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

Introduction: Stable 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). Methods: The 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. Results: The 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. Conclusions: The 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.