Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.