Synthesis of 13C-labeled derivatives of cysteine for magnetic resonance imaging studies of drug uptake and conversion to glutathione in rat brain

Effects of neurodegeneration have been linked to inefficient detoxification of free radicals due to lowered concentrations of antioxidants, especially glutathione, in the brain. In the biosynthesis of glutathione, cysteine concentration is generally the limiting factor. Glutathione and cysteine administrations are not effective treatments for neurodegeneration because glutathione inefficiently crosses cell membranes and cysteine is neurotoxic at high concentrations. Prodrugs of glutathione and cysteine may have more favorable uptake and/or toxicity profiles. Three such prodrugs were synthesized with a 13C‐label such that in vivo uptake of each and conversion to glutathione in the brain could be monitored by magnetic resonance imaging. L‐[3‐13C]‐Cysteine was treated with sodium acetate trihydrate and acetic anhydride to give 2(R)‐N‐acetyl‐[3‐13C]‐cysteine ([13C]‐NAC; 96%). Addition of triphosgene to L‐[3‐13C]‐cysteine provided 4(R)‐[5‐13C]‐2‐oxothiazolidine‐4‐carboxylic acid ([13C]‐OTZ; 65%). A four‐step pathway was used to synthesize ethyl γ‐L‐glutamyl‐[3‐13C]‐L‐cysteinate ([13C]‐GCEE). L‐[3‐13C]‐Cysteine was esterified (100% yield) and then cyclized with acetaldehyde to give ethyl 2(R,S)‐methyl‐[5‐13C]‐thiazolidine‐4(R)‐carboxylate (73%) as a mixture of two diastereomers (65:35). The thiazolidine was silylated (bis(trimethylsilyl)trifluoroacetamide) and reacted with N‐phthaloyl‐L‐glutamic anhydride. Treatment with hydrazine afforded ethyl N‐[γ‐4′(S)‐glutamyl]‐2(R,S)‐methyl‐[5‐13C]‐thiazolidine‐4(R)‐carboxylate (48%; 73:27 mixture of diastereomers). This was converted to the desired product, [13C]‐GCEE (49%), using mercury (II) acetate and hydrogen sulfide. Three prodrugs of glutathione and cysteine were synthesized with 13C labeling such that in vivo uptake of each and conversion to glutathione in the brain could be monitored by magnetic resonance imaging. L‐[3‐13C]‐Cysteine was used to generate: N‐acetyl‐[3‐13C]‐cysteine ([13C]‐NAC); 4(R)‐[5‐13C]‐2‐oxothiazolidine‐4‐carboxylic acid ([13C]‐OTZ); and ethyl γ‐L‐glutamyl‐[3‐13C]‐L‐cysteinate ([13C]‐GCEE).