Cell line–dependent metabolic fate of curcumin analogs assessed by liquid chromatography with quadrupole time-of-flight mass spectrometry and subcellular microscopy

Curcumin exhibits broad biological activity, including anticancer effects, but its therapeutic potential is limited because of poor stability, rapid metabolism, and nonspecific activity, prompting the development of structurally modified analogs with improved drug-like properties. However, how such modifications influence intracellular metabolism and subcellular behavior remains insufficiently understood. In this study, the intracellular fate of the reference molecule curcumin and 3 promising curcumin-based benzothiazepine analogs with enhanced selective anticancer activity was investigated in cancerous (HCT-116) and noncancerous (IPEC-J2) intestinal cell lines. An untargeted liquid chromatography with quadrupole time-of-flight mass spectrometry approach was applied to characterize degradation and metabolic products in cell lysates and extracellular medium, complemented by fluorescence-based imaging to explore their subcellular distribution. Interestingly, all compounds were detected intracellularly in both cell lines but displayed distinct structure- and cell-dependent metabolic patterns. Sulfation was predominant in HCT-116 cells, whereas methylation was more abundant in IPEC-J2 cells. In addition, phase II modifications-methylation and sulfation-occurred preferentially at phenolic moieties. Fluorescence imaging revealed mainly cytosolic, nuclear-excluded distribution for all compounds, with limited evidence for selective organelle accumulation. These findings demonstrate that curcumin-based benzothiazepine analogs undergo distinct cell-dependent metabolic processing, highlighting intracellular metabolism as a potential contributor to their previously reported selective anticancer activity. SIGNIFICANCE STATEMENT: By combining liquid chromatography with quadrupole time-of-flight mass spectrometry and subcellular fluorescence microscopy, this study provides insight into the intracellular fate of curcumin and 3 analogs, which differed both between compounds and between cancerous HCT-116 and noncancerous IPEC-J2 cells. These findings highlight the importance of structural modifications and cell-dependent metabolic processing when evaluating bioactive compounds because such mechanisms may substantially influence biological activity and selectivity.