title: A novel P450-catalyzed transformation of the 2,2,6,6-tetramethyl piperidine moiety to a 2,2-dimethyl pyrrolidine in human liver microsomes: characterization by high resolution quadrupole-time-of-flight mass spectrometry and 1H-NMR.
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 C(3)H(6) 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 using (1)H-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.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, Merck & Co. Inc., Rahway, NJ 07065, USA.
authors.forename: Wenji
authors.initials: W
authors.lastname: Yin
authors.forename: George A
authors.initials: GA
authors.lastname: Doss
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Stearns
authors.forename: Ashok G
authors.initials: AG
authors.lastname: Chaudhary
authors.forename: Cornelis E
authors.initials: CE
authors.lastname: Hop
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Franklin
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
mesh_terms: D001711:Biotransformation; D002384:Catalysis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D010880:Piperidines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D010881:Piperidones / Q000737:chemistry; D011759:Pyrrolidines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D014216:Triacetoneamine-N-Oxyl / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D010880:Piperidines; D010881:Piperidones; D011759:Pyrrolidines; D014216:Triacetoneamine-N-Oxyl; C002273:tempidon