Measurement of Atropisomer Racemization Kinetics Using Segmented Flow Technology

When stable atropisomers are encountered by drug discovery teams, they can have important implications due to potential differences in their biological activity, pharmacokinetics, and toxicity. Knowledge of an atropisomer's activation parameters for interconversion is required to facilitate informed decisions on how to proceed. Herein, we communicate the development of a new method for the rapid measurement of atropisomer racemization kinetics utilizing segmented flow technology. This method leverages the speed, accuracy, low sample requirement, safety, and semiautomated nature of flow instrumentation to facilitate the acquisition of kinetics data required for experimentally probing atropisomer activation parameters. Measured kinetics data obtained for the atropo isomerization of AMPA antagonist CP-465021 using segmented flow and traditional thermal methods were compared to validate the method

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

The rationale for using M₁ selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M₁ muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M₁ muscarinic acetylcholine receptor activation. Five brain penetrant M₁-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M₁ activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M₁ muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use

Design and Synthesis of γ- and δ‑Lactam M₁ Positive Allosteric Modulators (PAMs): Convulsion and Cholinergic Toxicity of an M₁‑Selective PAM with Weak Agonist Activity

Recent data demonstrated that activation of the muscarinic M₁ receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M₂ and M₃ activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M₁ PAMs that address this hypothesis. The lead molecule from this series, compound <b>1</b> (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M₁-selective PAM with minimal agonist activity. Compound <b>1</b> was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M₁ is sufficient to elicit cholinergic AEs

Design and Synthesis of γ- and δ‑Lactam M₁ Positive Allosteric Modulators (PAMs): Convulsion and Cholinergic Toxicity of an M₁‑Selective PAM with Weak Agonist Activity

Recent data demonstrated that activation of the muscarinic M₁ receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M₂ and M₃ activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M₁ PAMs that address this hypothesis. The lead molecule from this series, compound <b>1</b> (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M₁-selective PAM with minimal agonist activity. Compound <b>1</b> was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M₁ is sufficient to elicit cholinergic AEs

Discovery of the Potent and Selective M1 PAM-Agonist <i>N</i>‑[(3<i>R</i>,4<i>S</i>)‑3-Hydroxytetrahydro‑2<i>H</i>‑pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4-yl)benzyl]­pyridine-2-carboxamide (PF-06767832): Evaluation of Efficacy and Cholinergic Side Effects

It is hypothesized that selective muscarinic M₁ subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer’s disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M₁ with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M₁ activation. This manuscript describes the development of a series of M₁-selective pyridone and pyridine amides and their key pharmacophores. Compound <b>38</b> (PF-06767832) is a high quality M₁ selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M₂/M₃ subtype activity, compound <b>38</b> still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M₁ activation contributes to the cholinergic liabilities that were previously attributed to activation of the M₂ and M₃ receptors