Design, synthesis and evaluation of novel 2-phenyl-3-(1H-pyrazol-4-yl)pyridine positive allosteric modulators for the M₄ mAChR

Translation of muscarinic acetylcholine receptor (mAChR) agonists into clinically used therapeutic agents has been difficult due to their poor subtype selectivity. M₄ mAChR subtype-selective positive allosteric modulators (PAMs) may provide better therapeutic outcomes, hence investigating their detailed pharmacological properties is crucial to advancing them into the clinic. Herein, we report the synthesis and comprehensive pharmacological evaluation of M₄ mAChR PAMs structurally related to 1e, Me–C-c, [¹¹C]MK-6884 and [¹⁸F]12. Our results show that small structural changes to the PAMs can result in pronounced differences to baseline, potency (pEC₅₀) and maximum effect (E_max) measures in cAMP assays when compared to the endogenous ligand acetylcholine (ACh) without the addition of the PAMs. Eight selected PAMs were further assessed to determine their binding affinity and potential signalling bias profile between cAMP and β-arrestin 2 recruitment. These rigorous analyses resulted in the discovery of the novel PAMs, 6k and 6l, which exhibit improved allosteric properties compared to the lead compound, and probative in vivo exposure studies in mice confirmed that they maintain the ability to cross the blood-brain barrier, making them more suitable for future preclinical assessment.