PROTEIN TYROSINE KINASE MODULATORS AND METHODS OF USE
20180009782 · 2018-01-11
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
C07D405/12
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
A61K31/495
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C07D401/12
CHEMISTRY; METALLURGY
A61K31/395
HUMAN NECESSITIES
C07D413/12
CHEMISTRY; METALLURGY
International classification
C07D401/12
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
Abstract
Heterocyclic pyrimidine compounds that modulate mutant-selective epidermal growth factor receptor (EGFR) and ALK kinase activity are disclosed. More specifically, the invention provides pyrimidines which inhibit, regulate and/or modulate kinase receptor, particularly in selectively modulation of various EGFR mutant activity and ALK kinase activity have been disclosed. Pharmaceutical compositions comprising the pyrimidine derivative, and methods of treatment for diseases associated with protein kinase enzymatic activity, particularly EGFR or ALK kinase activity including non-small cell lung cancer comprising administration of the pyrimidine derivative are disclosed.
Claims
1-67. (canceled)
68. A method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, which comprises a compound of Formula I or its pharmaceutically acceptable salt: ##STR00176## wherein X is NH; Y is halogen; Z is O; R.sub.1 is N(R.sub.8)(R.sub.9), wherein each R.sub.8 and R.sub.9 is independently H or alkyl; R.sub.2 is H; R.sub.3 is H; R.sub.4 is C.sub.1-6 alkoxy; R.sub.5 is H or C.sub.1-6 alkyl, R.sub.7 is —NR.sub.22C(O)CR.sub.23═CR.sub.10R.sub.11, wherein each R.sub.10, R.sub.11, R.sub.22 or R.sub.23 is independently H or alkyl, R.sub.6 is ##STR00177## wherein each A.sub.1 or A.sub.2 is, independently, CH or N; and R.sub.14 is alkyl.
69. The method of claim 68, wherein each R.sub.6 is ##STR00178##
70. The method of claim 68, wherein R.sub.1 is —NHCH.sub.3 or —NCH.sub.3CH.sub.3.
71. The method of claim 68, wherein R.sub.4 is —OCH.sub.3.
72. The method of claim 68, wherein R.sub.5 is H.
73. The method of claim 68, wherein R.sub.7 is ##STR00179##
74. The method of claim 68, wherein the compound is N-(5-(5-chloro-4-(2-(2-(dimethylamino)-2-oxoacetyl)phenylamino)pyrimidin-2-ylamino)-4-methoxy-2-(4-methylpiperazin-1-yl)phenyl)acrylamide; N-(5-(5-chloro-4-(2-(2-(methylamino)-2-oxoacetyl)phenylamino)pyrimidin-2-ylamino)-4-methoxy-2-(4-methylpiperazin-1-yl)phenyl)acrylamide; or N-(5-(5-chloro-4-(2-(2-(dimethylamino)-2-oxoacetyl)phenylamino)pyrimidin-2-ylamino)-4-methoxy-2-(1-methylpiperidin-4-yl)phenyl)acrylamide.
75. The method of claim 68, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
76. The method of claim 75, wherein a weight ratio of the compound of formula I to the excipient is from about 0.0001 to about 10.
77. The method of claim 68, where in the disease is selected from cancer, cancer metastasis, cardiovascular disease, an immunological disorder, an ocular disorder, or a combination thereof.
78. The method of claim 77, where in the cancer is selected from non-small cell lung cancer (NSCLS), glioblastoma, pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, epithelial cancer, ovarian cancer, prostate cancer, adenocarcinoma, or a combination thereof.
79. The method of claim 68, wherein the subject is a human.
80. The method of claim 68, wherein the disease is caused by a kinase regulation disorder.
81. The method of claim 80, wherein the kinase comprises EGFR, ALK, an ALK fusion protein, Flt3, Jak3, Blk, Bmx, Btk, HER2 (ErbB2), HER4 (ErbB4), Itk, Tec, Txk, or a combination thereof.
82. The method of claim 81, wherein the EGFR comprises an EGFR mutant.
83. The method of claim 82, wherein the EGFR mutant comprises one or more mutations selected from: (i) L858R, (ii) T790M, (iii) both L858R and T790M, (iv) delE746_A750, (v) both delE746_A750 and T790M, or (vi) the combination thereof.
84. The method of claim 68, wherein the subject suffers from an EGFR-driven cancer, which is characterized by presence of one or more EGFR mutations selected from: (i) L858R, (ii) T790M, (iii) both L858R and T790M, (iv) delE746_A750, (v) both delE746_A750 and T790M, or (vi) the combination thereof.
85. The method of claim 81, wherein the ALK fusion protein comprises MEL4-ALK or NPM-ALK kinase.
86. A method of inhibiting kinase activity, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, which comprises a compound of Formula I or its pharmaceutically acceptable salt: ##STR00180## wherein X is NH; Y is halogen; Z is O; R.sub.1 is N(R.sub.8)(R.sub.9), wherein each R.sub.8 and R.sub.9 is independently H, or alkyl; R.sub.2 is H; R.sub.3 is H; R.sub.4 is C.sub.1-6 alkoxy; R.sub.5 is H or C.sub.1-6 alkyl, R.sub.7 is —NR.sub.22C(O)CR.sub.23═CR.sub.10R.sub.11, wherein each R.sub.10, R.sub.11, R.sub.22 or R.sub.23 is independently H or alkyl, R.sub.6 is ##STR00181## wherein each A.sub.1 or A.sub.2 is, independently, CH or N; and R.sub.14 is alkyl.
87. The method of claim 86, wherein the kinase is selected from the group consisting of EGFR, ALK, an ALK fusion protein, Flt3, Jak3, Blk, Bmx, Btk, HER2 (ErbB2), HER4 (ErbB4), Itk, Tec, and Txk.
Description
EXAMPLES
[0300] The following Examples are provided to better illustrate the present invention. All parts and percentages are by weight and all temperatures are degrees Celsius, unless explicitly stated otherwise. The following abbreviations have been used in the examples: [0301] ATP: Adenosine triphosphate; [0302] DIPEA: N,N-Diisopropylethylamine; [0303] DMF: N,N-Dimethylformamide; [0304] DMA: N,N-Dimethyacetamide; [0305] DMAP: 4-N,N-Dimethylamiopryidine; [0306] DMSO: Dimethyl sulfoxide; [0307] DEAD: Diethyl azodicarboxylate; [0308] HATU: O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; [0309] DIPEA: N,N-Diisopropylethylamine; [0310] EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; [0311] TBAB: Tetrabutyl ammonium bromide; [0312] TEA: Triethylamine; [0313] EtOAc: Ethyl acetate; [0314] GSR: Glutathione-S-Transferase; [0315] Crk: CT10 (Chicken Tumor Retrovirus 10); [0316] min: Minute; [0317] h or hr: Hour; [0318] rt or RT: room temperature; [0319] SDS, Sodium Dodecyl Sulfate; [0320] SDS-PAGE, Sodium Dodecyl Sulfate PolyAcrylamide Electrophoresis Gel
[0321] TLC, Thin layer chromatography.
Example 1 Synthesis of Compound 1
[0322] ##STR00023##
[0323] A mixture of Compound 1a (25 g, 0.11 mol), Compound 1b (20 g, 0.11 mol) and K.sub.2CO.sub.3 (37.5 g, 0.22 mol) in DMF (200 ml) was stirred at 70° C. for 3 hrs. Then H.sub.2O (300 ml) was added to the mixture and extracted with EA (200 ml×3), the aqueous layer was acidified with HCl (1 mol/L) to pH 3-4. The precipitate was collected by filtration and washed with methanol (5 ml). After dried under air for 5 hrs, 23 g of Compound 1c was obtained.
[0324] (COCl).sub.2 (7.2 ml in 10 ml of DCM) was added to a solution of Compound 1c (5.0 g) and 2 drops of DMF in DCM at ice bath under N.sub.2 with stirring. After 3.5 hrs, the solvent was removed. The residue was dissolved in DCM and then added 10 ml of EtOH. After stirring for 1 hr, the reaction mixture was quenched with water. The organic layer was separated and dried, and concentrated. The residue was purified by chromatography to give 4.7 g of Compound 1d as a yellow solid.
[0325] A mixture of Compound 1d (200 mg) and Compound 1e was stirred in sealed tube at 130° C. for 1 hr. After the solvent was removed, the residue was purified to give Compound 1. MS: 552.2 (M+H).sup.+.
Example 2 Synthesis of Compound 2
[0326] ##STR00024##
[0327] A mixture of Compound 1 and NaOH (0.5 ml, 2 mol/L) in MeOH was stirred at room temperature for 1 hr. Then the solution was acidified with HCl to about pH 5. After filtration, the solid was dried to give the Compound 2 (37 mg). MS: 524.2 (M+H).sup.+.
Example 3 Synthesis of Compound 3
[0328] ##STR00025##
[0329] A mixture of Compound 2 (140 mg) and NH.sub.2OH (72 mg), PyBOP (180 mg) in 10 mL of DMF was stirred at 45° C. for 3 hrs. Then, the reaction solution was purified by pre-HPLC to give 30 mg of compound 3 as a solid. MS: 539.2 (M+H).sup.+.
Example 4 Synthesis of Compound 4
[0330] ##STR00026##
[0331] (COCl).sub.2 (7.2 ml in 10 ml of DCM) was added to a solution of Compound 1c (5.0 g) and 2 drops of DMF in 50 Ml of DCM at ice bath under N.sub.2 with stirring. After 3.5 hrs, the solvent was removed. The residue was dissolved in DCM and NH.sub.3 (aqs.) added into the solution. After stirring for 1 hr, the precipitate was collected by filtration to give 6.0 g of Compound 1f as a white solid.
[0332] Following the same procedure as Example 1 using Compound 1f instead of 1d with Compound 1e to yield Compound 4. MS: 523.2 (M+H).sup.+.
TABLE-US-00001 Physical Data Ex (MS) No Chemical Name Structure (M + H).sup.+ 5 ethyl 2-(2-(5-chloro-2-(4-(4- (dimethylamino)piperidin- 1-yl)-2-methoxyphenylamino) pyrimidin-4-ylamino)phenyl) acrylate
Example 40. Synthesis of Compound 40
[0333] ##STR00062##
[0334] A three-necked round bottom flask equipped with mechanical stirrer, addition funnel and thermometer was charged with Compound 40a (10.0 g), TEA (12.0 ml) and DCM (30.0 ml). And then a solution of acryloyl chloride (5.9 g) in DCM (20.0 ml) was added dropwise (via addition funnel) after the reaction mixture was cooled to −10° C. The reaction mixture was stirred at −10° C. until the reaction was complete detected by TLC (PE:EA=3:1). Then, the reaction was quenched by water (30.0 ml) and filtered to give the crude product of Compound 40b (8.5 g) used for the next reaction directly.
[0335] A three-necked round bottom flask equipped with mechanical stirrer, addition funnel and thermometer was charged with Compound 40b (2.0 g), SnCl.sub.2 (14.1 g) and MeOH (60.0 ml). Then, the reaction mixture was stirred at 80° C. until the reaction was complete detected by TLC (DCM:MeOH=10:1). After, the reaction mixture was concentrated under reduced pressure and partitioned between ethyl acetate (50.0 ml) and the aqueous solution of K.sub.2CO.sub.3 and filtered. The filtrate was separated and the organic phase was washed with water, dried over Na.sub.2SO.sub.4 and evaporated to dryness to give Compound 40c (1.5 g) as a yellow solid.
[0336] A mixture of Compound 1c (1.03 g), dimethylamine hydrochloride (0.54 g), HATU (1.51 g), and TEA (1.00 g) in DCM (20 ml) was stirred at 25° C. for 6 hrs. Then, the reaction mixture was concentrated under reduced pressure and the residue was purified by chromatography to give 0.64 g Compound 40d as a yellow solid.
[0337] Compound 40d (200 mg), Compound 40c (96 mg), con-HCl (3d) and n-BuOH (60 ml) was stirred at 130° C. in sealed tube until the reaction was complete detected by TLC (DCM:MeOH=10:1). Then, the reaction mixture was washed with water and concentrated under reduced pressure. After, the residue was purified by column chromatography to give 500 mg Compound 40 as a yellow solid. MS: 464.1 (M+H).sup.+. H-NMR (DMSO-d6,400 MHz): 10.25 (s, 1H), 9.12 (s, 1H), 8.34 (s, 1H), 7.98 (s, 1H), 7.39-7.33 (m, 2H), 7.24-7.19 (m, 2H), 6.54-6.51 (m, 2H), 6.49-6.47 (m, 1H), 6.54-6.47 (m, 1H), 6.25-6.21 (dd, 1H), 5.74-5.71 (dd, 1H), 3.00 (s, 3H), 2.90 (s, 3H).
Example 41. Synthesis of Compound 41
Method 1
[0338] ##STR00063##
[0339] A mixture of 4-methoxy-3-nitroaniline (1.52 g, 9.04 mmol), triethylamine (1.37 g, 13.54 mmol) and DCM (60 ml) was cooled to 0° C., acryloyl chloride (0.90 g, 9.94 mmol) solution in DCM (20 mL) was added dropwise, the resulting mixture was stirred at 0-5° C. for 20 mins. The progress of the reaction was monitored by TLC. Reaction was quenched with water (50 ml), the aqueous solution was extracted with DCM (30 ml×2). The combined organic phase was dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to give 1.52 g Compound 41b as a yellow solid.
[0340] A mixture of Compound 41b (1.50 g, 6.75 mmol), 1,4-Diazabicyclo[2.2.2]octane (2.27 g, 20.25 mmol), paraformaldehyde (1.01 g, 33.75 mmol), 1,4-dioxane (100 ml) and water (60 ml) was heated to 80° C. for 15 h. Water (100 ml) was added, the resulting mixture was extracted with EA (100 ml×2). The combined organic phase was washed with brine (100 ml), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure, the residue was purified by column chromatography to give 1.25 g Compound 41c as a yellow solid.
[0341] A mixture of Compound 41c (1.20 g, 4.76 mmol) and DCM (130 ml) was cooled to 0° C., phosphorus tribromide (1.54 g, 5.71 mmol) solution in DCM (20 ml) was then added dropwise and quenched with water (50 ml) after 15 mins, the aqueous solution was extracted with DCM (30 ml×2). The combined organic phase was washed with brine (50 ml×2), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure and the residue (Compound 41d) was used for the next step without further purification.
[0342] To a solution of Compound 41d (1.35 g) in DCM (100 ml) was added morpholine (1.86 g, 21.40 mmol). The resulting mixture was stirred at ambient for 15 mins. Water (50 ml) was added, the organic solution was washed with brine (30 ml), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to give 1.28 g Compound 41e as a brown solid.
[0343] A mixture of Compound 41e (1.25 g, 3.89 mmol), iron power (4.34 g, 77.80 mmol), EtOH (50 ml), saturated aqueous solution of ammonium chloride (20 ml) was heated to 65° C. for 30 mins. After cooling the mixture was filtered, brine (20 ml) was added and the product was extracted with EA (50 ml×3,). The combined organic phase was washed with brine (50 ml), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to give 0.94 g Compound 41f as a brown solid.
[0344] A mixture of Compound 41f (508 mg, 1.74 mmol), Compound 40d (705 mg, 2.09 mmol), p-toluenesulfonic acid (449 mg, 2.61 mmol) and n-butyl alcohol (100 ml) was heated to 100° C. for 14 hours. The mixture was cooled and concentrated under reduced pressure, the residue was basified with aqueous solution of sodium carbonate (50 ml), extracted with EA (50 ml, 30 ml×2), the combined organic phase was washed with brine (50 ml×2), dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure, the residue was purified by column chromatography to give 198 mg Compound 41 as a yellow solid. MS: 593.2 (M+H)+. HNMR (DMSO-d6, 400 MHz): 11.36 (s, 1H), 11.05 (s, 1H), 8.89 (d, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 7.92 (s, 1H), 7.61 (d, 1H), 7.54-7.56 (m, 1H), 7.49 (t, 1H), 7.14 (t, 1H), 7.04 (d, 1H), 6.02 (s, 1H), 5.54 (s, 1H), 3.77 (s, 3H), 3.55 (s, 4H), 3.24 (s, 2H), 2.99 (s, 3H), 2.88 (s, 3H), 2.35 (s, 4H).
Method 2
[0345] ##STR00064##
[0346] To a solution of Compound 41a (2.0 g) in DCM (25 ml) was added dropwise morpholine (0.6 g) in DCM (5 ml) at 0° C. with stirring. After that, the reaction mixture was stirred for another 3 hrs at 0-5° C. Then the reaction mixture was concentrated under reduced pressure to give 1.5 g Compound 41b as a white solid which was used for the next reaction directly.
[0347] A flask was charged with Compound 41b (1.45 g), LiOH (0.46 g), MeOH (10 ml) and water (5 ml). Then, the reaction mixture was stirred at room temperature for 1 hr. The reaction mixture was adjusted to PH=1-2 by con-HCl after most reaction solvent was removed under reduce pressure. And then, the appeared solid was filtered and the filter cake was dried to give 1.5 g Compound 41c.
[0348] A three-necked round bottom flask equipped with mechanical stirrer, addition funnel and thermometer was charged with Compound 41c (1.2 g) and DCM (25 ml). And then a solution of oxalyl chloride (1.1 g) in DCM (15 ml) was added dropwise (via addition funnel) at 0° C. while keeping inner temperature between 0-5° C. Then, the reaction mixture was warmed to 15° C. and then kept at 15° C. with stirring until the reaction was complete detected by TLC(DCM:MeOH=10:1). Reaction solvent was removed under reduce pressure to give 1.3 g Compound 41d used for the next reaction directly.
[0349] A three-necked round bottom flask equipped with mechanical stirrer, addition funnel and thermometer was charged with 4-methoxy-3-nitroaniline (0.8 g) and DCM (25 ml) and cooled to 0° C. After a solution of Compound 41d (1.3 g) in DCM (15 ml) was added dropwise (via addition funnel) while keeping inner temperature between 0-5° C. Then, the reaction mixture was stirred with warming naturally until the reaction was complete detected by TLC(DCM:MeOH=10:1) The reaction mixture was washed with water and organic phase was concentrated under reduced pressure to a residue, and then the residue was purified by column chromatography to give 0.8 g Compound 41e.
[0350] A three-necked round bottom flask equipped with mechanical stirrer and thermometer was charged with Compound 41e (98 mg), Fe(347 mg), and saturated aqueous solution of NH.sub.4Cl (15 ml). Then the reaction mixture was heated to 80° C. and stirred until the reaction was complete detected by TLC(DCM:MeOH=10:1). After, most reaction solvent was removed under reduce pressure to afford a residue. Then, the residue was partitioned between ethyl acetate (20 ml) and water (10 ml) 3 times and the combined organic phase was washed with brine and dried over Na.sub.2SO.sub.4 to give 62 mg Compound 41f.
[0351] Compound 40d (74 mg), Compound 41f (60 mg), TsOH (36 mg) and n-BuOH (15 ml) was stirred at 90° C. in sealed tube until the reaction was complete detected by TLC (DCM:MeOH=10:1). Then, the reaction mixture was washed with water and the organic phase was concentrated under reduced pressure. After that, the residue was purified by column chromatography to give 8 mg Compound 41 as a yellow solid. MS: 5930.2 (M+H).sup.+.
TABLE-US-00002 Physical Data EX (MS) No. Chemical Name Structure (M + H).sup.+ 42 1-(2-(2-(4-(4-acetylpiperazin- 1-yl)-2-methoxyphenylamino)- 5-chloropyrimidin-4-ylamino) phenyl)-2-morpholinoethane- 1,2-dione
Example 72. Synthesis of Compound 72
[0352] ##STR00095##
[0353] To a solution of crotonic acid (5.05 g, 58.65 mmol) and a drop of DMF in DCM (80 mL), was added oxalyl dichloride (11.12 g, 87.98 mmol) dropwise and the mixture was stirred at 15° C. for 30 min, then the solvent was concentrated under reduced pressure. The residue A (Compound 72a) was used for the next step without further purification.
[0354] To a solution of Compound 72b (4.92 g, 29.33 mmol) and DIPEA (3.80 g, 29.33 mmol) in DCM (100 mL) at 0° C., was A solution in DCM (30 mL) dropwise, and the resulting mixture was stirred at 0˜5° C. for 30 min. Reaction was quenched with water (50 mL), the organic phase was concentrated under reduced pressure and the residue was purified by chromatography to give 4.21 g Compound 72c as a yellow solid.
[0355] A mixture of Compound 72c (402 mg, 1.72 mmol), NBS (334 mg, 1.88 mmol), AIBN (40 mg, 0.26 mmol) and benzene (70 mL) was heated to 80° C. for 5 hours. The mixture was concentrated under reduced pressure, and the residue of Compound 72d was used for the next step without further purification.
[0356] Compound 72d in DCM (30 mL) was added piperidine (1 mL), the resulting mixture was stirred at ambient temperature for 2 mins, evaporated to under reduced pressure, the residue was purified by chromatography to give 380 mg Compound 72e as a light yellow solid.
[0357] A mixture of Compound 72e (205 mg, 0.64 mmol), iron power (715 mg, 12.80 mmol) in EtOH (20 mL) and saturated aqueous solution of ammonium chloride (5 mL) was heated to 65° C. for 30 min. After cooled, water was added to filtrate and the product was extracted with EA (50 mL, 30 mL), the combined organic layers was washed with brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to give 108 mg Compound 72f as a brown solid.
[0358] A mixture of Compound 72f (105 mg, 0.36 mmol), Compound 40d (145 mg, 0.43 mmol), p-toluenesulfonic acid (93 mg, 0.54 mmol) in n-butyl alcohol (20 mL) was heated to 100° C. with stirring for 13 hours. The mixture was cooled and concentrated under reduced pressure, the residue was basified with aqueous solution of sodium carbonate (20 ml), extracted with EA (50 mL, 30 mL), the combined organic extracts was washed with brine (30 mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure, the residue was purified by chromatography to give 38 mg Compound 72 as a yellow solid. MS: 591.2 (M+H).sup.+.
TABLE-US-00003 Physical EX Data (MS) No. Chemical Name Structure (M + H).sup.+ 73 N-(3-(5-chloro-4-(2-(2- (dimethylamino)-2- oxoacetyl)phenylamino) pyrimidin-2-ylamino)- 4-methoxyphenyl)-2- (piperidin-1-ylmethyl) acrylamide
Example 74 Synthesis of Compound 74
[0359] ##STR00097## ##STR00098##
[0360] A solution of Compound 74a (50 g) and Pd/C (10 g) in MeOH (100 ml) under H.sub.2 was stirred at room temperature for 4 hrs. After filtered, the filtrate was concentrated under reduced pressure to give 41 g Compound 74b.
[0361] To a solution of Compound 74b (8.05 g) dissolved in con.H.sub.2SO.sub.4 (40 ml), was added KNO.sub.3 (5.77 g) with stirring for 2 hrs at ice bath. The reaction mixture was poured into NaOH solution, and the appeared solid was filtered to give 8.5 g Compound 74c as a yellow solid.
[0362] A mixture of Compound 74c (19.7 g), 1-methylpiperazine (15.9 g), and K.sub.2CO.sub.3 (21.94 g) in DMF was stirred at 80□ for 8 hrs. Water was added to the reaction mixture and extracted with EA, the organic layer was washed with brine, dried over Na.sub.2SO.sub.4, concentrated under reduced pressure to give 10.2 g Compound 74d as a yellow solid.
[0363] To a solution of Compound 74d (1.02 g) and TEA (0.5 g) in DCM (20 ml), was added dropwise acryloyl chloride (0.38 g) at ice bath with stirring for 1 hr. The mixture was quenched with water and the organic layer was separated, after dried, it was concentrated under reduced pressure to give 0.98 g Compound 74e as a solid.
[0364] Compound 74e (0.98 g), iron powder (0.85 g) and NH.sub.4Cl (0.82 g) were dissolved in ethanol and water, then the mixture was stirred at 50□ for 2 hrs. After filtered, the filtrated was extracted with EA, dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure to give 0.58 g Compound 74f as a solid.
[0365] A mixture of Compound 74f (16.5 g), Compound 40d (23.2 g) and TsOH (11.75 g) in BuOH (250 ml) was stirred at 70□ for 8 hrs. After the solvent was removed, the residue was purified by chromatography to give 11.3 g Compound 74 as a light yellow solid. MS: 592.2 (M+H).sup.+. HNMR (DMSO-d6, 400 MHz): 8.84-8.82 (d, J=7.76 Hz, 1H), 8.24 (s, 1H), 8.09-8.07 (d, J=6 Hz, 1H), 7.63-7.61 (d, J=8 Hz, 1H), 7.53 (s, 1H), 7.16-7.12 (t, J=7.6 Hz, 1H), 6.87 (s, 1H), 6.63-6.56 (dd, J=10 Hz, J=17.2 Hz, 1H), 6.18-6.13 (d, J=16.8 Hz, 1H), 5.71-5.68 (d, J=10.4 Hz, 1H), 3.77 (s, 3H), 3.00 (s, 3H), 2.89 (s, 6H), 2.55-2.50 (m, 5H), 2.25-2.25 (m, 3H).
TABLE-US-00004 Physical EX Data (MS) No. Chemical Name Structure (M + H).sup.+ 75 N-(5-(5-chloro-4-(2-(2- (methylamino)-2- oxoacetyl)phenylamino) pyrimidin-2-ylamino)- 4-methoxy-2-(4- methylpiperazin-1-yl) phenyl)acrylamide
Example 79. Synthesis of Compound 79
[0366] ##STR00103##
[0367] The mixture of Compound 72d (0.5 g) and DCM (50 ml) was added morpholine (1 ml), the resulting mixture was stirred at ambient temperature for 5 mins, evaporated to dryness, the residue was purified by chromatography to give 350 mg Compound 79b as a light yellow solid.
[0368] A mixture of Compound 79b (152 mg, 0.47 mmol), iron power (525 mg, 9.40 mmol), EtOH (20 ml) and saturated aqueous solution of ammonium chloride (10 ml) was heated to 65° C. for 15 mins. After cooling the mixture was filtered, brine (30 ml) was added and the product was extracted with EA (50 ml, 30 ml). The combined organic phase was washed with brine (30 ml), dried over anhydrous Na.sub.2SO.sub.4, concentrated under reduced pressure to give 102 mg Compound 79c as a brown solid.
[0369] A mixture of Compound 79c (102 mg, 0.34 mmol), Compound 40d (138 mg, 0.41 mmol), p-toluenesulfonic acid (87 mg, 0.51 mmol) and n-butyl alcohol (15 ml) was heated to 105° C. for 7 hrs. The mixture was cooled and concentrated in vacuo, the residue was basified with aqueous solution of sodium carbonate (20 ml), extracted with EA (50 ml, 30 ml), the combined organic phase was washed with brine (30 ml), dried over anhydrous Na.sub.2SO.sub.4 and evaporated to dryness, the residue was purified by chromatography to give 92 mg Compound 79 as a yellow solid. MS: 594.9 (M+H).sup.+. HNMR (DMSO-d6, 400 MHz): 11.35 (s, 1H), 9.93 (s, 1H), 8.85 (d, 1H), 8.61 (s, 1H), 8.28 (s, 1H), 7.93 (s, 1H), 7.63 (d, 1H), 7.48 (m, 2H), 7.13 (t, 1H), 7.04 (d, 1H), 6.65-6.68 (m, 1H), 6.24 (d, 1H), 3.77 (s, 3H), 3.59 (s, 4H), 3.11 (s, 2H), 3.01 (s, 3H), 2.90 (s, 3H), 2.38 (s, 4H).
TABLE-US-00005 Physical Data EX (MS) No. Chemical Name Structure (M + H).sup.+ 80 (E)-N-(3-(5-chloro-4- (2-(2-(dimethylamino)-2- oxoacetyl)phenylamino) pyrimidin-2-ylamino)-4- methoxyphenyl)-4- (dimethylamino)but- 2-enamide
Example 115. Synthesis of Compound 115
[0370] ##STR00139##
[0371] To a stirred solution of N, N-dimethylethanolamine (8.58 g, 96 mmol) in DMF (80 ml) at 0□, NaH (1.54 g, 64 mmol) was added slowly. After the mixture was stirred at 0-5□ for 30 minutes, Compound 115a (5.01 g, 32 mmol) in DMF was added dropwise. Then the reaction mixture was stirred with warming naturally to the room temperature for 4 hours. The reaction was complete detected by TLC (PE:EA=1:1). Then water (80 ml) was added to the solution to quench the overdose NaH. Afterwards, the mixture was extracted with ethyl acetate (80 ml×2). The combined organic phase was washed with brine (80 ml) and dried over sodium sulfate and concentrated under reduced pressure to give 6.49 g Compound 115b.
[0372] To a stirred solution of Compound 115b (6.49 g, 28.81 mmol) and triethylamine (4.8 ml, 34.58 mmol) in DCM (55 ml) at 0□, a solution of acryloyl chloride (2.3 ml, 31.68 mmol) in DCM (10 ml) was added dropwise. The reaction mixture was stirred at 0□ for 30 mins and then the reaction solution was washed with water (60 ml) and the organic phase was concentrated under reduced pressure to give 7.32 g Compound 115c.
[0373] A mixture of Compound 115c (7.32 g, 25.93 mmol) in ethanol (15 ml), iron powder (8.84 g, 155.57 mmol) and ammonium chloride (2.77 g, 51.86 mmol) in water (70 ml) was heated to 90° C. for 30 mins. Then the reaction solution was filtered and the filtrate was extracted with ethyl acetate (80 ml×2) and the combined organic phase was washed with brine (80 ml) and dried over sodium sulfate and concentrated under reduced pressure to give 2.31 g Compound 115d.
[0374] A mixture of Compound 115d (2.30 g, 9.23 mmol), Compound 40d (3.13 g, 9.23 mmol) and hydrochloric acid (0.3 ml) in n-Butanol (30 ml) was heated to 110□ for 6 hours. The progress was monitored by TLC (DCM:MeOH=10:1). Then the reaction mixture was cooled to room temperature and concentrated under reduced pressure to afford crude compound. The crude product was purified by column chromatography to give 1.71 g Compound 115 as a yellow solid. MS: 551.2 (M+H).sup.+. H-NMR (DMSO-d6, 400 MHz): 8.35 (s, 1H), 8.29 (s, 1H), 7.88-7.82 (m, 1H), 7.47 (s, 1H), 7.32-7.15 (m, 4H), 7.05-7.02 (m, 1H), 6.25-6.21 (m, 1H), 5.70-5.67 (m, 1H), 4.33-4.32 (t, 3H), 3.60-3.55 (t, 3H), 3.00 (s, 6H), 2.90 (s, 6H).
TABLE-US-00006 Physical EX Data (MS) No. Chemical Name Structure (M + H).sup.+ 116 N-(5-(5-chloro-4-(2-(2- (dimethylamino)-2- oxoacetyl)phenylamino) pyrimidin-2-ylamino)-2- (difluoromethoxy) phenyl)acrylamide
Example 124. Synthesis of Compound 124
[0375] ##STR00148##
[0376] To a stirred solution of 3-hydroxy tetrahydrofuran (203 mg) in DMF (10 ml) at 0□, NaH (92 mg) was added slowly. After the mixture was stirred at 0-5□ for 30 minutes, the compound 124a (300 mg) in DMF was added dropwised. Then the reaction mixture was stirred with warming naturally to the room temperature. The reaction was complete detected by TLC (PE:EA=1:1) after 4 hours. Then 15 ml H.sub.2O was added to the solution. Afterwards, the mixture was extracted by EA (15 ml×2). The combined organic phase was combined and washed with aqueous solution of NaCl and dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give crude Compound 124b. The crude product was purified by column chromatography to give 226 mg Compound 124b.
[0377] To a stirred solution of Compound 124b (220 mg) and TEA (0.2 ml) in DCM (15 ml) at 0□, a solution of acryloyl chloride (0.1 ml) in DCM (2.0 ml) was added dropwised. The reaction mixture was stirred at 0° C. for 30 minutes. Then the reaction was complete detected by TLC (PE:EA=1:1). The reaction solution was washed by water (20 ml) and the organic phase was concentrated under reduced pressure to give 220 mg Compound 124c.
[0378] To a stirred solution of Compound 124c (220 mg) in ethanol (5 ml), Fe powder (500 mg) and saturated aqueous solution of NH.sub.4Cl (15 ml) was added. Then the reaction mixture was heated to 90° C. and stirred until the reaction was complete detected by TLC (DCM:MeOH=10:1). The reaction solution was filtered and the filtrate was partitioned between ethyl acetate (20 ml) and water (10 ml) 2 times. Then the combined organic phase was washed with aqueous solution of NaCl and dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give 160 mg Compound 124d.
[0379] A mixture solution of Compound 124d (150 mg), 40 d (205 mg) and con-HCl (0.05 ml) in n-Butanol was heated to 85° C. with stirring for 3 hrs. The reaction was complete detected by TLC (DCM:MeOH=10:1). Then, after the reaction mixture was cooled to 20° C., it was filtered and the filter cake was dried to give 115 mg Compound 124. MS: 550.2 (M+H).sup.+. H-NMR (DMSO-d6, 400 MHz): 11.38 (s, 1H), 9.81 (s, 1H), 9.19 (s, 1H), 8.90 (s, 1H), 8.40 (s, 1H), 8.22 (s, 1H), 7.64-7.69 (m, 2H), 7.32-7.36 (m, 1H), 7.23-7.27 (t, 1H), 7.00 (d, 1H), 6.64-6.71 (dd, 1H), 6.18-6.22 (dd, 1H), 5.71-5.74 (dd, 1H), 5.02 (d, 1H), 3.87-3.94 (m, 2H), 3.73-3.78 (m, 2H), 3.00 (s, 3H), 2.90 (s, 3H), 2.19 (m, 1H), 2.05-2.10 (m, 1H).
TABLE-US-00007 Physical Data EX (MS) No. Chemical Name Structure (M + H).sup.+ 125 N-(3-(5-chloro-4-(2-(2- (dimethylamino)-2-oxoacetyl) phenylamino)pyrimidin-2- ylamino)phenyl)-2-(((2- (dimethylamino)ethyl) (methyl)amino)methyl) acrylamide
Example 143. Synthesis of Compound 143
[0380] ##STR00167## ##STR00168##
[0381] A mixture of Compound 143a (5.12 g, 23.59 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-car-boxylate (7.30 g, 23.59 mmol), tetrakis (triphenylphosphine) palladium(0) (1.36 g, 1.18 mmol) and K.sub.2CO.sub.3 (8.15 g, 58.98 mmol) in DMF (200 ml) was degassed with five vacuum/nitrogen cycles. The mixture was heated to 80° C. with stirring under nitrogen for 16.5 hours. After cooled to 10° C., water (300 ml) was added, and the resulting mixture was extracted with ethyl acetate (300 ml, 150 ml×2). The combined organic extracts were washed with brine (200 ml×2), dried over anhydrous Na.sub.2SO.sub.4 and then evaporated under reduced pressure. The residue was purified by chromatography to give 4.82 g Compound 143b as a yellow solid.
[0382] A mixture of Compound 143b (4.81 g, 15.08 mmol), acetic anhydride (5 ml, 52.89 mmol) in DCM (300 ml) was stirred at 25° C. for 20 hours. After concentrated in under reduced pressure, the residue was dissolved in saturated aqueous solution of sodium bicarbonate (200 ml) with stirring for 12 hours at ambient temperature. The precipitated solid was collected by filtration to give 5.28 g Compound 143c.
[0383] A mixture solution of Compound 143c (5.27 g, 14.58 mmol) and trifluoroacetic acid (13 ml, 175 mmol) in DCM was stirred at 40° C. for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, the residue (Compound 143d) was used for the next step without further purification.
[0384] A mixture of Compound 143d (3.80 g, 14.54 mmol), formaldehyde solution (5 ml, 178.90 mmol), acetic acid (1 ml, 17.48 mmol), sodium triacetoxyborohyride (9.25 g, 43.65 mmol) and DCM (150 ml) was stirred at ambient temperature for 20 mins. Saturated aqueous solution of K.sub.2CO.sub.3 (100 ml) was added, the resulting mixture was extracted with DCM (100 ml×2). The combined organic layers were washed with brine (100 ml), dried over anhydrous Na.sub.2SO.sub.4, then concentrated under reduced pressure and the residue (Compound 143e) was used for the next step without further purification.
[0385] To a solution of Compound 143e (3.81 g, 13.84 mmol) in EA (100 ml) and EtOH (100 ml) was added palladium 10% on carbon (1.05 g). The resulting mixture was stirred at ambient under hydrogen atmosphere for 2 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated to give 3.21 g Compound 143f.
[0386] A mixture of Compound 143f (3.21 g, 12.99 mmol), Compound 40d (4.40 g, 12.99 mmol), p-toluenesulfonic acid (2.68 g, 15.59 mmol) and n-butyl alcohol (150 ml) was heated to 100° C. with stirring for 7 hours. The mixture was cooled and concentrated under reduced pressure, the residue was purified by chromatography to give 5.02 g Compound 143 g as a light yellow solid.
[0387] To a stirred solution of Compound 143 g (1.98 g, 3.60 mmol) in THF (50 ml) was added con.hydrochloric acid (15 ml), the resulting mixture was stirred at 65° C. for 17 hours. Reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to 0-5° C. and saturated aqueous solution of K.sub.2CO.sub.3 was added to adjust pH value to 9-10, then extracted with DCM (100 ml×2), the combined organic extracts was washed with brine (100 ml), dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure. Crude product was purified by chromatography to give 0.28 g Compound 143h as a yellow solid.
[0388] To a solution of Compound 143h (255 mg, 0.5 mmol), triethylamine (100 mg, 1.0 mmol) in DCM (100 ml) was added acryloyl chloride (69 mg, 0.75 mmol) dropwise and the mixture was stirred at 0-5° C. Reaction was quenched with saturated aqueous solution of potassium carbonate (200 ml), extracted with DCM (100 ml×3), the combined organic extracts was washed with brine (100 ml×2), dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure, the residue was purified by Pre-TLC to give 138 mg Compound 143 as a yellow solid. MS: 561.2 (M+H).sup.+. HNMR (DMSO-d6, 400 MHz): 11.32 (s, 1H), 9.61 (s, 1H), 9.02 (d, 1H), 8.33 (s, 1H), 7.63-7.80 (m, 3H), 7.50 (d, 1H), 7.20-7.23 (m, 2H), 6.46-6.53 (m, 1H), 6.19-6.23 (dd, 1H), 5.72-5.75 (dd, 1H), 3.01 (s, 3H), 2.91 (s, 3H), 2.87 (s, 2H), 2.61-2.67 (m, 1H), 2.21 (s, 3H), 1.98 (m, 2H), 1.63-1.65 (m, 4H).
TABLE-US-00008 Physical Data EX (MS) No. Chemical Name Structure (M + H).sup.+ 144 N-(5-(5-chloro-4-(2-(2- (dimethylamino)-2-oxoacetyl) phenylamino)pyrimidin-2- ylamino)-4-methoxy-2-(1- methylpiperidin-4-yl) phenyl)acrylamide
Pharmacological Testing
Example A. Kinase Assays (Single Dose Inhibition)
[0389] Assays were conducted for an in vitro kinase panel having EGFR WT, L858R, T790M, L858R/T790M and ALK. Assay conditions included 10 μM ATP and 100 nM test compounds.
[0390] Assay Protocol:
[0391] All reactions are initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting. This assay is performed by Millipore. The experiment is carried out in duplicate. The value for the control sample (DMSO) was set to 100%, and the values for the compound-treated samples were expressed as activity relative to the control sample.
[0392] Kinase-Specific Assay Conditions
[0393] Alk (h) is incubated with 8 mM MOPS pH7.0, 0.2 mM EDTA, 250 μM KKKSPGEYVNIEFG, 10 mM MgAcetate, [γ-.sup.33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required) and 0.1 μM test compound.
[0394] EGFR (h) and EGFR (L858R) (h) are incubated with 8 mM MOPS pH7.0, 0.2 mM EDTA, 10 mM MnCl.sub.2, 0.1 mg/mL poly(Glu, Tyr)4:1, 10 mM MgAcetate, [γ-.sup.33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required) and 0.1 μM test compound.
[0395] EGFR (T790M) (h) and EGFR (T790M, L858R) (h) are incubated with 8 mM MOPS pH7.0, 0.2 mM EDTA, 250 μM GGMEDIYFEFMGGKKK, 10 mM MgAcetate, [γ-.sup.33P-ATP](specific activity approx. 500 cpm/pmol, concentration as required) and 0.1 μM test compound.
TABLE-US-00009 TABLE 1 Activity (control) % EGFR EGFR EGFR ALK EGFR (L858R) (T790M) (T790M, L858R) Example (h) (h) (h) (h) (h) CO-1686 @ 0.1 μM 49 92 97 21 7 4 @ 0.1 μM 8 65 8 4 2 40 @ 0.1 μM 28 106 103 79 24 53 @ 0.1 μM 5 109 60 21 4 61 @ 0.1 μM 5 101 70 54 13 64 @ 0.1 μM 47 106 100 79 39 65 @ 0.1 μM 19 118 102 80 25 66 @ 0.1 μM 35 107 104 66 24 67 @ 0.1 μM 13 105 85 50 9 41 @ 0.1 μM 21 76 39 2 3 72 @ 0.1 μM 14 102 28 54 9 73 @ 0.1 μM 17 67 37 3 3 74 @ 0.1 μM 9 110 73 62 12 78 @ 0.1 μM 36 68 37 2 3 79 @ 0.1 μM 7 92 25 16 2 80 @ 0.1 μM 10 101 43 42 7 102 @ 0.1 μM 5 98 57 40 5 87 @ 0.1 μM 5 88 44 8 1 89 @ 0.1 μM 8 67 10 5 1 94 @ 0.1 μM 6 100 84 45 9 97 @ 0.1 μM 23 74 46 2 6 101 @ 0.1 μM 35 110 94 83 41 123 @ 0.1 μM 17 115 94 84 31 111 @ 0.1 μM 8 109 76 59 11 112 @ 0.1 μM 21 85 66 14 1 113 @ 0.1 μM 19 88 78 16 3 114 @ 0.1 μM 10 91 52 47 8 115 @ 0.1 μM 6 73 45 12 3 117 @ 0.1 μM 8 113 88 65 14 118 @ 0.1 μM 3 82 29 18 2 120 @ 0.1 μM 8 115 76 62 14 124 @ 0.1 μM 16 109 100 78 20 125 @ 0.1 μM 20 106 54 18 2 126 @ 0.1 μM 19 93 47 5 2 129 @ 0.1 μM 8 104 75 24 3 130 @ 0.1 μM 13 113 98 84 22 131 @ 0.1 μM 9 104 104 82 27 132 @ 0.1 μM 16 103 114 77 30 135 @ 0.1 μM 80 106 103 96 95 136 @ 0.1 μM 29 99 104 92 54
Example B. Kinase Assays (IC.SUB.50.)
[0396] Assays were conducted for an in vitro kinase panel having EGFR WT, L858R, T790M, L858R/T790M and ALK. Assay conditions included 10 pt curves with 1 μM (EGFR L858R, T790M, L858R/T790M and ALK) or 10 μM (EGFR WT) top concentration (duplicates) and Km ATP.
[0397] Kinase Assay Protocol:
[0398] Bar-coded Corning, low volume NBS, black 384-well plate (Corning Cat. #4514)
[0399] 1. 2.5 μL—4× Test Compound or 100 nL 100× plus 2.4 μL kinase buffer;
[0400] 2. 5 μL—2× Peptide/Kinase Mixture;
[0401] 3. 2.5 μL—4×ATP Solution;
[0402] 4. 30-second plate shake;
[0403] 5. 60-minute Kinase Reaction incubation at room temperature;
[0404] 6. 5 μL—Development Reagent Solution;
[0405] 7. 30-second plate shake;
[0406] 8. 60-minute Development Reaction incubation at room temperature;
[0407] 9. Read on fluorescence plate reader and analyze the data.
[0408] Kinase-Specific Assay Conditions:
[0409] ALK
[0410] The 2×ALK/Tyr 01 mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA. The final 10 μL Kinase Reaction consists of 4.25-96 ng ALK and 2 μM Tyr 01 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent B is added.
[0411] EGFR (ErbB1)
[0412] The 2×EGFR (ErbB1)/Tyr 04 mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 4 mM MnCl.sub.2, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction consists of 1.1-8 ng EGFR (ErbB1) and 2 μM Tyr 04 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 2 mM MnCl.sub.2, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent B is added.
[0413] EGFR (ErbB1) L858R
[0414] The 2×EGFR (ErbB1) L858R/Tyr 04 mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 4 mM MnCl.sub.2, 1 mM EGTA, 2 mM DTT. The final 10 μL Kinase Reaction consists of 0.2-3.36 ng EGFR (ErbB1) L858R and 2 μM Tyr 04 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 2 mM MnCl.sub.2, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent B is added.
[0415] EGFR (ErbB1) T790M
[0416] The 2×EGFR (ErbB1) T790M/Tyr 04 mixture is prepared in 50 mM HEPES pH 6.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% NaN.sub.3. The final 10 μL Kinase Reaction consists of 3.9-34.8 ng EGFR (ErbB1) T790M and 2 μM Tyr 04 in 50 mM HEPES pH 7.0, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA, 0.01% NaN.sub.3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent B is added.
[0417] EGFR (ErbB1) T790M L858R
[0418] The 2×EGFR (ErbB1) T790M L858R/Tyr 04 mixture is prepared in 50 mM HEPES pH 6.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% NaN.sub.3. The final 10 μL Kinase Reaction consists of 0.36-2.96 ng EGFR (ErbB1) T790M L858R and 2 μM Tyr 04 in 50 mM HEPES pH 7.0, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA, 0.01% NaN.sub.3. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:64 dilution of Development Reagent B is added.
[0419] Compounds formula (I) included potent inhibitors of EGFR mutants in kinase assays. For example, for the resistant mutant EGFR L858R/T790M, previously known inhibitors gefitinib, icotinib, and CO-1686 had IC.sub.50 values between 16.6 nM to >1 μM, while many compounds of formula (I) exhibited IC.sub.50 values in the range of 4.08 to 22.8 nM. Thus, compounds of formula (I) could provide the necessary inhibitors for EGFR-driven cancers.
TABLE-US-00010 TABLE 2 IC.sub.50/nm EGFR EGFR (ErbB1) EGFR (ErbB1) T790M (ErbB1) EGFR Example ALK L858R L858R T790M (ErbB1) Icotinib 2.02 >1000 >1000 1.23 Gefitinib 0.458 412 179 <0.508 CO-1686 145 108 16.6 13.7 101 4 27.9 44.3 4.08 7.52 45.1 61 12.2 >1000 22.8 38.2 756 67 17.3 500 15.4 18.9 273 74 9.33 422 15.5 15.8 513 87 15.1 225 14.7 21.3 354 115 8.62 547 11.7 18.7 397 118 10.8 158 5.13 4.92 77.6
Example C. Cell Proliferation Assay
[0420] NSCLC cell lines were used to examine the activity of compounds of formula (I) against 3 general forms of EGFR: wild type EGFR (the naturally occurring form, WT), EGFR with an activating mutation (delE746_A750 [Del]; this form is sensitive to first generation EGFR inhibitors), and EGFR with both an activating mutation and a T790M resistance mutation (L858R/T790M; the addition of the T790M mutation makes this form resistant to first generation EGFR inhibitors).
[0421] Effects of test compounds on in vitro proliferation were measured by MTS cell viability assay.
[0422] Cell Culture
[0423] H1975 (EGFR L858R/T790M), HCC827 (EGFR Del), A549 (EGFR WT) and A431 (EGFR WT) NSCLC cells were all obtained from ATCC.
[0424] H1975 and HCC827 cells were maintained in RPMI 1640 (Gibco) supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM glutamine.
[0425] A549 cells were maintained in Ham's F12K medium (Gibco) supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM glutamine.
[0426] A431 cells were maintained in DMEM (Gibco) supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM glutamine.
[0427] MTS Cell Viability Assay:
[0428] 1. Seed cells at density of 2×103 cells per well of 96-wells plates, grow for 24 hours;
[0429] 2. Prepare test compounds in each well with a final medium volume of 200 μl;
[0430] 3. Incubate for 3 days of exposure;
[0431] 4. Prepare reagents following the instructions in the Cell Proliferation Assay kit (Promega);
[0432] 5. Change to serum-free medium with a final volume of 100 l/well. Prepare a set of wells with medium only for background subtraction;
[0433] 6. Add 20 μl MTS solution containing PMS to each well (final concentration of MTS will be 0.33 mg/ml);
[0434] 7. Incubate 1 to 4 hours at 37° C. in a humidified, 5% CO.sub.2 atmosphere.
[0435] 8. Record absorbance at 490 nm using VICTOR™X5 plate reader (PerkinElmer).
[0436] All experimental points were set up in three wells and all experiments were repeated at least three times.
[0437] The Compound of Example 40, 65, 66, 67, 74, 112 and 118 demonstrated potent activity against both activated and resistant T790M forms of EGFR in vitro cellular assays. For example, in one set of studies, proliferation of HCC827 cells expressing EGFR Del were inhibited with GI.sub.50s of 20 nM by Example 67 and 118; and proliferation of H1975 cells expressing EGFR-L858R/T790M were inhibited with GI.sub.50s (20 nM and 10 nM) similar to that of HCC827 cells.
[0438] In contrast, Example 67 and 118 were essentially inactive against EGFR WT in cellular assays, i.e., inhibited proliferation with GI.sub.50s >1500 nM in A549 cells (EGFR WT), showing a great selectivity between EGFR WT and mutants. However the second generation EGFR TKI afatinib induced similar inhibition of proliferation on HCC827 cells (EGFR Del) and A431 cells (EGFR WT).
TABLE-US-00011 TABLE 3 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 Example (H1975)/μM (HCC827)/μM (A549)/μM (A431)/μM CO-1686 0.08 0.09 2.00 0.45 WZ4002 0.15 0.03 >3 NA 24 >3 >3 NA NA 18 2.86 >3 NA NA 3 0.84 0.94 >3 NA 17 0.86 1.23 >3 0.26 16 1.13 0.90 >3 NA 1 >3 >3 NA NA 2 >3 >3 NA NA 4 >3 2.63 >3 NA 15 >3 >3 NA NA 10 >3 >3 NA NA 42 >3 >3 >3 NA 43 >3 >3 >3 NA 44 1.23 2.36 >3 NA 45 >3 >3 >3 NA 46 0.81 1.63 >3 NA 47 >3 >3 >3 NA 48 1.95 1.94 NA NA 25 >3 >3 NA NA 49 >3 >3 NA NA 53 0.21 0.54 >3 0.05 40 0.01 0.07 0.64 NA 54 >3 >3 NA NA 55 >3 >3 NA NA 56 >3 >3 NA NA 57 0.10 0.27 >3 NA 58 0.98 0.53 0.37 NA 59 0.19 >3 >3 NA 60 0.15 0.87 2.08 NA 61 0.01 0.17 0.74 0.01 62 >3 2.33 >3 NA 63 0.64 >3 >3 NA 64 0.09 0.45 >3 NA 65 0.02 0.12 >3 NA 66 0.05 0.12 >3 NA 67 0.02 0.02 1.58 0.11 68 0.65 1.20 >3 NA 69 0.31 1.20 >3 NA 70 0.31 1.05 1.37 NA 71 0.99 0.78 1.19 NA 74 0.05 0.03 0.62 0.05 87 0.01 0.02 0.05 0.06 118 0.01 0.02 1.89 0.01 112 0.01 0.03 0.04 0.10 126 0.01 0.02 NA 0.21 131 0.01 0.05 NA 0.75
[0439] The compounds of the present invention are preferably formulated as pharmaceutical compositions administered by a variety of routes. Most preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing the same are well known in the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al, eds., 19.sup.th ed., Mack Publishing Co., 1995). The compounds of Formula I are generally effective over a wide dosage range.
[0440] For example, dosages per day normally fall within the range of about 1 mg to about 200 mg total daily dose, preferably 1 mg to 150 mg total daily dose, more preferably 1 mg to 50 mg total daily dose. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed. The above dosage range is not intended to limit the scope of the invention in any way. It will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.