Synthesis and evaluation of the dopamine D2 and D3 receptor agonists, (R,S)-¹⁸F-5-OH-FPPAT and (R,S)-¹⁸F-7-OH-FHXPAT

Abstract

80

Objectives: Dopamine D2 and D3 receptors have been associated with a number of neurological and psychiatric disorders. Development of agonist-based imaging methodologies of this receptor is important for studies of normal and abnormal brain function, and dopamine competition studies. Our goals are: (1) Synthesize and evaluate ¹⁸F-7-OH-FHXPAT as a potential tracer for D3 receptors; (2) Improve and automate reported 3-step radiosynthesis of ¹⁸F-5-OH-FPPAT to 2-steps (Shi et al., NMB, 2004).

Methods: Two precursor molecules, 2-(N-propyl-N-5’-bromopentyl)amino-5-tetrahydroxypyranyltetralin (for ¹⁸F-5-OH-FPPAT) and 2-(N-propyl-N-5’-bromohexyl)amino-7-tetrahydroxypyranyltetralin (for ¹⁸F-7-OH-FHXPAT) were prepared using the respective tetralones. Radiolabeling of the bromo-precursors were carried out using ¹⁸F-fluoride, Kryptofix/K₂CO₃ in acetonitrile at 95^oC for 30 min. Product mixture was purified on RP-HPLC (CH₃CN:H₂O 0.25%Et₃N, 68%:32%; flow rate 2.5 ml/min). Brain slices (10μm thick) from male Sprague-Dawley rats were used for in vitro binding studies. Slides were preincubated at RT for 10 min in Tris-HCl buffer (pH 7.4). Slices were incubated with 5-6 μCi/cc of ¹⁸F-5-OH-FPPAT or ¹⁸F-7-OH-FHXPAT at 37^oC for 1 hr. For competition studies, brain slices were incubated with dopamine. Nonspecific binding was measured using 10μM sulpiride. Slides were rinsed twice for 2 min in ice-cold buffer, dipped in ice water, blown dry and laid out for autoradiography. Slides with tissue samples were exposed to a multipurpose storage phosphor screen for 24 hours, developed, and quantified. Specific binding to D2 and D3 receptors in striatum (St), cortex (Ctx), and cerebellum (Cer) were measured.

Results: Radiosynthesis of the THP-protected bromo-precursors proceeded in high radiochemical yields (approx.30%) and the THP was deprotected under mild acidic conditions. This procedure avoided a previously reported hydride reduction step. The products were obtained in specific activities of >2000 Ci/mmol. Binding characteristics of the 5-hydroxy and 7-hydroxy derivatives were distinctly different. While ¹⁸F-5-OH-FPPAT bound predominantly to the striata an area rich in D2 receptors (St/Cer=17), binding of ¹⁸F-7-OH-FHXPAT was more wide-spread. Binding was seen in the striata (St/Cer=2) which was sensitive to increasing dopamine. Regions in the cerebellum (possibly D3 sites) were more evident with ¹⁸F-7-OH-FHXPAT. A greater degree of binding in the cortex and hippocampus was seen with ¹⁸F-7-OH-FHXPAT compared to ¹⁸F-5-OH-FPPAT.

Conclusions: A two step radiosynthesis has been successfully carried out for previously described ¹⁸F-5-OH-FPPAT as well as a new radiotracer, ¹⁸F-7-OH-FHXPAT. Further investigation on the nature of their binding (D2 versus D3) is underway.