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MAK683 ( N -((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3- c ]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [ 14 C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O -hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.

Background and Objectives: MET401 is a potent and selective c-Met inhibitor with a novel triazolopyrimidine scaffold. The aim of this study was to determine the pharmacokinetic profile of MET401 in preclinical species, and to identify the metabolic soft spot and enzyme involved, in order to help medicinal chemists to modify the compound to improve the pharmacokinetic profile., Methods: A metabolite identification study was performed in different liver fractions from various species. Chemical inhibition with selective cytochrome P450 (CYP) and molybdenum hydroxylase inhibitors was carried out to identify the enzyme involved. The deuterium substitution strategy was adopted to reduce metabolism. Pharmacokinetic studies were performed in rats to confirm the effect., Results: Although M-2 is a minor metabolite in liver microsomal incubations, it became the predominant metabolite in incubations with liver S9, cytosol, hepatocytes and rat pharmacokinetic study. M-2 was synthesized enzymatically and the structure was identified as a mono-oxidation on the triazolopyrimidine moiety. The M-2 formation was ascribed to aldehyde oxidase (AO)-mediated metabolism based on the following evidence-M-2 production was NADPH independent, pan-CYP inhibitor 1-aminobenzotriazole and xanthine oxidase inhibitor allopurinol did not inhibit M-2 formation, and AO inhibitors menadione and raloxifene inhibited M-2 formation. The deuterated analog MET763 demonstrated an improved pharmacokinetic profile with lower clearance, longer terminal half-life and double oral exposure compared with MET401 in rats., Conclusions: These results indicate that the main metabolic pathway of MET401 is AO-mediated metabolism, which leads to poor in vivo pharmacokinetic profiles in rodents. The deuterium substitution strategy could be used to reduce AO-mediated metabolism liability.

Some quinoline-containing c-Met kinase inhibitors are aldehyde oxidase (AO) substrates. 3-Substituted quinoline triazolopyridine analogs were synthesized to understand the electron-donating and steric hindrance effects on AO-mediated metabolism. Metabolic stability studies for these quinoline analogs were carried out in liver cytosol from mice, rats, cynomolgus monkeys, and humans. Several 3-N-substituted analogs were found to be unstable in monkey liver cytosolic incubations (half-life, <10 minutes), and five of them (63, 53, 51, 11, and 71) were chosen for additional mechanistic studies. Mono-oxygenation on the quinoline ring was identified by liquid chromatography tandem mass spectrometry. Metabolite formation was inhibited by the AO inhibitors menadione and raloxifene, but not by the xanthine oxidase inhibitor allopurinol. It was found that small electron-donating groups at the 3-quinoline moiety made the analogs more susceptible to AO metabolism, whereas large 3-substituents could reverse the trend. Although species differences were observed, this trend was applicable to all species tested. Small electron-donating substituents at the 3-quinoline moiety increased both affinity (decreased Michaelis constant) and V max maximum velocity toward AO in kinetic studies, whereas large substituents decreased both parameters probably as a result of steric hindrance. Based on our analysis, a common structural feature with high AO liability was proposed. Our finding could provide useful information for chemists to minimize potential AO liability when designing quinoline analogs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)