Characterization of Ubrogepant: A Potent and Selective Antagonist of the Human Calcitonin Gene‒Related Peptide Receptor.

A growing body of evidence has implicated the calcitonin gene-related peptide (CGRP) receptors in migraine pathophysiology. With the recent approval of monoclonal antibodies targeting CGRP or the CGRP receptor, the inhibition of CGRP-mediated signaling has emerged as a promising approach for preventive treatments of migraine in adults. However, there are no small-molecule anti-CGRP treatments available for treating migraine. The current studies aimed to characterize the pharmacologic properties of ubrogepant, an orally bioavailable, CGRP receptor antagonist for the acute treatment of migraine. In a series of ligand binding assays, ubrogepant exhibited a high binding affinity for native (K i=0.067 nM) and cloned human (K i=0.070 nM) and rhesus CGRP receptors (K i=0.079 nM), with relatively lower affinities for CGRP receptors from rat, mouse, rabbit and dog. In functional assays, ubrogepant potently blocked human α-CGRP stimulated cAMP response (IC50 of 0.08 nM) and exhibited highly selective antagonist activity for the CGRP receptor compared with other members of the human calcitonin receptor family. Furthermore, the in vivo CGRP receptor antagonist activity of ubrogepant was evaluated in a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans. Results demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV with a mean EC50 of 3.2 and 2.6 nM in rhesus monkeys and humans, respectively. Brain penetration studies with ubrogepant in monkeys showed a CSF/plasma ratio of 0.03 and low CGRP receptor occupancy. In summary, ubrogepant is a competitive antagonist with high affinity, potency, and selectivity for the human CGRP receptor. SIGNIFICANCE STATEMENT: Ubrogepant is a potent, selective, orally delivered, small-molecule competitive antagonist of the human calcitonin generelated peptide receptor. In vivo studies using a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV, indicating a predictable pharmacokinetic-pharmacodynamic relationship.

Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Differential anti-thrombotic benefit and bleeding risk profiles of antagonists of protease-activated receptor 1 and 4 in Cynomolgus Macaques.

Platelet activation plays a crucial role in hemostasis and thrombosis. Thrombin, the most potent stimulus of platelet activation, mediates platelet activation via the protease activated receptors (PARs). The platelet PAR repertoire in mediating thrombin's action differs across species. Only nonhuman primate (NHP) platelet activation is known to be similar to humans, mediated by PAR1 and PAR4, hence limiting translational in vivo studies of PAR's role in thrombosis and hemostasis to NHPs. Earlier studies have demonstrated a range of distinct in vitro activities of PAR1 and 4 in platelet activation yet the implications of these events in vivo is unclear. The objective of this study is to investigate and compare the roles of PAR1 and PAR4 in hemostasis and thrombosis in a relevant animal species. NHP models for pharmacokinetic, ex vivo platelet aggregation responses, FeCI3 injury-mediated arterial thrombosis and template bleeding were developed in Cynomolgus Macaques. Potent and selective small molecule antagonists of PAR1 and PAR4 were characterized in an array of in vitro assays, and subsequently examined head-to-head in the NHP models. Treatment of NHPs with antagonists of PAR1 or PAR4 both resulted in strong inhibition of ex vivo platelet aggregation. At doses that led to similar inhibition of platelet aggregation, animals treated with the PAR4 antagonist showed similar levels of anti-thrombotic efficacy, but longer bleeding times, compared to animals treated with the PAR1 antagonist. These findings suggest that PAR1 antagonism will likely produce a larger therapeutic index (ie. a larger anti-thrombotic efficacy over bleeding risk margin) than PAR4 antagonism.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.