title: Isolation and structure elucidation of unexpected in-process impurities during tetrazole ring formation of an investigational drug substance.
abstract: During the formation of a tetrazole ring on an investigational drug, two in-process impurities were detected and analyzed by LC-MS, which suggested that both impurities were drug-related with the same mass-to-charge ratio. To understand and control their formation, both impurities were isolated from the mother liquor of the reaction using a multi-step isolation procedure to obtain a sufficient amount for high-resolution mass spectrometry (HRMS) and NMR structural analysis. HRMS suggested a protonated mass of 577.32 Da for both impurities; however, MS fragmentation patterns provided limited information on their structures. NMR analysis indicated the presence on an additional NH functional group in both isolates with similar spatial and bond correlations to one of the dimethylcarbamoyl moieties and the corresponding aromatic ring. A phenyldimethylcarbamoylamino moiety was supported by the NMR and HRMS data and could be explained based on the 'Schmidt-like' reaction mechanism, which was an unexpected reaction pathway. Because the reaction conditions were fixed because of safety concerns, the crystallization protocol was redesigned to reduce the levels of these impurities significantly. Copyright © 2016 John Wiley & Sons, Ltd.
authors: Department of Attribute Sciences, Amgen Inc., Thousand Oaks, CA, USA.
authors: Maria Victoria
authors: http://orcid.org/0000-0002-8429-9534
authors: MV
authors: Silva Elipe
authors: Currently at St. Jude Medical, Sylmar, CA, USA.
authors: Chul
authors: C
authors: Yoo
authors: Currently at ASK-Gene Pharma, Inc., Camarillo, CA, USA.
authors: Fang
authors: F
authors: Xia
authors: Currently at Kythera Biopharmaceuticals, Westlake Village, CA, USA.
authors: Jason
authors: J
authors: Simiens
authors: Department of Attribute Sciences, Amgen Inc., Thousand Oaks, CA, USA.
authors: Kevin
authors: K
authors: Crossley
authors: Department of Attribute Sciences, Amgen Inc., Thousand Oaks, CA, USA.
authors: John R
authors: JR
authors: Huckins
authors: Currently at Gilead Sciences, Foster City, CA, USA.
authors: Hong-Xun
authors: HX
authors: Guo
authors: Department of Attribute Sciences, Amgen Inc., Thousand Oaks, CA, USA.
authors: Jason
authors: J
authors: Tedrow
authors: Currently at Kythera Biopharmaceuticals, Westlake Village, CA, USA.
authors: Kirby
authors: K
authors: Wong-Moon
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D061565:Cycloaddition Reaction; D004340:Drug Contamination; D015507:Drugs, Investigational / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D013777:Tetrazoles / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D015507:Drugs, Investigational; D013777:Tetrazoles
keywords: 13C NMR; 15N NMR; 1H NMR; HRMS; NMR; Schmidt-like reaction mechanism; drug development; in-process impurities; investigational drug; isolation