In our cell-painting set-up, the compounds were tested at different concentrations (10, 30, and 50 μM) in U2OS cells and a total of 579 morphological parameters were analyzed. An induction score is calculated from this analysis which represents the number of parameters for which the change is significant in comparison to the DMSO control. Compounds that generate induction levels greater than a threshold of 5% are considered to be active, while those below are considered inactive. Interestingly, most compounds that were observed to have high membrane permeability according to CHI-IAM and Caco-2 were found to be active (Table , and Fig. ). The exception to this rule was SA-B2, which only showed activity at the highest concentration of 50 μM. Both cLogP and tPSA did not correlate with either the induction values measured at either the 30 or 50 μM compound concentration (Fig. and). Next, we compared the profiles of all active compounds to analyze their similarity. We selected the profiles of each compound at the lowest concentration that reached the activity threshold to avoid conditions that led to significant decreases in cell viability. The direction of the change of each parameter is compared between the different compounds to derive a biosimilarity score (in %) and the profiles are considered to be similar when this is > 75%. By using this threshold, the only compound that could be considered similar to Sanguinamide A, was SA-B1 with a biosimilarity of 82% (see Fig. ). The more hydrophobic SA-A2 and SA-B3 had the lowest similarity (64 and 67%, resp.), while SA-B2 was close at 74%. When using the other compounds as the starting point for comparison, it became clear that the hydrophobic compounds SA-A2 and SA-B2 were highly similar, as well as all the new analogues within the SA-B series.