(2S)-1-acetylpyrrolidine-2-carboxylic acid

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

Procedure: To a suspension of 5.75 g (25 m mol) 6-(4-aminophenyl)-1,2-dihydro-2-oxonicotinic acid and 175 ml of dichloromethane, 10.2 ml (75 m mol) triethylamine and 9.5 ml (75 m mol) chlorotrimethylsilane are added and the reaction mixture stirred at room temperature for 1 hour. To a solution of 7.86 g (50 m mol) of N-Acetyl-L-proline in 100 ml of dichloromethane at -10° C., 5.5 ml (50 m mol) of N-methylmorpholine and 6.5 ml (50 m mol) of isobutyl chloroformate are added and stirred at -10° C. for 1/2 hour. To this cold mixed anhydride mixture is added the silylated 6-(4-aminophenyl)-1,2-dihydro-2-oxonicotinic acid and the resulting mixture is stirred for 3 hrs in an ice bath and overnight at room temperature. Next, 25 ml of isopropanol is added to the reaction mixture and stirred. The reaction mixture is evaporated and the residue is triturated with ice and water. The solid is collected by filtration and washed with water, isopropanol and ether to yield 4.82 g of 6-[4-(N-Acetyl-L-prolylamino)phenyl]-1,2-dihydro-2-oxonicotinic acid, m.p. 288°-290° dec. From the filtrate an additional 3.94 g of product is obtained. are added stirred at -10° C. for 1/2 hour the resulting mixture is stirred for 3 hrs in an ice bath and overnight at room temperature stirred The reaction mixture is evaporated the residue is triturated with ice and water The solid is collected by filtration washed with water, isopropanol and ether

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

Procedure: To a suspension of 5.75 g (25 m mol) 6-(4-aminophenyl)-1,2-dihydro-2-oxonicotinic acid and 175 ml of dichloromethane, 10.2 ml (75 m mol) triethylamine and 9.5 ml (75 m mol) chlorotrimethylsilane are added and the reaction mixture stirred at room temperature for 1 hour. To a solution of 7.86 g (50 m mol) of N-Acetyl-L-proline in 100 ml of dichloromethane at -10° C., 5.5 ml (50 m mol) of N-methylmorpholine and 6.5 ml (50 m mol) of isobutyl chloroformate are added and stirred at -10° C. for 1/2 hour. To this cold mixed anhydride mixture is added the silylated 6-(4-aminophenyl)-1,2-dihydro-2-oxonicotinic acid and the resulting mixture is stirred for 3 hrs in an ice bath and overnight at room temperature. Next, 25 ml of isopropanol is added to the reaction mixture and stirred. The reaction mixture is evaporated and the residue is triturated with ice and water. The solid is collected by filtration and washed with water, isopropanol and ether to yield 4.82 g of 6-[4-(N-Acetyl-L-prolylamino)phenyl]-1,2-dihydro-2-oxonicotinic acid, m.p. 288°-290° dec. From the filtrate an additional 3.94 g of product is obtained. are added stirred at -10° C. for 1/2 hour the resulting mixture is stirred for 3 hrs in an ice bath and overnight at room temperature stirred The reaction mixture is evaporated the residue is triturated with ice and water The solid is collected by filtration washed with water, isopropanol and ether

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

Procedure: A mixture of 7.07 g (45 mmol) of N-acetyl-L-proline, 5.85 ml (45 mmol) isobutyl chloroformate, 4.95 ml 45 mmol) N-methyl morpholine, and 90 ml of dichloromethane is stirred at -10° to -20° for 30 minutes. A mixture of 6.13 g (30 mmol) of 6-amino-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid, 12.6 ml (90 mmol) triethylamine, 11.4 ml (90 mmol) chlorotrimethylsilane, and 150 ml of dichloromethane is stirred at room temperature for 30 minutes and then is cooled to -20°. The cold mixed anhydride solution is added to the cold silylated quinoline and is stirred with cooling for 4 hours and at room temperature ovenight. The reaction mixture is evaporated to dryness and the residue is treated with 1 N hydrochloric acid and the solid is filtered. The solid is digested with ethanol and filtered to give 6.43 g of 6-[[(1-acetyl- 2-pyrrolidinyl)-carbonyl]amino]-1,4-dihydro-4-oxo-quinolinecarboxylic acid. is stirred at room temperature for 30 minutes is cooled to -20° is stirred The reaction mixture is evaporated to dryness the residue is treated with 1 N hydrochloric acid the solid is filtered filtered

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

Procedure: A solution of 4.71 g (30 mmol) of N-acetyl-L-proline and 60 ml of dichloromethane is cooled to -18° and 3.3 ml (30 mmol) of N-methyl morpholine and 3.9 ml (30 mmol) of isobutyl chloroformate is added, and the reaction mixture is stirred for 30 minutes at -10° to -20°. A mixture of 4.08 g (20 mmol) of 6-amino-1,2-dihydro-2-oxo-3-quinolinecarboxylic acid, 150 ml of dichloromethane, 8.4 ml (60 mmol) of triethylamine, and 7.6 ml (60 mmol) of chlorotrimethylsilane is stirred at room temperature for 35 minutes. The silylation mixture is cooled to -20° and the mixed anhydride of proline is added and stirred with cooling overnight. The reaction mixture is evaporated to dryness, and the residue is treated with ethanol to yield 4.42 g of 6-[[(1-acetyl-2-pyrrolidinyl)carbonyl]-amino]-1,2-dihydro-2-oxo-3-quinolinecarboxylic acid, mp 303° dec. is stirred at room temperature for 35 minutes The silylation mixture is cooled to -20° stirred with cooling overnight The reaction mixture is evaporated to dryness the residue is treated with ethanol

Conditions: temp:150.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Proline (1 g, 8.68 mmol) and acetic acid (20 mL) were mixed together and heated at 150° C. in a microwave for two hours. The excess acetic acid was removed using a rotary evaporator. The residue was stirred in MTBE at ambient temperature until a solid precipitated. The mixture was filtered and the solid was washed with MTBE. The solid was dried in a vacuum oven at 60° C. The product was obtained as a white solid (1.12 g, 82.5%). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.77-1.95 (m, 4H), 1.97 (s, 2H), 2.0-2.19 (m, 1H), 3.35 (dt, 1H), 3.41-3.58 (m, 2H), 4.19 (dd, 1H). The excess acetic acid was removed a rotary evaporator precipitated The mixture was filtered the solid was washed with MTBE The solid was dried in a vacuum oven at 60° C

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

Procedure: The mixture of compound 4-(2,3-difluorobenzylamino)-6-(4-(piperidin-4-yl)phenylamino)nicotinamide (Example 209) (50 mg, 0.11 mmol), (S)-acetyl-pyrrolidine-2-carboxylic acid (35 mg, 0.22 mmol), DIEA (180 μL, 1.1 mmol) in 4 mL NMP was stirred at RT. To it was added BOP (210 mg, 0.55 mmol). The mixture was stirred for overnight, quenched with TFA, and subjected to reverse phase preparative HPLC to isolate the title compound (64 mg). MS found for C31H34F2N6O3 as (M+H)+ 577.5. UV: λ=259 nm. 1H NMR (a pair of rotomers): (CD3OD) δ 8.13 (with 8.15, 1H, s), 7.36 (with 7.32, 2H, d, J=8.8 Hz), 7.25 (1H, m), 7.16 (1H, m), 7.13-7.07 (3H, m), 5.83 (with 5.79, 1H, s), 4.95 (1H, m), 4.65 (1H, m), 4.56 (with 4.55, 2H, s), 4.20 (1H, m), 3.66 (2H, m), 3.30 (1H, m), 2.92 (1H, m), 2.79 (1H, m), 2.27 (1H, m), 2.12 (with 2.10, 3H, s), 2.07-1.85 (5H, m), 1.68 (2H, m) ppm. The mixture was stirred for overnight quenched with TFA

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

Procedure: To a suspension of L-Pro (247 mg, 2.15 mmol) in pyridine (1 ml) was added acetic anhydride (I ml), and the mixture was stirred at room temperature for 5 min. The solvent was removed in vacuo and the resulting oil was partitioned between ethyl acetate and 1N hydrochloric acid. The organic layer was dried over sodium sulfate, and concentrated, and the resulting solid was recrystallized from ethyl acetate to give N-acetyl-L-proline (167 mg, 44%), a white powder: m.p. 108° C. [lit 115° C. (from CHCl3)99 ]; [α]D25 -171° (c, 0.91, CHCl3) [(lit107 -118° (c, 1, H2O)] Anal. Calcd. for C7H11NO3 : C, 53.49; H, 7.05, N, 8.91. Found: C, 53.57; H, 7.09; N, 8.76. The solvent was removed in vacuo the resulting oil was partitioned between ethyl acetate and 1N hydrochloric acid The organic layer was dried over sodium sulfate concentrated the resulting solid was recrystallized from ethyl acetate

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

Procedure: To a suspension of L-Pro (247 mg, 2.15 mmol) in pyridine (1 ml) was added acetic anhydride (I ml), and the mixture was stirred at room temperature for 5 min. The solvent was removed in vacuo and the resulting oil was partitioned between ethyl acetate and 1N hydrochloric acid. The organic layer was dried over sodium sulfate, and concentrated, and the resulting solid was recrystallized from ethyl acetate to give N-acetyl-L-proline (167 mg, 44%), a white powder: m.p. 108° C. [lit 115° C. (from CHCl3)99 ]; [α]D25 -171° (c, 0.91, CHCl3) [(lit107 -118° (c, 1, H2O)] Anal. Calcd. for C7H11NO3 : C, 53.49; H, 7.05, N, 8.91. Found: C, 53.57; H, 7.09; N, 8.76. The solvent was removed in vacuo the resulting oil was partitioned between ethyl acetate and 1N hydrochloric acid The organic layer was dried over sodium sulfate concentrated the resulting solid was recrystallized from ethyl acetate

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

Procedure: 1 ml of a 30 mM solution of N-choloracetyl-N-methyl-L-alanine in 0.1M tris-HC1, pH 7.0 was introduced into a reaction container thermostatted to 30° C. and mixed with 1.99 ml 0.1M tris --HCl pH 7.0. The reaction was started by adding 0.01 ml N-acyl-L-proline acylase from Comamonas TESTOSTERONI DSM 5416 corresponding to 0.23 units and 0.2 μg protein. 1 unit is defined as the amount of enzyme which converts 1 μmole of N-acetyl-L-proline per minute at 30° C. and pH 7.0. Specimens were taken at various points in time to detect the course of the reaction, compounded with 2N HCl 1:2 and subsequently analyzed by HPLC using an RP 8-column (250×4 mm). After elution with 7% (v/v) acetonitrile in 0.01M heptane sulfonic acid (adjusted to pH 2.2 with 85% H3PO4) at a flow rate of 2.2 ml/min. and a column temperature of 40° C., the decrease of N-chloroacetyl-N-methyl-L-alanine was determined by measuring the UV absorption at 210 nm. to 30° C. and mixed with 1.99 ml 0.1M at 30° C. the course of the reaction After elution with 7% (v/v) acetonitrile in 0.01M heptane sulfonic acid (adjusted to pH 2.2 with 85% H3PO4) at a flow rate of 2.2 ml/min. a column temperature of 40° C. the UV absorption at 210 nm

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

Procedure: Following the procedure of Example 30, triethylamine (0.21 mL, 1.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.15 g, 0.78 mmol), N-hydroxybenzotriazole (0.11 g, 0.78 mmol), N-acetyl-L-proline (0.094 g, 0.60 mmol), and 7,8,9,10-tetrahydro-6H-benzo[b]-cyclohepta[d]furan-2-ylamine (0.12 g, 0.60 mmol) 1-acetyl-N-7,8,9,10-tetrahydro-6H-benzo[b]cyclohepta[d]furan-2-yl-L-prolinamide (0.13 g). MS (ESI) m/z 341 ([M+H]+); MS (ESI) m/z 339.1 ([M−H]−); HRMS: calcd for C20H24N2O3+H+, 341.18597; found (ESI, [M+H]+), 341.1863.

Conditions: temp:4.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In 200 ml of THF was dissolved 15.7 g of acetyl-proline. To this solution were added under cooling with edible salt and ice 11.5 g of HOSu and 20.6 g of DCC, and the mixture was stirred for 21 hours at 4° C. The reaction liquid was filtered and the filtrate was evaporated whereby a semi-solid substance was obtained. This substance was recrystallized from isopropyl alcohol to obtain a white solid substance which was then dissolved in 400 ml of THF. To this solution was added 7.5 g of L-prolinol, and the mixture was stirred for 72 hours at room temperature. After removal of THF by distillation, the residue was dissolved in 300 ml of ethyl acetate and the solution was washed with 100 ml of 1-M Na2CO3, dehydrated with anhydrous Na2SO4 and evaporated until dryness. To the resultant oily substance were added 35.8 g of WBCD.HCl and 160 ml of redistilled DMSO, and the mixture was stirred for 10 minutes. Subsequently, 7.1 ml of a DMSO solution of 2-M anhydrous H3PO4 was added and the mixture was stirred for further 2 hours. The reaction was stopped with 300 ml of a buffer solution of 1-M potassium phosphate of pH 7.5. The reaction mixture was extracted with 350 ml of ethyl acetate, and the extract was dried over anhydrous Na2SO4. The ethyl acetate was distilled off and the residue was dissolved in 300 ml of ethanol. To this solution was added 200 g of NaHSO3 in 350 ml of water, and the mixture was stirred vigorously for 10 minutes. After concentration of the ethanol up to about 300 ml, unreacted materials were eliminated twice with 100 ml of ether, and solid Na2CO3 was added until the pH value of the liquid became 9. After 5 minutes, the reaction liquid was extracted twice with 100 ml of ether, and the ether was then distilled off to leave an oily substance. Yield 5.5 g (23.5%). To this solution were added The reaction liquid was filtered the filtrate was evaporated whereby a semi-solid substance was obtained This substance was recrystallized from isopropyl alcohol to obtain a white solid substance which the mixture was stirred for 72 hours at room temperature After removal of THF by distillation the residue was dissolved in 300 ml of ethyl acetate the solution was washed with 100 ml of 1-M Na2CO3 evaporated until dryness To the resultant oily substance were added 35.8 g of WBCD HCl and 160 ml of redistilled DMSO, and the mixture was stirred for 10 minutes Subsequently, 7.1 ml of a DMSO solution of 2-M anhydrous H3PO4 was added the mixture was stirred for further 2 hours The reaction mixture was extracted with 350 ml of ethyl acetate the extract was dried over anhydrous Na2SO4 The ethyl acetate was distilled off the residue was dissolved in 300 ml of ethanol To this solution was added 200 g of NaHSO3 in 350 ml of water the mixture was stirred vigorously for 10 minutes was added until the pH value of the liquid After 5 minutes the reaction liquid was extracted twice with 100 ml of ether the ether was then distilled off to leave an oily substance

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

Procedure: In Ar atmosphere, 2-bromo-1-(4-bromophenyl)ethanone (2 g, 7.2 mmol) was dissolved in acetonitrile (18 ml) under Ar atmosphere, added with (S)-1-acetylpyrrolidine-2-carboxylic acid (1.1 g, 7.2 mmol) and DIPEA (1.4 g, 10.8 mmol) at 0° C., and agitated at a room temperature for 4 hours. The reaction termination was confirmed by TLC (Hex:EA=2:1) and performed by vacuum evaporation to remove the solvent. The concentrated solution was added with ethylacetate (20 mL) and washed with H2O. The aqueous layer was extracted with ethylacetate (10 mL) and the organic layer was dried with Na2SO4, filtered and concentrated with vacuum evaporation. The product was transferred to a pressure tube, dissolved in xylene (20 mL) and agitated at 160° C. for 2 hours with addition of NH4OAc (2.8 g, 10.8 mmol). The reaction termination was confirmed by TLC (HX:EA=1:1), cooled to a room temperature and concentrated with vacuum evaporation. The concentrated solution was added with dichloromethane (DCM, 20 mL) and washed with H2O (10 mL). The aqueous layer was extracted with DCM (10 mL), and the organic layer was collected. The collected organic layer was dried with Na2SO4, filter and concentrated. The concentrated solution was purified with column chromatograph(Hex/EA=1:1), to obtain the subject compound (1.93 g. 80%). performed by vacuum evaporation to remove the solvent The concentrated solution was added with ethylacetate (20 mL) washed with H2O The aqueous layer was extracted with ethylacetate (10 mL) the organic layer was dried with Na2SO4 filtered concentrated with vacuum evaporation The product was transferred to a pressure tube dissolved in xylene (20 mL) cooled to a room temperature concentrated with vacuum evaporation The concentrated solution was added with dichloromethane (DCM, 20 mL) washed with H2O (10 mL) The aqueous layer was extracted with DCM (10 mL) the organic layer was collected The collected organic layer was dried with Na2SO4 filter concentrated The concentrated solution was purified with column chromatograph(Hex/EA=1:1), to obtain the subject compound (1.93 g. 80%)