Novel Derivatives of 3-Amino-4-hydroxy-benzenesulfonamide: Synthesis, Binding to Carbonic Anhydrases, and Activity in Cancer Cell 2D and 3D Cultures

A series of novel derivatives of 3-amino-4-hydroxybenzenesulfonamide was synthesized. As the analyzed compounds possess a sulfonamide group, the affinity of these compounds for human carbonic anhydrases (CAs) was measured by fluorescent thermal shift assay, and compound selectivity for different isoenzymes was identified. The crystal structures of the complexes of compound <b>25</b> with CAI and CAII were determined. Additionally, the activity of compounds on the viability of three cancer cell lines-human glioblastoma U-87, triple-negative breast cancer MDA-MB-231, and prostate adenocarcinoma PPC-1-was established using the MTT assay and compared to CAIX-selective and non-selective comparative compounds U-104 and acetazolamide. The half-maximal concentration (EC₅₀) was determined for the identified most active compounds, and their selectivity over fibroblasts was established. Compound <b>9</b> (inhibitor of multi-CAs) and compound <b>21</b> (not binding to CAs), considered the most promising candidates, were tested in cancer cell 3D cultures (cancer spheroids) by assessing their effect on spheroid growth and viability. Both compounds reduced the viability of spheroids from all cancer cell lines. U-87 and PPC-1 spheroids became looser in the presence of compound <b>9</b>, while the growth of MDA-MB-231 spheroids was slower compared to the control. Compound <b>21</b> reduced the growth of U-87 and MDA-MB-231 3D cultures, with no significant effect on PPC-1 spheroids.