MACROCYCLIC COMPOUND AS CDK INHIBITOR, PREPARATION METHOD THEREFOR, AND USE THEREOF IN MEDICINE

Abstract

The present invention relates to a macrocyclic compound as a CDK inhibitor, a preparation method therefor and the use thereof in medicine. Specifically, the present invention relates to a novel macrocyclic compound represented by a general formula (I), a preparation method therefor, a pharmaceutical composition containing the compound, the use thereof as a therapeutic agent, particularly as a CDK inhibitor, and the use thereof in treating cancers, inflammation, viral infections, cardiac hypertrophy or HIV, wherein each substituent of the general formula (I) is the same as that defined in the description.

##STR00001##

Claims

1. A compound of formula (I): ##STR00028## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: A and B are each independently selected from the group consisting of CH and N atom; U, V, W, X and Y are each independently selected from the group consisting of CH and N atom; Q is selected from the group consisting of a bond, C.sub.1-4 alkylene and —C(═O)—, wherein the C.sub.1-4 alkylene is optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, haloC.sub.1-4 alkyl, C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; G is selected from the group consisting of a bond, —O—, —N(R.sup.7)—, —C(═O)—, C.sub.1-4 alkylene, C.sub.2-4 alkenyl, —S—, —SO—, —SO.sub.2— and 3 to 6 membered heterocyclyl, wherein the C.sub.1-4 alkylene, C.sub.2-4 alkenyl and 3 to 6 membered heterocyclyl are each independently optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, haloC.sub.1-4 alkyl, C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; J is selected from the group consisting of a bond, —O—, —N(R.sup.7)—, —C(═O)—, C.sub.1-4 alkylene, C.sub.2-4 alkenyl, —S—, —SO—, —SO.sub.2— and 3 to 6 membered heterocyclyl, wherein the C.sub.1-4 alkylene, C.sub.2-4 alkenyl and 3 to 6 membered heterocyclyl are each independently optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, haloC.sub.1-4 alkyl, C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; L is selected from the group consisting of a bond, —C(═O)—, C.sub.1-4 alkylene, C.sub.2-4 alkenyl, —S—, —SO—, —SO.sub.2— and 3 to 6 membered heterocyclyl, wherein the C.sub.1-4 alkylene, C.sub.2-4 alkenyl and 3 to 6 membered heterocyclyl are each independently optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, haloC.sub.1-4 alkyl, C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; R.sup.1 is selected from the group consisting of H atom, C.sub.1-4 alkyl, cyano, C.sub.2-4 alkenyl and C.sub.2-4 alkynyl, wherein the C.sub.1-4 alkyl, C.sub.2-4 alkenyl and C.sub.2-4 alkynyl are each independently optionally substituted by one or more substituents selected from the group consisting of halogen, amino, nitro, hydroxy and cyano; R.sup.2 is selected from the group consisting of H atom and C.sub.1-4 alkyl, wherein the C.sub.1-4 alkyl is optionally substituted by one or more substituents selected from the group consisting of halogen, amino, nitro, hydroxy and cyano; or, R.sup.1 and R.sup.2 together with the atom to which they are attached form a C.sub.3-6 cycloalkyl or 3 to 6 membered heterocyclyl, wherein the C.sub.3-6 cycloalkyl or 3 to 6 membered heterocyclyl are each independently optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, haloC.sub.1-4 alkyl, C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; R.sup.3 and R.sup.6 are each independently selected from the group consisting of H atom and C.sub.1-4 alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano; R.sup.4 and R.sup.5 are each independently selected from the group consisting of H atom, C.sub.1-4 alkyl, halogen, hydroxy and C.sub.1-4 alkoxy, wherein the C.sub.1-4 alkyl and C.sub.1-4 alkoxy are each independently optionally substituted by one or more substituents selected from the group consisting of halogen, amino, nitro, hydroxy and cyano; and R.sup.7 is selected from the group consisting of H atom and C.sub.1-4 alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkoxy, haloC.sub.1-4 alkoxy, halogen, amino, nitro, hydroxy and cyano.

2. The compound of formula (I) according to claim 1, wherein -G-J-L- is selected from the group consisting of —NH—CH.sub.2—CH.sub.2—, —O—CH.sub.2—CH.sub.2—, —NH—C(═O)—CH.sub.2— and —C(═O)—NH—CH.sub.2—.

3. The compound of formula (I) according to claim 1, wherein Q is a bond or methylene.

4. The compound of formula (I) according to claim 1, wherein V, X and Y are each independently CH.

5. The compound of formula (I) according to claim 1, wherein W is an N atom.

6. The compound of formula (I) according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from the group consisting of H atom and C.sub.1-4 alkyl, or R.sup.1 and R.sup.2 together with the atom to which they are attached form a C.sub.3-6 cycloalkyl.

7. The compound of formula (I) according to claim 1, wherein R.sup.3 and R.sup.6 are each independently a C.sub.1-4 alkyl.

8. The compound of formula (I) according to claim 1, wherein R.sup.4 and R.sup.5 are each independently a halogen e.

9. The compound of formula (I) according to claim 1, selected from the group consisting of: ##STR00029##

10. A method for preparing the compound of formula (I) according to claim 1, comprising a step of: ##STR00030## subjecting a compound of formula (IA) to an intramolecular cyclization reaction at the presence of a condensing agent to obtain the compound of formula (I), wherein: LG.sub.1 and LG.sub.2 are each independently a leaving group; and A, B, U, V, W, X, Y, G, J, L, and R.sup.1 to R.sup.6 are as defined in claim 1.

11. The method according to claim 10, wherein the condensing agent is selected from the group consisting of 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, N,N′-diisopropylcarbodiimide, 0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; and LG.sub.1 and LG.sub.2 are each independently selected from the group consisting of H atom, OH, halogen, mesylate, triflate and tosylate.

12. The method according to claim 10, wherein G is —NH—, LG.sub.1 is a H atom, J is —C(═O)—, and -LG.sub.2 is OH.

13. A pharmaceutical composition, comprising a therapeutically effective amount of the compound of formula (I) according to claim 1 and pharmaceutically acceptable carrier(s), diluent(s) or excipient(s).

14. A method for treating a CDK-related disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the compound of formula (I) according to claim 1, wherein the CDK-related disease is selected from the group consisting of cancer, inflammation, viral infection, cardiac hypertrophy and HIV.

15. The method according to claim 14, wherein the cancer is selected from the group consisting of bladder cancer, head and neck cancer, breast cancer, stomach cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, laryngeal cancer, lymphatic system cancer, hematopoietic system cancer, genitourinary tract cancer, gastrointestinal cancer, ovarian cancer, prostate cancer, gastric cancer, bone cancer, small cell lung cancer, glioma, colorectal cancer and pancreatic cancer; the inflammation is related to rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis, chronic inflammation and organ transplant rejection; and the viral infection is related to HIV virus, human papilloma virus, herpes virus, pox virus, Epstein-Barr virus, Sindbis virus or adenovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0099] By reading the following examples, the person skilled in the art will better understand the present invention. The following examples merely serve to illuminate the present invention.

[0100] In the examples of the present invention, the experiment methods that do not specify the specific conditions are generally conducted in accordance with conventional conditions, or in accordance with conditions recommended by the material or product manufacturers. The reagents without a specific source are commercially available conventional reagents.

[0101] The structures of the compounds were identified by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in 10.sup.−6 (ppm). The solvent for determination was deuterated-chloroform (CDCl.sub.3), and the internal standard was tetramethylsilane (TMS). The following abbreviations are used: s for singlet, bs for broad singlet, d for doublet, t for triplet, qdt for quartet, m for multiplet or massive, dd for double of doublet, etc.

[0102] Liquid chromatograph-mass spectrometer: Agilent LCMS1260/MSD6120, column: Agilent ZORBAX SB-C18, 2.1*50 mm, 1.8 μm, mobile phase: A: H.sub.2O (0.1% FA), B: acetonitrile, gradient elution, 0.5 mL/min, 45.0° C., ionization mode: API-ES, polarity: positive.

[0103] Nuclear magnetic resonance spectrometer: Bruker ARX-500 and Bruker ARX-400.

[0104] MTT detection instrument: Thermo Scientific Multiskan GO full-wavelength microplate reader.

[0105] Qingdao GF254 silica gel plate is used as the thin-layer silica gel chromatography (TLC) plate. The dimension of the silica gel plate used in TLC is 0.15 mm to 0.2 mm, and the dimension of the silica gel plate used in product purification is 0.4 mm to 0.5 mm.

[0106] Yantai Huanghai 200 to 300 mesh silica gel is generally used as a carrier for silica gel column chromatography.

[0107] Unless otherwise stated, the reactions were carried out under argon atmosphere or nitrogen atmosphere.

[0108] Unless otherwise stated, the solution refers to an aqueous solution.

[0109] Unless otherwise stated, the reaction temperature is room temperature.

[0110] The reaction process in the examples was monitored by thin layer chromatography (TLC).

Example 1

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benz o[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 1

[0111] ##STR00017## ##STR00018## ##STR00019## ##STR00020##

Step 1

Methyl 4-(benzylamino)-6-chloronicotinate 102

[0112] Methyl 4,6-dichloronicotinate 101 (6 g, 29.1 mmol), benzylamine (3.4 g, 32 mmol) and triethylamine (9.5 g, 87.3 mmol) were dissolved in 30 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 200 mL of water was added. The reaction solution was stirred for 30 minutes and filtered, and the filter cake was washed with water and dried under reduced pressure. The title compound was obtained as a white solid (7.6 g, yield: 94.1%).

[0113] MS (ESI): 277.7 [M+1].sup.+.

Step 2

(4-(Benzylamino)-6-chloropyridin-3-yl)methanol 103

[0114] Lithium aluminum hydride (2.1 g, 54.9 mmol) was suspended in 70 mL of tetrahydrofuran, and the solution was cooled to below −20° C., followed by the addition of methyl 4-(benzylamino)-6-chloronicotinate 102 (7.6 g, 27.4 mmol). The reaction solution was reacted at −20° C. for 2 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate=3:1). After the reaction was completed, 2.1 mL of water, 3.2 mL of 10% sodium hydroxide solution and 10.2 mL of water were added successively. The reaction solution was stirred for 30 minutes, dried and filtered, and the filtrate was concentrated to dryness by rotary evaporation. The title compound was obtained as a white solid (7.5 g, yield: 100%).

[0115] MS (ESI): 249.7 [M+1].sup.+.

Step 3

4-(Benzylamino)-6-chloronicotinaldehyde 104

[0116] (4-(Benzylamino)-6-chloropyridin-3-yl)methanol 103 (7.5 g, 30.2 mmol) was dissolved in 100 mL of dichloromethane, and the solution was cooled to below 0° C., followed by the addition of DMP (Dess-Martin oxidant, 15.3 g, 36.3 mmol) in batches. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (ethyl acetate). After the reaction was completed, 100 mL of 10% NaOH solution was added. The reaction solution was stirred for 30 minutes and separated into two phases. The aqueous phase was extracted by dichloromethane. The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (PE:EA=3:1) to obtain the title compound as a yellow oil (6.5 g, yield: 87.1%).

[0117] MS (ESI):247.7 [M+1].sup.+.

Step 4

N-Benzyl-2-chloro-5-((4-ethylpiperazin-1-yl)methyl)pyridin-4-amine 105

[0118] 4-(Benzylamino)-6-chloronicotinaldehyde 104 (6.5 g, 26.3 mmol) and N-ethylpiperazine (3.6 g, 31.6 mmol) were dissolved in 50 mL of dichloromethane, and the solution was cooled to below 0° C., followed by the addition of sodium triacetoxyborohydride (8.3 g, 39.45 mmol) in batches. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (ethyl acetate). After the reaction was completed, 100 mL of saturated sodium bicarbonate solution was added. The reaction solution was stirred for 30 minutes and separated into two phases. The aqueous phase was extracted by dichloromethane (100 mL×3). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate:methanol=10:1) to obtain the title compound as a colorless oil (7.8 g, yield: 85.9%).

[0119] MS (ESI):348.5 [M+1].sup.+.

Step 5

N.SUP.4.-Benzyl-5-((4-ethylpiperazin-1-yl)methyl)pyridine-2,4-diamine 106

[0120] The intermediate N-benzyl-2-chloro-5-((4-ethylpiperazin-1-yl)methyl)pyridin-4-amine 105 (6 g, 17.4 mmol), benzophenone imine (3.7 g, 20.8 mmol), sodium tert-butoxide (2.34 g, 24.36 mmol), Pd.sub.2(dba).sub.3 (1.6 g, 1.74 mmol) and BINAP (3.24 g, 5.22 mmol) were dissolved in 100 mL of toluene under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 14 hours. The reaction was monitored by TLC (DCM:MeOH=10:1). After the reaction was completed, 100 mL of methyl tert-butyl ether was added. The reaction solution was stirred for 30 minutes, filtered, and washed, and the filtrate was concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was dissolved in 50 mL of THE and 20 mL of methanol, followed by the addition of 10 mL of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 2 hours. The reaction was monitored by TLC (dichloromethane:methanol=10:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Saturated sodium bicarbonate solution was added, and the reaction solution was extracted by ethyl acetate. The aqueous phase was further extracted by dichloromethane. The organic phases were combined, dried and concentrated to dryness by rotary evaporation. The title compound was obtained as a white solid (3 g, yield: 52.9%).

[0121] MS (ESI):326.2 [M+1].sup.+.

Step 6

Methyl 3-aminobutanoate 108

[0122] Thionyl chloride (8.4 mL, 116.4 mmol) was added dropwise to 40 mL of methanol and stirred for 1 hour at 0° C. 3-Aminobutyric acid 107 (4 g, 38.8 mmol) was added to the reaction system, which was then stirred at room temperature for 5 hours. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Methanol was added, and then the solution was concentrated to dryness, and this process was repeated twice. The title compound was obtained as a colorless oil (5 g, yield: 100%).

Step 7

Methyl 3-((5-bromo-3-fluoro-2-nitrophenyl)amino)butanoate 109

[0123] 1,3-Difluoro-5-bromo-2-nitrobenzene (2 g, 8.4 mmol) and methyl 3-aminobutanoate 108 (0.98 g, 8.4 mmol) were dissolved in 20 mL of DMF, followed by the addition of DIEA (3.25 g, 25.2 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 50 mL of water was added, and the reaction solution was extracted with ethyl acetate (3×25 mL). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=50:1) to obtain the title compound as a yellow solid (2.4 g, yield: 84.5%).

[0124] MS (ESI):336.1 [M+1].sup.+.

Step 8

Methyl 3-((2-amino-5-bromo-3-fluorophenyl)amino)butanoate 110

[0125] Methyl 3-((5-bromo-3-fluoro-2-nitrophenyl)amino)butanoate 109 (2 g, 5.9 mmol) was dissolved in 15 mL of DMF, followed by the addition of stannous chloride (6.7 g, 29.8 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 50 mL of water was added, and the reaction solution was extracted with ethyl acetate (3×25 mL). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation. The title compound was obtained as an orange oil (2.2 g, yield: 100%).

[0126] MS (ESI):306.2 [M+1].sup.+.

Step 9

Methyl 3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 111

[0127] Methyl 3-((2-amino-5-bromo-3-fluorophenyl)amino)butanoate 110 (2.2 g, 5.9 mmol) was dissolved in 9 mL of trimethyl orthoformate and 6 mL of acetic acid. The reaction solution was reacted at 90° C. for 8 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Saturated sodium bicarbonate solution was added, and the reaction solution was extracted with ethyl acetate (3×15 mL). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was dissolved in 10 mL of a solution of petroleum ether and ethyl acetate (1:10), stirred for one hour and filtered. The filter cake was washed with petroleum ether, and dried under reduced pressure to obtain the title compound as a white solid (1.2 g, yield: 61.2%).

[0128] MS (ESI):330.1 [M+1].sup.+.

Step 10

Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butan oate 113

[0129] Methyl 3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 111 (2.5 g, 8 mmol), pinacol diborate (2.3 g, 9.1 mmol), potassium acetate (2.4 g, 24 mmol) and Pd(dppf)Cl.sub.2 (326 mg, 0.4 mmol) were dissolved in 70 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the intermediate 2,4-dichloro-5-fluoropyrimidine (1.7 g, 10.4 mmol), cesium carbonate (7.8 g, 24 mmol), Pd(dppf)Cl.sub.2 (326 mg, 0.4 mmol) and 7 mL of water were added to the reaction solution. The reaction solution was reacted at 110° C. for 14 hours. The reaction was monitored by LC-MS. When the raw material no longer decreased, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with ethyl acetate (3×25 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain the title compound as a yellow solid (1 g, yield: 32.5%).

[0130] MS (ESI):381.1 [M+1].sup.+.

Step 11

Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 114

[0131] Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butan oate 113 (0.7 g, 1.8 mmol), N.sup.4-benzyl-5-((4-ethylpiperazin-1-yl)methyl)pyridine-2,4-diamine (0.72 g, 2.2 mmol), Pd.sub.2(dba).sub.3 (82 mg, 0.09 mmol), 2-dicyclohexylphosphorus-2,4,6-triisopropylbiphenyl (X-phos) (128 mg, 0.27 mmol) and potassium carbonate (0.745 g, 5.4 mmol) were suspended in 70 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 12 hours. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with dichloromethane (3×25 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol=5:1) to obtain the title compound as a brown solid (0.5 g, yield: 38.9%).

[0132] MS (ESI): 670.7 [M+1].sup.+.

Step 12

Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 115

[0133] Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 114 (0.5 g, 0.7 mmol) was dissolved in 4 mL of concentrated sulfuric acid at 0° C., and stirred for 30 minutes. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was poured into crushed ice, and 10% sodium hydroxide solution was added to adjust pH=8-9. The reaction solution was extracted with ethyl acetate (3×20 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the title compound as a brown solid (0.4 g, yield: 100%).

[0134] MS (ESI):580.6 [M+1].sup.+.

Step 13

3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoic acid 116

[0135] Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 115 (0.4 g, 6.9 mmol) was dissolved in 5 mL methanol and 5 mL of 10% sodium hydroxide solution. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. Concentrated hydrochloric acid was added to adjust pH=4-5. The reaction solution was concentrated to dryness by rotary evaporation and dried. 50 mL of ethanol was added, and the solution was stirred for 5 hours, filtered, and washed. The filtrate was concentrated to dryness by rotary evaporation to obtain the title compound as a yellow solid (0.8 g, yield: 100%).

[0136] MS (ESI):566.6 [M+1].sup.+.

Step 14

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benz o[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 1

[0137] 3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoic acid 116 (0.8 g, 0.7 mmol) and triethylamine (212 mg, 2.1 mmol) were dissolved in 50 mL of dichloromethane, followed by the addition of HATU (323 mg, 0.85 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. Water was added, and the reaction solution was extracted with dichloromethane (3×20 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by preparative silica gel plate (DCM:MeOH=5:1) to obtain the title compound as a white solid (32 mg).

[0138] MS (ESI):548.6 [M+1].sup.+.

[0139] .sup.1H NMR (400 MHz, CDCl.sub.3)) δ 10.41 (s, 1H), 10.21 (s, 1H), 9.73 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.59 (d, J=11.8 Hz, 1H), 5.23 (s, 1H), 4.02 (m, 1H), 3.65-3.35 (m, 2H), 3.10-2.55 (m, 9H), 2.47-2.08 (m, 5H), 1.79 (d, J=6.4 Hz, 3H), 1.03 (t, J=6.5 Hz, 3H).

Example 2

(R)-4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 2

[0140] ##STR00021##

Step 1

Methyl (R)-3-aminobutanoate 202

[0141] Thionyl chloride (5.1 g, 43 mmol) was added dropwise to 40 mL of methanol, and the solution was stirred for 1 hour at 0° C. (R)-3-Aminobutanoic acid 201 (3 g, 29 mmol) was added to the reaction system, which was then stirred at room temperature for 5 hours. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Methanol was added, and then the solution was concentrated to dryness, and this process was repeated twice. The title compound was obtained as a colorless oil (4.0 g, yield: 100%). The resulting product was used directly in the next step.

Step 2

Methyl (R)-3-((5-bromo-3-fluoro-2-nitrophenyl)amino)butanoate 203

[0142] 1,3-Difluoro-5-bromo-2-nitrobenzene (6.8 g, 29 mmol) and methyl (R)-3-aminobutanoate 202 (4.0 g, 29 mmol) were dissolved in 50 mL of DMF, followed by the addition of DIEA (11.2 g, 87 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 100 mL of water was added, and the reaction solution was extracted with ethyl acetate (3×25 mL). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=50:1) to obtain the title compound as a yellow solid (9 g, yield: 90.3%).

[0143] MS (ESI):335.2 [M+1].sup.+.

Step 3

Methyl (R)-3-((2-amino-5-bromo-3-fluorophenyl)amino)butanoate 204

[0144] Methyl (S)-3-((5-bromo-3-fluoro-2-nitrophenyl)amino)butanoate 203 (9 g, 26.8 mmol) was dissolved in 50 mL of DMF, followed by the addition of stannous chloride (30.3 g, 34 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 100 mL of water was added, and the reaction solution was extracted with ethyl acetate (3×50 mL). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation. The title compound was obtained as an orange oil (10 g, yield: 100%).

[0145] MS (ESI):306.2 [M+1].sup.+.

Step 4

Methyl (R)-3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 205

[0146] Methyl (S)-3-((2-amino-5-bromo-3-fluorophenyl)amino)butanoate 204 (10 g, 26.8 mmol) was dissolved in 45 mL of trimethyl orthoformate and 30 mL of acetic acid. The reaction solution was reacted at 90° C. for 8 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Saturated sodium bicarbonate solution was added, and the reaction solution was extracted with ethyl acetate (3×25 mL).

[0147] The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was dissolved in 20 mL of a solution of ethyl acetate and petroleum ether (1:10), stirred for one hour and filtered. The filter cake was washed with petroleum ether, and dried under reduced pressure to obtain the title compound as a white solid (6.2 g, yield: 69.7%).

[0148] MS (ESI):330.1 [M+1].sup.+.

Step 5

Methyl (R)-3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 207

[0149] Methyl (R)-3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 205 (5 g, 15.1 mmol), pinacol diborate (4.6 g, 18.2 mmol), potassium acetate (4.84 g, 45.3 mmol) and Pd(dppf)Cl.sub.2 (0.64 g, 0.75 mmol) were dissolved in 120 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the intermediate 2,4-dichloro-5-fluoropyrimidine (3.2 g, 19.6 mmol), cesium carbonate (14.7 g, 45.3 mmol), Pd(dppf)Cl.sub.2 (0.61 g, 0.7 mmol) and 12 mL of water were added to the reaction solution. The reaction solution was reacted at 110° C. for 14 hours. The reaction was monitored by LC-MS. When the raw material no longer decreased, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with ethyl acetate (3×40 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain the title compound as a yellow solid (3.1 g, yield: 53.6%).

[0150] MS(ESI):381.1[M+1].sup.+.

Step 6

Methyl (R)-3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 208

[0151] Methyl (R)-3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 207 (1.1 g, 2.95 mmol), N.sup.4-benzyl-5-((4-ethylpiperazin-1-yl)methyl)pyridine-2,4-diamine (0.8 g, 2.46 mmol), Pd.sub.2(dba).sub.3 (112 mg, 0.12 mmol), X-phos (175 mg, 0.37 mmol) and potassium carbonate (1.02 g, 7.38 mmol) were suspended in 100 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 12 hours. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with dichloromethane (3×30 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol=5:1) to obtain the title compound as a brown solid (0.4 g, yield: 20.3%).

[0152] MS (ESI):670.7 [M+1].sup.+.

Step 7

Methyl (R)-3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 209 Methyl

[0153] (R)-3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 208 (0.4 g, 0.6 mmol) was dissolved in 4 mL of concentrated sulfuric acid at 0° C., and the solution was stirred for 30 minutes. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was poured into crushed ice, and 10% sodium hydroxide solution was added to adjust pH=8-9. The reaction solution was extracted with ethyl acetate (3×30 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the title compound as a brown solid (0.4 g, yield: 100%).

Step 8

(R)-3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoic acid 210 Methyl

[0154] (R)-3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoate 209 (0.4 g, 6.9 mmol) was dissolved in 5 mL methanol and 5 mL of 10% sodium hydroxide solution. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. Concentrated hydrochloric acid was added to adjust pH=4-5. The reaction solution was concentrated to dryness by rotary evaporation and dried. 50 mL of ethanol was added, and the solution was stirred for 5 hours, filtered, and washed. The filtrate was concentrated to dryness by rotary evaporation to obtain the title compound as a yellow solid (0.5 g, yield: 100%).

[0155] MS(ESI):566.6[M+1].sup.+.

Step 9

(R)-4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 2

[0156] (R)-3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoro pyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)butanoic acid 210 (0.5 g, 0.69 mmol) and triethylamine (212 mg, 2.1 mmol) were dissolved in 50 mL of dichloromethane, followed by the addition of HATU (323 mg, 0.85 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. Water was added, and the reaction solution was extracted with dichloromethane (3×30 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by preparative silica gel plate (dichloromethane:methanol=5:1) to obtain the title compound as a white solid (24 mg, yield: 7.4%).

[0157] MS(ESI):548.6[M+1].sup.+.

[0158] .sup.1H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 10.21 (s, 1H), 9.73 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.59 (d, J=11.8 Hz, 1H), 5.23 (s, 1H), 3.85 (d, J=13.2 Hz, 1H), 3.65-3.35 (m, 2H), 3.10-2.55 (m, 9H), 2.47-2.08 (m, 5H), 1.79 (d, J=6.4 Hz, 3H), 1.03 (t, J=6.5 Hz, 3H).

Example 3

(S)-4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 3

[0159] ##STR00022##

[0160] In accordance with the method described in Example 2, methyl (S)-3-aminobutyrate was used as the starting material, accordingly, (S)-4.sup.5-((4-Ethylpiperazin-1-yl)methyl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8-dimethyl-1.sup.1H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 3 was prepared as a white solid.

[0161] MS(ESI):548.6[M+1].sup.+.

[0162] .sup.1H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 10.21 (s, 1H), 9.73 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.59 (d, J=11.8 Hz, 1H), 5.23 (s, 1H), 3.85 (d, J=13.2 Hz, 1H), 3.65-3.35 (m, 2H), 3.10-2.55 (m, 9H), 2.47-2.08 (m, 5H), 1.79 (d, J=6.4 Hz, 3H), 1.03 (t, J=6.5 Hz, 3H).

Example 4

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.-methyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 4

[0163] ##STR00023##

Step 1

Methyl Aminobutyrate 402

[0164] Thionyl chloride (10 g, 84 mmol) was added dropwise to 40 mL of methanol and stirred for 1 hour at 0° C. Aminobutyric acid (5 g, 56 mmol) was added to the reaction system and stirred at room temperature for 5 hours. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. MeOH was added, and then the solution was concentrated to dryness, and this process was repeated twice. The title compound was obtained as a colorless oil (6 g, yield: 100%). The resulting product was used directly in the next step.

Step 2

Methyl 3-((5-bromo-3-fluoro-2-nitrophenyl)amino)propanoate 403

[0165] 1,3-Difluoro-5-bromo-2-nitrobenzene (13.3 g, 56 mmol) and methyl 3-aminopropanoate 402 (6 g, 56 mmol) were dissolved in 100 mL of DMF, followed by the addition of DIEA (21.6 g, 168 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (PE:EA=5:1). After the reaction was completed, 150 mL of water was added, and the reaction solution was extracted with 150 mL of ethyl acetate twice. The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (PE:EA=50:1) to obtain the title compound as a orange solid (14 g, yield: 77.8%).

[0166] MS (ESI): 322.1 [M+1].sup.+.

Step 3

Methyl 3-((2-amino-5-bromo-3-fluorophenyl)amino)propanoate 404

[0167] Methyl 3-((5-bromo-3-fluoro-2-nitrophenyl)amino)propanoate 403 (14 g, 43.6 mmol) was dissolved in 150 mL of DMF, followed by the addition of stannous chloride (49 g, 218 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (petroleum ether: ethyl acetate=5:1). After the reaction was completed, 500 mL of water was added, and the reaction solution was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation. The title compound was obtained as a pale yellow oil (12 g, yield: 100%).

[0168] MS (ESI): 292.2 [M+1].sup.+.

Step 4

Methyl 3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 405

[0169] Methyl 3-((2-amino-5-bromo-3-fluorophenyl)amino)propanoate 404 (12 g, 41 mmol) was dissolved in 90 mL of trimethyl orthoacetate and 54 mL of acetic acid. The reaction solution was reacted at 90° C. for 8 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1). After the reaction was completed, the reaction solution was concentrated to dryness by rotary evaporation. Saturated sodium bicarbonate solution (about 100 mL) was added, and the reaction solution was extracted with ethyl acetate (50 ml×3). The organic phases were combined, washed with water, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was dissolved in 10 mL of a solution of ethyl acetate and petroleum ether (1:10), and the solution was stirred for one hour, filtered, washed with petroleum ether, and dried under reduced pressure to obtain the title compound as a white solid (6.8 g, yield: 52.3%).

[0170] MS (ESI): 316.1 [M+1].sup.+.

Step 5

Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 407

[0171] Methyl 3-(6-bromo-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 405 (5 g, 15.9 mmol), pinacol diborate (4.8 g, 19.1 mmol), potassium acetate (4.7 g, 47.7 mmol) and Pd(dppf)Cl.sub.2 (650 mg, 0.8 mmol) were dissolved in 100 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the intermediate 2,4-dichloro-5-fluoropyrimidine (3.4 g, 20.4 mmol), cesium carbonate (15.5 g, 47.7 mmol), Pd(dppf)Cl.sub.2 (650 mg, 0.8 mmol) and 15 mL of water were added to the reaction solution. The reaction solution was reacted at 110° C. for 14 hours. The reaction was monitored by LC-MS. When the raw material no longer decreased, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water (about 100 ml) was added, and the solution was extracted with ethyl acetate (50 ml×3). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=1:1) to obtain the title compound as a brown solid (2.8 g, yield: 48.2%).

[0172] MS (ESI): 367.1 [M+1].sup.+.

Step 6

Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 408

[0173] Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 407 (2.8 g, 7.6 mmol), N.sup.4-benzyl-5-((4-ethylpiperazin-1-yl)methyl)pyridine-2,4-diamine (2 g, 6.3 mmol), Pd.sub.2(dba).sub.3 (580 mg, 0.63 mmol), X-phos (900 mg, 1.89 mmol) and potassium carbonate (2.6 g, 18.9 mmol) were suspended in 70 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 12 hours. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water (about 70 ml) was added, and the solution was extracted with ethyl acetate (50 ml×3). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH=5:1) to obtain the title compound as a brown oil (1.5 g, yield: 30.9%).

[0174] MS (ESI): 656.7 [M+1].sup.+.

Step 7

Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 409

[0175] Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 408 (1.5 g, 2.1 mmol) was dissolved in 4 mL of concentrated sulfuric acid at 0° C., and the solution was stirred for 30 minutes. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was poured into crushed ice (about 100 g), and 10% sodium hydroxide solution was added to adjust pH=8-9. The reaction solution was extracted with ethyl acetate (50 ml×3). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the title compound as a brown solid (0.9 g, yield: 63.6%).

[0176] MS (ESI): 566.6 [M+1].sup.+.

Step 8

3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoic acid 410

[0177] Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoate 409 (0.9 g, 1.6 mmol) was dissolved in 5 mL methanol and 5 mL of 10% sodium hydroxide solution. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. Concentrated hydrochloric acid was added to adjust pH=4-5. The reaction solution was concentrated to dryness by rotary evaporation and dried. 50 mL of ethanol was added, and the solution was stirred for 5 hours, filtered, and washed. The filtrate was concentrated to dryness by rotary evaporation to obtain the title compound as an off-white solid (1.6 g, yield: 100%).

[0178] MS (ESI): 552.6 [M+1].sup.+.

Step 9

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.-methyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 4

[0179] 3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1H-benzo[d]imidazol-1-yl)propanoic acid 410 (1.6 g, 0.9 mmol) and triethylamine (270 mg, 2.7 mmol) were dissolved in 50 mL of dichloromethane, followed by the addition of HATU (510 mg, 1.3 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. Water (about 50 ml) was added, and the reaction solution was separated into two phases. The aqueous phase was extracted with ethyl acetate (50 ml×3). The organic phase was dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by preparative silica gel plate (DCM:MeOH=5:1) to obtain the title compound as a white solid (35 mg).

[0180] MS (ESI): 534.6 [M+1].sup.+.

[0181] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.68 (s, 1H), 10.11 (s, 1H), 9.52 (s, 1H), 8.67 (d, J=4.1 Hz, 1H), 8.39 (s, 1H), 8.08 (s, 1H), 7.58 (d, J=11.8 Hz, 1H), 4.71 (s, 2H), 3.97-3.41 (m, 2H), 3.03-2.60 (m, 9H), 2.48-1.93 (m, 6H), 1.02 (s, 3H).

Example 5

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-.SUP.1.H-3,5-diaza-1(6,1)-benz o[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphane 5

[0182] ##STR00024##

[0183] LiAlH.sub.4 (10 mg, 0.26 mmol) was added to a solution of 4.sup.5-((4-ethylpiperazin-1-yl)methyl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8-dimethyl-.sup.1H-3,5-diaza-1(6,1)-benzo [d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 1 (100 mg, 0.182 mmol) in THE (5 mL) at 0° C. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. Saturated aqueous ammonium chloride solution (about 10 ml) was added at 0° C., and the reaction solution was separated into two phases. The aqueous phase was extracted with ethyl acetate (10 ml×3). The organic phase was dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by preparative silica gel plate (DCM:MeOH=5:1) to obtain the title compound as a white solid (60 mg).

[0184] MS (ESI): 534.2 [M+1]+.

[0185] .sup.1H NMR (400 MHz, CDCl.sub.3)) δ 10.41 (s, 1H), 10.21 (s, 1H), 9.73 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.59 (d, J=11.8 Hz, 1H), 5.23 (s, 1H), 3.85 (d, J=13.2 Hz, 1H), 3.32-3.20 (m, 2H), 3.10-2.55 (m, 9H), 2.47-2.08 (m, 5H), 2.00-1.84 (m, 2H), 1.79 (d, J=6.4 Hz, 3H), 1.03 (t, J=6.5 Hz, 3H).

Example 7

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-2.SUP.5.-fluoro-8-methyl-1.SUP.1.H-3,5-diaza-1(6,1)-indola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 7

[0186] ##STR00025##

Step 1

Methyl 3-(6-bromo-1H-indol-1-yl)butanoate 7-1

[0187] 6-Bromoindole (1.0 g, 5.1 mmol), methyl 3-bromobutanoate (1.38 g, 7.65 mmol) and sodium hydroxide (0.43 g, 7.65 mmol) were added to N,N-dimethylformamide (DMF, 10 mL). The reaction solution was reacted under a nitrogen atmosphere at 70° C. for 10 hours. Water (50 mL)/ethyl acetate (50 mL) were added to the reaction solution and stirred for extraction. The organic phase was dried, filtered and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether: 1/30-1/10) to obtain compound 7-1 as a white solid (1.2 g, yield: 80%). MS (ESI):296.1 [M+1].sup.+.

Step 2

Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-3

[0188] Methyl 3-(6-bromo-1H-indol-1-yl)butanoate 7-1 (2.4 g, 8 mmol), pinacol diborate (2.3 g, 9.1 mmol), potassium acetate (2.4 g, 24 mmol) and Pd(dppf)Cl.sub.2 (326 mg, 0.4 mmol) were dissolved in 70 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the intermediate 2,4-dichloro-5-fluoropyrimidine (1.7 g, 10.4 mmol), cesium carbonate (7.8 g, 24 mmol), Pd(dppf)Cl.sub.2 (326 mg, 0.4 mmol) and 7 mL of water were added to the reaction solution. The reaction solution was reacted at 110° C. for 14 hours. The reaction was monitored by LC-MS. When the raw material no longer decreased, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with ethyl acetate (3×25 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain the title compound as a yellow solid (1 g, yield: 32.5%).

[0189] MS (ESI):348.1 [M+1].sup.+.

Step 3

Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-4

[0190] Methyl 3-(6-(2-chloro-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-3 (0.63 g, 1.8 mmol), N.sup.4-benzyl-5-((4-ethylpiperazin-1-yl)methyl)pyridine-2,4-diamine (0.72 g, 2.2 mmol), Pd.sub.2(dba).sub.3 (82 mg, 0.09 mmol), 2-dicyclohexylphosphorus-2,4,6-triisopropylbiphenyl (X-phos) (128 mg, 0.27 mmol) and potassium carbonate (0.745 g, 5.4 mmol) were suspended in 70 mL of dioxane under a nitrogen atmosphere. The reaction solution was reacted at 100° C. for 12 hours. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was concentrated to dryness by rotary evaporation. Water was added, and the solution was extracted with dichloromethane (3×25 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol=5:1) to obtain the title compound as a brown solid (0.45 g, yield: 40%).

[0191] MS (ESI): 637.1 [M+1].sup.+.

Step 4

Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-5

[0192] Methyl 3-(6-(2-((4-(benzylamino)-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-4 (0.45 g, 0.7 mmol) was dissolved in 4 mL of concentrated sulfuric acid at 0° C., and the solution was stirred for 30 minutes. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. The reaction solution was poured into crushed ice, and 10% sodium hydroxide solution was added to adjust pH=8-9. The reaction solution was extracted with ethyl acetate (3×20 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the title compound as a brown solid (0.4 g, yield: 100%).

[0193] MS (ESI):547.1 [M+1].sup.+.

Step 5

3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoic acid 7-6

[0194] Methyl 3-(6-(2-((4-amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoate 7-5 (0.38 g, 6.9 mmol) was dissolved in 5 mL methanol and 5 mL of 10% sodium hydroxide solution. The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (dichloromethane:methanol=5:1). After the reaction was completed, the reaction was stopped. Concentrated hydrochloric acid was added to adjust pH=4-5. The reaction solution was concentrated to dryness by rotary evaporation and dried. 50 mL of ethanol was added, and the solution stirred for 5 hours, filtered, and washed. The filtrate was concentrated to dryness by rotary evaporation to obtain the title compound as a yellow solid (0.38 g, yield: 100%).

[0195] MS (ESI):551.1 [M+1].sup.+.

Step 6

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8-dimethyl-1H-3,5-diaza-1(6,1)-indola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 7

[0196] 3-(6-(2-((4-Amino-5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-1H-indol-1-yl)butanoic acid 7-6 (0.38 g, 0.7 mmol) and triethylamine (212 mg, 2.1 mmol) were dissolved in 50 mL of dichloromethane, followed by the addition of HATU (323 mg, 0.85 mmol). The reaction solution was stirred at room temperature overnight. The reaction was monitored by TLC (DCM:MeOH=5:1). After the reaction was completed, the reaction was stopped. Water was added, and the reaction solution was extracted with dichloromethane (3×20 mL). The organic phases were combined, dried and concentrated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by preparative silica gel plate (DCM:MeOH=5:1) to obtain the title compound 7 as a white solid (120 mg).

[0197] MS (ESI):515.2 [M+1].sup.+.

[0198] .sup.1H NMR (400 MHz, CDCl.sub.3)) δ 10.23 (s, 1H), 10.01 (s, 1H), 9.21 (s, 1H), 8.45-8.20 (m, 2H), 8.12 (d, J=11.8 Hz, 1H), 7.82 (s, 1H), 7.59 (d, J=11.8 Hz, 1H), 7.33 (d, J=12.1 Hz, 1H), 6.41 (d, J=12.1 Hz, 1H), 5.23 (s, 1H), 4.5 (br, 1H), 4.06 (m, 1H), 3.65 (s, 2H), 2.84-2.62 (m, 2H), 2.47-2.08 (m, 8H), 1.68 (d, J=6.2 Hz, 3H), 1.01 (t, J=6.5 Hz, 3H).

Example 8

4.SUP.5.-((4-Ethylpiperazin-1-yl)methyl)-1.SUP.4.,2.SUP.5.-difluoro-1.SUP.2.,8,8-trimethyl-1.SUP.1.H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 8

[0199] ##STR00026##

[0200] In accordance with the method described in Example 1, 3-aminobutyric acid 107 in Step 6 was replaced with 3-amino-3-methylbutyric acid, accordingly, 4.sup.5-((4-Ethylpiperazin-1-yl)methyl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8,8-trimethyl-1.sup.1H-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-6-one 8 was prepared as a white solid.

[0201] MS (ESI):562.2 [M+1].sup.+.

[0202] .sup.1H NMR (400 MHz, CDCl.sub.3)) δ 10.31 (s, 1H), 10.01 (s, 1H), 9.73 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.62 (d, J=11.8 Hz, 1H), 3.75 (s, 2H), 3.35 (q, J=6.5 Hz, 2H), 2.64 (s, 2H), 2.51 (s, 3H), 2.47-2.35 (m, 8H), 1.43 (s, 6H), 1.03 (t, J=6.5 Hz, 3H).

Example 9

5′-((4-Ethylpiperazin-1-yl)methyl)-4′,5′-difluoro-2′-methylspiro[cyclopropane-1,8′-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan]-6′-one 9

[0203] ##STR00027##

[0204] In accordance with the method described in Example 1, 3-aminobutyric acid 107 in Step 6 was replaced with 2-(1-aminocyclopropyl)acetic acid, accordingly, 5′-((4-ethylpiperazin-1-yl)methyl)-4′,5′-difluoro-2′-methylspiro[cyclopropane-1,8′-3,5-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan]-6′-one 9 was prepared as a white solid.

[0205] MS (ESI):560.1 [M+1].sup.+.

[0206] .sup.1H NMR (400 MHz, CDCl.sub.3)) δ 10.34 (s, 1H), 10.24 (s, 1H), 9.71 (s, 1H), 8.89-8.47 (m, 2H), 8.12 (s, 1H), 7.62 (d, J=11.8 Hz, 1H), 3.75 (s, 2H), 3.35 (q, J=6.5 Hz, 2H), 2.84 (s, 2H), 2.62 (s, 3H), 2.47-2.35 (m, 8H), 1.03 (t, J=6.5 Hz, 3H), 0.76-0.74 (m, 2H), 0.52-0.48 (m, 2H).

Examples 6, 10 and 11

[0207] In accordance with the method described in Examples 1 to 3, appropriate compounds were used as starting materials, accordingly, the following compounds were prepared: [0208] 4.sup.5-((4-ethylpiperazin-1-yl)methyl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8-dimethyl-1′H-5-oxa-3-aza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphane 6; [0209] 4.sup.5-((4-ethylpiperazin-1-yl)methyl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8-dimethyl-1.sup.1H-3,6-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-5-one 10; [0210] 4.sup.5-(4-ethylpiperazin-1-yl)-1.sup.4,2.sup.5-difluoro-1.sup.2,8-dimethyl-1.sup.1H-3,6-diaza-1(6,1)-benzo[d]imidazola-2(4,2)-pyrimidina-4(2,4)-pyridinacyclooctaphan-5-one 11.

Test Example 1

Determination of Inhibitory Effect on CDK4/Cyclin D1

[0211] The determination of IC.sub.50 of CDK4/Cyclin D1 was conducted according to the following process. A 96-well filter plate (Millipore MADVN6550) was used. The total volume was 0.1 ml, containing buffer solution A (20 mM TRIS (tris[hydroxymethyl]aminomethane) (pH7.4), 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2), 2 μM ATP (containing 0.25 Ci[32P]ATP), 20 ng CDK4, 1 g retinoblastoma protein and the test compound diluted in buffer solution A in an appropriate ratio. Buffer solution A alone without the test compound was used as the control without inhibitory effect. Buffer solution A containing excess EDTA was used to determine the background 32P level in the absence of enzymatic activity. All components except ATP were added to the wells, and the plate was shaked on a plate mixer for 2 minutes. [32P]ATP was added to initiate the reaction, and the plate was incubated at 25° C. for 15 minutes. 0.1 ml of 20% trichloroacetic acid (TCA) was added to stop the reaction. The plate was kept at 4° C. for at least 1 hour to allow the substrate to precipitate. The wells were washed five times with 0.2 ml of 10% TCA, and the binding of 32P was measured using a R plate counter (wallac Inc., Gaithersburg, Md.). IC.sub.50 of the test compound was determined by median effect method (Chou, T-C and Talalay P. Applications of the median effect principle for the assessment of low-dose risk of carcinogens and for the quantitation of synergism and antagonism of chemotherapeutic agents. In New Avenues in Developmental Cancer Chemotherapy (Eds. Harrap, K. T. and Connors, T. A.), pp. 37-64. Academic Press, New York, 1987).

[0212] Experimental method: Inhibitory activity on CDK4/6 kinase was determined by Caliper Mobility Shift method

[0213] 1. Preparation of 1-fold kinase buffer solution:

[0214] 1) Preparation of 1-fold CDK4 kinase buffer solution

[0215] 800 μL of 1000 mM HEPES stock solution (pH 7.5) and 40 μL of 10% Triton X-100 stock solution were added to 39160 μL of ultra pure water, and the resulting solution was mixed well.

[0216] 2) Preparation of 1-fold CDK6 kinase buffer solution

[0217] 50 mL of 1000 mM HEPES stock solution (pH 7.5) and 50 μL of 30% Brij-35 stock solution were added to 949.95 mL of ultra pure water, and the resulting solution was mixed well.

[0218] 2. Preparation of stop solution

[0219] 25 mL of 4% Coating Reagent #3 (provided with the 12-sipper chip used in Caliper device) stock solution, 50 mL of 1000 mM HEPES stock solution (pH 7.5), 50 mL of 0.5 M EDTA stock solution and 0.25 mL of 30% Brij-35 stock solution were added to 374.75 mL of ultra pure water, and the resulting solution was mixed well.

[0220] 3. Preparation of 2.5-fold kinase solution

[0221] 1) Preparation of 2.5-fold CDK4/D3 kinase solution

[0222] 7 μL of CDK4/D3 enzyme solution and 9 μL of 1M DTT stock solution were added to 1784 μL of 1-fold CDK4 kinase buffer solution, and the resulting solution was mixed well.

[0223] 2) Preparation of 2.5-fold CDK6/D3 kinase solution 18 μL of CDK6/D3 enzyme solution and 14 μL of 1M DTT stock solution were added to 2768 μL of 1-fold CDK6 kinase buffer solution, and the resulting solution was mixed well.

[0224] 4. Preparation of 2.5-fold polypeptide solution

[0225] 1) Preparation of 2.5-fold CDK4/D3 polypeptide solution

[0226] 10 L of 100 mM ATP stock solution, 45 μL of 1M MgCl2 stock solution and 45 μL of FAM-labelled polypeptide 8 were added to 1700 μL of 1-fold CDK4 kinase buffer solution, and the resulting solution was mixed well.

[0227] 2) Preparation of 2.5-fold CDK6/D3 polypeptide solution

[0228] 23 μL of 100 mM ATP stock solution, 75 μL of 1M MgCl2 stock solution and 75 μL of FAM-labelled polypeptide 8 were added to 2827 μL of 1-fold CDK6 kinase buffer solution, and the resulting solution was mixed well.

[0229] 5. Preparation of 5-fold test compound solution:

[0230] A 10 mM solution of the test compound in DMSO was diluted with DMSO to obtain a solution with a concentration of 50 μM, which was used as the stock solution. Said stock solution was subjected to 4-fold gradient dilution with DMSO to obtain solutions with a concentration of 12. μM, 3.12 μM, 0.78 μM, 0.19 μM, 0.048 μM, 12.2 nM, 3 nM, 0.76 nM and 0.19 nM, respectively. Each of the resulting solution was 10-fold diluted with 1-fold kinase buffer solution to obtain a 5-fold compound solution.

[0231] 6. CDK4/6 enzymatic reaction:

[0232] 1) 5 μL of the prepared 5-fold test compound solution and 10 μL of the prepared 2.5-fold kinase solution were added to the corresponding wells in a 384-well plate, and the plate was incubated at room temperature for 10 minutes.

[0233] 2) 10 μL of the prepared 2.5-fold polypeptide solution was added to the corresponding wells to start the enzymatic reaction, and the plate was incubated at 28° C. for 5 hours.

[0234] 7. Enzymatic assay:

[0235] 25 μL of stop solution was added to each corresponding well to stop the reaction.

[0236] 8. The data was read by the Caliper device, the inhibition rate was calculated by the following formula, and curve fitting was carried out by GraphPad5.0 software to obtain the IC.sub.50 value.

TABLE-US-00002 CDK4 CDK6 Example No. IC.sub.50 (nM) IC.sub.50 (nM) LY2834219 1.2 2.2 1 0.70 0.48 2 0.49 0.35 3 15 27 4 0.78 1.13 5 21 14 7 1.5 2.5 8 8 10 9 5 12

Test Example 2

In Vitro Cell Inhibitory Activity of the Compound of the Present Invention

[0237] Experimental method: Cell proliferation assay was carried out by BrdU method (BrdU cell proliferation assay kit, Cell Signaling Technology Co.)

[0238] 1. Preparation of reagents and compounds

[0239] Preparation of 1-fold washing solution:

[0240] A stock washing solution (concentration: 20-fold) was diluted with ultra pure water to obtain the 1-fold washing solution.

[0241] Preparation of 1-fold detection antibody solution:

[0242] A BrdU detection antibody stock solution (concentration: 100-fold) was diluted with a detection antibody diluent to obtain the 1-fold detection antibody solution.

[0243] Preparation of 1-fold HRP-labelled secondary antibody solution:

[0244] A stock solution of RP-labelled anti-mouse IgG antibody (concentration: 100-fold) was diluted with HRP-labelled antibody diluent to obtain the 1-fold HRP-labelled secondary antibody solution.

[0245] 10-fold BrdU solution:

[0246] A BrdU stock solution (concentration: 1000-fold) was diluted with the corresponding cell culture medium to obtain the 10-fold BrdU solution.

[0247] Preparation of test compound:

[0248] Preparation of test compound stock solution: 10 mM stock solution was prepared by 100% DMSO.

[0249] Preparation of gradient dilution solution of test compound: the 10 mM stock solution of test compound was subjected to 4-fold serial gradient dilution with DMSO to obtain solutions with a concentration of 2.5 mM, 62 μM, 156 PM, 3 μM, 9. μM and 2. μM, respectively. 2 μL of DMSO-diluted compound was added to 198 μL of culture medium containing 10% FBS to obtain 10-fold test compound, wherein the maximum concentration of the test compound was 10 μM, the DMSO concentration was 1%, with a total of 7 concentration gradients.

[0250] Preparation of culture medium:

[0251] MCF-7 culture medium: DMEM+10% FBS+0.01 mg/mL insulin

[0252] 2. Experimental procedures

[0253] (1) Cells grown to 80% (in exponential growth phase) were digested with pancreatin, and were collected by centrifugation. MDA-MB-435S cells and U87MG cells were re-suspended in a FBS-free culture medium, counted and inoculated to a 96-well plate; MDA-MB-435S cells were inoculated at 3000 cells/well/81 μL, and U87MG cells were inoculated at 4000 cells/well/81 μL. MCF-7 cells were resuspended in a culture medium containing 1% FBS, counted and inoculated to a 96-well plate at 4000 cell/well/82 μL. The cells were incubated in a cell incubator at 37° C.

[0254] (2) After 24 hours of incubation, FBS (9 μL) was added to each well of the MDA-MB-435S cells and U87MG cells, and 8 μL of FBS was added to each well of the MCF-7 cells, so as to obtain a final FBS concentration of 10%.

[0255] (3) A different concentration of 10-fold test compound (10 μL) was added to each well to obtain a final concentration of test compound of 10 μM, 2.5 μM, 625 nM, 156 nM, 39 nM, 9.8 nM or 2.5 nM, respectively, with 3 repeated wells/group, and the cells were incubated at 37° C. for 72 hours.

[0256] Solvent control: 0.1% DMSO

[0257] Blank control: only culture medium without cells

[0258] Normal cell control: normal cells without any treatment

[0259] (4) 10-fold BrdU solution (10 μL) was added to each well, and the cells were incubated in the incubator for 4 hours, followed by discarding the culture medium.

[0260] (5) Fixing/denaturation solution (10 μL) was added to each well, and the cells were incubated at room temperature for 30 minutes, followed by discarding the solution.

[0261] (6) 1-fold detection antibody solution (100 μL) was added to each well, the cells were incubated at room temperature for 1 hour, followed by discarding the solution, and the cells were washed with 1-fold washing solution (200 μL/well) three times.

[0262] (7) 1-fold HRP-labelled secondary antibody solution (100 μL) was added to each well, the cells were incubated at room temperature for 30 minutes, followed by discarding the solution, and the cells were washed with 1-fold washing solution (200 μL/well) three times.

[0263] (8) TMB substrate solution (100 μL) was added to each well, and the cells were incubated at room temperature for 30 minutes.

[0264] (9) Stop solution (100 μL) was added to each well, and the OD value at 450 nm was measured by a microplate reader.

[0265] 3. Data Processing

[0266] 1) Cell survival rate (%)=(OD.sub.test compound−OD.sub.blank control)/(OD.sub.normal cell control−OD.sub.blank control)×100%,

[0267] OD.sub.blank control: blank control value; OD.sub.normal cell control: normal cell control value;

[0268] 2) The data was processed by GraphPad Prism 5 software to obtain the curve and IC.sub.50 values.

TABLE-US-00003 MCF-7 Example No. IC.sub.50 (nM) LY2834219 120 1 142 2 42 3 4125 4 132