(2R)-2-acetamido-3-sulfanylpropanoic acid

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

Procedure: Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobin (bovine), methylene blue and Medium 199 sets were purchased from Sigma Chemical Co., St. Louis, Mo. N-acetylcysteine was obtained from Aldrich Chemical Co., Milwaukee, Wis. Captopril was kindly provided by Dr. Victor Dzau. Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn, N.J. Leukotriene D4 was purchased from Anaquest, BOC Inc., Madison, Wis. Antibiotic/antimycotic mixture (10,000 U/ml penicillin G sodium, 10,000 mg/ml, streptomycin sulfate, 25 mg/ml amphotericin B) was purchased from Gibco laboratories, Grand Island, N.Y. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, Mass.

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

Procedure: Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobin (bovine), methylene blue and Medium 199 sets were purchased from Sigma Chemical Co., St. Louis, Mo. N-acetylcysteine was obtained from Aldrich Chemical Co., Milwaukee, Wis. Captopril was kindly provided by Dr. Victor Dzau. Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn, N.J. Leukotriene D4 was purchased from Anaquest, BOC Inc., Madison, Wis. Antibiotic/antimycotic m nxture (10,000 U/ml penicillin G sodium, 10,000 mg/ml, streptomycin sulfate, 25 mg/ml amphotericin B) was purchased from Gibco laboratories, Grand Island, N.Y. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, Mass.

Conditions: temp:25.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: N-acetyl-L-cysteine (0.5 g, 3.06 mmol) was dissolved in 50 ml of dry dioxane. Chloromethyl benzamide (0.52 g, 3.06 mmol) was added and the colorless solution was stirred at 25° C. for 18 hours. The dioxane was evaporated under vacuum with minimal amount of heating to give a colorless oil: NMR (CDCl3 +DMSO) δ8.05-7.38 (m, 5, aryl), 4.58 (d, 2, --SCH2NH--), 2.01 (s, 3, --CH3). The dioxane was evaporated under vacuum with minimal amount of heating to give a colorless oil

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

Procedure: Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobin (bovine), methylene blue and Medium 199 sets were purchased from Sigma Chemical Co., St. Louis, Mo. N-acetylcysteine was obtained from Aldrich Chemical Co., Milwaukee, Wis. Captopril was kindly provided by Dr Victor Dzau. Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn, N.J. Leukotriene D4 was purchased from Anaquest, BOC Inc., Madison, Wis. Antibiotic/antimycotic mixture (10,000 U/ml penicillin G sodium, 10,000 mcg/ml, streptomycin sulfate, 25 mcg/ml amphotericin B) was purchased from Gibco Laboratories, Grand Island, N.Y. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, Mass.

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

Procedure: Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobin (bovine), methylene blue and Medium 199 sets were purchased from Sigma Chemical Co., St. Louis, Mo. N-acetylcysteine was obtained from Aldrich Chemical Co., Milwaukee, Wis. Captopril was kindly provided by Dr Victor Dzau. Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn, N.J. Leukotriene D4 was purchased from Anaquest, BOC Inc., Madison, Wis. Antibiotic/antimycotic mixture (10,000 U/ml penicillin G sodium, 10,000 mcg/ml, streptomycin sulfate, 25 mcg/ml amphotericin B) was purchased from Gibco Laboratories, Grand Island, N.Y. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, Mass.

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A reaction flask is charged with (S)-3-((S)-1-(4-bromophenyl)ethyl)-6-(2-hydroxy-2-methylpropyl)-6-phenyl-1,3-oxazinan-2-one (19.66 g, 45.47 mmol) (see Ex. 588 of WO/2009/017664), compound 1 (7.65 g, 50.02 mmol), [1,1′-bis(diphenylphosphino)ferroncene]dichloropalladium(II), complex with dichloromethane (37.40 mg, 0.046 mmol), potassium carbonate (18.85 g, 136.42 mmol) and 2-propanol (100 mL). The flask is evacuated and refilled with argon three times. The contents of the reactor are heated to 80° C. and held for about 6 hours at which time compound 1 is completely consumed. The contents of the reactor are then treated with a solution of N-acetyl-L-cysteine (0.75 g, 4.58 mmol) in 2-propanol (100 mL) and stirred at 80° C. for 2 hours. The resultant suspension is then filtered. (The suspension is maintained at 50° C. to 70° C. during the filtration step.) The resultant solids are then rinsed with 2-propanol (50 mL pre-heated to 70° C.). The combined filtrate and washes is heated to 70° C., maintained at reduced pressure until about 150 ml of volatiles are removed, cooled to 65° C., and seeded with compound 2. The resultant suspension is stirred at 60-65° C. for 0.5 hours, cooled to 0° C. over about 2 hours, and maintained at 0° C. for 1 hour. The resultant solids are collected by filtration and washed with cold 2-propanol (50 mL). The solids are then recrystallized from a mixture of 2-propanol (40 mL) and water (200 mL) to provide compound 2 (monohydrate) as a white solid. Yield: 19.38 g, 88.2% yield based on BI135541. Purity: 98.96 wt %. Analysis of the product agrees with the data reported in Example 48, Method 2 of WO/2009/134400 which describes the synthesis of the monohydrate of compound 1 by reacting 4-iodo-1-methylpyridin-2(1H)-one with (S)-6-(2-hydroxy-2-methylpropyl)-6-phenyl-3-((S)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethyl)-1,3-oxazinan-2-one. The flask is evacuated refilled with argon three times is completely consumed The resultant suspension is then filtered (The suspension is maintained at 50° C. to 70° C. during the filtration step ) The resultant solids are then rinsed with 2-propanol (50 mL pre-heated to 70° C.) The combined filtrate and washes is heated to 70° C. maintained at reduced pressure until about 150 ml of volatiles are removed cooled to 65° C. The resultant suspension is stirred at 60-65° C. for 0.5 hours cooled to 0° C. over about 2 hours maintained at 0° C. for 1 hour The resultant solids are collected by filtration washed with cold 2-propanol (50 mL) The solids are then recrystallized from a mixture of 2-propanol (40 mL) and water (200 mL)

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A reaction flask is charged with (S)-3-((S)-1-(4-bromophenyl)ethyl)-6-(2-hydroxy-2-methylpropyl)-6-phenyl-1,3-oxazinan-2-one (19.66 g, 45.47 mmol) (see Ex. 588 of WO/2009/017664), compound 1 (7.65 g, 50.02 mmol), [1,1′-bis(diphenylphosphino)ferroncene]dichloropalladium(II), complex with dichloromethane (37.40 mg, 0.046 mmol), potassium carbonate (18.85 g, 136.42 mmol) and 2-propanol (100 mL). The flask is evacuated and refilled with argon three times. The contents of the reactor are heated to 80° C. and held for about 6 hours at which time compound 1 is completely consumed. The contents of the reactor are then treated with a solution of N-acetyl-L-cysteine (0.75 g, 4.58 mmol) in 2-propanol (100 mL) and stirred at 80° C. for 2 hours. The resultant suspension is then filtered. (The suspension is maintained at 50° C. to 70° C. during the filtration step.) The resultant solids are then rinsed with 2-propanol (50 mL pre-heated to 70° C.). The combined filtrate and washes is heated to 70° C., maintained at reduced pressure until about 150 ml of volatiles are removed, cooled to 65° C., and seeded with compound 2. The resultant suspension is stirred at 60-65° C. for 0.5 hours, cooled to 0° C. over about 2 hours, and maintained at 0° C. for 1 hour. The resultant solids are collected by filtration and washed with cold 2-propanol (50 mL). The solids are then recrystallized from a mixture of 2-propanol (40 mL) and water (200 mL) to provide compound 2 (monohydrate) as a white solid. Yield: 19.38 g, 88.2% yield based on BI135541. Purity: 98.96 wt %. Analysis of the product agrees with the data reported in Example 48, Method 2 of WO/2009/134400 which describes the synthesis of the monohydrate of compound 1 by reacting 4-iodo-1-methylpyridin-2(1H)-one with (S)-6-(2-hydroxy-2-methylpropyl)-6-phenyl-3-((S)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethyl)-1,3-oxazinan-2-one. The flask is evacuated refilled with argon three times is completely consumed The resultant suspension is then filtered (The suspension is maintained at 50° C. to 70° C. during the filtration step ) The resultant solids are then rinsed with 2-propanol (50 mL pre-heated to 70° C.) The combined filtrate and washes is heated to 70° C. maintained at reduced pressure until about 150 ml of volatiles are removed cooled to 65° C. The resultant suspension is stirred at 60-65° C. for 0.5 hours cooled to 0° C. over about 2 hours maintained at 0° C. for 1 hour The resultant solids are collected by filtration washed with cold 2-propanol (50 mL) The solids are then recrystallized from a mixture of 2-propanol (40 mL) and water (200 mL)

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

Procedure: Glutathione, L-cysteine, DL-homocysteine, D-penicillin, hemoglobin Coovine), methylene blue and Medium 199 sets were purchased from Sigma Chemical Co., St. Louis, MO. N-acetylcysteine was obtained from Aldrich Chemical Co., Milwaukee, WI. Captopril was kindly provided by Dr. Victor Dzau. Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn,. N.J. Leukotriene D4 was purchased from Anaquest, BOC Inc., Madison, WI. Antibiotic/antimycotic mixture (10,000 U/ml penicillin G sodium, 10,000 mg/ml, streptomycin sulfate, 25 mg/ml amphotericin B) was purchased from Gibco laboratories, Grand Island, NY. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, MA.

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

Procedure: A solution of N-acetyl-L-cysteine (3.26 g) (commercially available) in methanol (10 ml) with concentrated hydrochloric acid (four drops) was refluxed for 16 hours under a nitrogen atomosphere.

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

Procedure: A solution of 4-bromobutyric acid (5.1 g, 30.6 mmoles) and 1,1′-carbonyldiimidazol (5.61 g, 34.6 mmoles)) in chloroform (50 ml) is left at room temperature under stirring for 1 hour. To the reaction mixture N-acetyl cysteine (5 g, 30.6 mmoles) dissolved in N,N-dimethylformamide (5 ml) and sodium ethylate (50 mg) is added under stirring. After 24 hours the solution 2 is washed with HCl 1% and brine, the organic phase is anhydrified with sodium sulphate and evaporated at reduced pressure. The obtained crude product is purified by chromatography on silica gel column, eluting with ethyl acetate/chloroform 7/3. N-acetyl-S-(4-bromobutyroyl) cysteine is obtained. under stirring After 24 hours the solution 2 is washed with HCl 1% and brine evaporated at reduced pressure The obtained crude product is purified by chromatography on silica gel column eluting with ethyl acetate/chloroform 7/3

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

Procedure: A solution of 4-bromobutyric acid (5.1 g, 30.6 mmoles) and 1,1′-carbonyldiimidazol (5.61 g, 34.6 mmoles)) in chloroform (50 ml) is left at room temperature under stirring for 1 hour. To the reaction mixture N-acetyl cysteine (5 g, 30.6 mmoles) dissolved in N,N-dimethylformamide (5 ml) and sodium ethylate (50 mg) is added under stirring. After 24 hours the solution 2 is washed with HCl 1% and brine, the organic phase is anhydrified with sodium sulphate and evaporated at reduced pressure. The obtained crude product is purified by chromatography on silica gel column, eluting with ethyl acetate/chloroform 7/3. N-acetyl-S-(4-bromobutyroyl) cysteine is obtained. under stirring After 24 hours the solution 2 is washed with HCl 1% and brine evaporated at reduced pressure The obtained crude product is purified by chromatography on silica gel column eluting with ethyl acetate/chloroform 7/3

Conditions: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: One half gram (1.85 mmole) of chromium chloride (CrCl3) was dissolved in 30 ml of deionized water to get a green solution. 1.5 g (9.2 mmole) of N-acetyl cysteine (NAC) was dissolved in 40 ml of deionized water were added, and the mixture was stirred for 3 hours at 40° C. until the solution turned dark green-blue. to get a green solution were added