N-(5-{2-[ethyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-benzo[1,3]dioxol-2-ylmethyl)-succinamic acid 2,5-dioxo-pyrrolidin-1-yl ester → Methylenedioxyethamphetamine

HRID 2212468

Reaction equation

HRID 2212468 reaction equation
O=C1CCC(=O)N1OC(=O)CCC(NC[CH:16]1[O:20][C:19]2[CH:21]=[CH:22][C:23]([CH2:25][CH:26]([N:28](CC)[C:29](=O)[C:30](F)(F)F)[CH3:27])=[CH:24][C:18]=2[O:17]1)=O>P([O-])([O-])([O-])=O.[K+].[K+].[K+].CN(C=O)C>[CH3:30][CH2:29][NH:28][CH:26]([CH2:25][C:23]1[CH:22]=[CH:21][C:19]2[O:20][CH2:16][O:17][C:18]=2[CH:24]=1)[CH3:27]

Participants

4

Reactant 1

HCID 86595989N-(5-{2-[ethyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-benzo[1,3]dioxol-2-ylmethyl)-succinamic acid 2,5-dioxo-pyrrolidin-1-yl ester

Product 1

HCID 105039Methylenedioxyethamphetamine

Reagents, catalysts & solvents 2

HCID 6228DimethylformamideSolventHCID 62657Potassium Phosphate, TribasicSolvent

Procedure

MDEA-BSA conjugate 36 was synthesized as described in copending, commonly assigned U.S. Ser. No. 10/622,254 filed contemporaneously herewith. A solution of 500 mg of bovine serum albumin (BSA) in 6.7 mL of 50 mM potassium phosphate (pH 7.5) was cooled in an ice-bath. To the solution was added 8.5 mL of DM50 dropwise, and the reaction mixture was maintained below room temperature. To the protein solution was added a solution of 12 mg (0.022 mmol) of N-(5-{2-[ethyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-benzo[1,3]dioxol-2-ylmethyl)-succinamic acid 2,5-dioxo-pyrrolidin-1-yl ester (compound 2L in U.S. Ser. No. 10/622,254) in 1.5 mL of anhydrous DMF dropwise. The reaction mixture was allowed to stir at room temperature 48 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of 70% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 50% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 30% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours) at room temperature. The trifluoroacetamido group of the conjugate was deprotected by dialysis of the resulting conjugate against 10% ammonium hydroxide for 3 days (1 L each for approximately 24 hours each), followed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C. (1 L each). The protein concentration was determined to be 7.12 mg/mL using BioRad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248, 1976). A total of 45 mL of the conjugate was obtained.

Workup

  1. additionTo the solution was added 8.5 mL of DM50 dropwise, and the reaction mixture
  2. temperaturewas maintained below room temperature
  3. customThe resulting conjugate was placed in a dialysis tube
  4. custom(10,000 MW cut-off) and was dialyzed in 1 L of 70% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 50% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 30% DMSO in 50 mM potassium phosphate (at least 3 hours)
  5. custom1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours)
  6. customat room temperature
  7. customThe trifluoroacetamido group of the conjugate was deprotected by dialysis of the resulting conjugate against 10% ammonium hydroxide for 3 days (1 L each for approximately 24 hours each),
  8. customfollowed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C.
  9. concentrationThe protein concentration
  10. customA total of 45 mL of the conjugate was obtained

Sources & evidence

ORD record
ord-91bfb8dc7ac541e9ae035ad70237f987
Source dataset
uspto-grants-2006_09
Patent number
US07101980B2

Linked notes

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