(E)-4-ethoxy-4-oxobut-2-enoic acid

Conditions: temp:70.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: tert-Butyl cis(±)-4-{[(4-chloro-5-ethyl-1H-imidazol-2-yl)carbonyl]amino}-3-methoxypiperidine-1-carboxylate obtained by the method described in Example (1g) (133 mg, 0.34 mmol) was dissolved in methanol (2 mL). A 4 N hydrochloric acid/ethyl acetate solution (4 mL) was added, and the mixture was stirred at 70° C. for 30 minutes. Following concentration under reduced pressure, the residue was dissolved in DMA (5 mL). Fumaric acid monoethyl ester (54.42 mg, 0.38 mmol), WSC hydrochloride (197.41 mg, 1.03 mmol) and DMAP (41.94 mg, 0.34 mmol) were added, and the mixture was stirred at room temperature overnight. Dilute hydrochloric acid was added to the reaction solution, followed by extraction with ethyl acetate. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. concentration under reduced pressure the residue was dissolved in DMA (5 mL) the mixture was stirred at room temperature overnight followed by extraction with ethyl acetate Then, the organic layer was washed with brine dried over anhydrous sodium sulfate

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

Procedure: To (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate (Intermediate 9-2: 87.6 mg, 0.183 mmol) is added a solution of HCl in 1,4-dioxane (0.456 mL, 1.825 mmol) at room temperature. After stirring for 3 hours, the reaction mixture is concentrated under reduced pressure to give (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride. A mixture of (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride, fumaric acid monoethyl ester (33.4 mg, 0.220 mmol), EDCl (63.3 mg, 0.330 mmol), DIPEA (0.058 ml, 0.330 mmol) and HOAt (44.9 mg, 0.330 mmol) in DMF (1.8 ml) is allowed to stir at room-temperature for 3 hour. The reaction mixture is diluted with water, and then the products are extracted with EtOAc. The organic layer is washed with NH4OH, 1M HClaq and brine, dried over Na2SO4, filtered, and concentrated to give crude. The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100) to give (R,E)-ethyl 4-(4-(benzyloxy)-1-(3′-chlorobiphenyl-4-yl)-4-oxobutan-2-ylamino)-4-oxobut-2-enoate (72.9 mg); HPLC retention time=1.40 minutes (condition B); MS (m+1)=506.3; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.31 (t, J=7.1 Hz, 3 H) 2.58 (A of ABX, Jab=16.4 Hz, Jax=5.3 Hz, 1 H) 2.6 (B of ABX, Jab=16.4 Hz, Jbx=5.1 Hz, 1 H) 2.88 (A of ABX, Jab=13.6 Hz, Jax=8.1 Hz, 1 H) 3.03 (B of ABX, Jab=13.6 Hz, Jbx=6.3 Hz, 1 H) 4.24 (q, J=7.1 Hz, 2 H) 4.56-4.64 (m, 1 H) 5.12 (A of AB, J=12.1 Hz, 1 H) 5.18 (B of AB, J=12.1 Hz, 1 H) 6.57 (br d, J=9.1 Hz, 1 H) 6.77 (A of AB, J=15.4 Hz, 1 H) 6.81 (B of AB, J=15.4 Hz, 1 H) 7.19 (br d, J=8.1 Hz, 2 H) 7.29-7.47 (m, 10 H) 7.53-7.54 (m, 1 H). the products are extracted with EtOAc The organic layer is washed with NH4OH, 1M HClaq and brine dried over Na2SO4 filtered concentrated to give crude The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100)

Conditions: temp:70.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: tert-Butyl cis(±)-4-{[(4-chloro-5-ethyl-1H-imidazol-2-yl)carbonyl]amino}-3-methoxypiperidine-1-carboxylate obtained by the method described in Example (1g) (133 mg, 0.34 mmol) was dissolved in methanol (2 mL). A 4 N hydrochloric acid/ethyl acetate solution (4 mL) was added, and the mixture was stirred at 70° C. for 30 minutes. Following concentration under reduced pressure, the residue was dissolved in DMA (5 mL). Fumaric acid monoethyl ester (54.42 mg, 0.38 mmol), WSC hydrochloride (197.41 mg, 1.03 mmol) and DMAP (41.94 mg, 0.34 mmol) were added, and the mixture was stirred at room temperature overnight. Dilute hydrochloric acid was added to the reaction solution, followed by extraction with ethyl acetate. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. concentration under reduced pressure the residue was dissolved in DMA (5 mL) the mixture was stirred at room temperature overnight followed by extraction with ethyl acetate Then, the organic layer was washed with brine dried over anhydrous sodium sulfate

Conditions: temp:70.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: tert-Butyl cis(±)-4-{[(4-chloro-5-ethyl-1H-imidazol-2-yl)carbonyl]amino}-3-methoxypiperidine-1-carboxylate obtained by the method described in Example (1g) (133 mg, 0.34 mmol) was dissolved in methanol (2 mL). A 4 N hydrochloric acid/ethyl acetate solution (4 mL) was added, and the mixture was stirred at 70° C. for 30 minutes. Following concentration under reduced pressure, the residue was dissolved in DMA (5 mL). Fumaric acid monoethyl ester (54.42 mg, 0.38 mmol), WSC hydrochloride (197.41 mg, 1.03 mmol) and DMAP (41.94 mg, 0.34 mmol) were added, and the mixture was stirred at room temperature overnight. Dilute hydrochloric acid was added to the reaction solution, followed by extraction with ethyl acetate. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. concentration under reduced pressure the residue was dissolved in DMA (5 mL) the mixture was stirred at room temperature overnight followed by extraction with ethyl acetate Then, the organic layer was washed with brine dried over anhydrous sodium sulfate

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

Procedure: To (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate (87.6 mg, 0.183 mmol) is added a solution of HCl in 1,4-dioxane (0.456 mL, 1.825 mmol) at room temperature. After stirring for 3 hours, the reaction mixture is concentrated under reduced pressure to give (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride. A mixture of (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride, fumaric acid monoethyl ester (33.4 mg, 0.220 mmol), EDCl (63.3 mg, 0.330 mmol), DIPEA (0.058 ml, 0.330 mmol) and HOAt (44.9 mg, 0.330 mmol) in DMF (1.8 ml) is allowed to stir at room temperature for 3 hour. The reaction mixture is diluted with water, and then the products are extracted with EtOAc. The organic layer is washed with NH4OH, 1M HClaq and brine, dried over Na2SO4, filtered, and concentrated to give crude. The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100) to give (R,E)-ethyl 4-(4-(benzyloxy)-1-(3′-chlorobiphenyl-4-yl)-4-oxobutan-2-ylamino)-4-oxobut-2-enoate (72.9 mg); HPLC retention time=1.40 minutes (condition B); MS (m+1)=506.3; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.31 (t, J=7.1 Hz, 3H) 2.58 (A of ABX, Jab=16.4 Hz, Jax=5.3 Hz, 1H) 2.6 (B of ABX, Jab=16.4 Hz, Jbx=5.1 Hz, 1H) 2.88 (A of ABX, Jab=13.6 Hz, Jax=8.1 Hz, 1H) 3.03 (B of ABX, Jab=13.6 Hz, Jbx=6.3 Hz, 1H) 4.24 (q, J=7.1 Hz, 2H) 4.56-4.64 (m, 1H) 5.12 (A of AB, J=12.1 Hz, 1H) 5.18 (B of AB, J=12.1 Hz, 1H) 6.57 (br d, J=9.1 Hz, 1H) 6.77 (A of AB, J=15.4 Hz, 1H) 6.81 (B of AB, J=15.4 Hz, 1H) 7.19 (br d, J=8.1 Hz, 2H) 7.29-7.47 (m, 10H) 7.53-7.54 (m, 1H). the products are extracted with EtOAc The organic layer is washed with NH4OH, 1M HClaq and brine dried over Na2SO4 filtered concentrated to give crude The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100)

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

Procedure: To (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate (87.6 mg, 0.183 mmol) is added a solution of HCl in 1,4-dioxane (0.456 mL, 1.825 mmol) at room temperature. After stirring for 3 hours, the reaction mixture is concentrated under reduced pressure to give (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride. A mixture of (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride, fumaric acid monoethyl ester (33.4 mg, 0.220 mmol), EDCI (63.3 mg, 0.330 mmol), DIPEA (0.058 ml, 0.330 mmol) and HOAt (44.9 mg, 0.330 mmol) in DMF (1.8 ml) is allowed to stir at room temperature for 3 hour. The reaction mixture is diluted with water, and then the products are extracted with EtOAc. The organic layer is washed with NH4OH, 1M HCl aq and brine, dried over Na2SO4, filtered, and concentrated to give crude. The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100) to give (R,E)-ethyl 4-(4-(benzyloxy)-1-(3′-chlorobiphenyl-4-yl)-4-oxobutan-2-ylamino)-4-oxobut-2-enoate (72.9 mg); HPLC retention time=1.40 minutes (condition B); MS (m+1)=506.3; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.31 (t, J=7.1 Hz, 3 H) 2.58 (A of ABX, Jab=16.4 Hz, Jax=5.3 Hz, 1 H) 2.6 (B of ABX, Jab=16.4 Hz, Jbx=5.1 Hz, 1 H) 2.88 (A of ABX, Jab=13.6 Hz, Jax=8.1 Hz, 1 H) 3.03 (B of ABX, Jab=13.6 Hz, Jbx=6.3 Hz, 1 H) 4.24 (q, J=7.1 Hz, 2 H) 4.56-4.64 (m, 1 H) 5.12 (A of AB, J=12.1 Hz, 1 H) 5.18 (B of AB, J=12.1 Hz, 1 H) 6.57 (br d, J=9.1 Hz, 1 H) 6.77 (A of AB, J=15.4 Hz, 1 H) 6.81 (B of AB, J=15.4 Hz, 1 H) 7.19 (br d, J=8.1 Hz, 2 H) 7.29-7.47 (m, 10 H) 7.53-7.54 (m, 1 H). the products are extracted with EtOAc The organic layer is washed with NH4OH, 1M HCl aq and brine dried over Na2SO4 filtered concentrated to give crude The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100)

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

Procedure: To a solution of fumaric acid mono-ethyl ester (721 mg, 5 mmol) and tert-butanol (0.938 mL, 10 mmol) in 10 mL of DCM was added DMAP (122 mg, 1 mmol) followed by DCC (2.06 g, 10 mmol). The resulting mixture was stirred at rt for 16 h, taken to dryness under reduced pressure, stirred overnight with 50 mL acetone and filtered to remove DCU. The resulting filtrate was concentrated under reduced pressure, and the residue taken up in EtOAc. The EtOAc was washed twice with 30 mL 0.1 M KHSO4, and once with 30 mL saturated NaHCO3 and once with 30 mL of brine. The resulting EtOAc solution was treated with charcoal and dried over magnesium sulfate, concentrated under reduced pressure, and subjected to silica gel flash chromatography using a gradient of 0–15% EtOAc in hexanes as eluent to give essentially pure (one peak on HPLC) 4-1 (350 mg, 35% yield). filtered to remove DCU The resulting filtrate was concentrated under reduced pressure The EtOAc was washed twice with 30 mL 0.1 M KHSO4 The resulting EtOAc solution was treated with charcoal dried over magnesium sulfate concentrated under reduced pressure

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

Procedure: Maleic anhydride (100.0 g, 1.02 mole) was dissolved in anhydrous ethanol (60 ml, excess) by heating the mixture in an erlenmeyer flask on a steam bath. Thionyl chloride (1.0 ml) was added using a syringe and the mixture heated with stirring for ten minutes. To the turbid mixture was added carbon tetrachloride (100 ml) and the liquid quickly filtered through a Buchner funnel having a coarse glass frit. The solid was discarded. The filtrate was allowed to cool during which the time the ethyl hydrogen fumarate product (92 g, 63%) crystallized. by heating the mixture in an erlenmeyer flask on a steam bath the mixture heated the liquid quickly filtered through a Buchner funnel to cool during which the time the ethyl hydrogen fumarate product (92 g, 63%) crystallized

Conditions: temp:70.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: tert-Butyl cis(±)-4-{[(4-chloro-5-ethyl-1H-imidazol-2-yl)carbonyl]amino}-3-methoxypiperidine-1-carboxylate obtained by the method described in Example (1g) (133 mg, 0.34 mmol) was dissolved in methanol (2 mL). A 4 N hydrochloric acid/ethyl acetate solution (4 mL) was added, and the mixture was stirred at 70° C. for 30 minutes. Following concentration under reduced pressure, the residue was dissolved in DMA (5 mL). Fumaric acid monoethyl ester (54.42 mg, 0.38 mmol), WSC hydrochloride (197.41 mg, 1.03 mmol) and DMAP (41.94 mg, 0.34 mmol) were added, and the mixture was stirred at room temperature overnight. Dilute hydrochloric acid was added to the reaction solution, followed by extraction with ethyl acetate. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. Following concentration under reduced pressure, the residue was purified by silica gel column chromatography (elution solvent: dichloromethane/methanol=40/1, 20/1, 10/1) to obtain 147.3 mg of the title compound as a solid (100%). concentration under reduced pressure the residue was dissolved in DMA (5 mL) the mixture was stirred at room temperature overnight followed by extraction with ethyl acetate Then, the organic layer was washed with brine dried over anhydrous sodium sulfate concentration under reduced pressure the residue was purified by silica gel column chromatography (elution solvent: dichloromethane/methanol=40/1, 20/1, 10/1)

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

Procedure: 351 mg (1 mmol) of 5-aminomethyl-7-bromo-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline hydrochloride, (Example 2) 217 mg (1.5 equiv.) of fumaric acid monoethyl ester, 0.210 ml of triethylamine, 383 mg (2 equiv.) of N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride and 270 mg (2 equiv.) of 1-hydroxy-benzotriazole are stirred at 20° C. in dried dimethylformamide for 18 hours. The mixture is poured into 700 ml of water and 3 ml of 1 N hydrochloric acid and stirred for 15 minutes. The solid is filtered off, washed with water and dried. The solid is filtered off washed with water dried

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

Procedure: To (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate (Intermediate 9-2: 87.6 mg, 0.183 mmol) is added a solution of HCl in 1,4-dioxane (0.456 mL, 1.825 mmol) at room temperature. After stirring for 3 hours, the reaction mixture is concentrated under reduced pressure to give (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride. A mixture of (R)-benzyl 3-amino-4-(3′-chlorobiphenyl-4-yl)butanoate hydrochloride, fumaric acid monoethyl ester (33.4 mg, 0.220 mmol), EDCl (63.3 mg, 0.330 mmol), DIPEA (0.058 ml, 0.330 mmol) and HOAt (44.9 mg, 0.330 mmol) in DMF (1.8 ml) is allowed to stir at room-temperature for 3 hour. The reaction mixture is diluted with water, and then the products are extracted with EtOAc. The organic layer is washed with NH4OH, 1M HClaq and brine, dried over Na2SO4, filtered, and concentrated to give crude. The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100) to give (R,E)-ethyl 4-(4-(benzyloxy)-1-(3′-chlorobiphenyl-4-yl)-4-oxobutan-2-ylamino)-4-oxobut-2-enoate (72.9 mg); HPLC retention time=1.40 minutes (condition B); MS (m+1)=506.3; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.31 (t, J=7.1 Hz, 3 H) 2.58 (A of ABX, Jab=16.4 Hz, Jax=5.3 Hz, 1 H) 2.6 (B of ABX, Jab=16.4 Hz, Jbx=5.1 Hz, 1 H) 2.88 (A of ABX, Jab=13.6 Hz, Jax=8.1 Hz, 1 H) 3.03 (B of ABX, Jab=13.6 Hz, Jbx=6.3 Hz, 1 H) 4.24 (q, J=7.1 Hz, 2 H) 4.56-4.64 (m, 1 H) 5.12 (A of AB, J=12.1 Hz, 1 H) 5.18 (B of AB, J=12.1 Hz, 1 H) 6.57 (br d, J=9.1 Hz, 1 H) 6.77 (A of AB, J=15.4 Hz, 1 H) 6.81 (B of AB, J=15.4 Hz, 1 H) 7.19 (br d, J=8.1 Hz, 2 H) 7.29-7.47 (m, 10 H) 7.53-7.54 (m, 1 H). the products are extracted with EtOAc The organic layer is washed with NH4OH, 1M HClaq and brine dried over Na2SO4 filtered concentrated to give crude The obtained residue is purified by silica gel flash column chromatography (heptane/EtOAc=100:0 to 0:100)