(2R)-2-[methyl-[(2-methylpropan-2-yl)oxycarbonyl]amino]propanoic acid

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

Procedure: By the method of part A of Example 3 N-(tert-butyloxycarbonyl)-N-methyl-D-alanine (2.03 g.) was condensed with 3-(4-fluorophenyl)propylamine (prepared by condensing 4-fluorobenzaldehyde with cyanoacetic acid in refluxing pyridine using piperidine as catalyst and hydrogenating the resulting β-4-fluorophenyl)acrylonitrile in ethanolic ammonia under high pressure using Raney nickel catalyst, m.r. of methanesulfonate salt 129°-131° C.; 2.49 g.), affording N2 -(tert-butyloxycarbonyl)-N2 -methyl-N-[3-(4-fluorophenyl)propyl]-D-alaninamide as a syrup (2.77g.).

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

Procedure: By the method of part A of Example 3 N-(tert-butyloxycarbonyl)-N-methyl-D-alanine (2.03 g.) was condensed with 3-(4-fluorophenyl)propylamine (prepared by condensing 4-fluorobenzaldehyde with cyanoacetic acid in refluxing pyridine using piperidine as catalyst and hydrogenating the resulting β-4-fluorophenyl)acrylonitrile in ethanolic ammonia under high pressure using Raney nickel catalyst, m.r. of methanesulfonate salt 129°-131° C.; 2.49 g.), affording N2 -(tert-butyloxycarbonyl)-N2 -methyl-N-[3-(4-fluorophenyl)propyl]-D-alaninamide as a syrup (2.77g.).

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

Procedure: To 48 ml of a solution of Boc-D -Ala-OH (3.0 g, 15.9 mmol) in tetrahydrofuran (THF) was added 60% sodium hydride (1.9 g, 47.6 mmol) under ice-cooling and the mixture was stirred for 10 minutes. Then, methyl iodide (7.9 ml, 127 mmol) was added and the mixture was further stirred at room temperature for 22 hours. The reaction mixture was then concentrated under reduced pressure, ice-cooled, acidified with 10% citric acid-H2O, and extracted with ethyl acetate. The extract was washed with saturated NaCl-H2O, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was crystallized from hexane and the crystal crop was washed with hexane to provide the title compound. Yield 3.07 g (94.8%). cooling the mixture was further stirred at room temperature for 22 hours The reaction mixture was then concentrated under reduced pressure, ice- cooled extracted with ethyl acetate The extract was washed with saturated NaCl-H2O dried over magnesium sulfate concentrated under reduced pressure The residue was crystallized from hexane the crystal crop was washed with hexane

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

Procedure: To a solution of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (39.7 mg, 0.064 mmol) in anhydrous DMF (1 mL) was added at room temperature N-Boc-N-methyl-D-alanine (11.91 mg, 0.059 mmol), followed by N,N-diisopropylethylamine (0.0306 mL, 0.176 mmol) and finally a solution of N-(1-{[3-(aminomethyl)phenyl]methyl}-4-hydroxy-1H-indazol-3-yl)-5-chloro-2-thiophenesulfonamide formate salt (for a preparation see Intermediate 15) (29 mg, 0.059 mmol) in anhydrous DMF (1 mL). The reaction mixture was stirred at room temperature for 1 hour and then was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate. The organic layer was separated, washed with brine-water (1:1), passed through a hydrophobic frit, and evaporated in-vacuo to yield a colourless oil. LCMS (System B) RT=3.01 min, ES+ve m/z 634/636 (M+H)+ for desired BOC protected product. This product was suspended in a solution of hydrogen chloride in 1,4-dioxane (4M, 0.5 mL) and methanol was added until the reaction was in solution. The mixture was stirred at room temperature for 18 hours and then evaporated in-vacuo to yield a colourless oil. The residual oil was dissolved in MeOH-DMSO (0.5 mL) and purified by MDAP) on OA MDAP on Sunfire C18 column, eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B: 0.1% v/v solution of formic acid in acetonitrile) (25 min run). The appropriate fractions were combined and evaporated in-vacuo to yield the title compound as a colourless oil (15 mg, 44%). LCMS (System B) RT=1.63 min, ES+ve m/z 534/536 (M+H)+ was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate The organic layer was separated washed with brine-water (1:1) evaporated in-vacuo to yield a colourless oil was added until the reaction The mixture was stirred at room temperature for 18 hours evaporated in-vacuo to yield a colourless oil purified by MDAP) on OA MDAP on Sunfire C18 column eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B (25 min run) evaporated in-vacuo

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

Procedure: To a solution of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (39.7 mg, 0.064 mmol) in anhydrous DMF (1 mL) was added at room temperature N-Boc-N-methyl-D-alanine (11.91 mg, 0.059 mmol), followed by N,N-diisopropylethylamine (0.0306 mL, 0.176 mmol) and finally a solution of N-(1-{[3-(aminomethyl)phenyl]methyl}-4-hydroxy-1H-indazol-3-yl)-5-chloro-2-thiophenesulfonamide formate salt (for a preparation see Intermediate 15) (29 mg, 0.059 mmol) in anhydrous DMF (1 mL). The reaction mixture was stirred at room temperature for 1 hour and then was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate. The organic layer was separated, washed with brine-water (1:1), passed through a hydrophobic frit, and evaporated in-vacuo to yield a colourless oil. LCMS (System B) RT=3.01 min, ES+ve m/z 634/636 (M+H)+ for desired BOC protected product. This product was suspended in a solution of hydrogen chloride in 1,4-dioxane (4M, 0.5 mL) and methanol was added until the reaction was in solution. The mixture was stirred at room temperature for 18 hours and then evaporated in-vacuo to yield a colourless oil. The residual oil was dissolved in MeOH-DMSO (0.5 mL) and purified by MDAP) on OA MDAP on Sunfire C18 column, eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B: 0.1% v/v solution of formic acid in acetonitrile) (25 min run). The appropriate fractions were combined and evaporated in-vacuo to yield the title compound as a colourless oil (15 mg, 44%). LCMS (System B) RT=1.63 min, ES+ve m/z 534/536 (M+H)+ was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate The organic layer was separated washed with brine-water (1:1) evaporated in-vacuo to yield a colourless oil was added until the reaction The mixture was stirred at room temperature for 18 hours evaporated in-vacuo to yield a colourless oil purified by MDAP) on OA MDAP on Sunfire C18 column eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B (25 min run) evaporated in-vacuo

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

Procedure: A solution of Intermediate M (100 mg, 0.276 mmol), (R)-2-(tert-butoxycarbonyl-methyl-amino)-propionic acid (67 mg, 0.33 mmol), TEA (0.115 mL, 0.828 mmol), and TBTU (106 mg, 0.331 mmol) in DMF (2 mL) is heated at 60° C. overnight. The reaction is diluted with EtOAc, rinsed with sat. NaHCO3, and the organics dried and concentrated. The residue is purified by silica gel chromatography eluting with 0-10% MeOH/DCM. The pooled and concentrated product fractions are treated with 4M HCl in 1,4-dioxane (5 mL) and stirred overnight. The reaction is concentrated and purified by reverse phase HPLC eluting with 10-90% MeCN in water (0.1% TFA). The concentrated product fractions are dissolved in MeOH and eluted through a carbonate resin cartridge to afford the title compound 261. (LC/MS method 16: ES+ m/z 411.4 [M+H]+, Rt=0.33 min) rinsed with sat. NaHCO3 the organics dried concentrated The residue is purified by silica gel chromatography eluting with 0-10% MeOH/DCM The pooled and concentrated product fractions are treated with 4M HCl in 1,4-dioxane (5 mL) The reaction is concentrated purified by reverse phase HPLC eluting with 10-90% MeCN in water (0.1% TFA) The concentrated product fractions are dissolved in MeOH eluted through a carbonate resin cartridge

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

Procedure: To a solution of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (39.7 mg, 0.064 mmol) in anhydrous DMF (1 mL) was added at room temperature N-Boc-N-methyl-D-alanine (11.91 mg, 0.059 mmol), followed by N,N-diisopropylethylamine (0.0306 mL, 0.176 mmol) and finally a solution of N-(1-{[3-(aminomethyl)phenyl]methyl}-4-hydroxy-1H-indazol-3-yl)-5-chloro-2-thiophenesulfonamide formate salt (for a preparation see Intermediate 15) (29 mg, 0.059 mmol) in anhydrous DMF (1 mL). The reaction mixture was stirred at room temperature for 1 hour and then was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate. The organic layer was separated, washed with brine-water (1:1), passed through a hydrophobic frit, and evaporated in-vacuo to yield a colourless oil. LCMS (System B) RT=3.01 min, ES+ve m/z 634/636 (M+H)+ for desired BOC protected product. This product was suspended in a solution of hydrogen chloride in 1,4-dioxane (4M, 0.5 mL) and methanol was added until the reaction was in solution. The mixture was stirred at room temperature for 18 hours and then evaporated in-vacuo to yield a colourless oil. The residual oil was dissolved in MeOH-DMSO (0.5 mL) and purified by MDAP) on OA MDAP on Sunfire C18 column, eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B: 0.1% v/v solution of formic acid in acetonitrile) (25 min run). The appropriate fractions were combined and evaporated in-vacuo to yield the title compound as a colourless oil (15 mg, 44%). LCMS (System B) RT=1.63 min, ES+ve m/z 534/536 (M+H)+ was partitioned between saturated aqueous sodium hydrogen carbonate solution and ethyl acetate The organic layer was separated washed with brine-water (1:1) evaporated in-vacuo to yield a colourless oil was added until the reaction The mixture was stirred at room temperature for 18 hours evaporated in-vacuo to yield a colourless oil purified by MDAP) on OA MDAP on Sunfire C18 column eluting with solvents A/B (A: 0.1% v/v solution of formic acid in water, B (25 min run) evaporated in-vacuo

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

Procedure: [(tert-Butoxy)carbonyl]amino (2R)-2-{[(tert-butoxy)carbonyl](methyl)amino}propanoate is prepared from (2R)-2-{[(tert-butoxy)carbonyl](methyl)amino}propanoic acid and N-tert-butoxycarbonyl hydroxylamine according to Scheme 6. The compound exists as rotomers and is reported as such. (0.95 g, 60%), 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 7.81-7.95 (1H, m), 4.60-5.04 (1H, m), 2.85-2.93 (3H, m), 1.40-1.54 (18H, m), 1.22-1.33 (3H, m).

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

Procedure: By the method of part A of Example 3 N-(tert-butyloxycarbonyl)-N-methyl-D-alanine (2.03 g.) was condensed with 3-(4-fluorophenyl)propylamine (prepared by condensing 4-fluorobenzaldehyde with cyanoacetic acid in refluxing pyridine using piperidine as catalyst and hydrogenating the resulting β-4-fluorophenyl)acrylonitrile in ethanolic ammonia under high pressure using Raney nickel catalyst, m.r. of methanesulfonate salt 129°-131° C.; 2.49 g.), affording N2 -(tert-butyloxycarbonyl)-N2 -methyl-N-[3-(4-fluorophenyl)propyl]-D-alaninamide as a syrup (2.77g.).