(4S)-3-acetyl-1,3-thiazolidine-4-carboxylic acid

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

Procedure: To a solution of 331 mg (0.85 mmol) of 2-amino-3-((3-methyl-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-8-carbonyl)amino)propionic acid methyl ester in 8.5 ml of dichloromethane, 149 mg (0.85 mmol) of (S)-3-acetylthiazolidin-4-carboxylic acid, 376 mg (0.85 mmol) of BOP reagent and 0.16 ml (0.95 mmol) of N,N-diisopropylethylamine were added, and the resulting mixture was stirred overnight at room temperature. To the reaction mixture, 10 ml of semi-saturated saline was added, and the resulting mixture was extracted with ethyl acetate. Organic phases were combined, washed with 0.5M hydrochloric acid, with saturated aqueous sodium hydrogen carbonate solution and with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (chloroform:methanol=100:1-10:1) to obtain 305 mg of 2-(((S)-3-acetylthiazolidin-4-carbonyl)amino)-3-((3-methyl-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-8-carbonyl)amino)propionic acid methyl ester (yield: 65%). To the reaction mixture, 10 ml of semi-saturated saline was added the resulting mixture was extracted with ethyl acetate washed with 0.5M hydrochloric acid, with saturated aqueous sodium hydrogen carbonate solution and with saturated saline dried over anhydrous sodium sulfate concentrated The residue was purified by silica gel column chromatography (chloroform:methanol=100:1-10:1)

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

Procedure: EDC (211 mg, 1.1 mmol) was added to a stirred solution of N-acetyl-D-thioproline (175 mg, 1 mmol), O-benzyl tyrosine methyl ester hydrochloride (322 mg, 1 mmol), HOBT (149 mg, 1.1 mmol) and NMM (242 μl, 2.2 mmol) in DCM (10 ml) at 0°. The mixture was stirred at room temperature overnight then diluted with DCM (100 ml). The DCM solution was washed with 1M hydrochloric acid (30 ml), saturated aqueous NaHCO3 (30 ml) and water (30 ml), dried (Na2SO4) and evaporated in vacuo. The residue was purified by chromatography (SiO2 ; ethyl acetate) to give the title compound as a white foam (417 mg, 94%); δH (DMSO-d6, 300K) (2 rotameric species observed) 8.55 (d. J 7.8 Hz) and 8.28 (3, J 8.1 Hz) together (1H, NHCO), 7.43-7.25 (5H, m, Ph), 7.14-7.08 (2H, m, ArH), 6.92-6.88 (2H, m, ArH), 5.06 (2H, s, CH2Ph), 4.79-4.66 (m) and 4.45-4.42 (m) and 4.21 (d, J 9.7 Hz) together (4H, CHα-tyr, CHα-thiopro and NCH2S), 3.62 (s) and 3.61 (s) together (3H, CO2Me), 3.28-2.71 (4H, m, CHCH2Ar+CHCH2S) and 2.04 (s) and 1.82 (s) togher (3H, CH3CO); m/z (ESI, 15V) 443 (M+ +1). The DCM solution was washed with 1M hydrochloric acid (30 ml), saturated aqueous NaHCO3 (30 ml) and water (30 ml) dried (Na2SO4) evaporated in vacuo The residue was purified by chromatography (SiO2 ; ethyl acetate)

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

Procedure: EDC.HCl (760 mg, 3.96 mmol) was added to a solution of N-acetyl-D-thioproline (630 mg, 3.6 mmol), Intermediate 5 (880 mg, 3.6 mmol), HOBT (535 mg, 3.96 mmol) and NMM (834 μl, 7.6 mmol) in DMF (20 ml). The mixture was stirred at room temperature overnight. The DMF was evaporated in vacuo and the residue dissolved in ethyl acetate (150 ml) and water (50 ml). The organic phase was washed with 10% citric acid (50 ml), saturated aqueous NaHCO3 (50 ml) and water (50 ml), dried (Na2SO4) and evaporated in vacuo. The residue was purified by column chromatography (SiO2, methanol/DCM, 7:93) to give the title compound as a colourless gum (680 mg, 52%); δH (DMSO-d6, 400K) 7.40 (1H, br s, CONH), 6.93 (2H, d, J 8.6 Hz, ArH), 6.68 (2H, d, J 8.3 Hz, ArH), 4.84 (1H, dd, J 3.8, 7.4 Hz, CHαthiopro), 4.78 (1H, d, J 9.2 Hz, NCHAHBS), 4.40 (1H, d, J 9.2 Hz, NCHAHBS), 3.63 (3H, S, CO2CH3), 3.30 (1H, dd, J 7.4, 11.5 Hz, CHCHAHBS), 3.16 (1H, dd, J 3.8, 11.5 Hz, CHCHAHBS), 3.13 (1H, d, J 13.7 Hz, CHAHBAr), 3.03 (1H, d, J 13.7 Hz, CHAHBAr), 2.02 (3H, s, CH3CO) and 1.38 (3H, s, CCH3); m/z (ES, 15V) 367 (M+ +1). The DMF was evaporated in vacuo the residue dissolved in ethyl acetate (150 ml) The organic phase was washed with 10% citric acid (50 ml), saturated aqueous NaHCO3 (50 ml) and water (50 ml) dried (Na2SO4) evaporated in vacuo The residue was purified by column chromatography (SiO2, methanol/DCM, 7:93)

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

Procedure: EDC.HCl (760 mg, 3.96 mmol) was added to a solution of N-acetyl-D-thioproline (630 mg, 3.6 mmol), Intermediate 5 (880 mg, 3.6 mmol), HOBT (535 mg, 3.96 mmol) and NMM (834 μl, 7.6 mmol) in DMF (20 ml). The mixture was stirred at room temperature overnight. The DMF was evaporated in vacuo and the residue dissolved in ethyl acetate (150 ml) and water (50 ml). The organic phase was washed with 10% citric acid (50 ml), saturated aqueous NaHCO3 (50 ml) and water (50 ml), dried (Na2SO4) and evaporated in vacuo. The residue was purified by column chromatography (SiO2, methanol/DCM, 7:93) to give the title compound as a colourless gum (680 mg, 52%); δH (DMSO-d6, 400K) 7.40 (1H, br s, CONH), 6.93 (2H, d, J 8.6 Hz, ArH), 6.68 (2H, d, J 8.3 Hz, ArH), 4.84 (1H, dd, J 3.8, 7.4 Hz, CHαthiopro), 4.78 (1H, d, J 9.2 Hz, NCHAHBS), 4.40 (1H, d, J 9.2 Hz, NCHAHBS), 3.63 (3H, S, CO2CH3), 3.30 (1H, dd, J 7.4, 11.5 Hz, CHCHAHBS), 3.16 (1H, dd, J 3.8, 11.5 Hz, CHCHAHBS), 3.13 (1H, d, J 13.7 Hz, CHAHBAr), 3.03 (1H, d, J 13.7 Hz, CHAHBAr), 2.02 (3H, s, CH3CO) and 1.38 (3H, s, CCH3); m/z (ES, 15V) 367 (M+ +1). The DMF was evaporated in vacuo the residue dissolved in ethyl acetate (150 ml) The organic phase was washed with 10% citric acid (50 ml), saturated aqueous NaHCO3 (50 ml) and water (50 ml) dried (Na2SO4) evaporated in vacuo The residue was purified by column chromatography (SiO2, methanol/DCM, 7:93)

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

Procedure: EDC (2.11 g, 11 mmol) was added to a stirred solution of N-acetyl-D-thioproline (1.75 g, 10 mmol), tyrosine methyl ester (2.32 g, 10 mmol), HOBT (1.49 g, 11 mmol) and NMM (2.31 ml, 21 mmol) in DMF (50 ml) at 0°. The mixture was stirred at room temperature overnight. The DMF was evaporated in vacuo and the residue dissolved in ethyl acetate (400 ml), and water (50 ml). The organic phase was washed with 10% citric acid (100 ml), saturated aqueous NaHCO3 (100 ml), water (100 ml) and brine (100 ml), dried (Na2SO4) and evaporated in vacuo to give the title compound as a slightly yellow powdery solid (2.75 g, 78%), used without further purification. A small portion was crystallised from EtOAC to give a white microcrystalline solid, m.p. 189-190°. δH (DMSO-D6, 400K) 7.7 (1H, br s, CONH), 6.98 (2H, d, J 8.6 Hz, ArH), 6.69 (2H, d, J 8.5 Hz, ArH), 4.83 (1H, dd, J 3.9, 7.4 Hz, CHαthiopro), 4.77 (1H, d, J 9.2 NCHAHBS), 4.53 (1H, dt, J 5.8, 8.1 Hz, CHαtyr), 4.38 (1H, d, J 9.2 Hz, NCHAHBS), 3.65 (3H, s, C O2HC), 3.25 (1H, dd, J 7.3, 11.5 Hz, CHCHAHBS), 3.05-2.97 (2H, m, CHAHBAr+CHCHAHBS), 2.89 (1H, dd, J 8.2, 14.1 Hz, CHAHBAr) and 1.99 (3H, s, CH3CO) [phenolic OH not observed at 400K, for δH (DMSO-d6, 300K) 9.19 (1H, br s, ArOH)]; m/z ESI, 27V), 353 (M+ +1). The DMF was evaporated in vacuo the residue dissolved in ethyl acetate (400 ml) The organic phase was washed with 10% citric acid (100 ml), saturated aqueous NaHCO3 (100 ml), water (100 ml) and brine (100 ml) dried (Na2SO4) evaporated in vacuo

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

Procedure: EDC (4.22 g, 22 mmol) was added to a solution of N-acetyl-D-thioproline (3.50 g, 20 mmol), tyrosine tert.butyl ester (4.74 g, 20 mmol), HOBT (2.97 g, 22 mmol) and NMM (2.42 ml, 22 mmol) in DMF (80 ml) at 0°. The mixture was stirred at room temperature overnight. The DMF was evaporated in vacuo and the residue dissolved in ethyl acetate (600 ml) and water (50 ml). The organic phase was washed with 10% citric acid (150 ml), saturated aqueous NaHCO3 (150 ml), water (150 ml) and brine (150 ml), dried (Na2SO4) and evaporated in vacuo to give the title compound as an off-white solid (7.39 g, 94%); δH (DMSO-d6, 300K) (2 rotameric species observed) 9.17 (1H, br s, ArCH), 8.43 (3, J 8.0 Hz), and 8.09 (d, J 8.0 Hz) together (1H, CONH), 6.98 (2H, t, J 7.6, ArH), 6.64 (2H, d, J 7.3, ArH), 4.81-4.67 (m), 4.47 (d, J 8.7 Hz) and 4.35-4.21 (m), together (4H, CHα-tyr, CHαthiopro and NCH2S), 3.32 (dd, J 7.2, 11.6 Hz), 3.17 (dd, J 7.5, 11.6 Hz), 2.99-2.72 (m) together (4H, CH2Ar+CHCH2S), 2.06 (s) and 1.84 (s) together (3H, CH3CO) and 1.36 (9H, S, CO2tBu); m/z (ESI, 15V) 395 (M+ +1). The DMF was evaporated in vacuo the residue dissolved in ethyl acetate (600 ml) The organic phase was washed with 10% citric acid (150 ml), saturated aqueous NaHCO3 (150 ml), water (150 ml) and brine (150 ml) dried (Na2SO4) evaporated in vacuo

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

Procedure: from 4-(trifluoromethyl)-DL-phenylalanine methyl ester and N-acetyl-D-thioproline δH (DMSO-d6) 8.56-8.12 (1 H, m,NH), 7.68-7.55 (2 H, m, ArH), 7.51-7.37 (2 H, m, ArH),4.85-4.15 (4 H, m, NCH2S and 2×α-CH), 3.40-2.65 (4 H, m,SCH2CH and CH2Ar),2.06 (s) and 2.04 (s) and 1.80(s) and 1.71 (s); together (3 H, COCH3); m/z (ESI, 60 V), 391 (MH+).

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

Procedure: A solution of N-acetyl-D-thioproline-L-tyrosine methyl ester (1.76 g, 5 mmol), [prepared from N-acetyl-D-thioproline and tyrosine methyl ester by a similar method to the preparation of Intermediate 5] in DMF (10 ml) was added to a suspension of sodium hydride (60% in mineral oil, 210 mg, 5.25 mmol) in DMF (5 ml) at room temperature. After 10 min a solution of 2,4-dichloropyrimidine (782 mg, 5.25 mmol) in DMF (5 ml) was added. After 1 h water (1 ml) was added and the solvent evaporated in vacuo. The residue was dissolved in EtOAc (200 ml) and washed with water (3×50 ml) and brine (30 ml), dried (Na2SO4) and evaporated in vacuo. Chromatography (SiO2, MeOH/DCM 5:95) gave the title compound as a white foam (1.59 g, 68%). δH (DMSO-d6, 400K) 8.56 (1 H, d, J 5.7 Hz, PyrH), 7.9 (1 H, br d, CONH),7.32 (2 H, d,J 8.7 Hz, ArH), 7.15 (2 H, d, J 8.7 Hz, ArH), 6.99 (1 H, d, J 5.7 Hz, PyrH), 4.83 (1 H, dd, J 7.4, 3.9 Hz, CHαthiopro), 4.77 (1 H, d, J 9.2 Hz, NCHHBS), 4.64 (1 H,dt, J 8.5, 5.6 Hz, CHαtyr),4.39 (1 H, d, J 9.2 Hz, NCHAHBS), 3.67 (3 H, s, CO2Me), 3.26 (1 H, dd, J 11.6, 7.4 Hz, CHCHAHBS), 3.19 (1 H, dd, J 14.0, 5.7 Hz, CHCHAHBAr), 3.09-3.00 (2 H, m, CHCHAHBS+CHCHAHBAr) and 2.00 (3 H, s,COCH3); m/z (ESI, 15 V) 465 (MH+). the solvent evaporated in vacuo The residue was dissolved in EtOAc (200 ml) washed with water (3×50 ml) and brine (30 ml) dried (Na2SO4) evaporated in vacuo

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

Procedure: To a solution of 331 mg (0.85 mmol) of 2-amino-3-((3-methyl-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-8-carbonyl)amino)propionic acid methyl ester in 8.5 ml of dichloromethane, 149 mg (0.85 mmol) of (S)-3-acetylthiazolidin-4-carboxylic acid, 376 mg (0.85 mmol) of BOP reagent and 0.16 ml (0.95 mmol) of N,N-diisopropylethylamine were added, and the resulting mixture was stirred overnight at room temperature. To the reaction mixture, 10 ml of semi-saturated saline was added, and the resulting mixture was extracted with ethyl acetate. Organic phases were combined, washed with 0.5M hydrochloric acid, with saturated aqueous sodium hydrogen carbonate solution and with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (chloroform:methanol=100:1-10:1) to obtain 305 mg of 2-(((S)-3-acetylthiazolidin-4-carbonyl)amino)-3-((3-methyl-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-8-carbonyl)amino)propionic acid methyl ester (yield: 65%). To the reaction mixture, 10 ml of semi-saturated saline was added the resulting mixture was extracted with ethyl acetate washed with 0.5M hydrochloric acid, with saturated aqueous sodium hydrogen carbonate solution and with saturated saline dried over anhydrous sodium sulfate concentrated The residue was purified by silica gel column chromatography (chloroform:methanol=100:1-10:1)