Dual-masked cathepsin B-activated 5-aminolevulinic acid prodrugs with enhanced photodynamic cytotoxicity.

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) exploits tumor-selective accumulation of protoporphyrin IX (PpIX). However, its clinical utility is constrained by poor physicochemical properties and suboptimal tumor selectivity. To address these limitations, we rationally designed and synthesized 17 N-terminal dipeptide-modified 5-ALA prodrugs using a dual-masked strategy. This approach integrates lipophilic shielding at the carboxyl terminus with a cathepsin B (Cath B)-cleavable dipeptide at the amino terminus, enabling tumor-specific release of active 5-ALA. Structure-activity relationship analysis identified compound 4b as a lead candidate, exhibiting enhanced PpIX accumulation and phototoxicity against MDA-MB-231, HeLa, HCT116, and A549 cell lines. Notably, in MDA-MB-231 cells, 4b showed an IC50 of 13.8 μM, outperforming parent 5-ALA and being comparable to the clinically used 5-aminolevulinic acid hexyl ester (HAL). Furthermore, the dual-masked modification improved stability and membrane permeability. Collectively, these findings demonstrate that Cath B-activated dual-masked represents an effective strategy to boost tumor selectivity and maximize the therapeutic efficacy of ALA-PDT.