*4.25. SRB Assay*

Breast cancer cells were seeded in 96 well plates with different cell numbers depending on the cell line in triplicate and after 24 h treated with different concentrations of compounds **14**–**28**. Treatment ended after 96 h when cells were fixed with 10% trichloroacetic acid (Carl Roth GmbH, Karlsruhe, Germany) for 1h at 4 ◦C. Afterwards, cells were washed with ice water four times and stained with 4.4% SRB solution (Sigma–Aldrich) for 10 min at room temperature. After washing cells with 1% acetic acid (Carl Roth GmbH), cells were air-dried overnight and then dissolved with 300 μL 20 mM Tris base solution (Sigma-Aldrich). Excitation was measured at 540 nm with a Spark plate reader (Tecan Treading AG, Männedorf, Switzerland) and IC50 values were calculated by dose-response curve fitting using Origin 2019 (OriginLab Corp., Northampton, MA, USA).

#### *4.26. Cell Death*

For the determination of apoptotic and necrotic cell death after treatment with compound 28, Annexin V-Sytox Deep Red staining was performed. Therefore, MDA-MB-231 and HS578T cells were seeded in 6-well plates. After 24 h, the cells were treated with different concentrations of compound 28 (10 nM, 100 nM, 250 nM, 500 nM, 1 μM, and 2 μM) for 24 h, 48 h, and 72 h at 37 ◦C and 5% CO2. For analysis of cell death, detached cells were collected in tubes and living cells were detached by accutase (Biowest, Nuaillé, France) and collected in the same tube. After several washing steps cells were resuspended in 1x annexin V binding puffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) and stained with 5 μL Annexin V-FITC (BioLegend, San Diego, CA, USA) and 1 μL 100 μM Sytox Deep Red Nucleic Acid Stain (Invitrogen, Thermo Fisher Scientific) for 15 min. Afterward, 400 μL 1x annexin V binding puffer were added to each tube. Gating was realized by the use of unstained, single annexin V-FITC or single Sytox Deep Red Nucleic Acid-stained cells, respectively. For quantification of necrotic and apoptotic cells, 10,000 cells were analyzed by LSRFortessa™ flow cytometer (BD Biosciences, Heidelberg, Germany).
