Discovery of a Promising Fluorine-18 Positron Emission Tomography Radiotracer for Imaging Sphingosine-1-Phosphate Receptor 1 in the Brain

Abstract

Sphingosine-1-phosphate receptor 1 (S1PR1) is recognized as a novel therapeutic and diagnostic target in neurological disorders. We recently transferred the S1PR1 radioligand [¹¹C]CS1P1 into clinical investigation for multiple sclerosis. Herein, we reported the design, synthesis and evaluation of novel F-18 S1PR1 radioligands. We combined the structural advantages of our two lead S1PR1 radioligands and synthesized 14 new S1PR1 compounds, then performed F-18 radiochemistry on the most promising compounds. Compound 6h is potent (IC₅₀ = 8.7 nM) and selective for S1PR1. [¹⁸F]6h exhibited a high uptake in macaque brain (SUV > 3.0) and favorable brain washout pharmacokinetics in positron emission tomography (PET) study. PET blocking and displacement studies confirmed the specificity of [¹⁸F]6h in vivo. Radiometabolite analysis confirmed no radiometabolite of [¹⁸F]6h entered into the brain to confound the PET measurement. In summary, [¹⁸F]6h is a promising radioligand to image S1PR1 and worth translational clinical investigation for humans with brain disorders.

Figure 1.

Structures of the lead S1PR1 PET radiotracers and ponesimod.

Figure 2.

Design strategy of novel S1PR1 ligands from the lead ligands CS1P1 and TZ4877.

Figure 3.

Brain uptakes (SUV) of six F-18 S1PR1 radiotracers in nonhuman primate. Dynamic PET scan from 0 to 120 min was performed for each radiotracer in a male cynomolgus macaque. The brain uptakes of radiotracers [¹⁸F]5a–c displayed an increasing trend from 0 to 120 min; the brain uptakes of [¹⁸F]6a,[¹⁸F]f, and [¹⁸F]h peaked quickly post-injection; [¹⁸F]6h had the highest initial brain uptake with SUV of 3.24 at 5 min and then gradually washed out from the brain, suggesting it is the most favorable S1PR1 radiotracer tracer for brain imaging compared to the other five F-18 S1PR1 radiotracers.

Figure 4.

PET characterization of [¹⁸F]6h in the macaque brain. (A) Between-subject-animal average brain uptake of [¹⁸F]6h was calculated from 4 PET scans of four different animals at baseline condition (inter-animal); within-subject average brain uptake of [¹⁸F]6h was calculated from four different PET scans for a single animal at baseline condition (intra-animal average), data represents mean ± SEM; (B) Representative PET and co-registered MRI images of [¹⁸F]6h in the macaque brain; (C) The brain uptake of [¹⁸F]6h in the brain regions of interest; (D) blocking/displacement studies with non-radioactive 6h and ponesimod.

Figure 5.

Tissue distribution of [¹⁸F]6h in SD rats. The initial uptake of [¹⁸F]6h was high in lung, liver, kidney, pancreas, spleen, and small intestine, and low in blood, muscle, and bone. The tracer then gradually washed out from the major organs of interest, and accumulated in liver and small intestine by 60 min post-injection. The rat brain uptake of [¹⁸F]6h was relatively high with %ID/gram of 0.93 ± 0.02. No defluorination was observed. Data represents mean ± SE, n = 3.

Figure 6.

HPLC radiometabolite analysis of macaque plasma, and rat brain and plasma samples post injection of [¹⁸F]6h. Radiometabolite analysis was performed on samples collected at 2, 10, 30, 60, and 90 min post-injection. [¹⁸F]6h directly was incubated with macaque or rat plasma in vitro as control. (A) HPLC radiometabolite analysis in macaque plasma showed a major radioactive peak of parental [¹⁸F]6h with a retention time of ~9 min and two minor radiometabolites at ~12 min and ~15 min; (B) Negligible radiometabolite was observed in the rat brain sample; (C) Similar to macaque plasma, radiometabolites were observed in the rat plasma with a much faster metabolism rate.

Scheme 1

^aReagents and conditions: (a) NaBH₄, MeOH, RT; (b) NH₂OH·HCl, NaHCO₃, MeOH, reflux; (c) 3a and tert-butyl 3-(methylamino)propanoate for 2f, 3b and tert-butyl 3-(methylamino)propanoate for 2g, 3a and tert-butyl 3-aminopropanoate hydrochloride for 2h, Et₃N, MeOH, RT; (d) 2-(methylamino)ethanol for 2i, 3-methylamino-1-propanol for 2j, 4-(methylamino)butan-1-ol for 2k, 3-aminopropanol for 2l, Et₃N, MeOH, RT.

Scheme 2

^aReagents and conditions: (a) EDCI, HOBt, DMF, RT ~ 120 °C for 5a-e, 6a–e, and 6h; (b) EDCI, HOBt, DMF, RT ~ 120 °C; then KOH (5M), MeOH, H₂O, RT for 5f, and 6f–g.

Scheme 3

^aReagents and conditions: (a) [¹⁸F]KF, Kryptofix 222, TMEDA, DMSO, H₂O, 150 °C, 5 min; (b) NaBH₄, EtOH, RT, 2 min; (c) 2-aminoethanol, AcOH, EtOH, 100 °C, 5 min; (d) NaCNBH₃, RT, 2 min; (e) formalin, 100 °C, 5 min, then NaCNBH₃, RT, 2 min; (f) 3-aminopropanol, AcOH, EtOH, 100 °C, 5 min.

Scheme 4

^aReagents and conditions: (a) EDCI, HOBt, DMF, RT ~ 120 °C; (b) 1,2-bis(tosyloxy)ethane, K₂CO₃, MeCN, 80 °C; (c) MOMCl, DIPEA, DCM, RT; (d) [¹⁸F]KF, Kryptofix 222, K₂CO₃, MeCN, 110 °C, 15 min; (e) HCl (6 N), 5 min; (f) KOH (5 M), EtOH, 110 °C, 5 min; (g) Cs₂CO₃, DMSO, 110 °C, 15 min.