Structure-property relationships of benzyl-substituted urea-based ligands with Phe-Phe linker targeting...

Prostate-specific membrane antigen (PSMA) is a validated target for prostate cancer imaging and therapy, yet small structural changes in PSMA ligands can markedly affect cellular internalization and in vivo clearance. Here we prepared four DOTA-based PSMA-targeting conjugates that have a Glu-urea-Lys (DCL) as a vector and a constant -L-Phe-L-Phe-containing linker, while varying the ε-benzyl substituent (m-Cl, p-Br, p-COOH, or o-NO₂). All conjugates were efficiently radiolabeled with gallium-68 and compared with clinically used [⁶⁸Ga]Ga-PSMA-11 and [⁶⁸Ga]Ga-PSMA-617. Experimental lipophilicity (logD_o/w) confirmed a hydrophilic profile for the whole series. In vitro assays across four prostate cancer cell lines showed PSMA-dependent uptake: LNCaP and 22Rv1 cells demonstrated substituent-dependent accumulation and internalization, whereas PC-3 and DU-145 cells exhibited only low, nonspecific binding. The halogenated analogues [⁶⁸Ga]Ga-I (m-Cl) and [⁶⁸Ga]Ga-II (p-Br) provided the most favorable combination of total cell uptake and internalized fraction. Docking to PSMA reproduced the canonical binding mode and, together with calculated logP_calc. and TPSA, supported the observed lipophilicity trends. Finally, a pilot biodistribution study in DU-145 xenograft-bearing mice revealed measurable tumor activity for [⁶⁸Ga]Ga-I but also pronounced renal retention, highlighting the need for further optimization of pharmacokinetics. These results identify ε-benzyl substitution as an effective handle to tune PSMA-ligand behavior and provide lead structures for the next design cycle.