In Vitro Pharmacological Profile of a New Small Molecule Bradykinin B₂ Receptor Antagonist

We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B₂ receptor antagonists, and its superior profile to the prior art B₂ receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B₂ receptor (K_b values 0.24, 0.95, and 1.24 nM, respectively, calcium mobilization assay). Compound 3 is more potent than the prior art compounds and icatibant in this assay (K_b icatibant 2.81 nM). The compounds also potently inhibit BK-induced contraction of endogenous B₂ receptors in a human isolated umbilical vein bioassay. The potencies of Compound 3, Compound 2, Compound 1, and icatibant are (pA₂ values) 9.67, 9.02, 8.58, and 8.06 (i.e. 0.21, 0.95, 2.63, and 8.71 nM), respectively. Compound 3 and Compound 2 were further characterized. They inhibit BK-induced c-Fos signaling and internalization of recombinant human B₂ receptors in HEK293 cells, and do not antagonize the venous effects mediated by other G protein-coupled receptors in the umbilical vein model, including the bradykinin B₁ receptor. Antagonist potency of Compound 3 at cloned cynomolgus monkey, dog, rat, and mouse B₂ receptors revealed species selectivity, with a high antagonist potency for human and monkey B₂ receptors, but several hundred-fold lower potency for the other B₂ receptors. The in vitro off-target profile of Compound 3 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B₁ receptor. Compound 3 showed a lower intrinsic clearance in the microsomal stability assay than the prior art compounds. With an efflux ratio of 1.0 in the Caco-2 permeability assay Compound 3 is predicted to be not a substrate of efflux pumps. In conclusion, we discovered a novel chemical class of highly selective and very potent B₂ receptor antagonists, as exemplified by Compound 3. The compound showed excellent absorption in the Caco-2 assay, predictive of good oral bioavailability, and favourable metabolic stability in liver microsomes. Compound 3 has provided a significant stepping stone towards the discovery of the orally bioavailable B₂ antagonist PHA-022121, currently in phase 1 clinical development.