Application of spin labeling to drug assays. III. [¹⁵N, ²H₁₃]2,2,5,5‐tetramethylpyrroline‐1‐oxyl‐3‐carboxylic acid coupled to phenytoin

Phorone and [²H₁₄]phorone were cyclized with ¹⁵NH₃ or ¹⁵N²H₃ to [¹⁵N]‐ or [¹⁵N, ²H₁₇]‐4‐oxo‐2,2,6,6‐tetramethylpiperidine which were then brominated at C‐3 and C‐5. Ring contraction then gave 2,2,5,5‐tetramethylpyrroline‐3‐carboxamide, which with H₂O₂ gave the corresponding nitroxide. Hydrolysis in base of the doubly labeled nitroxide then gave [¹⁵N, ²H]2,2,5,5‐tetramethylpyrroline‐1‐oxyl‐3‐carboxylic acid. The latter, when coupled to phenytoin, afforded a spin‐labeled drug of high sensitivity for detection by ESR technique. If the synthesis was started with [²H₁₄]phorone, negligible loss of deuterium was noted in the final product even when protiated reagents were used.