AUTHOR=Wurzer Alexander , Vágner Adrienn , Horváth Dávid , Fellegi Flóra , Wester Hans-Jürgen , Kálmán Ferenc K. , Notni Johannes 

TITLE=Synthesis of Symmetrical Tetrameric Conjugates of the Radiolanthanide Chelator DOTPI for Application in Endoradiotherapy by Means of Click Chemistry

JOURNAL=Frontiers in Chemistry

VOLUME=6

YEAR=2018

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2018.00107

DOI=10.3389/fchem.2018.00107

ISSN=2296-2646

ABSTRACT=<p>Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide)<sub>4</sub>. A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II), and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx)<sub>4</sub> (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of Ca<sup>II</sup>-, Zn<sup>II</sup>, and Cu<sup>II</sup>-complexes of DOTPI (log<italic>K</italic><sub>(CaL)</sub> = 8.65, log<italic>K</italic><sub>(ZnL</sub> = 15.40, log<italic>K</italic><sub>(CuL)</sub> = 20.30) and DOTPI(Chx)<sub>4</sub> (log<italic>K</italic><sub>(CaL)</sub> = 8.99, log<italic>K</italic><sub>(ZnL)</sub> = 15.13, log<italic>K</italic><sub>(CuL)</sub> = 20.42) were found. Transchelation of Cu(II)-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates [<italic>k</italic><sub>d</sub> = 25 × 10<sup>−7</sup> and 5 × 10<sup>−7</sup> s<sup>−1</sup> for Cu(DOTPI) and Cu(DOTPI(Chx)<sub>4</sub>), respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II) removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H<sub>4</sub>EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO) based, strain-promoted click chemistry (SPAAC), which were labeled with Lu-177 and subsequently evaluated <italic>in vitro</italic> and in SCID mice bearing subcutaneous LNCaP tumor (PSMA+ human prostate carcinoma) xenografts. High affinities (3.4 and 1.4 nM, respectively) and persistent tumor uptakes (approx. 3.5% 24 h after injection) confirm suitability of DOTPI-based tetramers for application in targeted radionuclide therapy.</p>