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.
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.
Yin
Wenji
W
Department of Drug Metabolism, Merck Research Laboratories, Merck & Co. Inc., Rahway, NJ 07065, USA.
Doss
George A
GA
Stearns
Ralph A
RA
Chaudhary
Ashok G
AG
Hop
Cornelis E
CE
Franklin
Ronald B
RB
Kumar
Sanjeev
S
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
D010880:Piperidines; D010881:Piperidones; D011759:Pyrrolidines; D014216:Triacetoneamine-N-Oxyl; C002273:tempidon