tert-butyl (2R)-2-aminopropanoate

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

Procedure: [4-((2R,3R)-1-(4-fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetic acid (50 mg, 0.1 mmol), tert-butyl D-alaninate (23 mg, 0.12 mmol) and 4-methylmorpholine (31 mg, 0.31 mmol) were dissolved in DCM (1.5 ml) and stirred at room temperature for 5 minutes. TBTU (40 mg, 0.12 mmol) was added and the reaction mixture was stirred for 2 h. TFA (0.7 ml) was added and the solution was stirred for 90 minutes. TFA and DCM were removed under reduced pressure and the residue was dissolved in DCM and washed with water. The organic layer was dried over sodium sulphate and the solvent was removed under reduced pressure giving 55 mg (95%) of the title product. M/z: 553.0 (M−1). the reaction mixture was stirred for 2 h the solution was stirred for 90 minutes TFA and DCM were removed under reduced pressure the residue was dissolved in DCM washed with water The organic layer was dried over sodium sulphate the solvent was removed under reduced pressure

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

Procedure: [4-((2R,3R)-1-(4-fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetic acid (50 mg, 0.1 mmol), tert-butyl D-alaninate (23 mg, 0.12 mmol) and 4-methylmorpholine (31 mg, 0.31 mmol) were dissolved in DCM (1.5 ml) and stirred at room temperature for 5 minutes. TBTU (40 mg, 0.12 mmol) was added and the reaction mixture was stirred for 2 h. TFA (0.7 ml) was added and the solution was stirred for 90 minutes. TFA and DCM were removed under reduced pressure and the residue was dissolved in DCM and washed with water. The organic layer was dried over sodium sulphate and the solvent was removed under reduced pressure giving 55 mg (95%) of the title product. M/z: 553.0 (M−1). the reaction mixture was stirred for 2 h the solution was stirred for 90 minutes TFA and DCM were removed under reduced pressure the residue was dissolved in DCM washed with water The organic layer was dried over sodium sulphate the solvent was removed under reduced pressure

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethylamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under reduced pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under reduced pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1)

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

Procedure: [4-((2R,3R)-1-(4-fluorophenyl)-3-{[2-(4-fluorophenyl)-2-oxoethyl]thio}-4-oxoazetidin-2-yl)phenoxy]acetic acid (50 mg, 0.1 mmol), tert-butyl D-alaninate (23 mg, 0.12 mmol) and 4-methylmorpholine (31 mg, 0.31 mmol) were dissolved in DCM (1.5 ml) and stirred at room temperature for 5 minutes. TBTU (40 mg, 0.12 mmol) was added and the reaction mixture was stirred for 2 h. TFA (0.7 ml) was added and the solution was stirred for 90 minutes. TFA and DCM were removed under reduced pressure and the residue was dissolved in DCM and washed with water. The organic layer was dried over sodium sulphate and the solvent was removed under reduced pressure giving 55 mg (95%) of the title product. M/z: 553.0 (M−1). the reaction mixture was stirred for 2 h the solution was stirred for 90 minutes TFA and DCM were removed under reduced pressure the residue was dissolved in DCM washed with water The organic layer was dried over sodium sulphate the solvent was removed under reduced pressure

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethylamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under reduced pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under reduced pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1)

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethyamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane;ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane;ethyl acetate 3:1)

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethylamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under reduced pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under reduced pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1)

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethyamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane;ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane;ethyl acetate 3:1)

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethylamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under reduced pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under reduced pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1)

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

Procedure: To a solution of D-alanine tert-butylester (1.28 g) and N,N-diisopropylethylamine (1.22 ml) in acetonitrile (15 ml), was added a solution of ethyl 2-azido-4-bromobutanoate (1 g) and sodium iodide (0.02 g) in acetonitrile (5 ml). The mixture was heated at 60° C. for 60 h and then concentrated under reduced pressure to give a brown oil. This oil was partitioned between DCM and water. The separated organic layer was washed further with water and dried (over magnesium sulphate), and concentrated under reduced pressure. The residual brown oil was purified using Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1) to give the title compound (0.204 g) as a mixture of two diastereoisomers. concentrated under reduced pressure to give a brown oil This oil was partitioned between DCM and water The separated organic layer was washed further with water dried (over magnesium sulphate) concentrated under reduced pressure The residual brown oil was purified Biotage™ chromatography (silica, eluting with cyclohexane:ethyl acetate 3:1)

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

Procedure: To a solution of 500 mg 5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carboxylic acid in 5 ml DMF were added 186 mg HOBt, 232 mg EDC, 0.50 ml DIPEA and 220 mg D-Ala-OtBu×HCl. After stirring for 12 h it was concentrated and the residue dissolved in ethyl acetate. It was extracted with aqueous LiCl (4% w/w), aqueous NaHCO3 and 0.1 M HCl. The organic layer was dried over MgSO4 and concentrated to furnish the crude coupling product as colorless oil. was concentrated the residue dissolved in ethyl acetate It was extracted with aqueous LiCl (4% w/w), aqueous NaHCO3 and 0.1 M HCl The organic layer was dried over MgSO4 concentrated to furnish the crude coupling product as colorless oil

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

Procedure: To a stirring solution of tert-butyl D-alaninate (5.60 g, 30.80 mmol) in DMF (50 mL) were added DIEA (8.29 g, 64.18 mmol) and (S)-3-(4-(5-bromopyrimidin-2-yl)phenyl)-2-(5-(tert-butyl)thiophene-2-carboxamido)propanoic acid (12.5 g, 25.67 mmol). The solution was cooled to 0° C. at ice bath and then HATU (9.06 g, 38.50 mmol) in 15 mL DMF solution was slowly added. The reaction was stirred 1 hour at 0° C. and then warmed to RT with stirring for 2 hours. The reaction solution was extracted with DCM (3×50 mL) and aqueous NaHCO3 (3×30 mL). The combined organics were dried over MgSO4 and evaporated. The final compound was purified by column chromatography (40% DCM in hexane) to afford 14.7 g (94%) of tert-butyl ((S)-3-(4-(5-bromopyrimidin-2-yl)phenyl)-2-(5-(tert-butyl)thiophene-2-carboxamido)propanoyl)-D-alaninateas solid powder. LCMS-ESI (m/z) calculated for C29H35BRN4O4S: 614.2. found 615.3 [M+H]+, tR=3.914 min. (Method 16). 1H NMR (400 MHz, CDCl3) δ 8.83 (d, J=3.6 Hz, 2H), 8.36 (d, J=8.2 Hz, 2H), 7.39 (d, J=8.2 Hz, 2H), 7.34 (d, J=3.8 Hz, 1H), 6.81 (d, J=3.8 Hz, 1H), 6.66 (d, J=7.6 Hz, 1H), 6.34 (d, J=7.2 Hz, 1H), 4.88 (d, J=5.9 Hz, 1H), 4.41 (t, J=7.2 Hz, 1H), 3.31 (dd, J=13.6, 5.8 Hz, 1H), 3.20 (dd, J=13.6, 7.8 Hz, 1H), 1.51-1.32 (m, 18H), 1.27 (d, J=7.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 172.02, 171.31, 162.28, 162.13, 161.42, 158.55, 142.27, 136.34, 134.66, 130.20, 128.82, 127.92, 123.07, 118.63, 80.90, 54.45, 48.86, 39.59, 39.38, 32.39, 28.04, 17.68. warmed to RT with stirring for 2 hours The reaction solution was extracted with DCM (3×50 mL) and aqueous NaHCO3 (3×30 mL) The combined organics were dried over MgSO4 evaporated The final compound was purified by column chromatography (40% DCM in hexane) to afford 14.7 g (94%) of tert-butyl ((S)-3-(4-(5-bromopyrimidin-2-yl)phenyl)-2-(5-(tert-butyl)thiophene-2-carboxamido)propanoyl)-D-alaninateas solid powder