Toward Novel [18F]Fluorine-Labeled Radiotracers for the Imaging of α-Synuclein Fibrils

The accumulation of α-synuclein aggregates (α-syn) in the human brain is an occurrence common to all α-synucleinopathies. Non-invasive detection of these aggregates in a living brain with a target-specific radiotracer is not yet possible. We have recently discovered that the inclusion of a methylenedioxy group in the structure of diarylbisthiazole (DABTA)-based tracers improves binding affinity and selectivity to α-syn. Subsequently, complementary in silico modeling and machine learning (ML) of tracer–protein interactions were employed to predict surface sites and structure–property relations for the binding of the ligands. Based on this observation, we developed a small focused library of DABTAs from which 4-(benzo[d][1,3]dioxol-5-yl)-4′-(3-[ 18 F]fluoro-4-methoxyphenyl)-2,2′-bithiazole [ 18 F]d 2 , 6-(4′-(3-[ 18 F]fluoro-4-methoxyphenyl)-[2,2′-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [ 18 F]d 4 , 4-(benzo [ d ][1,3]dioxol-5-yl)-4′-(6-[ 18 F]fluoropyridin-3-yl)-2,2′-bithiazole [ 18 F]d 6 , and 6-(4′-(6-[ 18 F]fluoropyridin-3-yl)-[2,2′-bithiazol]-4-yl)-[1,3]dioxolo[4,5- b ]pyridine [ 18 F]d 8 were selected based on their high binding affinity to α-syn and were further evaluated. Binding assay experiments carried out with the non-radioactive versions of the above tracers d 2 , d 4 , d 6 , and d 8 showed high binding affinity of the ligands to α-syn: 1.22, 0.66, 1.21, and 0.10 nM, respectively, as well as excellent selectivity over β-amyloid plaques (Aβ) and microtubular tau aggregates (>200-fold selectivity). To obtain the tracers, their precursors were radiolabeled either via an innovative ruthenium-mediated (S N Ar) reaction ( [ 18 F]d 2 and [ 18 F]d 4 ) or typical S N Ar reaction ( [ 18 F]d 6 and [ 18 F]d 8 ) with moderate-to-high radiochemical yields (13% – 40%), and high molar activity > 60 GBq/μmol. Biodistribution experiments carried out with the tracers in healthy mice revealed that [ 18 F]d 2 and [ 18 F]d 4 showed suboptimal brain pharmacokinetics: 1.58 and 4.63 %ID/g at 5 min post-injection (p.i.), and 1.93 and 3.86 %ID/g at 60 min p.i., respectively. However, [ 18 F]d 6 and [ 18 F]d 8 showed improved brain pharmacokinetics: 5.79 and 5.13 %ID/g at 5 min p.i.; 1.75 and 1.07 %ID/g at 60 min p.i.; and 1.04 and 0.58 %ID/g at 120 min p.i., respectively. The brain uptake kinetics of [ 18 F]d 6 and [ 18 F]d 8 were confirmed in a dynamic PET study. Both tracers also showed no brain radiometabolites at 20 min p.i. in initial in vivo stability experiments carried out in healthy mice. [ 18 F]d 8 seems very promising based on its binding properties and in vivo stability, thus encouraging further validation of its usefulness as a radiotracer for the in vivo visualization of α-syn in preclinical and clinical settings. Additionally, in silico and ML-predicted values correlated with the experimental binding affinity of the ligands.