A Binuclear Ruthenium–Phenformin Compound: Synthesis, Characterization, and Inhibition of the DHFR Enzyme

The Ru-phenformin compound Ru80 was synthesized and characterized by NMR and UV/Vis spectroscopies, as well as by SCXRD, HRMS, and elemental analysis. Enzymatic assays confirm that Ru80 is effective in reducing the catalytic activity of DHFR enzyme. Ru80 retains its inhibitory activity under macromolecularly crowded conditions designed to mimic cellular environments, requiring slightly higher concentrations to achieve comparable levels of inhibition. Thermodynamic analysis demonstrates that Ru80 significantly weakens the binding affinity of NADPH cofactor to DHFR enzyme. Enzyme kinetic analysis also suggests a mixed (noncompetitive) inhibition mechanism where Ru80 binds to the free enzyme as well as the enzyme substrate complex. Ru80 binds to DHFR enzyme in a time-dependent and nonspecific manner.