title: Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate.
abstract: 2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,β-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,β-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.
authors.affiliation: Drug Safety Sciences, Johnson and Johnson Pharmaceutical Research and Development, 1000 Route 202 South, New Jersey 08869, USA. hlim5@its.jnj.com
authors.forename: Heng-Keang
authors.initials: HK
authors.lastname: Lim
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Carlo
authors.initials: C
authors.lastname: Sensenhauser
authors.forename: Kevin
authors.initials: K
authors.lastname: Cook
authors.forename: Robert
authors.initials: R
authors.lastname: Preston
authors.forename: Tynisha
authors.initials: T
authors.lastname: Thomas
authors.forename: Brian
authors.initials: B
authors.lastname: Shook
authors.forename: Paul F
authors.initials: PF
authors.lastname: Jackson
authors.forename: Stefanie
authors.initials: S
authors.lastname: Rassnick
authors.forename: Kenneth
authors.initials: K
authors.lastname: Rhodes
authors.forename: Vedwatee
authors.initials: V
authors.lastname: Gopaul
authors.forename: Rhys
authors.initials: R
authors.lastname: Salter
authors.forename: Jose
authors.initials: J
authors.lastname: Silva
authors.forename: David C
authors.initials: DC
authors.lastname: Evans
mesh_terms: D058916:Adenosine A1 Receptor Antagonists / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D058917:Adenosine A2 Receptor Antagonists / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000737:chemistry*; D007192:Indenes / Q000737:chemistry / Q000633:toxicity*; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D009152:Mutagenicity Tests; D011743:Pyrimidines / Q000737:chemistry* / Q000633:toxicity*; D011759:Pyrrolidines / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000737:chemistry* / Q000378:metabolism; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics
chemical_list: C562225:2-amino-4-phenyl-8-pyrrolidin-1-ylmethylindeno(1,2-d)pyrimidin-5-one; D058916:Adenosine A1 Receptor Antagonists; D058917:Adenosine A2 Receptor Antagonists; D018736:DNA Adducts; D007097:Imines; D007192:Indenes; D007477:Ions; D011743:Pyrimidines; D011759:Pyrrolidines; D043682:Receptor, Adenosine A1; D043705:Receptor, Adenosine A2A; D004247:DNA; C030986:pyrimidine; C032519:pyrrolidine