(E)-4-(dimethylamino)but-2-enoic acid;hydrochloride

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution of (E)-4-(dimethylamino)but-2-enoic acid hydrochloride (1.28 g, 7.73 mmol, 2.1 equiv) in THF (18 ml) and a catalytic amount of DMF was cooled to 5° C. while oxalyl chloride (0.67 ml, 0.007 mol, 1.9 equiv) was added slowly. The reaction mixture was then warmed to room temperature and stirred for 3 hours. A solution of 6-amino-7-ethoxy-4-(3-ethynylphenylamino)quinoline-3-carbonitrile (1.2 g, 3.66 mmol, 1.00 equiv) in N-methyl-2-pyrrolidinone (15 ml) was added dropwise over 10 minutes. The mixture was stirred for overnight, quenched with water (200 ml). Aqueous sodium hydroxide was added to bring the pH to 11. The mixture was stirred for 1 hour which formed precipitate. The resulting precipitate was filtered and washed with water and the wet solid was dried to give 1.43 g product. MS (ESI) m/z: 563 (M+1). was added slowly The mixture was stirred for overnight quenched with water (200 ml) Aqueous sodium hydroxide was added The mixture was stirred for 1 hour which formed precipitate The resulting precipitate was filtered washed with water the wet solid was dried

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution of sodium hydroxide (6.2 g, 0.155 mol, 4 equiv) in water (40 ml) was added to a stirred solution of (E)-methyl 4-(dimethylamino)but-2-enoate hydrochloride (7 g, 0.039 mol, 1.00 equiv) in methanol (150 ml). The reaction mixture was stirred for 4 hours. 6N hydrochloric acid was added to bring the pH to 2. The reaction mixture was concentrated under reduced pressure, and ethanol (50 ml) was added and the solid was removed by filtration. The filtrate was concentrated under reduced pressure to thick oil. The oil was crystallized in isopropanol and acetone to give the product 3.6 g. MS (ESI) m/z: 563 (M+1). The reaction mixture was concentrated under reduced pressure, and ethanol (50 ml) was added the solid was removed by filtration The filtrate was concentrated under reduced pressure to thick oil The oil was crystallized in isopropanol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of (E)-4-(dimethylamino)but-2-enoic acid (5) (185 mg, 1.12 mmol) and a drop of DMF in dry DCM (3 mL) was added oxalyl dichloride (427 mg, 3.36 mmol) slowly. The mixture was stirred at room temperature for 2 h and the solvent was removed in vacuo. The freshly generated acid chloride was dissolved in dry DCM (5 mL) and was added dropwise into a solution of tert-butyl 3-iodo-1-(5-(methylamino)pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylcarbamate (64) (400 mg, 0.86 mmol) in DCM (8 mL) at 0° C. The resulting mixture was stirred at room temperature for 3 h. TFA (2 mL) was introduced and the reaction mixture was stirred for 5 h. The solvent was removed and the residue was purified by column chromatography (silca gel, 0 to 7% methanol in dichloromethane) to provide (E)-N-(6-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-3-yl)-4-(dimethylamino)-N-methylbut-2-enamide (65) (300 mg, 73%) as a white solid. LC-MS (ESI): m/z (M+1) 479. the solvent was removed in vacuo The freshly generated acid chloride was dissolved in dry DCM (5 mL) The resulting mixture was stirred at room temperature for 3 h the reaction mixture was stirred for 5 h The solvent was removed the residue was purified by column chromatography (silca gel, 0 to 7% methanol in dichloromethane)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of (E)-4-(dimethylamino)but-2-enoic acid hydrochloride salt (5) (66 mg, 0.4 mmol) in acetonitrile (5 mL) was added 1 drop of DMF before introducing (COCl)2 (66 mg, 0.52 mmol). The resulting mixture was stirred at RT for 2 h and then concentrated. The residue was dissolved in DCM (2 mL) before adding a solution of tert-butyl 3-iodo-1-(4-(methylamino)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylcarbamate (30) (170 mg, 0.438 mmol) in DCM (2 mL). The resulting solution was stirred at RT overnight before adding TFA (10 mL). The reaction mixture was stirred for 3 hr. The reaction mixture was concentrated and quenched with sat. aqueous NaHCO3. The mixture was extracted with 85% DCM/IPA (3×). The organic solution was dried over Na2SO4 and concentrated to provide (E)-N-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)phenyl)-4-(dimethylamino)-N-methylbut-2-enamide (32) (170 mg, 100%) which was used in the next step directly without purification. LC-MS (ESI): m/z (M+1) 478.3. concentrated The residue was dissolved in DCM (2 mL) The resulting solution was stirred at RT overnight The reaction mixture was stirred for 3 hr The reaction mixture was concentrated quenched with sat. aqueous NaHCO3 The mixture was extracted with 85% DCM/IPA (3×) The organic solution was dried over Na2SO4 concentrated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: HATU (10.3 mg) and 5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizine-4,8-diamine (8.8 mg) obtained in Step 11 of Example 1 were added to a solution of trans-4-dimethylaminocrotonic acid hydrochloride (4.5 mg) in DMF (0.5 ml) at room temperature. After being stirred for 1 hour at the same temperature, the mixture was poured into a saturated aqueous sodium bicarbonate solution, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was then distilled off under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography (produced by Wako, NH2 silica-gel 60 F254 plate, developing solvent: chloroform/methanol) to obtain the title compound as a light-yellow solid (6.4 mg). followed by extraction with ethyl acetate The organic layer was dried over anhydrous sodium sulfate the solvent was then distilled off under reduced pressure The resulting residue was purified by preparative thin-layer chromatography (produced by Wako, NH2 silica-gel 60 F254 plate, developing solvent: chloroform/methanol)

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To (E)-4-(dimethylamino)but-2-enoic acid hydrochloride (110 mg, 0.664 mmol) in acetonitrile (1.5 mL) was added oxalyl dichloride (75.9 mg, 0.598 mmol) followed by one drop of DMF. After heating to 60° C. for 30 minutes, the mixture was cooled to room temperature and concentrated to 0.5 mL total volume. The resulting solution was used without further purification. the mixture was cooled to room temperature concentrated to 0.5 mL total volume The resulting solution was used without further purification

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To (E)-4-(dimethylamino)but-2-enoic acid hydrochloride (110 mg, 0.664 mmol) in acetonitrile (1.5 mL) was added oxalyl dichloride (75.9 mg, 0.598 mmol) followed by one drop of DMF. After heating to 60° C. for 30 minutes, the mixture was cooled to room temperature and concentrated to 0.5 mL total volume. The resulting solution was used without further purification. the mixture was cooled to room temperature concentrated to 0.5 mL total volume The resulting solution was used without further purification

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1.74 ml (2.54 g, 0.02 moles) portion of oxalyl chloride was added to 3.31 grams (0.02 moles) of (E)-4-(dimethylamino)-2-butenoic acid hydrochloride in 75 ml of acetonitrile. To this was added a small drop of dimethylformamide. The reaction was heated and stirred in an oil bath at 63° for 20 minutes, giving an orange solution. This solution as concentrated in vacuo without the application of heat to about half its original volume. This solution was cooled in an ice bath and a solution of 4.45 g (0.01 moles) of the 6-amino-4-[4-(benzyloxy)-3-chloroanilino]-7-ethoxy-3-quinolinecarbonitrile in 50 ml of N-methylpyrrolidone was added in a stream. The reaction was cooled and stirred for 2 hours. The reaction was poured onto 100 ml of saturated aqueous sodium bicarbonate in ice. On standing the resulting gum solidified and the solid was filtered. This solid was chromatographed on silica gel. The column was washed with 3 liters of 1:19 methanol-ethyl acetate, then the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate. Concentration of the eluate gave a solid, which was filtered to give 2.96 grams of the title compound. From the filtrate was obtained an additional 1.0 grams of product. Total: 3.96 g. The reaction was heated giving an orange solution This solution as concentrated in vacuo without the application of heat to about half its original volume This solution was cooled in an ice bath The reaction was cooled stirred for 2 hours the solid was filtered This solid was chromatographed on silica gel The column was washed with 3 liters of 1:19 methanol-ethyl acetate the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate Concentration of the eluate gave a solid, which was filtered

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of (E)-4-(dimethylamino)but-2-enoic acid (5) (185 mg, 1.12 mmol) and a drop of DMF in dry DCM (3 mL) was added oxalyl dichloride (427 mg, 3.36 mmol) slowly. The mixture was stirred at room temperature for 2 h and the solvent was removed in vacuo. The freshly generated acid chloride was dissolved in dry DCM (5 mL) and was added dropwise into a solution of tert-butyl 3-iodo-1-(5-(methylamino)pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylcarbamate (64) (400 mg, 0.86 mmol) in DCM (8 mL) at 0° C. The resulting mixture was stirred at room temperature for 3 h. TFA (2 mL) was introduced and the reaction mixture was stirred for 5 h. The solvent was removed and the residue was purified by column chromatography (silica gel, 0 to 7% methanol in dichloromethane) to provide (E)-N-(6-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-3-yl)-4-(dimethylamino)-N-methylbut-2-enamide (65) (300 mg, 73%) as a white solid. LC-MS (ESI): m/z (M+1) 479. the solvent was removed in vacuo The freshly generated acid chloride was dissolved in dry DCM (5 mL) The resulting mixture was stirred at room temperature for 3 h the reaction mixture was stirred for 5 h The solvent was removed the residue was purified by column chromatography (silica gel, 0 to 7% methanol in dichloromethane)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of (E)-4-(dimethylamino)but-2-enoic acid hydrochloride salt (5) (66 mg, 0.4 mmol) in acetonitrile (5 mL) was added 1 drop of DMF before introducing (COCl)2 (66 mg, 0.52 mmol). The resulting mixture was stirred at RT for 2 h and then concentrated. The residue was dissolved in DCM (2 mL) before adding a solution of tert-butyl 3-iodo-1-(4-(methylamino)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylcarbamate (30) (170 mg, 0.438 mmol) in DCM (2 mL). The resulting solution was stirred at RT overnight before adding TFA (10 mL). The reaction mixture was stirred for 3 hr. The reaction mixture was concentrated and quenched with sat. aqueous NaHCO3. The mixture was extracted with 85% DCM/IPA (3×). The organic solution was dried over Na2SO4 and concentrated to provide (E)-N-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)phenyl)-4-(dimethylamino)-N-methylbut-2-enamide (32) (170 mg, 100%) which was used in the next step directly without purification. LC-MS (ESI): m/z (M+1) 478.3. concentrated The residue was dissolved in DCM (2 mL) The resulting solution was stirred at RT overnight The reaction mixture was stirred for 3 hr The reaction mixture was concentrated quenched with sat. aqueous NaHCO3 The mixture was extracted with 85% DCM/IPA (3×) The organic solution was dried over Na2SO4 concentrated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1.74 ml (2.54 g, 0.02 moles) portion of oxalyl chloride was added to 3.31 grams (0.02 moles) of (E)-4-(dimethylamino)-2-butenoic acid hydrochloride in 75 ml of acetonitrile. To this was added a small drop of dimethylformamide. The reaction was heated and stirred in an oil bath at 63° for 20 minutes, giving an orange solution. This solution as concentrated in vacuo without the application of heat to about half its original volume. This solution was cooled in an ice bath and a solution of 4.45 g (0.01 moles) of the 6-amino-4-[4-(benzyloxy)-3-chloroanilino]-7-ethoxy-3-quinolinecarbonitrile in 50 ml of N-methylpyrrolidone was added in a stream. The reaction was cooled and stirred for 2 hours. The reaction was poured onto 100 ml of saturated aqueous sodium bicarbonate in ice. On standing the resulting gum solidified and the solid was filtered. This solid was chromatographed on silica gel. The column was washed with 3 liters of 1:19 methanol-ethyl acetate, then the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate. Concentration of the eluate gave a solid, which was filtered to give 2.96 grams of the title compound. From the filtrate was obtained an additional 1.0 grams of product. Total: 3.96 g. The reaction was heated giving an orange solution This solution as concentrated in vacuo without the application of heat to about half its original volume This solution was cooled in an ice bath The reaction was cooled stirred for 2 hours the solid was filtered This solid was chromatographed on silica gel The column was washed with 3 liters of 1:19 methanol-ethyl acetate the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate Concentration of the eluate gave a solid, which was filtered

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1.74 ml (2.54 g, 0.02 moles) portion of oxalyl chloride was added to 3.31 grams (0.02 moles) of (E)-4-(dimethylamino)-2-butenoic acid hydrochloride in 75 ml of acetonitrile. To this was added a small drop of dimethylformamide. The reaction was heated and stirred in an oil bath at 63° for 20 minutes, giving an orange solution. This solution as concentrated in vacuo without the application of heat to about half its original volume. This solution was cooled in an ice bath and a solution of 4.45 g (0.01 moles) of the 6-amino-4-[4-(benzyloxy)-3-chloroanilino]-7-ethoxy-3-quinolinecarbonitrile in 50 ml of N-methylpyrrolidone was added in a stream. The reaction was cooled and stirred for 2 hours. The reaction was poured onto 100 ml of saturated aqueous sodium bicarbonate in ice. On standing the resulting gum solidified and the solid was filtered. This solid was chromatographed on silica gel. The column was washed with 3 liters of 1:19 methanol-ethyl acetate, then the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate. Concentration of the eluate gave a solid, which was filtered to give 2.96 grams of the title compound. From the filtrate was obtained an additional 1.0 grams of product. Total: 3.96 g. The reaction was heated giving an orange solution This solution as concentrated in vacuo without the application of heat to about half its original volume This solution was cooled in an ice bath The reaction was cooled stirred for 2 hours the solid was filtered This solid was chromatographed on silica gel The column was washed with 3 liters of 1:19 methanol-ethyl acetate the product was eluted with 3 liters of 1:5:94 triethylamine-methanol-ethyl acetate Concentration of the eluate gave a solid, which was filtered