Abstract
We describe herein a novel metabolic fate of the 2,2,6,6-tetramethyl-piperidine (2,2,6,6-TMPi) moiety to a ring-contracted 2,2-dimethyl pyrrolidine (2,2-DMPy) in human liver microsomal incubations. The existence of this pathway was demonstrated for three compounds (I-III) of varied structures suggesting that this may be a general biotransformation reaction for the 2,2,6,6-TMPi moiety. The 2,2-DMPy metabolites formed in incubations of the three compounds with human liver microsomes were characterized by online high performance liquid chromatography coupled to a high resolution hybrid quadrupole-time-of-flight mass spectrometer. Suggested elemental composition obtained from accurate mass measurements of the molecular ions and fragment ions of the metabolites clearly indicated the loss of a mass equivalent to C3H6 from the parent 2,2,6,6-TMPi functionality. Additional accurate tandem mass spectrometry data indicated that one of the original two gem-dimethyl groups was intact in the metabolite structure. Proof of a ring-contracted 2,2-DMPy structure was obtained using1H-NMR experiments on a metabolite purified from liver microsomal incubations, which showed only two geminal methyl groups, instead of four in the parent compound. Two-dimensional correlation spectroscopy and decoupling experiments established aliphatic protons arranged in a pyrrolidine ring pattern. The fact that the formation of 2,2-DMPy metabolites in human liver microsomes was NADPH-dependent suggested that this novel metabolic reaction was catalyzed by the cytochrome P450 (P450) enzyme(s). Immunoinhibition studies in human liver microsomal incubations using anti-P450 monoclonal antibodies and experiments with insect cell microsomes containing individually expressed recombinant human P450 isozymes indicated that multiple P450 isozymes were capable of catalyzing this novel metabolic transformation.
Footnotes
- Abbreviations used are::
- 2,2,6,6-TMPi
- 2,2,6,6-tetramethyl-piperidine
- 2,2-DMPy
- 2,2-dimethyl pyrrolidine
- P450
- cytochrome P450
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- Q-Tof
- quadrupole-time-of-flight
- MS
- mass spectrometry
- CID
- collision-induced dissociation
- HPLC
- high performance liquid chromatography
- Received August 19, 2002.
- Accepted November 12, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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