(2,5-dioxopyrrolidin-1-yl) (2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoate

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

Procedure: A suspension of 3.40 g (13.5 mmol) of Cbz-valine and 1.56 g (13.5 mmol) of N-hydroxysuccinimide in 200 ml of dichloromethane was treated with 2.86 g (14.9 mmol) of N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride and stirred at ambient temperature for 4 h. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water; dried over Na2SO4, and concentrated in vacuo to provide 4.00 g (85%) of the desired compound. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water dried over Na2SO4 concentrated in vacuo

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2Cl2 (1000 mL) to give compound 2b as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2b. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: A suspension of 3.40 g (13.5 mmol) of Cbz-valine and 1.56 g (13.5 mmol) of N-hydroxysuccinimide in 200 ml of dichloromethane was treated with 2.86 g (14.9 mmol) of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and stirred at ambient temperature for 4 h. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water; dried over Na2SO4, and concentrated in vacuo to provide 4.00 g (85%) of the desired compound. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water dried over Na2SO4 concentrated in vacuo

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

Procedure: A suspension of 3.40 g (13.5 mmol) of Cbz-valine and 1.56 g (13.5 mmol) of N-hydroxysuccinimide in 200 ml of dichloromethane was treated with 2.86 g (14.9 mmol) of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and stirred at ambient temperature for 4 h. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water; dried over Na2SO4, and concentrated in vacuo to provide 4.00 g (85%) of the desired compound. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO3, and water dried over Na2SO4 concentrated in vacuo

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

Procedure: Commercially available reagents and solvents were obtained as follows: HPLC-grade solvents, Fisher; anhydrous solvents, Aldrich; diisopropyl azodicarboxylate (DIAD, 95%), Lancaster; 4-aminobenzyl alcohol, Alfa Aesar; Z-val-OSu, Advanced ChemTech; L-citrulline, Novabiochem; (1S, 2R)-(+)-norephedrine and other commercially available reagents, Aldrich. Cbz-val-cit-PAB-OH (1) (Dubowchik, G. M.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 1. A Model Study of Structural Requirements for Efficient Release of Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3341–3346; Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3347–3352) and combretastatin A-4 (VIIIb) (Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.; Hogan, F. Antineoplastic Agents. 291. Isolation and Synthesis of Combretastatin A-4, A-5, and A-6. J Med. Chem. 1995, 38, 1666–1672) were synthesized as previously described. 1H NMR spectra were recorded on a Varian Gemini 300 MHz spectrophotometer. Flash column chromatography was performed using 230–400 mesh ASTM silica gel from EM Science. Analtech silica gel GHLF plates were used for thin-layer chromatography. HPLC was performed using a Waters Alliance system with a photodiode array detector. Combustion analyses were determined by Quantitative Technologies, Inc., Whitehouse, N.J. Commercially available reagents and solvents were obtained

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

Procedure: Commercially available reagents and solvents were obtained as follows: HPLC-grade solvents, Fisher; anhydrous solvents, Aldrich; diisopropyl azodicarboxylate (DIAD, 95%), Lancaster; 4-aminobenzyl alcohol, Alfa Aesar; Z-val-OSu, Advanced ChemTech; L-citrulline, Novabiochem; (1S, 2R)-(+)-norephedrine and other commercially available reagents, Aldrich. Cbz-val-cit-PAB-OH (1) (Dubowchik, G. M.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 1. A Model Study of Structural Requirements for Efficient Release of Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3341–3346; Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3347–3352) and combretastatin A-4 (VIIIb) (Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.; Hogan, F. Antineoplastic Agents. 291. Isolation and Synthesis of Combretastatin A-4, A-5, and A-6. J Med. Chem. 1995, 38, 1666–1672) were synthesized as previously described. 1H NMR spectra were recorded on a Varian Gemini 300 MHz spectrophotometer. Flash column chromatography was performed using 230–400 mesh ASTM silica gel from EM Science. Analtech silica gel GHLF plates were used for thin-layer chromatography. HPLC was performed using a Waters Alliance system with a photodiode array detector. Combustion analyses were determined by Quantitative Technologies, Inc., Whitehouse, N.J. Commercially available reagents and solvents were obtained

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2Cl2 (1000 mL) to give compound 2 as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2Cl2 (1000 mL) to give compound 2 as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2 Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2 Cl2 (1000 mL) to give compound 2b as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2b. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2 Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2 Cl2 (1000 mL) to give compound 2b as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2b. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: L-Serine (104.19 g, 991 mmol) was dissolved in water (1440 mL) in a 4-L Erlenmeyer flask. Solid NaHCO3 (83.25 g, 991 mmol was added and the mixture was stirred at room temperature to give a clear solution. Cbz-Val-OSu (300.0 g, 861 mmol) was added as a solution in 1,4-dioxane (1500 mL), with additional 1,4-dioxane (220 mL) used to rinse. The resulting cloudy mixture became clear after 1.5 h of stirring at 25° C. After 44 h, the mixture was divided into two equal portions. Methanol (700 mL) and 12 N aqueous HCl (42 mL, 504 mmol) was added to each portion, followed by EtOAc (1000 mL) and a solution of NaCl (100 g) dissolved in water (600 mL). The layers were separated and the organic layer was washed with saturated aqueous NaCl (350 mL). The aqueous layers were extracted in succession with EtOAc (1000 mL). The organic layers resulting from work-up of both portions of the reaction were combined, dried (Na2SO4), filtered, and evaporated to give a white solid (351 g). This material was suspended in CH2 Cl2 (1500 mL) and stirred for 2 h. The mixture was filtered and the crystals were washed with CH2 Cl2 (1000 mL) to give compound 2b as white crystals (262.3 g, 90% yield). 1H NMR (300 MHz, DMSO-d6): consistent with proposed structure for compound 2b. MS: m/z=339.1 (M+1). to give a clear solution to rinse of stirring at 25° C After 44 h The layers were separated the organic layer was washed with saturated aqueous NaCl (350 mL) The aqueous layers were extracted in succession with EtOAc (1000 mL) The organic layers resulting from work-up of both portions of the reaction dried (Na2SO4) filtered evaporated

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

Procedure: Commercially available reagents and solvents were obtained as follows: HPLC-grade solvents, Fisher; anhydrous solvents, Aldrich; diisopropyl azodicarboxylate (DIAD, 95%), Lancaster; 4-aminobenzyl alcohol, Alfa Aesar; Z-val-OSu, Advanced ChemTech; L-citrulline, Novabiochem; (1S, 2R)-(+)-norephedrine and other commercially available reagents, Aldrich. Cbz-val-cit-PAB-OH (1) (Dubowchik, G. M.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 1. A Model Study of Structural Requirements for Efficient Release of Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3341–3346; Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3347–3352) and combretastatin A-4 (VIIIb) (Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.; Hogan, F. Antineoplastic Agents. 291. Isolation and Synthesis of Combretastatin A-4, A-5, and A-6. J Med. Chem. 1995, 38, 1666–1672) were synthesized as previously described. 1H NMR spectra were recorded on a Varian Gemini 300 MHz spectrophotometer. Flash column chromatography was performed using 230–400 mesh ASTM silica gel from EM Science. Analtech silica gel GHLF plates were used for thin-layer chromatography. HPLC was performed using a Waters Alliance system with a photodiode array detector. Combustion analyses were determined by Quantitative Technologies, Inc., Whitehouse, N.J. Commercially available reagents and solvents were obtained