tert-butyl (2S)-pyrrolidine-2-carboxylate;hydrochloride

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

Procedure: A] Starting from H-Pro-OtBu.HCl (0.315 g, 1.5 mmoles) and Z-Pro-OH (0.397 g, 1.59 mmoles) the procedure of Example 1, C-D] was substantially applied. There was obtained 0.4 g of H-Pro-Pro-OtBu.

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

Procedure: 3-Nitrobenzoyl chloride was reacted with L-proline tert-butyl ester hydrochloride, then hydrogenated to produce (S)-1-(3-amino-phenyl)-pyrrolidin-2-carboxylic acid tert-butyl ester. This was reacted with 5-(2-adamantan-1-yl-ethyl)-2-cyclohexyl-1H-imidazole-4-carboxylic acid (Example 252) according to the procedure of Example 20, step d. The tert-butyl ester was deprotected by treating the chloroform solution of the ester with 4.0 M solution of hydrogen chloride in dioxan to afford the title compound as the hydrochloride salt. 1H NMR (300 MHz, d6-DMSO) (mixture of tautomers) 14.10 (1H, br s), 10.95 (1H, br m), 8.00 and 7.79 (1H, 2×m), 7.88 (1H, m), 7.41 (1H, m), 7.26 and 7.11 (1H, 2×), 4.43 (1H, m), 4.12 (1H, m), 3.48 (1H,m), 2.97 (3H, m), 2.28-1.29 (31H, m). Found: C, 64.88; H, 7.78; N, 8.92%; C34H45ClN4O4.1.0 mol H2O requires: C, 65.11; H, 7.55; N, 8.93%.

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

Procedure: To a mixture of L-proline-tert-butyl ester hydrogen chloride salt 75 (0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into ethyl acetate (100 mL) and was washed with 0.2 N HCl (50 mL), water (50 mL), brine (50 mL) and dried over MgSO4. Chromatography was conducted on a 2 mm radial chromatotron plate eluting with 10% ethyl acetate in hexanes. Product-containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (8 mL) and treated with piperidine (2 mL). The mixture was stirred for 1 h, concentrated under reduced pressure and purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane. This gave 200 mg (26%) of the dipeptide 74: 1H-NMR (400 MHz, CDCl3) δ 4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.57 (m, 4H), 3.2 (m, 1H), 2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H). was washed with 0.2 N HCl (50 mL), water (50 mL), brine (50 mL) dried over MgSO4 eluting with 10% ethyl acetate in hexanes Product-containing fractions were concentrated under reduced pressure dissolved in dichloromethane (8 mL) treated with piperidine (2 mL) The mixture was stirred for 1 h concentrated under reduced pressure purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane

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

Procedure: 4.46 g. of carbobenzoxy-β-alanine is dissolved in 20 ml. of tetrahydrofuran and the resulting solution cooled to -10° C. With constant stirring of the solution, 2.4 ml. of N-methylmorpholine followed by 2.6 ml. of isobutylchloroformate are added and the resulting reaction mixture is stirred thereafter for approximately 5 minutes at -10° C. 4.16 g. of L-proline t-butyl ester hydrochloride in 20 ml. of dimethylformamide and 2.8 ml. of triethylamine is added to the reaction mixture which is thereafter stirred overnight, while slowly warming to room temperature. The solvent of the reaction mixture is evaporated in vacuo at 30°-35°. The residue is dissolved in 800 ml. of ethyl acetate which is washed with (2×150 ml.) a 10% citric acid solution, with (1×150 ml.) water, with (2×150 ml.) saturated solution of sodium bicarbonate and then with (2×100 ml.) water. The organic layer is dried with anhydrous magnesium sulfate and the solvent evaporated to yield carbobenzoxy-β-alanyl-L-proline t-butyl ester. the resulting reaction mixture is stirred is thereafter stirred overnight The solvent of the reaction mixture is evaporated in vacuo at 30°-35° The residue is dissolved in 800 ml of ethyl acetate which is washed with (2×150 ml.) a 10% citric acid solution The organic layer is dried with anhydrous magnesium sulfate the solvent evaporated

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

Procedure: 4.46 g. of carbobenzoxy-β-alanine is dissolved in 20 ml. of tetrahydrofuran and the resulting solution cooled to -10° C. With constant stirring of the solution, 2.4 ml. of N-methylmorpholine followed by 2.6 ml. of isobutylchloroformate are added and the resulting reaction mixture is stirred thereafter for approximately 5 minutes at -10° C. 4.16 g. of L-proline t-butyl ester hydrochloride in 20 ml. of dimethylformamide and 2.8 ml. of triethylamine is added to the reaction mixture which is thereafter stirred overnight, while slowly warming to room temperature. The solvent of the reaction mixture is evaporated in vacuo at 30°-35°. The residue is dissolved in 800 ml. of ethyl acetate which is washed with (2×150 ml.) a 10% citric acid solution, with (1×150 ml.) water, with (2×150 ml.) saturated solution of sodium bicarbonate and then with (2×100 ml.) water. The organic layer is dried with anhydrous magnesium sulfate and the solvent evaporated to yield carbobenzoxy-β-alanyl-L-proline t-butyl ester. the resulting reaction mixture is stirred is thereafter stirred overnight The solvent of the reaction mixture is evaporated in vacuo at 30°-35° The residue is dissolved in 800 ml of ethyl acetate which is washed with (2×150 ml.) a 10% citric acid solution The organic layer is dried with anhydrous magnesium sulfate the solvent evaporated

Conditions: temp:-10.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 4.5 g of CBZ-sarcosine is dissolved in 100 ml tetrahydrofuran and 2.4 ml of n-methylmorpholine is added. The reaction mixture is cooled to -10° C. and stirred while 2.6 ml of isobutylchloroformate is added. After 5 minutes of additional stirring at -10° C. 4.15 g L-proline-t-butylester hydrochloride in 100 ml of tetrahydrofuran and 3.0 ml of n-methylmorpholine are added to the reaction mixture. The reaction mixture is stirred overnight and slowly warmed to room temperature. The solvent is evaporated under reduced pressure at 30° C. and the residue is dissolved in 600 ml ethylacetate. The ethylacetate layer is washed with (3×150 ml), lN sodium hydroxide, water (1×200 ml) 10% citric acid (3×150 ml), and water (3×150 ml). The organic layer is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to provide carbobenzoxysarcosyl-proline-t-butyl ester (CBZ-Sar-L-Pro-O-t-But). 6.5 g of this material is dissolved in 15 ml of methylene chloride and 25 ml of trifluoroacetic acid is then added. The reaction mixture is kept at room temperature for 45 minutes and then the solvent is evaporated under reduced pressure at 30° C. The residue is triturated with hexane (3×50 ml) and the hexane removed by decantation. The residue is evaporated from (2×50 ml) of benzene to provide an oily residue which is carbobenzoxysarcosyl-L-proline (CBZ-Sar-L-Pro). 5.4 g of this material and 3.45 g of N-hydroxysuccinimide are dissolved in 100 ml of tetrahydrofuran and cooled to 0° C. To this cooled reaction mixture is added 4.12 g of dicyclohexylcarbodiimide. The reaction mixture is stirred overnight at 4° C. and the solvent is evaporated under reduced pressure. The residue is dissolved in 400 ml of ethylacetate and the dicyclohexyl urea is filtered off. The filtrate is washed with lN-hydrochloric acid (3×100 ml), water (1×100 ml), saturated sodium bicarbonate solution (2×100 ml) and water (2×100 ml). The organic layer is dried over anhydrous magnesium sulfate, filtered and the solvent is evaporated under reduced pressure to provide carbobenzoxy-sarcosyl-L-proline-succinimidyl ester. is added The reaction mixture is stirred overnight slowly warmed to room temperature The solvent is evaporated under reduced pressure at 30° C. the residue is dissolved in 600 ml ethylacetate The ethylacetate layer is washed with (3×150 ml), lN sodium hydroxide, water (1×200 ml) 10% citric acid (3×150 ml), and water (3×150 ml) The organic layer is dried over anhydrous magnesium sulfate filtered evaporated under reduced pressure

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

Procedure: A solution of 175 mg of {4-[5-(2-fluoro-phenoxy)-oxazolo[5,4-d]pyrimidin-2-yl]-2,6-dimethyl-phenoxy}-acetyl chloride in 2 ml of dichloromethane was added slowly to a suspension of 102 mg of proline tert-butyl ester hydrochloride and 170 μl of triethylamine in 5 ml of dichloromethane. After stirring for 2 h, the solvent was removed in vacuo. 1.6 ml of trifluoroacetic acid were added and the mixture was stirred for 2 h at room temperature. Then 10 ml water were added and the aqueous layer was extracted three times with 15 ml of a dichloromethane/isopropanol mixture (3:1). After drying with sodium sulfate and filtration, the solvents were removed in vacuo and the title compound was isolated via preparative HPLC. Yield: 56 mg of a white solid. the solvent was removed in vacuo 1.6 ml of trifluoroacetic acid were added the mixture was stirred for 2 h at room temperature Then 10 ml water were added the aqueous layer was extracted three times with 15 ml of a dichloromethane/isopropanol mixture (3:1) After drying with sodium sulfate and filtration the solvents were removed in vacuo

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

Procedure: Prepared using General Procedure 7. A stirred solution of (S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-(heptyloxy)phenyl)pyrimidin-2-yl)phenyl)propanoic acid (419 mg, 0.738 mmol), (S)-tert-butyl pyrrolidine-2-carboxylate HCl (153 mg, 0.738 mmol) and TEA (257 μl, 1.845 mmol) in DMF (6 mL) was cooled to 0° C. and HATU (295 mg, 0.775 mmol) was slowly added over 5 minutes. The reaction was stirred at room temperature for 2 h, then diluted with 1M citric acid (30 mL) and iso-hexanes (20 mL). EA (100 mL) was added and the organic layer was isolated, washed with brine (100 mL), dried with MgSO4. The solvent was removed and the crude product was purified by chromatography 0-20% ACN in DCM to afford 436 mg (81%) of (S)-tert-butyl 1-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(5-(4-(heptyloxy)phenyl)pyrimidin-2-yl)phenyl)propanoyl)pyrrolidine-2-carboxylate INT-25 as a viscous oil. LCMS-ESI (m/z) calculated for C43H52N4O6: 720.4; no m/z observed, tR=11.45 min (Method 10). Prepared the organic layer was isolated washed with brine (100 mL) dried with MgSO4 The solvent was removed the crude product was purified by chromatography 0-20% ACN in DCM

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

Procedure: Bis(3-phthalimidopropyl)acetic acid (Step B) (2.5 g, 5.5 mmol), proline tert-butyl ester hydrochloride (0.42 g, 2.0 mmol), and N-methylmorpholine (1.19 g, 5.4 mmol) in 7 mL of DMF are cooled to 0° C. and treated with TBTU (0.64 g, 2.0 mmol). The mixture is then stirred at room temperature for 18 hours. The reaction mixture is poured into 70 mL of saturated sodium bicarbonate solution and extracted with two 50 mL portions of ethyl acetate. The combined organics are washed with 1 M KHSO4, saturated aqueous bicarbonate and brine, and dried over Na2SO4. The volatiles are removed under reduced pressure and the residue is chromatographed over silica gel (4:1 chloroform, ethyl acetate eluent) to give 2.88 g of 1-[Bis(3-phthalimidopropyl)acetyl]-proline tert-butyl ester. extracted with two 50 mL portions of ethyl acetate The combined organics are washed with 1 M KHSO4, saturated aqueous bicarbonate and brine dried over Na2SO4 The volatiles are removed under reduced pressure the residue is chromatographed over silica gel (4:1 chloroform, ethyl acetate eluent)

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

Procedure: A] Starting from H-Pro-OtBu.HCl (0.315 g, 1.5 mmoles) and Z-Pro-OH (0.397 g, 1.59 mmoles) the procedure of Example 1, C-D] was substantially applied. There was obtained 0.4 g of H-Pro-Pro-OtBu.

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

Procedure: A] Starting from H-Pro-OtBu.HCl (0.315 g, 1.5 mmoles) and Z-Pro-OH (0.397 g, 1.59 mmoles) the procedure of Example 1, C-D] was substantially applied. There was obtained 0.4 g of H-Pro-Pro-OtBu.

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

Procedure: 3-Nitrobenzoyl chloride was reacted with L-proline tert-butyl ester hydrochloride, then hydrogenated to produce (S)-1-(3-amino-phenyl)-pyrrolidin-2-carboxylic acid tert-butyl ester. This was reacted with 5-(2-adamantan-1-yl-ethyl)-2-cyclohexyl-1H-imidazole-4-carboxylic acid (Example 252) according to the procedure of Example 20, step d. The tert-butyl ester was deprotected by treating the chloroform solution of the ester with 4.0 M solution of hydrogen chloride in dioxan to afford the title compound as the hydrochloride salt. 1H NMR (300 MHz, d6-DMSO) (mixture of tautomers) 14.10 (1H, br s), 10.95 (1H, br m), 8.00 and 7.79 (1H, 2×m), 7.88 (1H, m), 7.41 (1H, m), 7.26 and 7.11 (1H, 2×), 4.43 (1H, m), 4.12 (1H, m), 3.48 (1H,m), 2.97 (3H, m), 2.28-1.29 (31H, m). Found: C, 64.88; H, 7.78; N, 8.92%; C34H45ClN4O4.1.0 mol H2O requires: C, 65.11; H, 7.55; N, 8.93%.