Binding Mechanisms and Therapeutic Activity of Heterocyclic Substituted Arylazothioformamide Ligands and Their Cu(I) Coordination Complexes

Finding new sources of biologically active compounds for anticancer or antimicrobial therapies remains an active area of research. Azothioformamides (ATFs) with a 1,3 N=N-C=S heterodiene backbone are a new class of biologically active compounds that chelate metals (e.g., Cu) forming stable ATF metal coordination complexes. In this study, ATF ligands were prepared with pyrrolidine, piperidine, <i>N</i>-methylpiperazine, and morpholine substituents on the formamide as to add more heterocyclic drug-like character for biological studies. Formamide derivatives were then complexed with various Cu(I) salts to form coordination complexes. Cu(I) salts were selected as to create potential bioactive compounds with less toxicity. Binding association constants of each Cu(I) salt to ATF ligands were extrapolated from UV-vis titration studies and were corroborated with DFT calculations using a hybrid functional B3LYP method. It was observed that the smaller pyrrolidine functionalized ATFs bound to the Cu(I) salts had stronger binding than any of the larger six-membered-ring heterocycles with association values in the 10⁴ - 10⁵ M^-1 range. The ATF-Cu(I) salt coordination complexes were then evaluated for antimicrobial activity against two bacteria (<i>Staphylococcus aureus</i>, <i>Escherichia coli</i>), one yeast (<i>Candida albicans</i>), four human cancer lines (A-549, K-562, HT-1080, MDA-MB-231), and two normal human lines (MRC-5, HFF). The ATF ligands themselves were inactive against all microbes and most human lines except K-562 cells, which were sensitive to three of the four ligands (IC₅₀'s = 7.0-25.5 μM). Most ATF-Cu(I) complexes showed low to medium micromolar activity against <i>Candida albicans</i> (IC₅₀'s 2.6-24.8 μM) and <i>Staphylococcus aureus</i> (IC₅₀'s = 3.4-37.7 μM), with increasing activity corresponding to complexes with higher binding association constants. The antiproliferative properties of ATF-Cu(I) metal salt complexes against mammalian cells were mixed, with low to medium micromolar activity across all cell lines. Notably, several ATF-Cu(I) salt coordination complexes showed submicromolar activity against the HT-1080 fibrosarcoma line (0.52-0.69 μM). The results demonstrate promising activity of ATF-Cu(I) complexes, particularly with pyrrolidine as the formamide component. These studies suggest that the stronger binding association values correlate to higher levels of biological activity.