acetic acid;tert-butyl azetidine-3-carboxylate

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of tert-butyl azetidine-3-carboxylate acetic acid salt (92.0 g, 0.423 mol) in methanol (1.0 L) at room temperature was added 4-formylbenzonitrile (50.8 g, 0.381 mol). The reaction mixture was cooled to 0° C., and sodium cyanoborohydride (28.8 g, 0.458 mol) was added portion-wise (caution: potential cyanide generation). The reaction mixture was allowed to warm to room temperature and was stirred overnight. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with a 10% aqueous solution of sodium bicarbonate and extracted with ethyl acetate. The organic layer was collected, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by silica gel chromatography using a 20% mixture of ethyl acetate in petroleum ether afforded tert-butyl 1-(4-cyanobenzyl)azetidine-3-carboxylate (89%) (After chromatography, Int. 1-D contained a small amount of 4-hydroxymethylbenzonitrile but was taken forward to the next step without further purification). LC/MS M+1=273.18. 1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.22-3.31 (m, 3H), 3.48-3.56 (m, 2H), 3.66 (s, 2H), 7.39 (d, J=8.28 Hz, 2H), and 7.60 (d, J=8.28 Hz, 2H). to warm to room temperature The reaction mixture was concentrated under reduced pressure the residue was diluted with a 10% aqueous solution of sodium bicarbonate extracted with ethyl acetate The organic layer was collected washed with brine dried over anhydrous sodium sulfate Concentration under reduced pressure followed by purification by silica gel chromatography

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of tert-butyl azetidine-3-carboxylate acetic acid salt (92.0 g, 0.423 mol) in methanol (1.0 L) at room temperature was added 4-formylbenzonitrile (50.8 g, 0.381 mol). The reaction mixture was cooled to 0° C., and sodium cyanoborohydride (28.8 g, 0.458 mol) was added portion-wise (caution: potential cyanide generation). The reaction mixture was allowed to warm to room temperature and stirring continued overnight. After the reaction mixture was concentrated under reduced pressure the residue was diluted with 10% aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was collected, washed with brine and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by silica gel chromatography using 20% ethyl acetate in petroleum ether afforded tert-butyl 1-(4-cyanobenzyl)azetidine-3-carboxylate (89%) (After chromatography, Int.1-D contained a small amount of 4-hydroxymethylbenzonitrile but was taken forward to the next step without further purification). LC/MS M+1=273.18. 1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.22-3.31 (m, 3H), 3.48-3.56 (m, 2H), 3.66 (s, 2H), 7.39 (d, J=8.28 Hz, 2H), and 7.60 (d, J=8.28 Hz, 2H). to warm to room temperature After the reaction mixture was concentrated under reduced pressure the residue was diluted with 10% aqueous sodium bicarbonate solution extracted with ethyl acetate The organic layer was collected washed with brine dried over anhydrous sodium sulfate Concentration under reduced pressure followed by purification by silica gel chromatography

Conditions: temp:65.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A reaction vial containing a stir bar was charged with 3-bromo-4-(bromomethyl)benzonitrile (100 mg, 0.364 mmol), tert-butyl azetidine-3-carboxylate acetic acid salt, Int.1-C (95 mg, 0.436 mmol), DMF (1 mL), and DIPEA (0.381 mL, 2.182 mmol). The vial was flushed with argon, sealed and placed on a reaction block which was heated to 65° C. After 5 hours, the reaction was cooled to RT and directly purified by preparative HPLC (methanol/water plus 0.1% TFA as eluent). To the product fractions was added a solution of sodium bicarbonate (200 mg) in water (4 mL) and then the volatiles were removed in vacuo to afford a white solid which was slurried in methanol (10 mL). Hydroxylamine hydrochloride (37.6 mg, 0.541 mmol) was added and the reaction was heated overnight in an oil bath set to 50° C. Additional hydroxylamine hydrochloride (30 mg) along with sodium bicarbonate (150 mg) were added and heating continued at 50° C. for 8 hours. The volatiles were removed in vacuo and the residue purified by preparative HPLC (methanol/water plus 0.1% TFA as eluent). The pH of the product containing fractions was adjusted to ˜7 using sodium bicarbonate, the solvents were concentrated and the resulting aqueous solution was extracted with DCM. The organics were dried over anhydrous magnesium sulfate then concentrated to afford (Z)-tert-butyl 1-(2-bromo-4-(N′-hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (12.6 mg, 0.032 mmol). The compound had an HPLC retention time=0.72 min.−Column: BEH C18 2.1×50 mm 1.7 u (gradient: 2% MeCN/water plus 0.05% to 98% MeCN/water plus 0.05% TFA over 1 min.); LC/MS M+1=384/386. A reaction vial containing a stir bar The vial was flushed with argon sealed the reaction was cooled to RT directly purified by preparative HPLC (methanol/water plus 0.1% TFA as eluent) To the product fractions was added a solution of sodium bicarbonate (200 mg) in water (4 mL) the volatiles were removed in vacuo to afford a white solid which the reaction was heated overnight in an oil bath set to 50° C heating continued at 50° C. for 8 hours The volatiles were removed in vacuo the residue purified by preparative HPLC (methanol/water plus 0.1% TFA as eluent) The pH of the product containing fractions the solvents were concentrated the resulting aqueous solution was extracted with DCM The organics were dried over anhydrous magnesium sulfate then concentrated

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of tert-butyl azetidine-3-carboxylate acetic acid salt (92.0 g, 0.423 mol) in methanol (1.0 L) at room temperature was added 4-formylbenzonitrile (50.8 g, 0.381 mol). The reaction mixture was cooled to 0° C., and sodium cyanoborohydride (28.8 g, 0.458 mol) was added portion-wise. The reaction mixture was allowed to warm to room temperature and stirring continued overnight. After the reaction mixture was concentrated under reduced pressure the residue was diluted with 10% aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was collected, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by silica gel chromatography using 20% ethyl acetate in petroleum ether afforded tert-butyl 1-(4-cyanobenzyl)azetidine-3-carboxylate (89%) (After chromatography, Int.1-D contained a small amount of 4-hydroxymethylbenzonitrile. This material was taken forward to the next step without further purification). LC/MS M+1=273.18. 1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.22-3.31 (m, 3H), 3.48-3.56 (m, 2H), 3.66 (s, 2H), 7.39 (d, J=8.28 Hz, 2H), and 7.60 (d, J=8.28 Hz, 2H). to warm to room temperature After the reaction mixture was concentrated under reduced pressure the residue was diluted with 10% aqueous sodium bicarbonate solution extracted with ethyl acetate The organic layer was collected washed with brine dried over anhydrous sodium sulfate Concentration under reduced pressure followed by purification by silica gel chromatography

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of tert-butyl azetidine-3-carboxylate acetic acid salt (92.0 g, 0.423 mol) in methanol (1.0 L) at room temperature was added 4-cyanobenzaldehyde (50.8 g, 0.381 mol). The reaction mixture was cooled to 0° C., and sodium cyanoborohydride (28.8 g, 0.458 mol) was added portion-wise. The reaction mixture was allowed to warm to room temperature and was stirred overnight. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with a 10% aqueous solution of sodium bicarbonate, extracted with ethyl acetate, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by flash silica gel column chromatography using a 20% mixture of ethyl acetate in petroleum ether afforded tert-butyl 1-(4-cyanobenzyl)azetidine-3-carboxylate (89%) (the desired product and alcohol impurity formed were not separated by column completely, and the mixture was taken for next step). LC/MS M+1=273.18. 1H-NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 3.22-3.31 (m, 3H), 3.48-3.56 (m, 2H), 3.66 (s, 2H), 7.39 (d, J=8.28 Hz, 2H), and 7.60 (d, J=8.28 Hz, 2H). to warm to room temperature The reaction mixture was concentrated under reduced pressure the residue was diluted with a 10% aqueous solution of sodium bicarbonate extracted with ethyl acetate washed with brine dried over anhydrous sodium sulfate Concentration under reduced pressure followed by purification by flash silica gel column chromatography

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

Procedure: To a solution of methyl 7-formyl-4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxylate (Preparation 45C, 72 mg, 0.280 mmol) in methanol (5 mL) and 1,2-dichloroethane (1 mL) was added acetic acid (0.1 mL) and tert-butyl azetidine-3-carboxylate acetic acid salt (73.0 mg, 0.336 mmol). The contents were allowed to stir at room temperature for 40 min. and then sodium triacetoxyborohydride (178 mg, 0.840 mmol) was added. The reaction mixture was allowed to stir at room temperature overnight. Additional sodium triacetoxyborohydride (2 eq) was added and the reaction mixture was allowed to stir at room temperature for 2 hours. Additional sodium triacetoxyborohydride (4 eq) was added and the reaction mixture was allowed to stir at room temperature for 3 days. Additional amine (0.5 eq.), followed by additional triacetoxyborohydride (3 eq.) was added and the reaction mixtures was stirred at room temperature for 6 more hours to drive the reaction to about 90% conversion. The solvents were evaporated and the residue was re-dissolved into 80 ml ethyl acetate, washed twice with sat. sodium bicarbonate, dried over sodium sulfate, and concentrated to give 95 mg of product. The compound had an HPLC ret. time=0.75/2.0 min. (condition E); MS:(M+H)=399.3. to stir at room temperature overnight to stir at room temperature for 2 hours to stir at room temperature for 3 days was added the reaction mixtures was stirred at room temperature for 6 more hours the reaction to about 90% conversion The solvents were evaporated the residue was re-dissolved into 80 ml ethyl acetate washed twice with sat. sodium bicarbonate dried over sodium sulfate concentrated

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

Procedure: To a cloudy solution of 7-(bromomethyl)-3-(5-phenyl-4-(trifluoromethyl)isoxazol-3-yl)-4,5-dihydronaphtho[1,2-c]isoxazole (Preparation 6C, 550 mg, 1.157 mmol) and tert-butyl azetidine-3-carboxylate acetic acid (377 mg, 1.736 mmol) in anhydrous DMF (12 mL) was added triethylamine (0.645 mL, 4.63 mmol) dropwise at 0° C. under a nitrogen atmosphere. The reaction mixture was stirred at the same temperature for 1 hr before being concentrated. The solid residue was partitioned between dichloromethane (2 mL) and saturated aqueous sodium bicarbonate solution (6 mL). The aqueous layer was extracted with dichloromethane (2×2 mL). The combined dichloromethane solutions were dried over sodium sulfate, concentrated and purified by silica gel chromatography (20 to 75% ethyl acetate in heptane) to afford tert-butyl 1-((3-(5-phenyl-4-(trifluoromethyl)isoxazol-3-yl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-yl)methyl)azetidine-3-carboxylate (520 mg, 0.943 mmol, 81% yield) as a solid. To the solid was added TFA (3 mL) and the mixture stirred at room temperature for 70 min before being concentrated. Dichloromethane (15 mL) was added and the mixture concentrated again. The residue was mixed with water (4 ml) and neutralized to pH=5 with an aqueous sodium acetate solution. The resulting solid was filtered and washed with water (3×1 mL). To the solid was added methanol (1 mL) and water (0.5 mL), the contents were sonicated, and filtered. To the solid was added acetonitrile (0.5 mL) and water (6 mL). Lyophilization gave 1-((3-(5-phenyl-4-(trifluoromethyl)isoxazol-3-yl)-4,5-dihydronaphtho[1,2-c]isoxazol-7-yl)methyl)azetidine-3-carboxylic acid (420 mg, 0.805 mmol, 69.6% yield) as a white solid. The compound had an HPLC retention time=3.25 min. (condition C). LC/MS M+1=496.2. 1H NMR (400 MHz, MeOD+CDCl3) δ ppm 8.06 (1H, d, J=7.9 Hz), 7.77 (2H, d, J=7.3 Hz), 7.59-7.71 (3H, m), 7.51 (1H, s), 7.48 (1H, d, J=7.7 Hz), 4.42 (2H, s), 4.25-4.34 (4H, m), 3.55-3.65 (1H, m), 3.14 (4H, s). before being concentrated The solid residue was partitioned between dichloromethane (2 mL) and saturated aqueous sodium bicarbonate solution (6 mL) The aqueous layer was extracted with dichloromethane (2×2 mL) The combined dichloromethane solutions were dried over sodium sulfate concentrated purified by silica gel chromatography (20 to 75% ethyl acetate in heptane)