Copper-free Click Labeling with the ^99mTc-Tricarbonyl-Core Using DACN Derivatives

Diazacyclononyne (DACN) derivatives belong to the class of diazamacrocycles containing a strained alkyne bond. Therefore, DACNs are predestinated starting materials to be applied in copper-free strain-promoted click labeling with azide-functionalized bio(macro)molecules. The diazamacrocyclic scaffold is easy to prepare via a double Nicholas reaction. The following functionalization is convenient by the use of functionalized sulfonamides or by the alkylation of the secondary amines provided by the molecule.
To make the DACN derivatives suitable for radiolabeling with technetium-99m, two dipicolylamine (DPA) derivatives were prepared allowing the use of the ^99mTc-tricarbonyl core. The first DPA-ligand is functionalized with an activated ester to allow convenient labeling of, e.g., amines. The second is based on the DACN macrocycle, allowing the copper-free click labeling. Two PSMA-binding derivatives based on PSMA-617 were chosen for final radiolabeling, one with a terminal amine function the other with an azide function.
For the conventional radiolabeling with e.g. primary amines, the DPA chelator was functionalized with a benzoate moiety which was further transferred into the corresponding activated TFP ester using tetrafluorophenol in three steps and an overall yield of 64%. For the click labeling procedure, the functionalized DACN macrocycle was prepared from a sulfonyl-modified diamide (4 steps) which was reacted with butyne-1,3-diol under Nicholas conditions. Finally, the DPA moiety was connected to the DACN via the propylsulfonyl linker. Both derivatives were reacted with (Et₄N)₂[ReBr₃(CO)₃] in methanol to obtain both reference complexes (^natRe-DPA-TFP ester: 91% yield, ^natRe-DPA-DACN: 76% yield).
[^99mTc][Tc(CO)₃]⁺ was prepared according to an established protocol using ^99mTc-pertechnetate and K₂[H₃BCO₂] (in-house-produced tricarbonyl kit). Prior to its use, the solution was brought to pH 5.5 using MES buffer. The radiolabeling of the DPA-TFP derivative was accomplished with [^99mTc][Tc(CO)₃]⁺ at 40°C for 30 min in MES buffer (pH 5.5) obtaining the ^99mTc-DPA-TFP ester with a radiochemical yield of 89%. The DPA-DACN derivative was labeled under the same conditions yielding ^99mTc-DPA-DACN with 83% RCY. The connection of the ^99mTc-labeled TFP ester to the primary amine of the PSMA targeting derivative was realized with 23% RCY at 100°C after 2.5 hours. Two additional by-products were observed by radio-HPLC indicating hydrolysis of the ^99mTc-DPA-TFP ester. In contrast, by-products were not found using the click reaction of the ^99mTc-labelled DPA-DACN-derivative for the conjugation with the azide-functionalized PSMA derivative. The labeling was performed using the same conditions, leading to 79% RCY after 4 hours. Both ^99mTc-radiolabeled DPA-building blocks and both ^99mTc-PSMA targeting derivatives were evidenced by comparison of the retention times of their respective ^natRe-derivatives.