SignificanceThe development of selective antagonists for muscarinic acetylcholine receptors is challenging due to high homology in orthosteric binding sites among subtypes. Starting from a single amino acid difference in the orthosteric pockets in M2 muscarinic acetylcholine receptor (M2R) and M3R, we developed an M3R-selective antagonist using molecular docking and structure-based design. The resulting M3R antagonist showed up to 100-fold selectivity over the M2R in affinity and 1,000-fold selectivity in vivo. The docking-predicted geometry was further confirmed by a 3.1 Å crystal structure of M3R in complex with the selective antagonist. The potential of structure-based design to develop selective drugs with reduced off-target effects is supported by this study.

18F-Labeled building blocks from the type of [18F]fluorophenylazocarboxylic-tert-butyl esters offer a rapid, mild, and reliable method for the 18F-fluoroarylation of biomolecules. Two series of azocarboxamides were synthesized as potential radioligands for dopamine D3 and the μ-opioid receptor, revealing compounds 3d and 3e with single-digit and sub-nanomolar affinity for the D3 receptor and compound 4c with only micromolar affinity for the μ-opioid receptor, but enhanced selectivity for the μ-subtype in comparison to the lead compound AH-7921. A “minimalist procedure” without the use of a cryptand and base for the preparation of 4-[18F]fluorophenylazocarboxylic-tert-butyl ester [18F]2a was established, together with the radiosynthesis of methyl-, methoxy-, and phenyl-substituted derivatives ([18F]2b–f). With the substituted [18F]fluorophenylazocarbylates in hand, two prototype azocarboxylates radioligands were synthesized by 18F-fluoroarylation, namely the methoxy azocarboxamide [18F]3d as the D3 receptor radioligand and [18F]4a as a prototype structure of the μ-opioid receptor radioligand. By introducing the new series of [18F]fluorophenylazocarboxylic-tert-butyl esters, the method of 18F-fluoroarylation was significantly expanded, thereby demonstrating the versatility of 18F-labeled phenylazocarboxylates for the design of potential radiotracers for positron emission tomography .

Muscarinic M 3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M 2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M 3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M 3 subtype selectivity over M 2 , while 3′-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl-and two quinuclidiniumsubstituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with K i values from 0.069 to 0.084 nM, as well as high selectivity over the M 2 subtype (46-to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

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