In this study, we describe the systematic modification of the peptide Sanguinamide A by exchanging its backbone thiazole for well-defined aromatic groups. The goal was to investigate the effect of small changes to these backbone modifications by changing their polarity or by adding extra methylene groups to either side to modify macrocycle flexibility. The resulting library of macrocycles was analyzed for membrane permeability using immobilized artificial membrane chromatography and the Caco-2 assay. To gain insight into the conformational changes in environments with varying polarity, the peptides were analyzed using variable temperature NMR and 2D-NMR were used to determine the structure fo selected peptides. Finally, the biological activity of the peptides was assessed using a target agnostic cell painting assay.