Choice of an Optimal Modular Strategy for the Synthesis of DOTA-Containing Heterobivalent Agents Targeting PSMA and GRPr

Heterodimeric approaches have emerged as a promising method for simultaneously targeting multiple receptors on tumor cells using a single molecule. Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPr) holds the potential to improve the accuracy of prostate cancer diagnosis. The aim of this study was to develop a convenient and simple modular strategy for the creation of heterobivalent (HBV) conjugates targeting PSMA/GRPr receptors. For this purpose, we developed and compared six alternative routes for the stereoselective synthesis of HBV conjugates designed to deliver the chelating agent DOTA to PSMA/GRPr receptors. The comparison of these alternative synthetic pathways took into account such factors as efficiency, complexity, synthesis, and purification details, as well as yields of the target compounds. Optimal conditions for the stereoselective synthesis of HBV ligands to PSMA and GRPr, which could serve as molecular platforms for the targeted delivery of therapeutic or diagnostic agents to these receptors, were revealed. For synthesized HBV ligand <b>26</b>^<b>x</b> and its HBV conjugate with DOTA <b>27</b>, the complete signal assignment in ¹H, ¹³C, and ¹⁵N NMR spectra was achieved using 2D NMR techniques. Based on these data, comprehensive signal assignments were provided for all final compounds in their NMR spectra. The final HBV conjugate <b>27</b> was labeled with Lu-177, with yields >99%, and the obtained radiotracer was studied in vitro for its binding specificity, with determining of the <i>K</i>_D and <i>B</i>_max using LNCaP (PSMA+) and PC-3 (GRPr+) cell lines.