Structure-property relationships of benzyl-substituted urea-based ligands with Phe-Phe linker targeting prostate-specific membrane antigen labeled with gallium-68.

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 (logDo/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 logPcalc. 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.