2-[(2S)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidin-2-yl]acetic acid

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

Procedure: DIEA (0.055 mL, 0.32 mmol) was added to a solution of (S)-3-(5-phenyl-4-(piperazin-1-yl)-1H-pyrazolo[3,4-b]pyridin-3-yloxy)propane-1,2-diol dihydrochloride (0.050 g, 0.079 mmol, see Example 15), (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (0.018 g, 0.079 mmol) and TBTU (0.031 g, 0.095 mmol) in DCM (1 mL) and stirred at room temperature for 1 hour. The reaction was then concentrated to dryness, and the residue was dissolved in THF/MeOH (2 mL, 1:1), and an aqueous LiOH solution (1 mL, 2 M) was added. The mixture was stirred for 10 minutes. Ethyl acetate (20 mL) was then added, and the organic layer was separated, washed with brine (10 mL), and dried over sodium sulfate. After removal of the solvent, the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1) to give tert-butyl(R)-3-(4-chlorophenyl)-1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-1-oxopropan-2-ylcarbamate as a solid. The solid was then dissolved in DCM (1 mL), and TFA (0.4 mL) was added. The mixture was stirred at room temperature for 1 hour and concentrated to dryness. The resulting residue was dissolved in DCM (0.5 mL), and HCl in ether (1 mL, 2 N)) was added. The resulting solid was collected by filtration to give (1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-2-((S)-pyrrolidin-2-yl)ethanone dihydrochloride (0.019 g, 43%). MS APCI (+) m/z 481.2 detected. The reaction was then concentrated to dryness the residue was dissolved in THF/MeOH (2 mL, 1:1) an aqueous LiOH solution (1 mL, 2 M) was added The mixture was stirred for 10 minutes Ethyl acetate (20 mL) was then added the organic layer was separated washed with brine (10 mL) dried over sodium sulfate After removal of the solvent the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1)

Conditions: temp:-20.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Diisopropylethylamine (575 μl) was added to a solution of 2-[(2S)-1-tert-butoxycarbonylpyrrolidin-2-yl]acetic acid (688 mg) in tetrahydrofuran (15 ml, and the mixture was cooled to −20° C. Isobutyl chloroformate (428 μl) was added thereto, and the mixture was stirred at −20° C. for 45 minutes. The resulting solid was filtered off, and the solid was washed with tetrahydrofuran. Sodium borohydride (227 mg) was added to the filtrate, and the mixture was stirred at room temperature for 5 minutes. Then, methanol (5 ml) was added thereto, and the mixture was stirred at room temperature for 5 minutes. Ethyl acetate and water were added to the reaction solution to separate the aqueous and organic layers. The aqueous layer was subjected to extraction with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (n-hexane-ethyl acetate) to obtain tert-butyl (2S)-2-(2-hydroxyethyl)pyrrolidine-1-carboxylate (606 mg). The resulting solid was filtered off the solid was washed with tetrahydrofuran Sodium borohydride (227 mg) was added to the filtrate the mixture was stirred at room temperature for 5 minutes Then, methanol (5 ml) was added the mixture was stirred at room temperature for 5 minutes Ethyl acetate and water were added to the reaction solution to separate the aqueous and organic layers The aqueous layer was subjected to extraction with ethyl acetate dried over anhydrous sodium sulfate concentrated under reduced pressure The obtained residue was purified by silica gel column chromatography (n-hexane-ethyl acetate)

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

Procedure: A solution of (S)-2-carboxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (2.00 g) in methanol (1.32 ml)/acetonitrile (40 ml) was treated with N,N-diisopropylethylamine (1.78 ml). Trimethylsilyldiazomethane (2M, 8.73 ml) was added dropwise and the resulting mixture was stirred at room temperature, under argon for 24 h. The mixture was diluted with ethyl acetate, washed with 2M HCl, saturated aqueous sodium hydrogen carbonate, brine then dried (magnesium sulfate). The solvent was removed under reduced pressure to afford the title compound (2.10 g) washed with 2M HCl, saturated aqueous sodium hydrogen carbonate, brine then dried (magnesium sulfate) The solvent was removed under reduced pressure

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

Procedure: A solution of (S)-2-carboxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (2.00 g) in methanol (1.32 ml)/acetonitrile (40 ml) was treated with N,N-diisopropylethylamine (1.78 ml). Trimethylsilyldiazomethane (2M, 8.73 ml) was added dropwise and the resulting mixture was stirred at room temperature, under argon for 24 h. The mixture was diluted with ethyl acetate, washed with 2M HCl, saturated aqueous sodium hydrogen carbonate, brine then dried (magnesium sulfate). The solvent was removed under reduced pressure to afford the title compound (2.10 g) washed with 2M HCl, saturated aqueous sodium hydrogen carbonate, brine then dried (magnesium sulfate) The solvent was removed under reduced pressure

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

Procedure: The title compound (3.2 g) was prepared from (S)-2-carboxymethyl-pyrrolidine-1-carboxylic acid tert butyl ester (2.02 g) and 2-bromo-4′-fluoroacetophenone (1.91 g) according to the method of description 36.

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

Procedure: The title compound (3.2 g) was prepared from (S)-2-carboxymethyl-pyrrolidine-1-carboxylic acid tert butyl ester (2.02 g) and 2-bromo-4′-fluoroacetophenone (1.91 g) according to the method of description 36.

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

Procedure: The title compound was prepared from (S)-2-carboxymethyl-pyrrolidine-1-carboxylic acid tert butyl ester (2.50) and 5-phenyltetrazole (1.59 g) according to the method of description 1.

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

Procedure: tert-Butyl (2S)-2-(2-methoxy-2-oxoethyl)-1-pyrrolidinecarboxylate (8.35 g, 34.3 mmol) was dissolved in 150 mL MeOH. Aqueous NaOH (50 mL, 1 M) was added and the reaction mixture was allowed to stand overnight. After concentration in vacuo to ca. 100 mL, the solution was added to 300 mL water and extracted with ether (2×). The carboxylate solution was acidified to pH 2 with 12 N HCl and extracted with ether (2×). The aqueous layer was saturated with salt and extracted a third time with ether. The combined organic layers were washed with water, brine and dried over MgSO4. Filtration and concentration provided the title acid as a white solid (7.28 g, 31.7 mmol, 92.6%): 1H NMR (300 MHz, CDCl3, mixture of rotamers) δ 4.16 (bs, 1H), 3.37 (bs, 2H), 3.0–2.8 (bs, 1H), 2.36 (dd, J=9.3, 15.3 Hz, 1H), 2.2–2.0 (m, 1H), 1.9–1.7 (m, 4H), 1.48 (s, 9H) ppm. After concentration in vacuo to ca. 100 mL the solution was added to 300 mL water extracted with ether (2×) extracted with ether (2×) extracted a third time with ether The combined organic layers were washed with water, brine dried over MgSO4 Filtration and concentration

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

Procedure: tert-Butyl (2S)-2-(2-methoxy-2-oxoethyl)-1-pyrrolidinecarboxylate (8.35 g, 34.3 mmol) was dissolved in 150 mL MeOH. Aqueous NaOH (50 mL, 1 M) was added and the reaction mixture was allowed to stand overnight. After concentration in vacuo to ca. 100 mL, the solution was added to 300 mL water and extracted with ether (2×). The carboxylate solution was acidified to pH 2 with 12 N HCl and extracted with ether (2×). The aqueous layer was saturated with salt and extracted a third time with ether. The combined organic layers were washed with water, brine and dried over MgSO4. Filtration and concentration provided the title acid as a white solid (7.28 g, 31.7 mmol, 92.6%): 1H NMR (300 MHz, CDCl3, mixture of rotamers) δ 4.16 (bs, 1H), 3.37 (bs, 2H), 3.0–2.8 (bs, 1H), 2.36 (dd, J=9.3, 15.3 Hz, 1H), 2.2–2.0 (m, 1H), 1.9–1.7 (m, 4H), 1.48 (s, 9H) ppm. After concentration in vacuo to ca. 100 mL the solution was added to 300 mL water extracted with ether (2×) extracted with ether (2×) extracted a third time with ether The combined organic layers were washed with water, brine dried over MgSO4 Filtration and concentration

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

Procedure: DIEA (0.055 mL, 0.32 mmol) was added to a solution of (S)-3-(5-phenyl-4-(piperazin-1-yl)-1H-pyrazolo[3,4-b]pyridin-3-yloxy)propane-1,2-diol dihydrochloride (0.050 g, 0.079 mmol, see Example 15), (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (0.018 g, 0.079 mmol) and TBTU (0.031 g, 0.095 mmol) in DCM (1 mL) and stirred at room temperature for 1 hour. The reaction was then concentrated to dryness, and the residue was dissolved in THF/MeOH (2 mL, 1:1), and an aqueous LiOH solution (1 mL, 2 M) was added. The mixture was stirred for 10 minutes. Ethyl acetate (20 mL) was then added, and the organic layer was separated, washed with brine (10 mL), and dried over sodium sulfate. After removal of the solvent, the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1) to give tert-butyl(R)-3-(4-chlorophenyl)-1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-1-oxopropan-2-ylcarbamate as a solid. The solid was then dissolved in DCM (1 mL), and TFA (0.4 mL) was added. The mixture was stirred at room temperature for 1 hour and concentrated to dryness. The resulting residue was dissolved in DCM (0.5 mL), and HCl in ether (1 mL, 2 N)) was added. The resulting solid was collected by filtration to give (1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-2-((S)-pyrrolidin-2-yl)ethanone dihydrochloride (0.019 g, 43%). MS APCI (+) m/z 481.2 detected. The reaction was then concentrated to dryness the residue was dissolved in THF/MeOH (2 mL, 1:1) an aqueous LiOH solution (1 mL, 2 M) was added The mixture was stirred for 10 minutes Ethyl acetate (20 mL) was then added the organic layer was separated washed with brine (10 mL) dried over sodium sulfate After removal of the solvent the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1)

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

Procedure: DIEA (0.055 mL, 0.32 mmol) was added to a solution of (S)-3-(5-phenyl-4-(piperazin-1-yl)-1H-pyrazolo[3,4-b]pyridin-3-yloxy)propane-1,2-diol dihydrochloride (0.050 g, 0.079 mmol, see Example 15), (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (0.018 g, 0.079 mmol) and TBTU (0.031 g, 0.095 mmol) in DCM (1 mL) and stirred at room temperature for 1 hour. The reaction was then concentrated to dryness, and the residue was dissolved in THF/MeOH (2 mL, 1:1), and an aqueous LiOH solution (1 mL, 2 M) was added. The mixture was stirred for 10 minutes. Ethyl acetate (20 mL) was then added, and the organic layer was separated, washed with brine (10 mL), and dried over sodium sulfate. After removal of the solvent, the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1) to give tert-butyl(R)-3-(4-chlorophenyl)-1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-1-oxopropan-2-ylcarbamate as a solid. The solid was then dissolved in DCM (1 mL), and TFA (0.4 mL) was added. The mixture was stirred at room temperature for 1 hour and concentrated to dryness. The resulting residue was dissolved in DCM (0.5 mL), and HCl in ether (1 mL, 2 N)) was added. The resulting solid was collected by filtration to give (1-(4-(3-((S)-2,3-dihydroxypropoxy)-5-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)piperazin-1-yl)-2-((S)-pyrrolidin-2-yl)ethanone dihydrochloride (0.019 g, 43%). MS APCI (+) m/z 481.2 detected. The reaction was then concentrated to dryness the residue was dissolved in THF/MeOH (2 mL, 1:1) an aqueous LiOH solution (1 mL, 2 M) was added The mixture was stirred for 10 minutes Ethyl acetate (20 mL) was then added the organic layer was separated washed with brine (10 mL) dried over sodium sulfate After removal of the solvent the residue was purified by column chromatography (ethyl acetate:MeOH, 20:1)

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

Procedure: A mixture of 4-(piperazin-1-yl)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-dipyrido[2,3-b;4′,3′-d]pyrrole-6-carbonitrile (100 mg, 0.2 mmol), (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (220 mg, 1.0 mmol), 1-hydroxybenzotriazole (50 mg, 0.4 mmol), (3-dimethylamino-propyl)-ethyl-carbodiimide hydrogen chloride (70 mg, 0.4 mmol), and triethylamine (0.1 mL, 0.7 mmol) in methylene chloride (2.5 mL) was stirred at ambient temperature overnight. The reaction mixture was diluted with methylene chloride (50 mL) and washed with saturated aqueous sodium bicarbonate solution (20 mL). The organic layer was separated, dried over sodium sulfate, filtered, concentrated in vacuo, and purified flash chromatography (silica, 4 g, ISCO, 0-70% ethyl acetate in heptane) to afford the title compound as a white foam, which was used in the next step without any further purification (140 mg). washed with saturated aqueous sodium bicarbonate solution (20 mL) The organic layer was separated dried over sodium sulfate filtered concentrated in vacuo purified flash chromatography (silica, 4 g, ISCO, 0-70% ethyl acetate in heptane)