Design, synthesis, and evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates for enhanced photodynamic therapy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy lacking effective targeted therapies. While 5-Aminolevulinic acid (5-ALA) is an FDA-approved prodrug for photodynamic therapy (PDT), its clinical efficacy against TNBC is severely restricted by poor lipophilicity and the rapid metabolic clearance of its active photosensitizer, protoporphyrin IX (PpIX), into heme. To overcome these limitations, we designed and synthesized 28 novel prodrug conjugates by covalently linking 5-ALA with the iron chelator 3-hydroxypyridinone (HPO) via an ester bond, exploring both R and S stereochemical configurations. Biological evaluations revealed that these conjugates possessed significantly improved lipophilicity and negligible dark toxicity, while exhibiting significantly enhanced phototoxicity to free 5-ALA. Notably, the R-configuration conjugates 13a and 13d emerged as the most potent candidates against MDA-MB-231 TNBC cells, demonstrating IC50 values of 51.16 ± 1.93 μM and 49.65 ± 2.50 μM, respectively (vs. >100 μM for 5-ALA). Mechanistic studies suggested that these conjugates induced nearly 4-fold higher intracellular PpIX fluorescence than 5-ALA, which is hypothesized to correlate with HPO-mediated labile iron chelation and subsequent preservation of the intracellular PpIX pool against ferrochelatase-mediated clearance. These findings highlight the promise of 5-ALA-HPO conjugates as synergistic, dual-action photosensitizers for advancing PDT in aggressive TNBC models in vitro.