Synthesis and Preliminary Evaluation of a Carbon-11 Labeled Probe for Imaging the Trace Amine-Associated Receptor 1 (TAAR1)

Objectives: The trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) that is activated by endogenous trace amines derived from circulating aromatic amino acids. Since the identification of TAAR1 in 2001, studies have consistently revealed a strong inverse association between TAAR1 and monoaminergic transporter activities. As such, TAAR1 has emerged as a key negative modulator of dopaminergic and adrenergic signaling. Selective TAAR1 agonists proved to be useful in hyper-dopaminergic conditions such as drug addiction and schizophrenia, while selective synthetic TAAR1 blockers (antagonists) increase the firing frequency of dopaminergic neurons in vitro and have been suggested for the treatment of hypo-dopaminergic conditions such as Parkinson’s disease. To date, there are no reports on TAAR1-directed probes for non-invasive receptor quantification in vivo. Accordingly, we sought to develop the first TAAR1 PET radioligand to allow non-invasive mapping of TAAR1.

Methods: Given (1) the relatively high TAAR1 binding affinity (K_i=2 nM), (2) the favorable lipophilicity and (3) the amenability for radiolabeling, the previously reported TAAR1 antagonist, N-(3-methoxyphenyl)-6-(pyrrolidin-1-yl)-5-(trifluoromethyl)nicotinamide (reference compound 4), was selected as a promising candidate for PET tracer development. Reference compound 4 and the desmethyl precursor were synthesized from commercially available 6-chloro-5-(trifluoromethyl)nicotinic acid and physicochemical profiling was performed. Carbon-11 labeling was accomplished with [¹¹C]MeI, whereas different nucleophiles were screened to optimize the radiolabeling conditions. Biodistribution and PET imaging studies were conducted under baseline and blocking conditions. To investigate the metabolic fate of [¹¹C]4, mouse plasma and brain samples were analyzed following tail-vein injection of the probe.

Results: Reference compound 4 and the precursor were synthesized in overall yields of 55% (2 steps) and 45% (3 steps), respectively. In silico physicochemical profiling predicted an appropriate brain-to-blood ratio (logBB = -0.08) for CNS-targeted PET. Further, compound 4 was found to be inactive in the hERG assay. Notably, the radiosynthesis proved to be challenging with Cs₂/K₂CO₃, despite high reaction temperatures and attempts to increase precursor amounts. In contrast, the use of NaOH led to a substantially improved RCY of 14% (non-decay corrected), with excellent radiochemical purities higher than 99% and molar activities greater 37 GBq/µmol. A LogD_7.4 value of 3.36 ± 0.22 (n =3) was measured by the shake-flask method. Despite the encouraging in vitro findings, only limited brain uptake was observed in vivo. Further, blockade experiments with commercially available TAAR1 antagonist, EPPTB, as well as with the non-radioactive reference, did not reveal any specific binding in the brain during small animal PET experiments. These results were further corroborated by ex vivo biodistribution studies in mice. Intriguingly, ex vivo metabolite analysis unveiled a relatively low level of intact parent tracer in the mouse plasma and brain at 30 min post injection, suggesting a rapid metabolism and the presence of radiometabolites in the brain.

Conclusions: We have successfully synthesized the first TAAR1-targeted PET probe. Despite promising in vitro performance characteristics, [¹¹C]4 did not exhibit in vivo specificity, possibly owing to the rapid metabolic degradation. Further studies are warranted to identify a suitable TAAR1 PET tracer, thereby facilitating the development of TAAR1-targeted drug candidates.