An improved synthesis and biological evaluation of a new cage-like bifunctional chelator, 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid, for 64Cu radiopharmaceuticals

IntroductionStable attachment of 64Cu2+ to a targeting molecule usually requires the use of a bifunctional chelator (BFC). Sarcophagine (Sar) ligands rapidly coordinate 64Cu2+ within the multiple macrocyclic rings comprising the cage structure under mild conditions, providing high stability in vivo. Previously, we have designed a new versatile cage-like BFC Sar ligand, 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid (AmBaSar), for 64Cu radiopharmaceuticals. Here we report the improved synthesis of AmBaSar, 64Cu2+ labeling conditions and its biological evaluation compared with the known BFC 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA).MethodsThe AmBaSar was synthesized in four steps starting from (1,8-diamine-Sar) cobalt(III) pentachloride ([Co(DiAmSar)]Cl5) using an improved synthetic method. The AmBaSar was labeled with 64Cu2+ in pH 5.0 ammonium acetate buffer solution at room temperature, followed by analysis and purification with HPLC. The in vitro stability of 64Cu-AmBaSar complex was evaluated in phosphate buffered saline (PBS), fetal bovine serum and mouse blood. The microPET imaging and biodistribution studies of 64Cu-AmBaSar were performed in Balb/c mice, and the results were compared with 64Cu-DOTA.ResultsThe AmBaSar was readily prepared and characterized by MS and 1H NMR. The radiochemical yield of 64Cu-AmBaSar was ≥98% after 30 min of incubation at 25°C. The 64Cu-AmBaSar complex was analyzed and purified by HPLC with a retention time of 17.9 min. The radiochemical purity of 64Cu-AmBaSar was more than 97% after 26 h of incubation in PBS or serum. The biological evaluation of 64Cu-AmBaSar in normal mouse demonstrated renal clearance as the primary mode of excretion, with improved stability in vivo compared to 64Cu-DOTA.ConclusionsThe new cage-like BFC AmBaSar was prepared using a simplified synthetic method. The 64Cu-AmBaSar complex could be obtained rapidly with high radiochemical yield (≥98%) under mild conditions. In vitro and in vivo evaluation of AmBaSar demonstrated its promising potential for preparation of 64Cu radiopharmaceuticals.