Structure–activity relationship of pyrazol-4-yl-pyridine derivatives and identification of a radiofluorinated probe for imaging the muscarinic acetylcholine receptor M 4

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M 4 ) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M 4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M 4 PET ligand that allows the non-invasive visualization of M 4 in the brain. Structure–activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 ― a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [ 18 F] 12, was synthesized in 28 ± 10% radiochemical yield, >37 GBq/μmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [ 18 F] 12 for the M 4 -rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [ 18 F] 12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [ 18 F] 12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [ 18 F] 12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M 4 PET radioligand with promising attributes. The availability of a clinically validated M 4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.