Overcoming the Genotoxicity of a Pyrrolidine Substituted Arylindenopyrimidine As a Potent Dual Adenosine A_2A/A₁ 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.

Metabolism and Excretion of Canagliflozin in Mice, Rats, Dogs, and Humans

“…Both systems were equipped with an electrospray ionization source operated in the positive ion mode. Accurate mass measurements using LTQ/Orbitrap were obtained by modification of a previously (Lim et al, 2007) or internal mass calibration by infusion of 10 pg/ml of tamoxifen (Lim et al, 2011). The source parameters were tuned for maximum sensitivity by infusion of 5 or 10 ng/ml canagliflozin in 50% acetonitrile/50% water directly into the mobile phase.…”

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…Instead, the most commonly utilized approach is to capture the reactive intermediate with a trapping agent, leading to formation of stable adducts of reactive metabolites that can be detected and characterized using LC-MS/MS techniques [4][5][6]. These methods have been extensively applied to efforts in the minimization of bioactivation for lead compounds at the drug discovery stage [7][8][9][10][11].…”

“…MDF processing of high resolution full MS data set provided unambiguous identification of both hard and soft reactive metabolites. A different approach was presented by Lim et al in their work of minimizing bioactivation of a drug candidate, in which S9 incubation in the presence of GSH and CN, along with their stable isotope analogues, was subject to HR-MS and isotope pattern dependent MS n acquisition on Orbitrap MS [11]. Mitchell et al used a peptide consisting of eleven amino acids including a terminus GSH and additional nucleophilic residues (e.g., lysine) for simultaneously trapping of both soft and hard electrophiles [31].…”

Bioactivation of the Cannabinoid Receptor Antagonist Rimonabant to a Cytotoxic Iminium Ion Metabolite

“…Case examples linking α-carbon oxidation of cyclic tertiary amines and iminium ions to covalent binding have been reported in the literature, 7−10 which include a report of genotoxicity caused by covalent modification of DNA. 11,12 These results raise the possibility that metabolic bioactivation of rimonabant could have toxicological consequences, especially in view of the key role played by reactive metabolites in the toxicity of many other drugs and foreign compounds. 13 In support of this possibility, recently it has been found that rimonabant exhibits potentiated in vitro toxicity to human liver derived THLE cell lines, which expressed P450 3A4 compared to THLE cells that expressed no P450 activity.…”