N-[(S)-(2,3,4,5,6-Pentafluorophenoxy)phenoxyphosphinyl]-L-alanine 1-methylethyl ester

A flask containing (S)-isopropyl 2-aminopropanoate hydrochloride (72.0 g, 430 mmol) was charged with phenyl phosphorodichloridate (64.2 mL, 430 mmol) and dichloromethane (DCM, 1200 mL). The mixture was cooled to -70 to -78°C with dry-ice acetone bath and then treated with drop wise addition of triethylamine (120 mL, 859 mmol) over a period of 30 minutes. The mixture was stirred at -70 to -78°C for 30 more minutes and then was allowed to warm to ambient temperature and stirred for lh. (0555) The reaction mixture was then cooled to 0-5 °C in ice-bath and added to a solution of 2, 3, 4, 5, 6-pentafluorophenol (79 g, 430 mmol) and triethylamine (59.9 mL, 430 mmol) in 100 mL DCM over a period of 30 minutes. The resulting mixture was stirred at -70 to -78°C for 30 more minutes, then was warmed to ambient temperature and stirred for 2h. (0556) The solids were filtered off and solid cake was washed with 200 mL ethyl acetate. (0557) The filtrate and washes were concentrated by vacuum distillation until a semi-solid residue remained. The semi-solid residue was dissolved in 500 mL ethyl acetate and washed with water and brine. The washes were re-extracted with 50 mL of ethyl acetate. The combined organic layer was dried over anhydrous MgSCn and concentrated to give crude racemic product 210 g (100percent yield). Based on the NMR characterization, the racemic product appears to be a 1 : 1 mixture of two diastereomers. (0558) Kinetic resolution of the racemic product to produce the desired SS diastereomer was accomplished by the following protocol. (0559) 1) The crude racemic mixture was slurried in 500 mL of 20percent ethyl acetate/ hexanes and was added to a solution of 5 g of pentafluorophenol, 10 mL of triethylamine, and 100 mg of dimethylaminopyridine in 20 mL of 20percent ethyl acetate/hexanes. The reaction mixture was warmed to 45-50 °C for 30 minutes, and the slurry was allowed to stir overnight. The white solid was collected by filtration and was washed with 200 mL of 20percent ethyl acetate/hexanes and 100 mL of hexanes. The product was dried at 40 °C under vacuum to give a white solid (weight: 98 g). Based on the NMR characterization, the product appears to be substantially the SS diastereomer. (0560) 2) The filtrate and washings from the above reaction were combined and concentrated to give a semi solid which was mainly the SS diastereomer as shown by NMR along with other impurities. This residue was dissolved in 150 mL ethyl acetate and washed with 50 mL of IN HC1, water and 5percent K2CO3 solution. The organic layer was dried and concentrated. The white residue was slurred in 100 mL of 20percent ethyl acetate /hexanes, and the solid was collected by filtration. The cake was then washed with 20percent ethyl acetate /hexanes, hexanes and dried. The weight of the resulting white solid was 22 g. Based on the - and Phenyl dichlorophosphate (62.88 g, 0.298 mol, 1 .0 eq) was added under nitrogen to a solution of L-alanine isopropylester hydrochloride (50.0 g, 0.298 mol) in DCM (310 ml_) at 0 °C - the addition was completed by wash with DCM (39 ml_). The mixture was cooled and triethylamine (63.35 g, 0.626 mol, 2.1 eq) was added over a period of 70 minutes with cooling keeping the temperature not higher than -14 °C, the addition was completed by wash with DCM (39 ml_). The mixture was stirred for one hour at -15 to -20 °C, then heated to -8 °C and a solution of pentafluorophenol (60.38 g, 0.328 mol, 1 .1 eq) and triethylamine (33.19 g, 0.328 mol, 1 .1 eq) in DCM (78 ml_) was added over a period of 42 minutes with cooling keeping the temperature not higher than 0 °C - the addition was completed by wash with DCM (39 ml_). The mixture was stirred for one hour at 0 °C and then over night at +5 °C. The formed precipitate was removed by filtration, and the filter cake washed with DCM (95 ml_). The combined filtrates were washed at 5 °C with water (2x190 ml_). The organic phase was distilled at 32 - 38°C at reduced pressure (650 - 600 mBar), and distillation was continued until a residual volume of approx. 170 ml_ partly crystallized mass was obtained. Ethyl acetate (385 ml_) was added, and the resulting clear solution was distilled at 43 - 45°C under reduced pressure (300 - 250 mBar). Distillation was continued until a residual volume of approx. 345 ml_ was obtained. The clear solution was cooled to 36°C, and crystallization is induced by addition of seed crystals of (S)-isopropyl 2- (((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (20 mg) prepared as described in J. Org. Chem., 201 1 , 76, 831 1 - 8319. The mixture was cooled to 27 °C over a period of one hour, then n-heptane (770 ml_) was added over a period of 47 minutes, and the mixture was stirred for an additional period of 37 minutes. Triethylamine (6.03 g, 0.2 eq) was added, and the mixture was stirred at 23 - 25 °C over night. The precipitate was isolated by filtration. The filter cake was washed with ethyl acetate:n-heptane (1 :9, 80 ml_) and dried to constant under reduced pressure (below 0.1 mBar) without heating, which gave the title compound (75.64 g, 56percent) as a white crystalline material. At 0 ° C under nitrogen atmosphere, phenyl dichlorophosphate (62.88 g, 0.298 mol, 1.0 eq.) was added to a solution of L-alanine isopropyl ester hydrochloride (50.0 g, 0.298 mol) in DCM (310 mL) was added - completion by washing with DCM (39 mL). The mixture was cooled and triethylamine (63.35 g, 0.626 mol, 2.1 eq.) Was added over a period of 70 minutes while keeping the temperature cool to -14 ° C and complete by washing with DCM (39 mL). The mixture was stirred at -15 to -20 ° C for one hour then warmed to -8 ° C and pentafluorophenol (60.38 g, 0.328 mol, 1.1 eq.) And triethylamine (33.19 g, 0.328 mol, 1.1 eq.) In DCM (78 mL) while cooling to keep the temperature below 0 ° C. - added by washing with DCM (39 mL). The mixture was stirred at 0 ° C for one hour and then at + 5 ° C overnight. The formed precipitate was removed by filtration, then the filter cake was washed with DCM (95 mL). The combined filtrates were washed with water (2x190 mL) at 5 ° C. The organic phase is distilled under reduced pressure (650-600 mbar) at 32-38 ° C and distillation is continued until a residual volume of about 170 mL to obtain a partially crystalline material. Ethyl acetate (385 mL) was added and the resulting clear solution was distilled under reduced pressure (300-250 mbar) at 43-45 ° C. Distillation was continued until a residual volume of about 345 mL was obtained. The clear solution was cooled to 36 ° C and was quenched by the addition of (S)-2-(((S)(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoic acid isopropyl ester (20 mg)as described in J. Org. Chem., 2011, 76, 8311-8319. The mixture was cooled to 27 ° C over a period of one hour, then n-heptane (770 mL) was added over a period of 47 minutes, and the mixture stirred for an additional 37 minutes. Triethylamine (6.03 g, 0.2 eq) was added and the mixture was stirred at 23-25 ° C overnight. The precipitate is isolated by filtration. The filter cake was washed with ethyl acetate: n-heptane (1: 9, 80 mL) and dried under reduced pressure (0.1 mbar or less) to constant weight without heating to give the title compound (75.64 g, 56percent) as a white crystalline material.L-alanine isopropyl ester hydrochloride (218.5 g) and toluene (1250 mL) were heated to reflux azeotropically till complete removal of water. The reaction mass was then cooled to 50°C and solvent was removed under reduced pressure. MDC was charged into the resultant mass was cooled to -60° to -50°C. Phenyldichloro phosphate (250.0 g) was charged into the reaction mass at -60°C to -50°C followed by trimethylamine (263.75 g) was added and the reaction mass was stirred for 2-3 hours at -60° to -50°C. Pentafluorophenol (240.0 g) was charged into the reaction mass followed by addition of triethylamine (143.39 g) at -60° to -50°C. The reaction mixture was allowed to warm to room temperature (RT) and stirred for 2-3 hours at RT. The reaction was monitored by HPLC (Distereomeric ratio of (Sp) : (Rp) = 35 : 65). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. Paraffin oil (1250 mL) was charged into the residual mass and the reaction mixture was heated to 60°C. The reaction mixture was stirred for 3 hours at 60°C. The reaction was monitored by HPLC (Distereomeric ratio of (Sp) : (Rp) = 99.67 : 0.37). The reaction mixture was filtered and washed with n-heptane (750 mL). The filtered solid was stirred with saturated bicarbonate solution (2500 mL) for 1 hour and filtered. The filtered solid was stirred with water (2500 mL) for 1 hour and filtered. The material was dried at 50-55°C in ATD to get pure (Sp)-isomer: (S)-2-[(S)-(2, 3, 4, 5, 6-pentafluoro-phenoxy)-phenoxy- phosphorylamino]propionic acid isopropyl ester (375 g) wherein (Rp)-isomer is 0.37 percent on chiral HPLC.Phenyl dichlorophosphate (50 g, 0.24 mol) and pentafluorophenol (43.6 g, 0.24 mol)Adding to 150 mL of dichloromethane, adding triethylamine (79.4 g, 0.79 mol) dropwise;The temperature was raised to reflux, and the temperature was lowered to room temperature after stirring for 7 to 8 hours.Add L-alanine isopropyl ester hydrochloride (40 g, 0.24 mol), Stir at room temperature for 4 to 5 hours; suction filtration, and concentrate the filtrate under reduced pressure.The concentrated residue was recrystallized from n-hexane and ethyl acetate:The residue can be dissolved in an appropriate amount of ethyl acetate, and then n-hexane is added dropwise at 30 to 40 ° C until turbidity occurs.Lowering the temperature to cause crystallization;Alternatively, the residue may be added to a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 1:4.Then heated to dissolve, and then cooled to crystallization; collect crystals, vacuum dry, N-[(S)-(2, 3, 4, 5, 6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester (compound of formula I)51.8g, the molar yield is 48percent, The HPLC purity was 99.1percent.(2S)-isopropyl-2-((chloro(phenoxy)phosphoryl)amino)propanoate (1.2 mol eq.) was dissolved in tetrahydrofuran (3.5 volumes). The reaction mass was cooled to -10°C. Solution of sodium salt of pentafluorophenol (1 mol eq.) in tetrahydrofuran (3.5 volumes) was added dropwise to the reaction mass at -10°C. After completion of the reaction solvent was distilled off. Ethyl acetate and water were added to the reaction mass. Reaction mass was stirred, ethyl acetate layer was separated and washed with sodium bicarbonate solution and brine. Ethyl acetate layer was concentrated under reduced pressure. Reaction mass was stripped with n- hepatane to get crude product. Crude product was dissolved in Methyl tert-butyl ether and n- heptane (1 : 1 ratio). The pH of reaction mass was adjusted to pH 8 by using triethylamine. Reaction mass was stirred overnight. Solid mass was filtered and washed with a mixture of methyl tertiary-butyl ether: n-heptane (1 : 1). The obtained product was dissolved in ethyl- acetate and washed with water and 20percent brine solution. Ethyl acetate layer was separated; solvent was distilled off under reduced pressure. Reaction mass was stripped with diisopropyl ether. Di-isopropyl ether was added to the reaction mass. Reaction mass was stirred at 45- 50°C. Reaction mass was cooled to 5-10°C and stirred. The titled compound was isolated by filtration and washed with di-isopropyl ether. The titled compound was dried under reduced pressure at 40°C. Yield 66.81percent.To a flask provided with mechanical stirrer, reflux condenser, thermometer and under nitrogen atmosphere is added a solution of (L)-alanine isopropyl ester hydrochloride (Q) (2.8 g, 16.8 mmol) obtained as described in and /Step b) The crude compound (12.4 g, 50 mmol) of the compound of formula II in step a)Adding 50 ml of methylene chloride, cooling the ice bath, controlling the temperature of 0 ° C, adding phenol slowly (4.7 g, 50 mmol)(10.1 g, 100 mmol) was added dropwise, and the mixture was gradually added to room temperature and allowed to react at room temperature for 1 hour. The reaction mixture was cooled in an ice bath and gradually added with five Fluorophenol (9.2 g, 50 mmol) and triethylamine (10.1 g, 100 mmol) in dichloromethane was added dropwise, Room temperature reaction for 5 hours. After completion of the reaction, 100 ml of ice water was added to the reaction solution, The dichloromethane phase was washed once with saturated aqueous sodium bicarbonate and once with saturated brine, The organic phases were combined and dried over anhydrous sodium sulfate. Filtering, and concentrating under reduced pressure to obtain the crude product;Step c) The concentrated crude product was recrystallized using n-hexane: ethyl acetate (4: 1)To obtain 10.8 g of N - [(S) - (2, 3, 4, 5, 6-pentafluorophenoxy)] phenoxyphosphoryl-L-alanine isopropyl ester, Yield 48percent, HPLC purity 99.3percent.Step 2. Preparation of PPAL-RS (0581) To a stirred solution of PPAL-RS in EtOAc (200 mL) and n-heptane (1.4 L) was added 2, 3, 4, 5, 6-pentafluorophenol (10.1 g) in triethylamine (6 g), and stirring was continued for about 4-8 h. After the R-isomer of the solid was less than 0.5percent, the solid was filtered. The solid was dissolved in EtOAc (4 L), washed with water (2×100 mL), brine (1 L), dried over anhydrous NaTo a solution of 8 (0.13 g, 0.35 mmol, 1.0 equiv.) in anhydrous THF (3.5 mL, 0.10 M), t-BuMgCl (1 M solutionin THF, 0.53 mL, 0.53 mmol, 1.5 equiv.) was added. The reaction mixture was stirred at room temperature for 30 minutes before theaddition of phosphoramidate4 (0.24 g, 0.53 mmol, 1.5 equiv.). After 16 hours, MeOH (0.3 mL)was added and the mixture was condensed under reduced pressure. Purification by C18 reverseflash chromatography (H2O/MeOH, gradient) provided 12 (178 mg, 80 %). Rf = 0.47(DCM/MeOH, 90:10); [alpha]25D +39 (c 1.1, MeOH); Formula: C26H31ClFN6O7P; MW: 624.99g/mol; IR (neat) numax 2980, 2244, 1729, 1592 cm-1; 1H NMR (500 MHz, CD3OD) delta 8.42 (d, J= 2.5 Hz, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.26 (d, J = 7.6 Hz, 2H), 7.19 (t, J = 7.4 Hz, 1H), 6.65(dd, J = 20.3, 3.5 Hz, 1H), 5.12 (dd, J = 52.4, 3.5 Hz, 1H), 4.94 (hept, J = 6.7 Hz, 1H), 4.40 -4.33 (m, 2H), 4.26 (dd, J = 6.9, 4.7 Hz, 1H), 4.19 (s, 3H), 3.91 (dq, J = 10.0, 7.1 Hz, 1H), 2.95(d, J = 17.2 Hz, 1H), 2.90 (d, J = 17.1 Hz, 1H), 1.37 (d, J = 3.5 Hz, 3H), 1.33 (d, J = 7.1 Hz, 3H), 1.20 (d, J = 3.6 Hz, 3H), 1.19 (d, J = 3.6 Hz, 3H) ppm (Labile proton was not observeddue to exchange with deuterated solvent); 13C NMR (126 MHz, CD3OD) delta 174.4 (d, J = 4.5Hz), 162.7, 154.6, 153.9, 152.2 (d, J = 6.8 Hz), 143.8 (d, J = 4.1 Hz), 130.8, 126.2, 121.5 (d, J= 4.9 Hz), 120.9, 118.0, 96.4 (d, J = 196.0 Hz), 85.6 (d, J = 16.9 Hz), 84.4 (d, J = 9.5 Hz), 70.2, 67.2, 55.8 (d, J = 2.9 Hz), 51.7, 47.8 (d, J = 19.5 Hz), 27.1 (d, J = 6.9 Hz), 21.93, 21.86, 20.5(d, J = 6.1 Hz), 14.4 (d, J = 10.3 Hz) ppm (J values result from 13C-F and 13C-P coupling andwere assigned when possible); HRMS (ESI) m/z: [M+Na]+ calcd for C26H31ClFN6NaO7P647.1557; found 647.1564 (+1.17 ppm).To a solution of 11 (54 mg, 0.12 mmol, 1.0 equiv.) in anhydrous THF (2.5 mL, 0.05 M), t-BuMgCl (1 M solution in THF, 0.20 mL, 0.20 mmol, 1.5 equiv.) was added. The reaction mixture was stirred for 30 minutes before the addition of phosphoramidate 4 (85 mg, 0.19 mmol, 1.5 equiv.). After 16 hours, MeOH (0.3 mL) was added and the mixture was condensed under reduced pressure. Purification by flash chromatography on silica gel (CHCl3/MeOH) followed by C18 reverse flash chromatography (H2O/MeOH) provided 14 (67 mg, 76 %). Rf = 0.64 (DCM/MeOH, 90:10); [alpha]25D +37 (c 0.67, CD3OD); Formula: C32H36ClFN7O6P; MW: 700.11 g/mol; IR (neat) numax 2979, 2249, 1728, 1619 cm-1; 1H NMR (500 MHz, CD3OD) delta 8.19 (d, J = 2.4 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.39 - 7.29 (m, 4H), 7.29 - 7.23 (m, 3H), 7.22 - 7.16 (m, 1H), 6.54 (dd, J = 20.6, 3.4 Hz, 1H), 5.07 (dd, J = 52.5, 3.4 Hz, 1H), 4.93 (hept, J = 6.2 Hz, 1H), 4.76 (s, 2H), 4.39 - 4.28 (m, 2H), 4.24 (dd, J = 7.1, 4.5 Hz, 1H), 3.91 (dq, J = 9.8, 7.1 Hz, 1H), 2.93 (d, J = 17.0 Hz, 1H), 2.88 (d, J = 17.1 Hz, 1H), 1.36 (d, J = 3.3 Hz, 3H), 1.32 (dd, J = 7.1, 1.0 Hz, 3H), 1.18 (appt, J = 6.5 Hz, 6H) ppm (Labile protons were not observed due to exchange with deuterated solvent); 13C NMR (126 MHz, CD3OD) delta 174.4 (d, J = 5.6 Hz), 156.5, 155.9, 152.2 (d, J = 7.0 Hz), 150.8, 141.1 (d, J = 3.3 Hz), 139.9, 130.8, 129.6, 128.9, 128.4, 126.3, 121.5 (d, J = 4.8 Hz), 119.0, 118.0, 96.4 (d, J = 196.5 Hz), 85.3 (d, J = 16.5 Hz), 84.2 (d, J = 8.4 Hz), 70.2, 67.3 (dd, J = 5.0, 2.2 Hz), 51.7, 47.8 (d, J = 19.2 Hz), 45.2, 27.0 (d, J = 7.2 Hz), 21.93, 21.87, 20.5 (d, J = 6.4 Hz), 14.4 (d, J = 11.0 Hz) ppm (J values result from 13C-F and 13C-P coupling and were assigned when possible); HRMS (ESI) m/z: [M+Na]+ calcd for C32H36ClFN7NaO6P 722.2029; found 722.2020 (-1.35 ppm).To a solution of 9 (63 mg, 0.18 mmol, 1.0 equiv.) inanhydrous THF (2.0 mL, 0.10 M), t-BuMgCl (1 M solutionin THF, 0.30 mL, 0.30 mmol, 1.5 equiv.) was added. The reactionmixture was stirred for 30 minutes before the addition of phosphoramidate4(0.13 g, 0.29 mmol, 1.5 equiv.). After 16 hours, MeOH (0.3 mL) was added and the mixturewas condensed under reduced pressure. Purification by C18 reverse flash chromatography(H2O/MeOH) provided 13 (98 mg, 87 %). [alpha]25D +46 (c 1.1, MeOH); Formula:C25H30ClFN7O6P; MW: 609.98 g/mol; IR (neat) numax 2981, 2243, 1731, 1615 cm-1; 1H NMR(500 MHz, CD3OD) delta 8.23 (d, J = 2.5 Hz, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 6.54 (dd, J = 20.6, 3.4 Hz, 1H), 5.07 (dd, J = 52.4, 3.5 Hz, 1H), 4.94 (hept, J = 6.1 Hz, 1H), 4.40 - 4.29 (m, 2H), 4.24 (dd, J = 7.1, 4.6 Hz, 1H), 3.92 (dq, J = 9.9, 7.1 Hz, 1H), 2.93 (d, J = 17.1 Hz, 1H), 2.88 (d, J = 17.1 Hz, 1H), 1.36 (d, J = 3.3 Hz, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.19 (appt, J = 5.6 Hz, 6H) ppm (Labile protons were not observed due to exchange with deuterated solvent); 13C NMR (126 MHz, CD3OD) delta 174.4 (d, J = 5.3 Hz), 158.1, 155.6, 152.2 (d, J = 6.9 Hz), 151.5, 141.5 (d, J = 4.9 Hz), 130.8, 126.3, 121.5 (d, J = 4.8 Hz), 118.6, 118.0, 96.4 (d, J = 196.5 Hz), 85.3 (d, J = 16.6 Hz), 84.2 (d, J = 8.2 Hz), 70.2, 67.3 (dd, J = 5.5, 2.5 Hz), 51.7, 47.8 (d, J = 19.3 Hz), 27.1 (d, J = 7.1 Hz), 21.94, 21.87, 20.5 (d, J = 6.4 Hz), 14.4 (d, J = 11.0 Hz) ppm (J values result from 13C-F and 13C-P coupling and were assigned when possible); HRMS (ESI) m/z: [M+Na]+ calcd for C25H30ClFN7NaO6P 632.1560; found 632.1545 (-2.33 ppm).To asolution of compound 9a (1.5 g, 4.39 mmol) in tetrahydrofuran(12.0 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (1.5 mL) at0 C, was added a tetrahydrofuran solution of tert-butylmagnesiumchloride (1 M, 4.39 mL, 4.39 mmol). The resulting suspension wasstirred at 0 C for 30 min and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (3.98 g, 8.78 mmol) wasadded. The resultant mixture was allowed to warm to room temperature.After 1.5 h an aqueous solution of half saturated NaHCO3 (75 mL)and then extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried over MgSO4 and concentrated. The crude productwas purified by flash chromatography on silica eluting with asolvent gradient of 0-5% methanol in dichloromethane to providecompound 16a (2.17 g, 81% yield) containing 6 wt% of 1, 3-dimethyltetrahydropyrimidin-2(1H)-one by NMR. 1H NMR (400 MHz, DMSO-d6) delta ppm 1.14 (d, J=6.3 Hz, 6Eta), 1.21 (d, J=7.1 Hz, 3Eta), 3.79 (m, 1Eta), 4.00 (m, 1Eta), 4.08 (m, 1Eta), 4.30 (m, 2Eta), 4.84 (m, 1Eta), 5.56 (d, J=8.2 Hz, 1Eta), 6.08 (dd, J=13.1, 10.1 Hz, 1Eta), 6.62 (s, 1Eta), 6.99 (d, J=5.5 Hz, 1Eta), 7.20 (m, 3Eta), 7.36 (m, 2Eta), 7.63 (d, J=8.1 Hz, 1Eta), 11.58 (s, 1Eta); 13C NMR (101 MHz, DMSO-d6) delta 20.26(d, J=6.5 Hz), 21.84, 21.89, 50.25, 64.58, 68.52, 77.00, 80.23, 84.59, 102.55, 120.51 (d, J=4.9 Hz), 125.14, 130.17, 150.72, 151.09 (d, J=6.5 Hz), 163.09, 173.05; 31P NMR (162 MHz, DMSO-d6) delta 3.28. MS(ESI) m/z 612.0 (Mu+Eta)+.General procedure: To asolution of compound 9a (1.5 g, 4.39 mmol) in tetrahydrofuran(12.0 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (1.5 mL) at0 C, was added a tetrahydrofuran solution of tert-butylmagnesiumchloride (1 M, 4.39 mL, 4.39 mmol). The resulting suspension wasstirred at 0 C for 30 min and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (3.98 g, 8.78 mmol) wasadded. The resultant mixture was allowed to warm to room temperature.After 1.5 h an aqueous solution of half saturated NaHCO3 (75 mL)and then extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried over MgSO4 and concentrated. The crude productwas purified by flash chromatography on silica eluting with asolvent gradient of 0-5% methanol in dichloromethane to providecompound 16a (2.17 g, 81% yield) containing 6 wt% of 1, 3-dimethyltetrahydropyrimidin-2(1H)-one by NMR. 1H NMR (400 MHz, DMSO-d6) delta ppm 1.14 (d, J=6.3 Hz, 6Eta), 1.21 (d, J=7.1 Hz, 3Eta), 3.79 (m, 1Eta), 4.00 (m, 1Eta), 4.08 (m, 1Eta), 4.30 (m, 2Eta), 4.84 (m, 1Eta), 5.56 (d, J=8.2 Hz, 1Eta), 6.08 (dd, J=13.1, 10.1 Hz, 1Eta), 6.62 (s, 1Eta), 6.99 (d, J=5.5 Hz, 1Eta), 7.20 (m, 3Eta), 7.36 (m, 2Eta), 7.63 (d, J=8.1 Hz, 1Eta), 11.58 (s, 1Eta); 13C NMR (101 MHz, DMSO-d6) delta 20.26(d, J=6.5 Hz), 21.84, 21.89, 50.25, 64.58, 68.52, 77.00, 80.23, 84.59, 102.55, 120.51 (d, J=4.9 Hz), 125.14, 130.17, 150.72, 151.09 (d, J=6.5 Hz), 163.09, 173.05; 31P NMR (162 MHz, DMSO-d6) delta 3.28. MS(ESI) m/z 612.0 (Mu+Eta)+.To a solution ofcompound40(34.8 mg, 0.134mmol) in a mixture oftetrahydrofuran(0.25 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (0.1 mL)at 0C was added a solutionofcichloro(2, 2, 6, 6-tetramethylpiperidinato)magnesate(1-) lithium(1:1) (86.5 muL, 0.087mmol)dropwise. The mixture was stirred at 0 C for 1 hour, and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(76.8 mg, 0.169mmol) was added. The mixture was allowed to slowly warm to room temperature and stirredovernight.Thesuspension was cooled to 0 C, and quenched with acetic acid (8 muL, 0.140mmol), warmed to room temperature, and diluted with water (0.1% TFA) and acetonitrile, thenPurified by reverse phase preparative HPLC on a Waters Nova-PakHR C18 6 mum 60A Prep-Pakcartridge column (40 mm×100 mm) using a gradient of 20% to 80% acetonitrile (A) and 0.1%trifluoroaceticacid in water (B) over 12 minutes and then 100% A for 5 minutes at a flow rate of 60 mL/minute to provide compound16i(45 mg, 0.081mmol, 60.4 % yield).1HNMR (400 MHz, DMSO-d6) delta ppm 1.16 (d, J= 6.2 Hz, 6H), 1.23 (d, J= 7.1 Hz, 3H), 1.37 (d, J= 23.6 Hz, 3H), 3.79 (tq, J= 10.2, 7.0 Hz, 1H), 3.92 - 3.97 (m, 1H), 4.03 (dd, J= 20.7, 5.1 Hz, 1H), 4.17 (dt, J= 11.8, 6.7 Hz, 1H), 4.26 (ddd, J= 10.8, 6.8, 3.6 Hz, 1H), 4.86 (h, J= 6.1 Hz, 1H), 5.56 (dd, J= 8.2, 2.2 Hz, 1H), 5.95 (d, J= 17.1 Hz, 1H), 6.02 (dd, J= 13.1, 10.0 Hz, 1H), 7.19 (dd, J= 15.6, 7.8 Hz, 3H), 7.37 (t, J= 7.9 Hz, 2H), 7.53 (dd, J= 8.1, 2.6 Hz, 1H), 11.47 (d, J= 2.3 Hz, 1H);MS (ESI+)m/z530.0 [Mu+Eta]+.Compound16gwas prepared using a method similar to that described for the synthesis of compound16a, using compound30and(S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate. Purification by column chromatography on silica gel eluting with 0-100% ethyl acetate inheptanesprovided the titledcompound (10.5 mg).1H NMR (400 MHz, DMSO-d6) delta ppm 0.80 (s, 3H), 1.05 (s, 3H), 1.15 (d, J= 6.2 Hz, 6H), 1.23 (d, J= 7.1 Hz, 3H), 3.70 (d, J= 8.8 Hz, 1H), 3.76 - 3.85 (m, 1H), 3.85 - 3.92 (m, 1H), 4.18 - 4.26 (m, 1H), 4.33 (ddd, J= 11.7, 6.1, 2.1 Hz, 1H), 4.86 (hept, J= 6.2 Hz, 1H), 5.49 (dd, J= 8.1, 2.2 Hz, 1H), 5.73 (s, 1H), 6.00 (dd, J= 12.9, 10.0 Hz, 1H), 7.18 (t, J= 7.4 Hz, 1H), 7.23 (d, J= 8.1 Hz, 2H), 7.34 - 7.41 (m, 2H), 7.61 (d, J= 8.1 Hz, 1H), 11.36 (d, J= 2.3 Hz, 1H);MS (ESI+)m/z525.9 (Mu+Eta)+, 548.1 (Mu+Na)+, 1072.9 (2M+Na)+.General procedure: To asolution of compound 9a (1.5 g, 4.39 mmol) in tetrahydrofuran(12.0 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (1.5 mL) at0 C, was added a tetrahydrofuran solution of tert-butylmagnesiumchloride (1 M, 4.39 mL, 4.39 mmol). The resulting suspension wasstirred at 0 C for 30 min and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (3.98 g, 8.78 mmol) wasadded. The resultant mixture was allowed to warm to room temperature.After 1.5 h an aqueous solution of half saturated NaHCO3 (75 mL)and then extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried over MgSO4 and concentrated. The crude productwas purified by flash chromatography on silica eluting with asolvent gradient of 0-5% methanol in dichloromethane to providecompound 16a (2.17 g, 81% yield) containing 6 wt% of 1, 3-dimethyltetrahydropyrimidin-2(1H)-one by NMR. 1H NMR (400 MHz, DMSO-d6) delta ppm 1.14 (d, J=6.3 Hz, 6Eta), 1.21 (d, J=7.1 Hz, 3Eta), 3.79 (m, 1Eta), 4.00 (m, 1Eta), 4.08 (m, 1Eta), 4.30 (m, 2Eta), 4.84 (m, 1Eta), 5.56 (d, J=8.2 Hz, 1Eta), 6.08 (dd, J=13.1, 10.1 Hz, 1Eta), 6.62 (s, 1Eta), 6.99 (d, J=5.5 Hz, 1Eta), 7.20 (m, 3Eta), 7.36 (m, 2Eta), 7.63 (d, J=8.1 Hz, 1Eta), 11.58 (s, 1Eta); 13C NMR (101 MHz, DMSO-d6) delta 20.26(d, J=6.5 Hz), 21.84, 21.89, 50.25, 64.58, 68.52, 77.00, 80.23, 84.59, 102.55, 120.51 (d, J=4.9 Hz), 125.14, 130.17, 150.72, 151.09 (d, J=6.5 Hz), 163.09, 173.05; 31P NMR (162 MHz, DMSO-d6) delta 3.28. MS(ESI) m/z 612.0 (Mu+Eta)+.General procedure: To asolution of compound 9a (1.5 g, 4.39 mmol) in tetrahydrofuran(12.0 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (1.5 mL) at0 C, was added a tetrahydrofuran solution of tert-butylmagnesiumchloride (1 M, 4.39 mL, 4.39 mmol). The resulting suspension wasstirred at 0 C for 30 min and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (3.98 g, 8.78 mmol) wasadded. The resultant mixture was allowed to warm to room temperature.After 1.5 h an aqueous solution of half saturated NaHCO3 (75 mL)and then extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried over MgSO4 and concentrated. The crude productwas purified by flash chromatography on silica eluting with asolvent gradient of 0-5% methanol in dichloromethane to providecompound 16a (2.17 g, 81% yield) containing 6 wt% of 1, 3-dimethyltetrahydropyrimidin-2(1H)-one by NMR. 1H NMR (400 MHz, DMSO-d6) delta ppm 1.14 (d, J=6.3 Hz, 6Eta), 1.21 (d, J=7.1 Hz, 3Eta), 3.79 (m, 1Eta), 4.00 (m, 1Eta), 4.08 (m, 1Eta), 4.30 (m, 2Eta), 4.84 (m, 1Eta), 5.56 (d, J=8.2 Hz, 1Eta), 6.08 (dd, J=13.1, 10.1 Hz, 1Eta), 6.62 (s, 1Eta), 6.99 (d, J=5.5 Hz, 1Eta), 7.20 (m, 3Eta), 7.36 (m, 2Eta), 7.63 (d, J=8.1 Hz, 1Eta), 11.58 (s, 1Eta); 13C NMR (101 MHz, DMSO-d6) delta 20.26(d, J=6.5 Hz), 21.84, 21.89, 50.25, 64.58, 68.52, 77.00, 80.23, 84.59, 102.55, 120.51 (d, J=4.9 Hz), 125.14, 130.17, 150.72, 151.09 (d, J=6.5 Hz), 163.09, 173.05; 31P NMR (162 MHz, DMSO-d6) delta 3.28. MS(ESI) m/z 612.0 (Mu+Eta)+.General procedure: To asolution of compound 9a (1.5 g, 4.39 mmol) in tetrahydrofuran(12.0 mL) and 1, 3-dimethyltetrahydropyrimidin-2(1H)-one (1.5 mL) at0 C, was added a tetrahydrofuran solution of tert-butylmagnesiumchloride (1 M, 4.39 mL, 4.39 mmol). The resulting suspension wasstirred at 0 C for 30 min and (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (3.98 g, 8.78 mmol) wasadded. The resultant mixture was allowed to warm to room temperature.After 1.5 h an aqueous solution of half saturated NaHCO3 (75 mL)and then extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried over MgSO4 and concentrated. The crude productwas purified by flash chromatography on silica eluting with asolvent gradient of 0-5% methanol in dichloromethane to providecompound 16a (2.17 g, 81% yield) containing 6 wt% of 1, 3-dimethyltetrahydropyrimidin-2(1H)-one by NMR. 1H NMR (400 MHz, DMSO-d6) delta ppm 1.14 (d, J=6.3 Hz, 6Eta), 1.21 (d, J=7.1 Hz, 3Eta), 3.79 (m, 1Eta), 4.00 (m, 1Eta), 4.08 (m, 1Eta), 4.30 (m, 2Eta), 4.84 (m, 1Eta), 5.56 (d, J=8.2 Hz, 1Eta), 6.08 (dd, J=13.1, 10.1 Hz, 1Eta), 6.62 (s, 1Eta), 6.99 (d, J=5.5 Hz, 1Eta), 7.20 (m, 3Eta), 7.36 (m, 2Eta), 7.63 (d, J=8.1 Hz, 1Eta), 11.58 (s, 1Eta); 13C NMR (101 MHz, DMSO-d6) delta 20.26(d, J=6.5 Hz), 21.84, 21.89, 50.25, 64.58, 68.52, 77.00, 80.23, 84.59, 102.55, 120.51 (d, J=4.9 Hz), 125.14, 130.17, 150.72, 151.09 (d, J=6.5 Hz), 163.09, 173.05; 31P NMR (162 MHz, DMSO-d6) delta 3.28. MS(ESI) m/z 612.0 (Mu+Eta)+.

Molecular Weight:453.3
XLogP3:4.5
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:11
Rotatable Bond Count:9
Exact Mass:453.07645045
Monoisotopic Mass:453.07645045
Topological Polar Surface Area:73.9
Heavy Atom Count:30
Complexity:608
Defined Atom Stereocenter Count:1
Undefined Atom Stereocenter Count:1
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes