Organocatalytic synthesis of novel pyrazoline and pyrimidine derivatives as potent thymidine kinase inhibitors targeting Staphylococcus aureus

A series of phenothiazine- and coumarin-based pyrazoline and pyrimidine derivatives were synthesized under metal-free conditions in excellent yields. The synthesized compounds were evaluated for their physicochemical properties and biological activities, including enzyme assays against thymidine kinase (TK) and anti-bacterial testing against S. aureus (ATCC 29,213), with ciprofloxacin used as a control. Several derivatives exhibited notable anti-bacterial activity, with compounds 5b and 5d demonstrating the strongest inhibition of TK. Fluorescence quenching studies indicated strong binding interactions and the binding constants (K) for compounds 5b and 5d were determined to be 7.38 x 106 M-1 and 8.39 x 106 M-1, respectively, indicating strong interactions with a single binding site. Molecular docking results indicated that compounds 5b and 5d achieved docking scores of-4.879 and-4.984 kcal/mol, along with favorable binding free energy (Delta Gbind) values of-48.47 and-45.28 kcal/mol, respectively. Also, the docking analysis showed that these molecules bound to the active pocket residues of TK by strong hydrogen bonding interactions. ADME analysis confirmed that both compounds adhered to Lipinski's rule of five, indicating good drug-likeness. Enzyme assays revealed that 5b and 5d inhibited TK with low IC50 values 13.04 and 13.88 mu M, respectively, suggesting their potential as potent TK inhibitors. Furthermore, molecular dynamics (MD) simulations were conducted to provide insight into the stability and binding behavior of these inhibitors within the active site of TK, reinforcing the experimental results and supporting their potential as promising therapeutic agents.