Structure–Activity Relationship of Cyanine Dyes in Relation to the Hepatic Uptake and Excretion

Background : Indocyanine green (ICG) is a clinically approved fluorescent dye that is used for lesion identification during hepatobiliary surgery. However, the chemical structure of ICG seems to be suboptimal for this excretion‐based diagnostic readout. In this study the effect of systematic structural variations in Cy5‐analogs were evaluated to elucidate the structure–activity relationship between hepatic uptake and excretion. Methods : Nine Cy5 dyes with variations in N ‐alkyl indole substitution (butyl sulfonate or methyl), and benzoannulation were synthesized in analogy to the ICG scaffold (Cy7.5‐(SO3‐)2). Photo‐, bio‐ and chemical properties were analyzed and combined to identify structure–activity relationships using Spearman's correlation and multiparametric analysis. Results : Chemical modifications were shown to alter (photo)chemical properties and constituted in clear biological effects; Sulfonate substitution supports hepatic excretion, while benzoannulation promoted nonspecific background accumulation. Cy5‐(SO3‐)2, rather than the ICG‐analog Cy5.5‐(SO3‐)2, was identified as lead. Conclusions : Systematic evaluation revealed key structural determinants that influence biliary excretion and allowed lead selection of dyes for hepatobiliary imaging. These insights provide a foundation for the rational design of optimized fluorescent agents for this application.