Design, synthesis, and discovery of cinnamoyl amide derivatives as potent NagZ inhibitors with antibacterial activity

β-N-acetylglucosaminidase (NagZ) plays an important role in the bacterial cell wall biosynthetic pathway. Inhibiting its activity could potentially impede bacterial growth. We report a study on the design and synthesis of cinnamoyl amides derived from rosmarinic acid (RA), and their enzymatic, antibacterial activity against NagZ and Pseudomonas aeruginosa respectively. In vitro enzyme activity determination showed that the best synthetic RA analogues displayed higher inhibitory activity than that of parent RA, in the same range than the most potent NagZ inhibitors reported so far. Remarkably, compounds 11h and Br-6 displayed interesting binding affinity values with K_i=3.3 ± 0.5 and 3.5 ± 1.0 μM, respectively. Docking simulations evidenced significant binding interactions of cinnamoyl amide derivatives with the active site of NagZ. Moreover, kinetic evaluations indicated these compounds displayed competitive behavior. Additionally, MICs of 11h and Br-6 combined with two β-Lactam antibiotics (imipenem and ceftazidime) were evaluated against P. aeruginosa by microdilution checkerboard assay, establishing that antibacterial agents show synergistic effects. In vivo antibacterial efficacy assay using a full-thickness skin defect model with P. aeruginosa infection confirmed these observations.