Structure-activity Relationship of Pyrazol-4-yl-pyridine Derivatives and Identification of a Radiofluorinated Probe for Imaging the Muscarinic Acetylcholine Receptor M

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M PET ligand that allows the non-invasive visualization of M in the brain. Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [F], was synthesized in 28 ± 10% radiochemical yield, >37 GBq/μmol and an excellent radiochemical purity >99%. Initial autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [F] for the M-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 [F] 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 [F] 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 [F] was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M PET radioligand with promising attributes. The availability of a clinically validated M PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.