Pteridine ketone derivative and applications thereof as EGFR, BLK, and FLT3 inhibitor

Abstract

Provided are a pteridine ketone derivative used as an EGFR, BLK, and FLT3 inhibitor and applications thereof. Specifically, provided are a compound of the following formula I, a pharmaceutical composition containing the compound of the formula I, and use of compound in preparing medicine for treating diseases mediated by EGFR, BLK, or FLT3 or inhibiting EGFR, BLK, and FLT3.

Claims

1. A compound having the structure of Formula II: ##STR00053## wherein Y is selected from N, CH; Z is selected from N, CR.sup.6; R.sup.1 is a hydrogen, a halogen, a C.sub.1-C.sub.6 alkoxyl, an optionally substituted C.sub.1-C.sub.6 alkyl, an optionally substituted aryl, or an optionally substituted aralkyl; each of R.sup.3 is independently selected from a halogen, a hydroxy, an optionally substituted acyloxy, an amino, an optionally substituted acylamino, an optionally substituted C.sub.1-C.sub.6 alkyl, CN, a carbamoyl, a carboxyl, an optionally substituted alkoxyformyl, an optionally substituted phenyl, an optionally substituted N-alkylpiperazinyl, an optionally substituted morpholinyl, an optionally substituted piperidinyl, an optionally substituted pyrrolyl, an optionally substituted pyrrolidinyl, NR.sub.aR.sub.b, or an optionally substituted pyridyl; each of R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently selected from a hydrogen, a halogen, a C.sub.1-C.sub.6 alkoxyl, a hydroxyl, an optionally substituted acyloxy, an amino, an optionally substituted acylamino, an optionally substituted C.sub.1-C.sub.6 alkyl, CN, a carbamoyl, a carboxyl, an optionally substituted alkoxyformyl, an optionally substituted phenyl, an optionally substituted N-alkylpiperazinyl, an optionally substituted morpholinyl, an optionally substituted piperidinyl, an optionally substituted pyrrolyl, an optionally substituted pyrrolidinyl, NR.sub.aR.sub.b, an optionally substituted pyridyl; each of R.sub.a and R.sub.b are independently selected from an alkyl and an alkenyl; and each of m and n is 0, 1, 2, 3 or 4; wherein optionally substituted means that the group modified by the term can be optionally substituted by 1-5 substituents selected from: a halogen, a (C.sub.1-3 alkyl)-CHO or CHO, a C.sub.1-6 straight chain or branched chain alkyl, a cyano, a nitro, an amino, a hydroxyl, a hydroxymethyl, a halogen-substituted alkyl, a halogen-substituted alkoxyl, a carboxyl, a C.sub.1-4 alkoxyl, an ethoxyformyl, N(CH.sub.3) and a C.sub.1-4 acyl; acyloxy refers to a group having the structure of the formula OC(O)R wherein R is selected from an alky, an alkenyl, and an alkynyl and R can be optionally substituted; acylamino refers to a group having the structure of the formula RNHC(O)R, wherein, R is selected from a bond or an alkyl, and R is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, an NR.sub.aR.sub.b-substituted alkyl, an NR.sub.aR.sub.b-substituted alkenyl, an NR.sub.aR.sub.b-substituted alkynyl, a halogen-substituted alkyl, a cyano-substituted alkenyl, ##STR00054## wherein R.sub.a and R.sub.b are selected from an alkyl and an alkenyl.

2. The compound of claim 1, wherein the compound has the structure of formula III: ##STR00055## wherein, R.sup.1 is a hydrogen, a halogen, a C.sub.1-C.sub.6 alkoxyl, an optionally substituted C.sub.1-C.sub.6 alkyl, an optionally substituted aryl, or an optionally substituted aralkyl; R.sup.3 is independently selected from a halogen, a hydroxyl, an optionally substituted acyloxy, an amino, an optionally substituted acylamino, an optionally substituted C.sub.1-C.sub.6 alkyl, CN, a carbamoyl, a carboxyl, an optionally substituted alkoxyformyl, an optionally substituted phenyl, an optionally substituted N-alkylpiperazinyl, an optionally substituted morpholinyl, an optionally substituted piperidinyl, an optionally substituted pyrrolyl, an optionally substituted pyrrolidinyl, NR.sub.aR.sub.b, or an optionally substituted pyridyl; each of R.sup.5, R.sup.6 and R.sup.7 is independently selected from a hydrogen, a halogen, a C.sub.1-C.sub.6 alkoxyl, a hydroxyl, an optionally substituted acyloxy, an amino, an optionally substituted acylamino, an optionally substituted C.sub.1-C.sub.6 alkyl, CN, a carbamoyl, a carboxyl, an optionally substituted alkoxyformyl, an optionally substituted phenyl, an optionally substituted N-alkyl-piperazinyl, an optionally substituted morpholinyl, an optionally substituted piperidinyl, an optionally substituted pyrrolyl, an optionally substituted pyrrolidinyl, NR.sub.aR.sub.b, or an optionally substituted pyridyl; each of R.sub.a and R.sub.b are independently selected from an alkyl and an alkenyl; and each of m and n is 0, 1, 2, 3 or 4.

3. The compound of claim 2, wherein R.sup.1 is selected from H, and a C.sub.1-C.sub.6 alkyl; R.sup.3 is selected from an amino, an acyloxy, a halogen, a hydroxyl, an alkyl, CN, a carboxyl, a morpholinyl, a N-alkyl-piperazinyl, a piperidinyl, a pyrrolyl, a pyrrolidinyl, a pyridyl, NR.sub.aR.sub.b, an acylamino, and a carbamoyl, wherein R.sub.a and R.sub.b are selected from an alkyl and an alkenyl; R.sup.5 is selected from H, an alkoxyl, a morpholinyl, a halogen, a N-alkyl-piperazinyl, a piperidinyl, a pyrrolyl, a pyrrolidinyl, a pyridyl, NR.sub.aR.sub.b, an acylamino, and a carbamoyl, wherein R.sub.a and R.sub.b can be selected from an alkyl and an alkenyl; R.sup.6 is H; and R.sup.7 is selected from H or an acylamino.

4. The compound of claim 3, wherein R.sup.5 is selected from a halogen, a 4-N-methylpiperazinyl, a N-morpholinyl, a N-piperidinyl, a N-pyrrolyl, a N-pyrrolidinyl, a N,N-diethyl-amino, a N,N-dimethylmethylamine group, and 4-pyridyl; R.sup.3 is selected from the group consisting of: ##STR00056## wherein X is a halogen; and R.sup.7 is H.

5. A compound selected from the group consisting of ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##

6. A pharmaceutical composition, wherein the pharmaceutical composition comprises the compound of claim 1, or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. A method of treating lung cancer or breast cancer, said method comprising administering to a subject in need thereof a compound of claim 1.

8. The compound of claim 2, wherein R.sup.6 is OCH.sub.3.

9. The compound of claim 1, wherein said compound comprises the structure of the formula: ##STR00064## ##STR00065## ##STR00066##

Description

EXAMPLE 1

(1) The particular method for steps a-f as said above is shown as follows:

Synthesis of tert-butyl (4-(2-chloro-5-nitropyrimidyl-4-amino)phenyl)carbamate (step a)

(2) ##STR00017##

(3) 2,4-dichloro-5-nitro-pyrimidine (95 mg, 0.49 mmol) was placed into a 10 mL round bottom flask, 3 mL of 1,4-dioxane was added, and stirred at room temperature. Tert-butyl (4-aminophenyl) carbamate (100 mg, 0.48 mmol) and N,N-diisopropylethylamine (69 mg, 0.53 mmol) were dissolved in 2 mL of 1,4-dioxane. The resulting solution was added dropwise into the reaction solution as said above. Upon completion of addition, the resulting mixture was stirred at room temperature for 0.5 h, and TLC showed that the raw material was completely conversed. The solvent was removed by rotary evaporation, and the crude product was separated through silica gel column chromatography (petroleum ether/ethyl acetate=10:1, V/V) to obtain tert-butyl (4-(2-chloro-5-nitropyrimidyl-4-amino)-phenyl)carbamate as orange solids (144 mg, yield 82%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.38 (s, 1H), 9.46 (s, 1H), 9.12 (s, 1H), 7.49 (d, J=8.6 Hz, 2H), 7.39 (d, J=8.6 Hz, 2H), 1.49 (s, 9H).

Synthesis of tert-butyl (4-(2-(4-methoxyphenylamino)-5-nitro-pyrimidyl-4-amino)phenyl) carbamate (step b)

(4) ##STR00018##

(5) Tert-butyl (4-(2-chloro-5-nitropyrimidyl-4-amino)phenyl) carbamate (50 mg, 0.14 mmol), p-anisidine (17 mg, 0.14 mmol), N,N-diisopropylethylamine (18 mg, 0.18 mmol) were placed into a 10 mL round bottom flask, 5 mL of 1,4-dioxane was added, and stirred at room temperature for 4 hours. TLC showed that the raw material was completely conversed. The solvent was removed by rotary evaporation, and the crude product was purified through silica gel column chromatography (petroleum ether/ethyl acetate=4:1, V/V) to obtain tert-butyl (4-(2-(4-methoxyphenylamino)-5-nitro-pyrimidyl-4-amino)phenyl) carbamate as yellow solids (51 mg, yield 82%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.30 (s, 1H), 10.26 (s, 1H), 9.45 (s, 1H), 9.04 (s, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.45 (d, J=8.8 Hz, 2H), 7.40 (d, J=8.6 Hz, 2H), 6.75 (d, J=8.6 Hz, 2H), 3.73 (s, 3H), 1.50 (s, 9H).

Synthesis of tert-butyl (4-(5-amino-2-(4-methoxyphenylamino)pyrimidyl-4-amino)phenyl) carbamate (Step c)

(6) ##STR00019##

(7) Tert-butyl (4-(2-(4-methoxy-phenylamino)-5-nitro-pyrimidyl-4-amino)phenyl) carbamate (45 mg, 0.10 mmol) was placed into a 50 mL round bottom flask. 20 mL of ethanol, and 5 mg of palladium on carbon (10% Pd) were added, hydrogen was filled, and the resulting reaction system was stirred at room temperature overnight. Upon the completion of reaction, the system was filtered, and the filtrate was spin-dried. The crude product was purified through silica gel column chromatography (dichloromethane/methanol=5:1, V/V) to give tert-butyl (4-(5-amino-2-(4-methoxylphenylamino)-pyrimidyl-4-amino)phenyl) carbamate as pale pink solids (30 mg, yield 83%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 9.23 (s, 1H), 8.42 (s, 1H), 8.10 (s, 1H), 7.62 (d, J=9.2 Hz, 2H), 7.56 (s, 1H), 7.53 (d, J=9.2 Hz, 2H), 7.40 (d, J=8.8 Hz, 2H), 6.77 (d, J=8.8 Hz, 2H), 3.70 (s, 3H), 1.48 (s, 9H).

Synthesis of tert-butyl (4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)-phenyl) carbamate (step d)

(8) ##STR00020##

(9) Tert-butyl (4-(5-amino-2-(4-methoxyphenylamino)pyrimidinyl-4-amino)phenyl) carbamate (30 mg, 0.07 mmol) was added into a 10 mL round bottom flask. 0.29 mL of glacial acetic acid and 5 mL of anhydrous ethanol were added, and then ethyl glyoxylate (50% in toluene) (16 mg, 0.08 mmol) was added. The resulting reaction mixture was heated to reflux and stirred overnight. Upon completion, solids precipitated. The solids were filtered, and the filter cake was washed with ethanol, ammonia water and deionized water, and dried, to give tert-butyl (4-(2-(4-methoxy-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) carbamate as yellow solids (18 mg, yield 76%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.08 (s, 1H), 9.64 (s, 1H), 8.84 (s, 1H), 8.03 (s, 1H), 7.65 (d, J=8.4 Hz, 2H), 7.30-7.28 (m, 4H), 6.61 (br, 2H), 3.67 (s, 3H), 1.52 (s, 9H).

Synthesis of 8-(4-aminophenyl)-2-(4-methoxyphenyl)-7(8H)-pteridin-one (Compound 001) (step e)

(10) ##STR00021##

(11) Tert-butyl (4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) carbamate (18 mg, 0.04 mmol) was placed a 5 mL round bottom flask. 2 mL of dichloromethane was added, and the resulting mixture was stirred at 0 C. 0.5 mL of trifluoroacetic acid was added, and the resulting mixture was stirred at 0 C. for 1 hour, and then at room temperature for another 1 hour. After the reaction was completed, a saturated sodium bicarbonate solution was added to neutralize the solution to alkaline, and the resulting mixture was extracted with dichloromethane (350 mL). The organic phase was washed with deionized water, and a saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was spin-dried to obtained 8-(4-aminophenyl)-2-(4-methoxyphenyl)-7(8H)-pteridin-one as yellow solids (14 mg, yield 99%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.04 (br, 1H), 8.81 (s, 1H), 8.00 (s, 1H), 7.40 (d, J=7.6 Hz, 2H), 6.98 (d, J=8.4 Hz, 2H), 6.73 (d, J=8.4 Hz, 2H), 6.67 (br, 2H), 5.44 (s, 2H), 3.70 (s, 3H). .sup.13C NMR (100 MHz, DMSO-d.sub.6): 159.19, 158.53, 157.17, 154.95, 151.76, 149.66, 146.68, 133.17, 129.22, 122.66, 121.04, 120.70, 114.37, 113.87, 55.55. HRMS (ESI) calculated for C.sub.19H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 361.1413. found 361.1414.

Synthesis of N-(4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl)acrylamide (Compound 002) (step f)

(12) ##STR00022##

(13) 8-(4-aminophenyl)-2-(4-methoxyphenyl)-7(8H)-pteridin-one (100 mg, 0.28 mmol) was placed in a 100 mL round bottom flask. 50 mL of dichloromethane, and triethylamine (28 mg, 0.28 mmol) were added, and the resulting mixture was stirred at 0 C. Acryloyl chloride (29 mg, 0.31 mmol) was dissolved in 5 mL of dichloromethane and added dropwise to the above reaction solution. Upon addition, the resulting reaction mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane/ethyl acetate=5:1, v/v) to obtain N-(4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide as yellow solids (34 mg, yield 30%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.42 (s, 1H), 10.07 (br, 1H), 8.84 (s, 1H), 8.04 (s, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.38 (d, J=8.8 Hz, 2H), 7.30 (br, 2H), 6.59 (br, 2H), 6.52 (dd, J=17.0, 10.0 Hz, 1H), 6.33 (dd, J=17.0, 1.8 Hz, 1H), 5.82 (dd, J=10.0, 1.8 Hz, 1H), 3.62 (s, 3H). .sup.13C NMR (100 MHz, DMSO-d.sub.6): 163.90, 159.28, 158.51, 156.68, 155.02, 151.44, 146.65, 139.72, 133.00, 130.18, 129.50, 127.72, 121.02, 120.61, 113.77, 55.40. HRMS (ESI) calculated for C.sub.22H.sub.19N.sub.6O.sub.3 [M+H].sup.+ 415.1519. found 415.1515.

(14) The following compounds were synthesized according to the above steps a-f:

N-(4-(2-(4-morpholino-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl)acrylamide (Compound 003)

(15) ##STR00023##

(16) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.44 (s, 1H), 10.00 (s, 1H), 8.82 (s, 1H), 8.02 (s, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.36 (d, J=8.4 Hz, 1H), 7.22 (br, 2H), 6.59 (br, 2H), 6.52 (dd, J=17.2, 10.2 Hz, 1H), 6.33 (d, J=17.2 Hz, 1H), 5.85 (d, J=10.2 Hz, 1H), 3.67 (br, 4H), 2.92 (br, 4H). HRMS (ESI) calculated for C.sub.25H.sub.24N.sub.7O.sub.3 [M+H].sup.+ 470.1941. found 470.1932.

N-(4-(2-(4-methoxyphenylamino)-6-methyl-7-oxo-8(7H)-pteridin-yl)phenyl)acrylamide (Compound 004)

(17) ##STR00024##

(18) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.44 (s, 1H), 9.90 (br, 1H), 8.77 (s, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 7.29 (br, 2H), 6.59 (br, 2H), 6.52 (dd, J=17.0, 10.0 Hz, 1H), 6.33 (dd, J=17.0, 1.9 Hz, 1H), 5.82 (dd, J=10.0, 1.9 Hz, 1H), 3.61 (s, 3H), 2.42 (s, 3H). HRMS (ESI) calculated for C.sub.23H.sub.21N.sub.6O.sub.3 [M+H].sup.+ 429.1675. found 429.1671.

8-(3-aminophenyl)-2-(4-methoxyphenyl)-7(8H)-pteridin-one (Compound 005)

(19) ##STR00025##

(20) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.06 (br, 1H), 8.83 (s, 1H), 8.01 (s, 1H), 7.41 (d, J=8.0 Hz, 2H), 7.22 (t, J=8.0 Hz, 1H), 6.75 (d, J=7.6 Hz, 1H), 6.67 (br, 2H), 6.53 (s, 1H), 6.48 (d, J=7.6 Hz, 1H), 5.35 (s, 2H), 3.69 (s, 3H). HRMS (ESI) calculated for C.sub.19H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 361.1413. found 361.1413.

N-(3-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 006)

(21) ##STR00026##

(22) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.42 (s, 1H), 10.10 (br, 1H), 8.85 (s, 1H), 8.05 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.78 (s, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.31 (br, 2H), 7.13 (d, J=8.0 Hz, 1H), 6.58 (br, 2H), 6.45 (dd, J=16.8, 10.4 Hz, 1H), 6.26 (dd, J=16.8, 1.6 Hz, 1H), 5.77 (dd, J=10.4, 1.6 Hz, 1H), 3.65 (s, 3H). HRMS (ESI) calculated for C.sub.22H.sub.19N.sub.6O.sub.3 [M+H].sup.+ 415.1519. found 415.1516.

N-(3-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) propionamide (Compound 007)

(23) ##STR00027##

(24) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.13 (s, 1H), 10.09 (s, 1H), 8.85 (s, 1H), 8.04 (s, 1H), 7.74 (d, J=8.0 Hz, 1H), 7.71 (s, 1H), 7.53 (t, J=8.0 Hz, 1H), 7.31 (br, 2H), 7.07 (d, J=8.0 Hz, 1H), 6.59 (br, 2H), 3.67 (s, 3H), 2.33 (q, J=7.6 Hz, 2H), 1.07 (t, J=7.6 Hz, 3H). HRMS (ESI) calculated for C.sub.22H.sub.21N.sub.6O.sub.3 [M+H].sup.+ 417.1675. found 417.1678.

N-(4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) propionamide (Compound 008)

(25) ##STR00028##

(26) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.15 (s, 1H), 10.08 (br, 1H), 8.85 (s, 1H), 8.04 (s, 1H), 7.80 (d, J=8.4 Hz, 2H), 7.35-7.33 (m, 4H), 6.61 (br, 2H), 3.67 (s, 3H), 2.41 (q, J=7.6 Hz, 2H), 1.14 (t, J=7.6 Hz, 3H). HRMS (ESI) calculated for C.sub.22H.sub.21N.sub.6O.sub.3 [M+H].sup.+ 417.1675. found 417.1674.

4-(dimethylamino)-N-(4-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl)-2-buteneamide (Compound 009)

(27) ##STR00029##

(28) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.45 (s, 1H), 10.10 (br, 1H), 8.85 (s, 1H), 8.05 (s, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.37 (d, J=8.8 Hz, 2H), 7.30 (br, 2H), 6.82 (td, J=15.4, 6.0 Hz, 1H), 6.60 (br, 2H), 6.40 (d, J=15.4 Hz, 1H), 3.63 (s, 3H), 3.27 (d, J=5.2 Hz, 2H), 2.33 (s, 6H). HRMS (ESI) calculated for C.sub.25H.sub.26N.sub.7O.sub.3 [M+H].sup.+ 472.2097. found 472.2095.

4-(dimethylamino)-N-(3-(2-(4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl)-2-buteneamide (Compound 010)

(29) ##STR00030##

(30) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.33 (s, 1H), 10.08 (br, 1H), 8.86 (s, 1H), 8.05 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.78 (s, 1H), 7.55 (t, J=8.0 Hz, 1H), 7.32 (br, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.74 (td, J=15.2, 5.6 Hz, 1H), 6.59 (br, 2H) 6.30 (d, J=15.2 Hz, 1H), 3.66 (s, 3H), 3.06 (d, J=5.6 Hz, 2H), 2.17 (s, 6H). HRMS (ESI) calculated for C.sub.25H.sub.24N.sub.7O.sub.3 [M+H].sup.+ 472.2097. found 472.2094.

4-(2 (4-methoxyphenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl acrylate (Compound 011)

(31) ##STR00031##

(32) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.15 (s, 1H), 8.87 (s, 1H), 8.06 (s, 1H), 7.51 (d, J=8.8 Hz, 2H), 7.45 (d, J=8.8 Hz, 2H), 7.31 (br, 2H), 6.69 (br, 2H), 6.60 (dd, J=17.2, 1.6 Hz, 1H), 6.51 (dd, J=17.2, 9.9 Hz, 1H), 6.22 (dd, J=9.9, 1.6 Hz, 1H), 3.67 (s, 3H). HRMS (ESI) calculated for C.sub.22H.sub.18N.sub.5O.sub.4 [M+H].sup.+ 416.1359. found 416.1359.

4-(dimethylamino)-N-(4-(7-oxo-2-(phenylamino)-8(7H)-pteridin-yl)phenyl)-2-buteneamide (Compound 012)

(33) ##STR00032##

(34) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.37 (s, 1H), 10.19 (br, 1H), 8.90 (s, 1H), 8.08 (s, 1H), 7.87 (d, J=8.4 Hz, 2H), 7.42 (d, J=7.6 Hz, 2H), 7.38 (d, J=8.4 Hz, 2H), 7.03 (br, 1H), 6.88 (t, J=7.6 Hz, 1H), 6.82 (td, J=15.4, 5.6 Hz, 1H), 6.37 (d, J=15.4 Hz, 1H), 3.14 (d, J=5.6 Hz, 2H), 2.24 (s, 6H). HRMS (ESI) calculated for C.sub.24H.sub.24N.sub.7O.sub.2 [M+H].sup.+ 442.1991. found 442.1989.

4-(dimethylamino)-N-(3-(7-oxo-2-(phenylamino)-8(7H)-pteridin-yl)phenyl)-2-buteneamide (Compound 013)

(35) ##STR00033##

(36) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.32 (s, 1H), 10.17 (s, 1H), 8.90 (s, 1H), 8.08 (s, 1H), 7.81-7.79 (m, 2H), 7.55 (t, J=8.0 Hz, 1H), 7.41 (d, J=7.2 Hz, 2H), 7.12 (d, J=8.0 Hz, 1H), 7.01 (br, 2H), 6.87 (t, J=7.2 Hz, 1H), 6.73 (td, J=15.2, 5.6 Hz, 1H), 6.28 (d, J=15.2 Hz, 1H), 3.05 (d, J=5.6 Hz, 2H), 2.16 (s, 6H). HRMS (ESI) calculated for C.sub.24H.sub.24N.sub.7O.sub.2 [M+H].sup.+ 442.1991. found 442.1996

N-(4-(7-oxo-2-(phenylamino)-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 014)

(37) ##STR00034##

(38) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.44 (s, 1H), 10.19 (br, 1H), 8.90 (s, 1H), 8.09 (s, 1H), 7.88 (d, J=8.4 Hz, 2H), 7.41-7.38 (m, 4H), 7.03 (br, 2H), 6.88 (t, J=7.2 Hz, 1H), 6.53 (dd, J=16.8, 10.4 Hz, 1H), 6.35 (dd, J=16.8, 1.6 Hz, 1H), 5.84 (dd, J=10.4, 1.6 Hz, 1H). HRMS (ESI) calculated for C.sub.21H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 385.1413. found 385.1405.

N-(3-(7-oxo-2-(phenylamino)-8(7H)-pteridin-yl)phenyl)acrylamide (Compound 015)

(39) ##STR00035##

(40) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.42 (s, 1H), 10.19 (s, 1H), 8.91 (s, 1H), 8.09 (s, 1H), 7.84-7.81 (m, 2H), 7.57 (t, J=8.0 Hz, 1H), 7.41 (br, 2H), 7.15 (d, J=7.6 Hz, 1H), 7.02 (br, 2H), 6.87 (t, J=7.6 Hz, 1H), 6.45 (dd, J=16.8, 10.4 Hz, 1H), 6.26 (dd, J=16.8, 1.6 Hz, 1H), 5.77 (dd, J=10.4, 1.6 Hz, 1H). HRMS (ESI) calculated for C.sub.21H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 385.1413. found 385.1413.

N-(4-(2-(4-chlorophenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 016)

(41) ##STR00036##

(42) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.46 (s, 1H), 10.34 (s, 1H), 8.92 (s, 1H), 8.11 (s, 1H), 7.88 (d, J=8.8 Hz, 2H), 7.41-7.36 (m, 4H), 7.06 (br, 2H), 6.53 (dd, J=16.8, 10.4 Hz, 1H), 6.36 (dd, J=16.8, 1.6 Hz, 1H), 5.84 (dd, J=10.4, 1.6 Hz, 1H). HRMS (ESI) calculated for C.sub.21H.sub.16N.sub.6O.sub.2Cl [M+H].sup.+ 419.1023. found 419.1031.

N-(3-(2-(4-chlorophenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 017)

(43) ##STR00037##

(44) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.44 (s, 1H), 10.34 (br, 1H), 8.93 (s, 1H), 8.11 (s, 1H), 7.84 (s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.59 (t, J=8.0 Hz, 1H), 7.43 (d, J=7.2 Hz, 2H), 7.15 (d, J=7.6 Hz, 1H), 6.46 (dd, J=16.8, 10.4 Hz, 1H), 6.26 (dd, J=16.8, 1.8 Hz, 1H), 5.77 (dd, J=10.12, 1.8 Hz, 1H). HRMS (ESI) calculated for C.sub.21H.sub.16N.sub.6O.sub.2Cl [M+H].sup.+ 419.1023. found 419.1027.

N-(3-(2-(4-morpholinophenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 018)

(45) ##STR00038##

(46) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.43 (s, 1H), 10.06 (s, 1H), 8.84 (s, 1H), 8.03 (s, 1H), 7.92 (br, 1H), 7.72 (s, 1H), 7.56 (t, J=7.6 Hz, 1H), 7.27 (br, 2H), 7.12 (d, J=7.2 Hz, 1H), 6.58 (br, 2H), 6.45 (dd, J=16.8, 10.4 Hz, 1H), 6.26 (d, J=16.8 Hz, 1H), 5.78 (d, J=10.4 Hz, 1H), 3.71 (br, 4H), 2.94 (br, 4H). HRMS (ESI) calculated for C.sub.25H.sub.24N.sub.7O.sub.3 [M+H].sup.+ 470.1941. found 470.1939.

N-(4-(2-(4-(4-methyl-1-piperazinyl)-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 019)

(47) ##STR00039##

(48) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.51 (s, 1H), 10.06 (s, 1H), 8.83 (s, 1H), 8.03 (s, 1H), 7.89 (d, J=8.4 Hz, 2H), 7.37 (d, J=8.4 Hz, 2H), 7.17 (d, J=6.4 Hz, 1H), 6.56-6.49 (m, 3H), 6.34 (d, J=16.8 Hz, 1H), 5.85 (d, J=10.8 Hz, 1H), 2.94 (br, 4H), 2.37 (br, 4H), 2.20 (s, 3H). HRMS (ESI) calculated for C.sub.26H.sub.27N.sub.8O.sub.2 [M+H].sup.+ 483.2257. found 483.2259.

N-(3-(2-(4-(4-methyl-1-piperazinyl)-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 020)

(49) ##STR00040##

(50) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.45 (s, 1H), 10.06 (s, 1H), 8.84 (s, 1H), 8.04 (s, 1H), 7.93 (br, 1H), 7.73 (s, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.25 (br, 2H), 7.12 (d, J=8.0 Hz, 1H), 6.57 (br, 2H), 6.46 (dd, J=16.8, 10.4 Hz, 1H), 6.27 (dd, J=16.8, 1.8 Hz, 1H), 5.78 (dd, J=10.4, 1.8 Hz, 1H), 2.98 (br, 4H), 2.42 (br, 4H), 2.22 (s, 3H). HRMS (ESI) calculated for C.sub.26H.sub.27N.sub.8O.sub.2 [M+H].sup.+ 483.2257. found 483.2259.

N-(3-(7-oxo-2-(4-(1-piperidinyl)phenylamino)-8(7H)-pteridinyl)phenyl) acrylamide (Compound 021)

(51) ##STR00041##

(52) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.44 (s, 1H), 10.03 (s, 1H), 8.83 (s, 1H), 8.02 (s, 1H), 7.94 (br, 1H), 7.73 (s, 1H), 7.55 (t, J=8.0 Hz, 1H), 7.24 (br, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.57 (br, 2H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.26 (dd, J=17.0, 1.8 Hz, 1H), 5.77 (dd, J=10.2, 1.8 Hz, 1H), 2.95 (br, 4H), 1.57 (br, 4H), 1.49 (br, 2H). HRMS (ESI) calculated for C.sub.26H.sub.26N.sub.7O.sub.2 [M+H].sup.+ 468.2148. found 468.2146.

N-(3-(7-oxo-2-(4-(1-pyrrolidinyl)phenylamino)-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 022)

(53) ##STR00042##

(54) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.40 (s, 1H), 9.92 (s, 1H), 8.79 (s, 1H), 7.99 (s, 1H), 7.90 (br, 1H), 7.74 (br, 1H), 7.54 (t, J=8.0 Hz, 1H), 7.20 (br, 2H), 7.10 (d, J=8.0 Hz, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.26 (dd, J=17.0, 1.8 Hz, 1H), 6.20 (br, 2H), 5.77 (dd, J=10.2, 1.8 Hz, 1H), 3.10 (br, 4H), 1.91 (br, 4H). HRMS (ESI) calculated for C.sub.25H.sub.24N.sub.7O.sub.2 [M+H].sup.+ 454.1991. found 454.1995.

N-(3-(2-(4-(diethylamino)phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 023)

(55) ##STR00043##

(56) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.42 (s, 1H), 9.92 (s, 1H), 8.80 (s, 1H), 8.00 (s, 1H), 7.92 (br, 1H), 7.73 (s, 1H), 7.53 (t, J=8.0 Hz, 1H), 7.19 (br, 2H), 7.09 (d, J=8.0 Hz, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.32 (br, 2H), 6.27 (dd, J=17.0, 1.8 Hz, 1H), 5.76 (dd, J=10.2, 1.8 Hz, 1H), 3.20 (br, 4H), 1.00 (t, J=6.8 Hz, 6H). HRMS (ESI) calculated for C.sub.25H.sub.26N.sub.7O.sub.2 [M+H].sup.+ 456.2148. found 456.2143.

N-(3-(2-(4-(acetylamino)-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 024)

(57) ##STR00044##

(58) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.43 (s, 1H), 10.16 (br, 1H), 9.78 (s, 1H), 8.87 (s, 1H), 8.06 (s, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.79 (s, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.32 (br, 2H), 7.23 (br, 2H), 7.15 (d, J=8.0 Hz, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.25 (dd, J=17.0, 1.8 Hz, 1H), 5.76 (dd, J=10.2, 1.8 Hz, 1H), 1.98 (s, 3H). HRMS (ESI) calculated for C.sub.23H.sub.20N.sub.7O.sub.3 [M+H].sup.+ 442.1628. found 442.1624.

4-(8-(3-acrylamidephenyl)-7-oxo-7,8-dihydropteridinyl-2-amino)benzamide (Compound 025)

(59) ##STR00045##

(60) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.43 (s, 1H), 10.40 (s, 1H), 8.95 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.71 (br, 1H), 7.61 (t, J=8.0 Hz, 1H), 7.55 (d, J=7.6 Hz, 2H), 7.47 (br, 2H), 7.18 (d, J=7.6 Hz, 2H), 6.44 (dd, J=17.0, 10.2 Hz, 1H), 6.25 (dd, J=17.0, 1.8 Hz, 1H), 5.76 (dd, J=10.2, 1.8 Hz, 1H). HRMS (ESI) calculated for C.sub.22H.sub.18N.sub.7O.sub.3 [M+H].sup.+ 428.1471. found 428.1476.

N-(3-(2-(4-methoxyphenylamino)-6-methyl-7-oxo-8(7H)-pteridin-yl)phenyl) acrylamide (Compound 026)

(61) ##STR00046##

(62) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.42 (s, 1H), 9.93 (br, 1H), 8.78 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.77 (s, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.31 (br, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.58 (br, 2H), 6.45 (dd, J=17.0, 10.2 Hz, 1H), 6.26 (d, J=17.0 Hz, 1H), 5.77 (d, J=10.2 Hz, 1H), 3.65 (s, 3H), 2.42 (s, 3H). HRMS (ESI) calculated for C.sub.23H.sub.21N.sub.6O.sub.3 [M+H].sup.+ 429.1675. found 429.1675.

N-(3-(8-(4-methoxyphenyl)-7-oxo-7,8-dihydro-pteridin-2-amino)phenyl) acrylamide (Compound 027)

(63) ##STR00047##

(64) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.17 (s, 1H), 10.01 (s, 1H), 8.89 (s, 1H), 8.07 (s, 1H), 7.63 (br, 1H), 7.33 (d, J=8.8 Hz, 2H), 7.27 (d, J=8.0 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 7.11 (d, J=8.8 Hz, 2H), 6.89 (br, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.25 (dd, J=17.0, 1.8 Hz, 1H), 5.74 (dd, J=10.2, 1.8 Hz, 1H), 3.85 (s, 3H). HRMS (ESI) calculated for C.sub.22H.sub.19N.sub.6O.sub.3 [M+H].sup.+ 415.1519. found 415.1519.

2-(3-aminophenylamino)-8-(4-methoxyphenyl)-7(8H)-pteridin-one (Compound 028)

(65) ##STR00048##

(66) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 9.91 (s, 1H), 8.85 (s, 1H), 8.04 (s, 1H), 7.35 (d, J=8.8 Hz, 2H), 7.16 (d, J=8.8 Hz, 2H), 6.68-6.65 (m, 3H), 6.16 (d, J=7.2 Hz, 1H), 4.63 (s, 2H), 3.86 (s, 3H). HRMS (ESI) calculated for C.sub.19H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 361.1413. found 361.1411.

N-(4-(8-(4-methoxyphenyl)-7-oxo-7,8-dihydro-pteridin-2-amino)phenyl) acrylamide (Compound 029)

(67) ##STR00049##

(68) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.19 (br, 1H), 10.03 (s, 1H), 8.87 (s, 1H), 8.05 (s, 1H), 7.36-7.34 (m, 6H), 7.16 (d, J=8.4 Hz, 2H), 6.41 (dd, J=17.0, 10.2 Hz, 1H), 6.23 (dd, J=17.0, 1.6 Hz, 1H), 5.72 (dd, J=10.2, 1.6 Hz, 1H), 3.92 (s, 1H). HRMS (ESI) calculated for C.sub.22H.sub.19N.sub.6O.sub.3 [M+H].sup.+ 415.1519. found 415.1524.

2-(4-aminophenylamino)-8-(4-methoxyphenyl)-7(8H)-pteridin-one (Compound 030)

(69) ##STR00050##

(70) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 9.87 (s, 1H), 8.77 (s, 1H), 7.97 (s, 1H), 7.32 (d, J=8.8 Hz, 2H), 7.13 (d, J=8.8 Hz, 2H), 7.08 (br, 2H), 6.24 (br, 2H), 4.84 (s, 2H), 3.88 (s, 3H). HRMS (ESI) calculated for C.sub.19H.sub.17N.sub.6O.sub.2 [M+H].sup.+ 361.1413. found 361.1417.

N-(4-(2-(2-methoxy)-4-(4-methoxy-1-piperazinyl)-phenylamino)-7-oxo-8(7H)-pteridinyl)phenyl)acrylamide (Compound 031)

(71) ##STR00051##

(72) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.43 (s, 1H), 8.80 (s, 1H), 8.42 (s, 1H), 8.03 (s, 1H), 7.85 (d, J=8.6 Hz, 2H), 7.34 (d, J=8.6 Hz, 2H), 7.25 (d, J=8.8 Hz, 1H), 6.54-6.48 (m, 2H), 6.33 (dd, J=17.0, 1.6 Hz, 1H), 6.02 (br, 1H), 5.84 (dd, J=10.2, 1.6 Hz, 1H), 3.76 (s, 3H), 3.02 (br, 4H), 2.43 (br, 4H), 2.23 (s, 3H). HRMS (ESI) calculated for C.sub.27H.sub.29N.sub.8O.sub.3 [M+H].sup.+ 513.2363. found 513.2362.

N-(3-(2-(2-methoxy-4-(4-methyl-1-piperazinyl)-phenylamino)-7-oxo-8(7H)-pteridin-yl)phenyl)acrylamide (Compound 032)

(73) ##STR00052##

(74) .sup.1H NMR (400 MHz, DMSO d.sub.6): 10.41 (s, 1H), 8.80 (s, 1H), 8.44 (br, 1H), 8.02 (s, 1H), 7.86 (br, 1H), 7.71 (s, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.30 (d, J=7.6 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H), 6.53 (s, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.26 (dd, J=17.0, 1.8 Hz, 1H), 6.02 (br, 1H), 5.78 (dd, J=10.2, 1.8 Hz, 1H), 3.76 (s, 3H), 3.04 (br, 4H), 2.44 (br, 4H), 2.23 (s, 3H). HRMS (ESI) Calculated for C.sub.27H.sub.29N.sub.8O.sub.3 [M+H].sup.+ 513.2363. Found 513.2361.

EXAMPLE 2

Assay on Bioactivity1

(75) Inhibitory effects in vitro of the compounds provided in the present invention on EGFR kinase activity was tested as follows:

(76) In vitro enzyme activity assay: wild-type and various mutants (T790M, L858R, L861Q, L858 R/T790M) EGFR, Z-Lyte Kinase Assay Kit were purchased from Invitrogen. 10 concentration gradients, from 5.110.sup.11 mol/L to 1.010.sup.6 mol/L, were set for all of the compounds to be tested.

(77) Concentrations of different kinases were determined based on the optimization of experiment, and the corresponding concentrations were: EGFR (PV3872, Invitrogen) 0.287 g/L, EGFR-T790M (PV4803, Invitrogen) 0.174 g/L, EGFR-L858R (PV4128, Invitrogen) 0.054 g/L, EGFR-L858R/T790M (PV4879, Invitrogen) 0.055 g/L.

(78) Compounds were diluted for 3 times in DMSO from 5.110.sup.9 M to 110.sup.4 M. 4 L of compound was dissolved in 96 L of water, to give a 4 compound solution. 40 M ATP was dissolved in 1.33 kinase buffer, and a kinase/peptide mixture comprising 2 kinase, 4 M tyrosine and four peptides was prepared for use. 10 L of kinase reaction system comprised 2.5 L of compound solution, 5 L of Kinase/peptide mixture, and 2.5 L of ATP solution. 5 L of phosphopeptide solution was used in place of kinase/peptide mixture as 100% phosphorylation control. 2.5 L of 1.33 kinase buffer was used to replace ATP solution as 100% inhibition control, and 2.5 L of 4% DMSO solution was used to replace compound solution as 0% inhibition control. After thoroughly mixing the solution within the plate, the plate was incubated at room temperature for 1.5 hours. 5 L of DevelopmentSolution was added into each well, and then the plate was incubated at room temperature for another 1 hour, and non-phosphorylated peptide was cleaved within this period. Finally, the reaction was quenched by adding 5 L of Stop Reagent. The Plate was measured with EnVision Multilabel Reader (Perkin Elmer). Experimental data were calculated by using GraphPad Prism version 4.0. Each experiment was repeated more than three times.

(79) The test results are shown in Table 1.

(80) TABLE-US-00001 TABLE 1 Compound Inhibitory activity on EGFR kinase (IC.sub.50, nM) No. T790M WT L858R T790M/L858R 001 >10000 >10000 >10000 >10000 002 665 446 546 606 003 8698 8011 4082 3297 004 >10000 3181 4877 >10000 005 >10000 >10000 87159 >10000 006 19.4 10.6 10.1 8.4 007 >10000 >10000 >10000 >10000 008 >10000 >10000 >10000 >10000 009 10.9 60.7 81.11 39.1 010 2253 8086 6084 2022 011 67 84.8 70.3 012 5980 5342 1920 013 86.6 101 39.5 014 16.1 26.4 113.2 015 14.7 12.4 5.28 016 7580 115.9 1491 017 67 84.8 70.3 018 7.94 5.83 3.02 019 1260 6382 738 020 1.47 1.2 0.824 021 19.7 11.7 5.49 022 23.3 12.6 5.33 023 23.1 16.3 5.57 024 15.8 13.4 5.12 025 12.7 9.64 4.39 026 7.24 5.93 16 027 2000 1710 1200 028 10603 18881 3534 029 >10000 1318 >10000 030 3415 23758 6751 031 429 376 229 032 3.67 2.36 1.17 WZ4002 9.58 2.6 1.02

Assay on Bioactivity2

(81) Cell proliferation and growth inhibition analysis: H1975 (non-small cell lung cancer cells, EGFR.sup.L858/T790M), HCC827 (non-small cell lung cancer cells, EGFR.sup.del E746-A750), A549 (non-small cell lung cancer cells, EGFR wild-type), BT474 (breast cancer cells, Her2 overexpression), SK-BR-3 (breast cancer cells, Her2 overexpression), MCF-7 (breast cancer cells, Her2 overexpression) cells were obtained from ATCC. Cell proliferation was evaluated by MTS assay. Cells were exposed to the process conditions for 72 hours, and the number of cells for each cell line used in the experiment was adjusted according to the absorbance value (absorbance value at 490 nm was 1.3-2.2). 6 concentration gradients (0.1 nM-10 M) were set for the compounds to be tested, with six parallel controls for each concentration.

(82) H1975, HCC827, A549, BT474, MCF-7, SK-BR-3 cells were cultured in the corresponding medium. Upon recovery, the cells were passaged at least two times and then used in experiments. Cells in Log phase were trypsinized and resuspended in the medium. H1975 (1000 cells per well), BT474 (1500 cells per well), MCF-7 (1500 cells per well), HCC827 (2000 cells per well), SK-BR-3 (2000 cells per well), A549 (2000 cells per well) were seeded into 96-well plates, the volume was 100 L, and 6 rows and 7 columns were set. The plate was placed in an incubator at 37 C. of 5% carbon dioxide overnight. Compounds were dissolved in DMSO to obtain the concentration of 10 M, and then gradually diluted to obtain the concentration of compound as 10 M, 0.1 M, 0.01 M, 0.001 M, 0.0001 M. 2 L of compound solution was added into 998 L of medium, and the resulting mixture was thoroughly mixed. 100 L of mixture was added into 96-well plate. 2 L of DMSO was used to replace the compound solution as 0% inhibition control. After the cells were cultured for 68 hours, 20 L of MTT (5 mg/mL) was added. After 4 hours, the supernatant was discarded and 150 L of DMSO was added. The resulting system was shaken for 10 minutes, and the plate was read with Synergy HT (Bio TeK) (OD490). The data was calculated by GraphPad Prism version 4.0, and IC.sub.50 was obtained by non-linear regression model using dose-response curve.

(83) The test results are shown in Table 2 and 3.

(84) TABLE-US-00002 TABLE 2 Inhibitory activity on cell proliferation Compound (IC.sub.50, M) No. HCC827 H1975 001 >10 >10 002 1.29 2.75 003 3.01 >10 004 3.33 >10 005 >10 >10 006 0.009 0.133 007 >10 >10 008 >10 >10 009 0.91 4.65 010 4.16 >10 011 2.29 5.6 012 4.42 18.9 013 0.914 3.51 014 68.1 77.8 015 0.015 0.437 016 68.7 20.8 017 0.163 0.82 018 0.017 0.216 019 0.676 8.87 020 0.002 0.043 021 0.02 0.238 022 0.073 0.531 023 0.031 0.477 024 0.229 6.08 025 0.604 7.3 026 0.013 1.22 027 0.408 3.05 028 15.9 82.8 029 0.543 0.953 030 4.65 15.3 031 0.466 2.88 032 0.004 0.038 WZ4002 0.014 0.039 Iressa 0.006 13

(85) TABLE-US-00003 TABLE 3 Inhibitory activity on cell proliferation Compound (IC.sub.50, M) No. A549 SK-BR-3 MCF-7 BT474 001 >10 >10 >10 >10 002 1.23 1.57 1.51 >10 003 >10 >10 >10 >10 004 >10 >10 >10 >10 005 >10 >10 >10 >10 006 0.73 2.35 15.0 2.24 007 >10 >10 >10 >10 008 6.12 >10 >10 >10 009 2.24 1.15 2.41 0.84 010 >10 7.17 3.60 3.43

Assay on Bioactivity3

(86) Kinase selectivity analysis: kinase selectivity experiment was performed by Shanghai ChemPartner using Caliper assay screening platform. All of kinases and other materials were purchased from commercial companies. Staurosporine and PI103 were used as control compounds in testing inhibitory activity of compound on different kinases.

(87) I. Mobility Shift Assay

(88) 1. Preparation of kinase matrix buffer and stop buffer for kinase test: 1) 1 kinase matrix buffer: 50 mM HEPES pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT; 2) stop buffer: 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA.

(89) 2. Preparation of compound solution: 1) the compound was diluted in 100% DMSO to a concentration, which was 50 times of the highest concentration used in test, and 100 L of the compound solution as said above was placed into each well of 96-well plate. 2) 30 L of compound solution was transferred into 60 L of 100% DMSO in the adjacent well, and the above operation was repeated for sequentially diluting the compound. 3) 100 L of 100% DMSO was added into two blank wells as no-compound and no-enzyme control, and the plate was marked as stock solution plate. 4) 10 L of solution from the stock solution plate was added into another 96-well plate as a temporary plate, and into each well, 90 L of 1 kinase buffer was added, the temporary plate was placed on a shaker for uniformly mixing the compound solution.

(90) 3. Preparation of assay plate: 5 L of solution from each well of the 96-well temporary plate was added into a 384-well plate, and the experiment was independently repeated.

(91) 4. Kinase reaction: 1) preparation of 2.5 enzyme solution by adding the kinase into 1 kinase matrix buffer. 2) preparation of 2.5 peptide solution: FAM labeled peptide, and ATP were added into 1 kinase matrix buffer. 3) 2.5 enzyme solution was transferred into the assay plate. 4) the assay plate comprised 5 L of the compound solution in 10% DMSO. 5) 10 L of 2.5 enzyme solution was added into each well of the 384-well assay plate. 6) The assay plate was incubated at room temperature for 10 minutes. 7) 2.5 peptide solution was transferred to the assay plate, and 10 L of peptide solution was added into each well of the 384-well assay plate. 8) kinase reaction and termination: after incubated at 28 C. for a certain time, 25 L of stop buffer was added to quench the reaction.

(92) 5. Caliper readings: experimental data were collected on Caliper.

(93) 6. Curve fitting: 1) conversion data were replicated from Caliper program. 2) the conversion value was converted into inhibition rate, inhibition rate %=(maximumconversion value)/(maximumminimum)*100, wherein maximum represents DMSO control, and minimum represents the low control.

(94) II. Kinase-Glo Analysis

(95) 1. Preparation of 1 kinase buffer for PI3Ka kinase test: 1 kinase buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl.sub.2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT.

(96) 2. Preparation of compounds for PI3Ka kinase test: 1) serial dilution of compound and preparation of stock solution plate. The compound was diluted in 100% DMSO to a concentration, which was 100 times of the highest concentration used in reaction, and 100 L of the compound solution as said above was transferred into each well of 96-well plate. 30 L of compound solution was transferred into 60 L of 100% DMSO in the adjacent well, and the above operation was repeated for sequentially diluting the compound. 100 L of 100% DMSO was added into two blank wells as no-compound and no-enzyme control, the plate was marked as stock solution plate. 2) Preparation of temporary plate. 4 L of solution from the stock solution plate was transferred to another 96-well plate, 96 L of 1 kinase buffer was added into each well, and the temporary plate was placed on a shaker and shaken for 10 mins for uniformly mixing the compound solution. 3) Preparation of assay plate. 2.5 L of solution from each well of the 96-well temporary plate was added into a 384-well plate, and the experiment was independently repeated.

(97) 3. Kinase PI3Ka reaction: 1) Preparation of 4 kinase solution. PI3Ka solution was prepared in 1 kinase buffer, the concentration of which was 4 times of the final concentration in the test. 2.5 L of 1 kinase solution was added into each well of the assay plate (except for control well, into which 2.5 L of kinase buffer was added), and the assay plate was shaken. 2) Preparation of 2 substrate solution. PIP2 substrate, ATP solution was prepared in 1 kinase reaction buffer, the concentration of which was 2 times of the final concentration in the test. 5 L of substrate solution was added into each well of the assay plate and shaken to evenly mix contents. 3) The kinase reaction was conducted by incubating at room temperature for 1 hour.

(98) 4. Kinase assay. Kinase-Glo Reagent was balanced to r.t., and 10 L of Kinase-Glo reagent was added into the assay plate to quench the reaction. Upon simple mixing, the plate was centrifuged, and slowly shaken on an oscillator for 15 minutes, and then data was read on a luminescence reader.

(99) 5. The data were read and collected on Flexstation.

(100) 6. Curve fitting: RLU data were replicated from Flexstation program. The data were converted into inhibition rate % Inhibition rate %=(RLU of sampleminimum)/(maximumminimum)*100, wherein maximum represents the RLU data of no-enzyme control, and minimum represents the RLU value of DMSO control group.

(101) III. BRAF Analysis

(102) 1. Preparation of 1 Kinase Buffer: 50 mM HEPES, pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA, 0.01% BRIJ-35.

(103) 2. Preparation of compounds for BRAF kinase test: 1) serial dilution of compound and preparation of stock solution plate. The compound was diluted in 100% DMSO to a concentration, which was 100 times of the highest concentration used in reaction, and 100 L of the compound solution as said above was transferred into wells of 96-well plate. 30 L of compound solution was transferred into 60 L of 100% DMSO in the adjacent well, and the above operation was repeated for sequentially diluting the compound. 100 L of 100% DMSO was added into two blank wells as no-compound and no-enzyme control, the plate was marked as stock solution plate. 2) Preparation of temporary plate. 4 L of compound solution from the stock solution plate was transferred to another 96-well plate, 96 L of 1 kinase buffer was added into each well, and the temporary plate was placed on a shaker and shaken for 10 mins for uniformly mixing the compound. 3) Preparation of assay plate. 2.5 L of solution from each well of the 96-well temporary plate was added into a 384-well plate, and the experiment was independently repeated.

(104) 3. Kinase BRAF reaction: 1) Preparation of 2 kinase solution. BRAF solution was prepared in 1 kinase buffer, the concentration of which was 2 times of the final concentration in the test. 5 L of kinase solution was added into each well of the assay plate (except for control well, into which 5 L of 1 kinase buffer was added), and the assay plate was shaken. 2) Preparation of 4 substrate solution. Fluorescein-MAP2K1, ATP substrate solution was prepared in 1 kinase buffer, the concentration of which was 4 times of the final concentration in the test. 2.5 L of substrate solution was added into each well of the assay plate to start the reaction, and the assay plate was shaken. 3) The kinase reaction was conducted by incubating at room temperature for 1 hour.

(105) 4. Kinase assay. Test solution was prepared in antibody dilution buffer, the concentration of which was 2 times of the following final concentration: antibody 2 nM, EDTA 10 M. 10 L of test solution was added into each well of the assay plate to quench the assay. Upon simple mixing, the plate was centrifuged, and incubated for at least 30 mins.

(106) 5. Read data: the data was read on Envision (excitation at 340 nM, emission at 520 nM, 495 nM).

(107) 6. Curve fitting: RLU data were replicated from Envision program. The rate of RFU 520 nM/495 nM was calculated and converted into inhibition rate % Inhibition rate %=(maximumrate of sample)/(maximumminimum)*100, wherein maximum represents DMSO control rate, and minimum represents the no-enzyme control rate. Curve fitting was performed through XLFit excel add-in version 4.3.1.

(108) Test results are shown in the following table.

(109) TABLE-US-00004 TABLE 4 % inhibition (10 M) % inhibition (1 M) Compound Compound Compound Compound Kinase 020 032 020 032 HER2 98 100 97 89 HER4 72 77 60 60 FLT1 58 11 17 9.4 FLT3 99 91 82 60 CDK2 49 48 18 25 BLK 100 99 99 85 JAK2 36 22 22 9.9 LCK 73 54 18 14 LYNA 40 20 11 6.1 cKit 3.2 4.8 12 9.3 PIM1 54 8.9 8.2 2.1 FGFR3 33 13 14 11 FGFR1 63 42 19 7.9 PDGFRa 35 18 4.9 1.2 PDGFRb 84 61 42 14 KDR 73 29 25 3.8 SRC 72 33 27 6.6 ABL 39 13 11 5.8 AUR B 87 69 52 32 C-MET 53 28 27 13 BRAF 21 5.2 7.0 0.35 PKACa 4.7 4.0 15 6.9 IKKB 2.0 4.3 9.4 3.3 IGF1R 36 37 21 15 GSK3b 34 16 16 12 P38a 21 1.2 2.8 5.6 ERK1 19 4.0 1.3 11

Assay on Bioactivity4

(110) Inhibitory effects in vitro of the compounds provided in the present invention on BLK and FLT3 kinase activity was tested as follows, wherein BLK, FLT3 were purchased from BPS, and staurosporine was used as a control compound:

(111) Preparation of 1 kinase matrix buffer and stop buffer. 1 kinase matrix buffer: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl.sub.2, 2 mM DTT; stop buffer: 100 mM HEPES, pH 7.5, 0.0015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA.

(112) Preparation of compound solution. The compound was dissolved in 100% DMSO to a concentration, which was 50 times of the final highest inhibiting-concentration. 100 L of the compound solution as said above was transferred into wells of 96-well plate. The above compound solution was sequentially diluted to the final desired concentration. In two blank wells of same 96-well plate, 100 L of 100% DMSO was added as no-compound and no-kinase control, and this plate was used as the original plate.

(113) Preparation of intermediate plate. 10 L of compound solution was transferred from the original plate to another 96-well plate as the intermediate plate; 90 L of 1 kinase buffer was added into each well of the intermediate plate, and the intermediate plate was shaken for 10 min.

(114) Preparation of assay plate. 5 L of solution from each well of intermediate plate was added into a 384-well plate, and repeated controls were set.

(115) Kinase reaction. 2.5 kinase solution and 2.5 peptide solution were prepared. 2.5 kinase solution was transferred into assay plate. Assay plate comprised 5 L of the compound solution in 10% DMSO. 10 L of 2.5 kinase solution was added in each well of 384-well assay plate. The plate was incubated at room temperature for 10 minutes. 2.5 peptide solution was added into each well. After incubated at 28 C. for an appropriate time, 25 L of stop buffer was added to quench the reaction. Experimental data were collected in Caliper. Curve was fitted. Experimental data were copied from Caliper program, and converted into inhibit rate Inhibition %=(MaxConversion)/(MaxMin)*100, wherein Max represents DMSO control, Min represents a lower control.

(116) Test results are shown in the following table 5.

(117) TABLE-US-00005 TABLE 5 Inhibitory activity on kinase (IC.sub.50, nM) Compound BLK FLT3 001 >10000 27 002 3037 128 003 4168 137 004 >10000 206 005 >10000 30 006 69 1591 007 >10000 1486 008 >10000 115 009 5312 152 010 106 4387 011 89 48 012 6321 160 013 171 3778 014 7479 129 015 124 1859 016 >10000 358 017 156 2825 018 41 798 019 796 51 020 <14 322 021 83 878 022 98 1084 023 83 1757 024 148 3548 025 76 1791 026 96 568 027 5656 119 028 7946 34 029 >10000 100 030 >10000 43 031 812 180 032 463 1169 Staurosporine 5.6 0.28

(118) The present invention is illustrated by the specific examples. However, it is appreciated that the scope of the invention should not be limited to these specific examples, while are defined in the claims. And any equivalent modification to the invention should fall within the scope of the invention.