Discovery tactics to mitigate toxicity risks due to reactive metabolite formation with 2-(2-hydroxyaryl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3h)-one derivatives, potent calcium-sensing receptor antagonists and clinical candidate(s) for the treatment of osteoporosis.

The synthesis and structure activity relationship studies on 5-trifluoromethylpyrido[4,3-dipyrimidin-4(3H)-ones as antagonists of the human calcium receptor (CaSR) have been recently disclosed [Didiuk et al. (2009) Bioorg. Med. Chem. Lett. 19, 4555-4559). On the basis of its pharmacology and disposition attributes, (R)-2-(2-hydroxypheny1)-3-(1-phenylpropan-2-y1)-5-(tritluoromethyl)pyrido[4,3d]pyrimidin-4(3H)-one (1) was considered for rapid advancement to first-in-human (FIH) trials to mitigate uncertainty surrounding the pharmacokinetic/pharmacodynamic (PK/PD) predictions for a short-acting bone anabolic agent. During the course of metabolic profiling, however, glutathione (GSH) conjugates of 1 were detected in human liver microsomes in an NADPH-dependent fashion. Characterization of the GSH conjugate structures allowed insight(s) into the bioactivation pathway, which involved CYP3A4mediated phenol ring oxidation to the catechol, followed by further oxidation to the elcctrophilic orthoquinone species. While the reactive metabolite (RM) liability raised concerns around the likelihood of a potential toxicological outcome, a more immediate program goal was establishing confidence in human PK predictions in the study. Furthermore, the availability of a clinical biomarker (serum parathyroid hormone) meant that PID could be assessed side by side with PK, an ideal scenario for a relatively unprecedented pharmacologic target. Consequently, progressing 1 into the clinic was given a high priority, provided the compound demonstrated an adequate safety profile to support FIFI studies. Despite forming identical RMs in rat liver microsomes, no clinical or histopathological signs prototypical of target organ toxicity were observed with 1 in in vivo safety assessments in rats. Compound I was also devoid of metabolism-based mutagenicity in in vitro (e.g., Salmonella Ames) and in vivo assessments (micronuclei induction in bone marrow) in rats. Likewise, metabolism-based studies (e.g., evaluation of (letoxicating routes of clearance and exhaustive PK/PD studies in animals to prospectively predict the likelihood of a low human efficacious close) were also conducted, which mitigated the risks of idiosyncratic toxicity to a large degree. In parallel, medicinal chemistry efforts were initiated to identify additional compounds with a complementary range of human PK predictions, which would maximize the likelihood of achieving the desired PD effect in the clinic. The back-up strategy also incorporated an overarching goal of reducing/ eliminating reactive metabolite formation observed with in. Herein, the collective findings from our discovery efforts in the CaSR program, which include the incorporation of appropriate derisking steps when dealing with RM issues are summarized.