Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography

Abstract

Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A(3) adenosine receptor (A(3)AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography.

Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N(6)-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats.

Results: We prepared an agonist ligand {[(76)Br](1'S,2'R,3'S,4'R,5'S)-4'-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/micromol and an antagonist ligand {[(76)Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147)} in 65% radiochemical yield with a specific activity of 22 GBq/micromol. The resultant products exhibited the expected high affinity (K(i) approximately 0.6 nM) and specific binding at the human A(3)AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A(3)AR, over a 2-h time course, which suggests the presence of a specific A(3)AR retention mechanism.

Conclusion: We were able to compare uptake of the [(76)Br]-labeled antagonist MRS5147 to [(76)Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A(3)AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

Figure 1

Panel A: Inhibition of in vitro binding of the agonist [⁷⁶Br]4 by various A₃AR ligands. Panel B: Inhibition of in vitro binding of antagonist [⁷⁶Br]6 by various A₃AR ligands. The human or rat A₃AR receptor was expressed in CHO cells. See legend to Table 1 for compound names.

Figure 1

Panel A: Inhibition of in vitro binding of the agonist [⁷⁶Br]4 by various A₃AR ligands. Panel B: Inhibition of in vitro binding of antagonist [⁷⁶Br]6 by various A₃AR ligands. The human or rat A₃AR receptor was expressed in CHO cells. See legend to Table 1 for compound names.

Figure 2

In vivo uptake in rat of A₃AR agonist [⁷⁶Br]4 at 15, 30, 60, and 120 min post injection in various tissues.

Figure 3

Tissue uptake of [⁷⁶Br]4 without and with MRS1523, an antagonist, administered at 30 min post-injection in rat.

Figure 4

In vivo uptake in rat of the A₃AR antagonist [⁷⁶Br]6 at 15, 60 and 120 min post-injection. Increasing uptake in the testes was observed over time. Both 60 and 120 min uptakes are statistically increased relative to the 15 min time point (p < 0.05)

Scheme 1

Synthesis of [⁷⁶Br] A₃AR ligands, agonist [⁷⁶Br]4 and antagonist [⁷⁶Br]6, and their stannyl precursors 9 and 8, respectively.

Chart 1

Structures of prototypical (1,2) and later-generation A₃AR agonists (3–5) and antagonist (6) based on the (N)-methanocarba ring system.