title: A novel biotransformation of alkyl aminopyrrolidine to aminopiperidine ring by human CYP3A.
abstract: The novel biotransformation of an aminopyrrolidine to an aminopiperidine during the metabolism of 5-(4-chlorophenyl)-3-methyl-2-((2R)-2-(((1-methylethyl)amino)methyl)-1-pyrrolidinyl)-6-(4-pyridinyl)-4(3H)-pyrimidinone (AMG657417) was investigated using the NADPH-fortified S9 fraction from human liver. The major metabolite (M18) had a protonated molecule (MH(+) m/z 438) identical to that of AMG657417 except that it eluted earlier on a reverse-phase high-performance liquid chromatography. The structure of M18 had been identified as 5-(4-chlorophenyl)-3-methyl-2-((1-(1-methylethyl)-3-piperidinyl)amino)-6-(4-pyridinyl)-4(3H)-pyrimidinone (I) by liquid chromatography-mass spectrometry and proton NMR. M18 was not observed when AMG657417 was incubated with either microsomal or cytosolic fraction from human liver, suggesting the involvement of both microsomal and cytosolic enzymes in the biotransformation. The reaction mechanisms have been elucidated by trapping the intermediates formed during the biotransformation. An aldehyde intermediate was initially produced by hydroxylation and opening of the pyrrolidine ring of the parent molecule, followed by intramolecular Schiff-base formation between the exocyclic isopropylamine nitrogen and the aldehyde carbonyl to form a piperidinyl iminium ion. The iminium ion was then reduced to the piperidine product. The presence of the aldehyde intermediate was verified by the formation of semicarbazide conjugates in human liver microsomal, S9, and recombinant CYP3A4 incubations of AMG657417. The presence of the piperidinyl iminium ion intermediate was confirmed by the formation of cyanide conjugates in the incubations in human liver S9. Two cyanide conjugates with identical protonated molecule and product ion mass spectra were observed, indicating the likelihood of diastereomer formation. A chemical inhibition study in NADPH-fortified S9 fraction indicated that the oxidation of AMG657417 was catalyzed almost exclusively by CYP3A.
authors.affiliation: Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320-1799, USA.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Chen
authors.forename: Gary L
authors.initials: GL
authors.lastname: Skiles
authors.forename: Magang
authors.initials: M
authors.lastname: Shou
authors.forename: Dean
authors.initials: D
authors.lastname: Hickman
authors.forename: Faye
authors.initials: F
authors.lastname: Hsieh
mesh_terms: D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism / Q000493:pharmacokinetics*; D011744:Pyrimidinones / Q000378:metabolism / Q000493:pharmacokinetics*; D011759:Pyrrolidines / Q000378:metabolism / Q000493:pharmacokinetics*; D012663:Semicarbazides / Q000378:metabolism
chemical_list: C570290:5-(4-chlorophenyl)-3-methyl-2-(2-(((1-methylethyl)amino)methyl)-1-pyrrolidinyl)-6-(4-pyridinyl)-4(3H)-pyrimidinone; D010880:Piperidines; D011744:Pyrimidinones; D011759:Pyrrolidines; D012663:Semicarbazides; C010059:carbamylhydrazine; D051544:Cytochrome P-450 CYP3A; D007654:Ketoconazole