Discovery and in vivo evaluation of a fluorine-18 pro-drug tracer for imaging sphingosine-1-phosphate receptor 2 in the brain.

Sphingosine-1-phosphate receptor 2 (S1PR2) plays an important role in demyelinating central nervous system (CNS) disease, such as multiple sclerosis (MS). To identify a suitable PET radiotracer for imaging S1PR2 in the brain, we synthesized thirteen new phthalazinyl-indazole containing S1PR2 ligands and determined their S1PR2 binding potencies in vitro. A racemic carboxylic acid and its enantiomeric isomers, (±)-5d, (-)-5d, (+)-5d, and 3-fluoropropoxyl analogues 5e, and (±)-5f are potent with IC50 values of 33.4, 13.4, 12.1, 27.7, and 16.4 nM for S1PR2, respectively. These compounds selectively bind S1PR2 over other S1PRs with IC50s >1000 nM. Three tracers (±)-[18F]5d, (-)-[18F]5d, (+)-[18F]5d were radiosynthesized with radiochemical yields of 38 %, 20 %, and 20 %, respectively, high radiochemical purity (>95 %), and high molar activity (>39 GBq/μmol, decay corrected to EOS). In addition, (±)-[18F]7a, a methyl ester pro-drug tracer of (±)-[18F]5d, was designed and synthesized with a radiochemical yield of 38 %, high radiochemical purity (>95 %), and high molar activity (>37 GBq/μmol, decay corrected to EOS). Our PET studies indicated (±)-[18F]5d had low mouse brain uptake with 0.35 %ID/g at 40-60 min post-injection, however, the pro-drug tracer (±)-[18F]7a had 11-fold higher brain uptake (4.10 %ID/g) than that of (±)-[18F]5d at 40-60 min post-injection. Ex vivo biodistribution revealed (±)-[18F]5d had consistently low brain uptake and no defluorination in vivo in SD rats. The radiometabolic analysis confirmed that (±)-[18F]7a was metabolized fast to form (±)-[18F]5d in rat plasma, and (±)-[18F]5d was ∼30 % of the total radioactivity at 45 min post-injection in rat brain. (±)-[18F]5d demonstrated relatively stable in nonhuman primate plasma in vivo. Furthermore, a PET brain study of (±)-[18F]7a showed that cuprizone-fed mice had reduced radioactive brain uptake compared with control mice, suggesting downregulation of S1PR2 expression in the brain of this demyelinating disease mouse model. Together, (±)-[18F]7a is a potential pro-drug PET radiotracer for imaging S1PR2 expression in the brain supporting further optimization of an S1PR2-specific radiotracer with appropriate in vivo radiolabeled metabolism kinetics.