amean standard error; .sup.b comparison of the tumor volume with the control group on Day 21; **P < 0.01 and ***P < 0.001, comparison with the medium control group at the same point of time.
Conclusion

(615) Tested Compound 18 has superior inhibitory effect on the tumor growth of gallbladder carcinoma xenografts model GL6899.

(616) Assay 4-10 In Vivo Pharmacodynamic Evaluation on Cholangiocarcinoma of the Compound of the Present Invention

(617) Material and Method

(618) Samples:

(619) Compound 18. See the chemical name, structural formula and preparation method of the compound in the preparation examples of the present invention.

(620) 10114, cholangiocarcinoma from a 61-year-old female patient.

(621) Experimental Animals:

(622) BALB/c nude mice of 6-8 weeks purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (SCXK (Shanghai) 2013-0018).

(623) Method

(624) Tumor Inoculation and Grouping

(625) Tumor tissues of human cholangiocarcinoma (10114) were cut into tumor blocks of volume of 3 mm3 mm3 mm which were inoculated subcutaneously in mice. The tumor volume was observed and measured. When the average tumor volume of the mice with tumor reached a certain volume, the mice with tumor cells were grouped and administered, the day for grouping being recorded as Day 0. The mice were randomly divided into group of Compound 18 with a dosage of 20 mg/kg and Vehicle medium group (sterile water for injection) according to the tumor volume. There were five animals in each group which were orally intragastrical administered once a day for 21 days in total. The tumor volume (TV) was measured with an ernier caliper twice a week. The efficacy was evaluated according to the tumor growth inhibition (TGI).

(626) Experimental Observation Index and Calculation:

(627) The calculation formulae for the tumor volume (TV), relative tumor growth inhibition TGI and relative tumor proliferation rate T/C (%) are the same as Assay 4-1.

(628) Statistical Analysis:

(629) The experimental data were analyzed using statistical software SPSS 20.0. The data associated with the tumor volume was analyzed with T-test. **P<0.05 compared with the control group represents significant statistical difference.

(630) Result:

(631) TABLE-US-00019 TABLE 17 The antitumor effect in human-derived cholangiocarcinoma (10114) xenograft model of the compound of the present invention Tumor Volume 21 days after the adminstration On Day 0 Tumor Volume Relative Tumor T/C TGI Groups Dosage (mm.sup.3) (mm.sup.3) Volume (%) (%) (%) Medium group 139.6 14.1 602.37 85.68 426.39 21.80 Compound 18 20 mg/kg 138.6 8.9 107.53 15.80*** 77.13 9.34 18.1 81.9 Note: **P < 0.01, ***P < 0.001, comparison with the medium control group.

CONCLUSION

(632) Tested Compound 18 of the present invention has superior inhibitory effect on the tumor growth in xenograft model 10114.