Studies Directed Toward the Synthesis of Novel Bitopic Ligands of the Muscarinic Acetylcholine Receptor Subtype 4

Purpose: Alzheimer’s disease (AD) is characterized by amyloid deposits that build up in the brain and cause neurodegeneration. It is estimated that 6.7 million Americans currently have AD and at age 45, the lifetime probability of developing AD are 1 in 5 for women and 1 in 10 for men. Cognitive and behavioral impairments in AD are associated with dysregulation of cholinergic signaling that occurs due to neuronal damage and a decreased availability of the acetylcholine neurotransmitter. Muscarinic acetylcholine receptors are a family of G protein-coupled receptors (GPCRs) with five subtypes (M₁₋₅). Clinical trials with the M₁/M₄-preferring orthosteric agonist xanomeline previously demonstrated promise in treating cognitive and behavioral impairments in AD patients; however, further advancement of the experimental drug was prevented due to adverse effects associated with activation of M₂ and/or M₃ receptors. Achieving subtype selectivity with orthosteric agonists remains challenging due to high homology at the binding site. On the other hand, positive allosteric modulators (PAMs) have demonstrated more promising selectivity profiles. We hypothesize that bitopic ligands that engage both the orthosteric and allosteric binding sites of M₄ will selectively and directly activate the receptor, offering a pharmacological profile advantageous for the treatment of the cognitive and behavioral symptoms associated with AD. We will utilize published crystal structures and molecular docking studies to design bitopic ligands of M₄ that consist of xanomeline covalently linked to known M₄ PAMs. The objective of the current study is to design and execute viable syntheses of select putative M₄ bitopic ligands to enable their pharmacological evaluation.

Methods: Autodock tools were used to prepare the model of M₄ using the published crystal structure (PDB code 5DSG), and potential ligands were docked with Autodock Vina. Compounds were synthesized using solution phase chemistry and microwave-assisted organic synthesis (MAOS) when possible. Automated normal and/or reverse phase chromatography were used to purify intermediates and final products as needed. All compounds were characterized to confirm structure and purity utilizing Bruker Fourier 300HD for ¹H and ¹³C nuclear magnetic resonance (NMR) and Agilent 6230 time-of-flight (TOF) LC/MS for high resolution mass spectrometry (HRMS).

Results: Molecular docking studies revealed multiple putative M₄ bitopic ligands predicted to have a high affinity for the receptor. Included among these compounds are analogs with xanomeline linked to known M₄ PAMs VU6003130 and VU0152100. Synthetic approaches to select analogs have been designed and their execution initiated.

Conclusions: Progress toward the synthesis of novel M₄ bitopic ligands has been made. Next steps for the project include completion of the synthesis of select ligands and their evaluation in functional assays to determine potency at M₄ and selectivity versus other family members.