Novel 11C-labeled positive allosteric modulators for imaging muscarinic acetylcholine receptor M4

1023 Objectives: We have identified a set of pyrazol-4-yl-pyridine compounds as positive allosteric modulators (PAMs) of muscarinic acetylcholine receptor subtype 4, which is involved in the potential treatment and/or prevention of neurological and psychiatric disorders and diseases.[1] The goal of the project was to synthesize a focused library of M4 PAMs based on pyrazol-4-yl-pyridine scaffold, determine potency and selectivity using functional assays, and to radiolabel the most potent and selective M4 PAMwith carbon-11 or fluorine-18. Methods: We designed an efficient synthetic route to access six M4 PAMs based on pyrazol-4-yl-pyridine scaffold.[2] Briefly, mesylate 2 was formed in 95% yield by reacting alcohol 1 and MsCl in the presence of Et3N, followed by N-alkylation of pyrazole boronic ester 3, to give boronic ester 4 in 75% yield. Stepwise Suzuki coupling reactions were carried out between bromide 5 and boronic acid ester 4 to generate 6 in 53% yield, followed by the coupling of chloride 6 and boronic acid ester 7a to afford desired M4 PAM 8 in 64% yield. The analogous procedure was applied to obtain amide precursor 9 for radiolabeling in 66% yield. N-Alkylation of 9 generated fluoro analogs 12 and 13 in 37-39% yields, respectively. Their 18F-radiolabeling precursors 14 and 15 were generated in 14-40% yields. Pyrazol-4-yl-pyridine 8 and its five derivatives (9-13) were subsequently screened for their in vitro potency and selectivity toward M1, M2, M3, M4 and M5 using cell-based calcium release functional assay. Preliminary 11C-labelings were carried out for two potent and selective M4 PAM 8 and 10 using its corresponding amide precursor 9 and 11, respectively with [11C]CH3I in the presence of KOH in DMSO (0.4 mL) at 130°C for 5 min. Results:The synthesis of standard 8 and its close analogs 9-13 for pyrazol-4-yl-pyridine were achieved in four steps with overall 20-25% yields. All six candidates showed high potency (EC50 values 37.8 nM for 8, 70.6 nM for 9, 33.0 nM for 10, 37.2 nM for 11, 97.3 nM for 12, and 201.7 nM for 13 in average two independent runs) at M4 and excellent selectivity (> 100 fold) for M4 over other subtypes, including M1, M2, M3 and M5. The reaction between [11C]CH3I and amide 9 or 11 generated radiolabeled pyrazol-4-yl-pyridine [11C]8 or [11C]10 in 2-3% isolated radiochemical yield (non-decay corrected from starting [11C]CO2) with greater than 40 GBq/µmol specific activity. No signs of radiolysis was observed up to 90 min after formulation (10% ethanol in saline). Conclusions: We have successfully prepared six potent and selective pyrazol-4-yl-pyridines for M4 PAM and conducted in vitro pharmacological evaluation. The radiolabeled [11C]8 and [11C]10 was produced in reasonable isolated yields and high specific activity. Further characterization including PET imaging studies, ex vivo whole body distribution and radiometabolite analysis will be performed in order to evaluate and develop potent and selective M4 PET tracers. References: [1] Neuropsychiatr Dis Treat. 2014; 10: 183-191; [2] WO2017112556. Acknowledgement: We thank the NIH PDSP program for M4 compound screening.