Design and synthesis of C-functionalized atridat to conjugate biomolecules for pre-targeted imaging: ⁶⁸Ga-ATRIDAT-BIOTIN

The high specificity and strong affinity (Kd =10-15 M) of avidin for biotin plays a significant role in the two-step pre targeting protocol based on administration of the avidin antibody conjugate followed by the radioactive biotin chelate. Indigenously synthesized tert-butyl 2,2'- (12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl) diacetate (ATRIDAT) is found to have a high stability constant with a wide range of metal ions. ATRIDAT was conjugated to biotin to obtain a specifically targeting molecule that binds avidin. Two step targeting could localize and image cancer cells in vivo which in turn can provide a high tumor targeting efficiency and modest signal amplification at the tumor site. Boc-protected diethyl amino malonate was conjugated with triethylene tetramine in high dilution method to give boc-protected macrocyclized product. This was then reacted with ter-butyl bromoacetate to increase the dentisity of the compound useful for metal chelation. After TFA cleavage, it was then reacted with NHS-activated biotin to give the desired product 2,2'- (11,13-dioxo-12- (5- ( (3aS,4S,6aR)-2- oxohexahydro-1H-thieno(3,4-d)imidazol-4-yl)pentanamido)-1,4,7,10- tetraaza-cyclotridecane-4,7-diyl)diacetic acid (ATRIDAT-Biotin). ⁶⁸Ga radiolabeling was performed and optimized to give more than 70% radiolabeling of chelate. Result and Conclusions: All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, ¹H, ¹³C NMR and mass spectroscopy. ATRIDATBiotin was obtained in appreciable yield from condensation of boc-protected amino diethyl ester and triethylene tetramine and then its reaction with biotin after successive steps. Cold labeling of ATRIDATBiotin was done with GaCl₃.3H₂O in water under nitrogen atmosphere. Radiolabeling was done using generator eluted Gallium chloride (⁶⁸Ga, 74 MBq) which was then added to the conjugate after pH adjustment to ~4.5 using sodium acetate. More than 70% radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological condition. Preliminary studies in vivo suggested appreciable uptake of ⁶⁸Ga-ATRIDAT-Biotin in tumor bearing mice. In conclusion a novel PET imaging agent based on two step pre-targeting with a new metal chelating system with biotin as biomarker has been developed which offers great possibilities in tumor imaging research applications for Ga-68 and Cu-64. (author)