Design, Synthesis, and Antibacterial Activity Evaluation and Mechanism of 1-<i>H</i>-Indole-3-pyrazolamide Derivatives Containing Piperazine

To develop antibacterial agents with novel mechanisms of action, a series of novel 3-pyrazolylindole derivatives incorporating piperazine moieties were designed and synthesized. The <i>in vitro</i> antibacterial activities of these compounds were evaluated against four phytopathogenic bacterial strains: <i>Xanthomonas oryzae</i> pv <i>oryzae</i> (<i>Xoo</i>), <i>Xanthomonas axonopodis</i> pv <i>citri</i> (<i>Xac</i>), <i>Pseudomonas syringae</i> pv <i>actinidiae</i> (<i>Psa</i>), and <i>Ralstonia solanacearum</i> (<i>Rs</i>). All of the synthesized derivatives exhibited moderate antibacterial activity. In particular, compound <b>P16</b> demonstrated potent inhibitory activity against <i>Xoo</i> and <i>Xac</i>, with EC₅₀ values of 2.54 ± 0.87 and 3.49 ± 0.04 μg/mL, respectively, which were significantly lower than those observed for the positive controls, paclobutrazol and copper thiazole. Compound <b>P16</b> showed protective and therapeutic efficacies of 86.41% and 94.56%, respectively, against <i>Xoo</i> infection. Furthermore, compound <b>P16</b> was shown to effectively suppress biofilm formation, bacterial motility, and extracellular polysaccharide production, induce morphological alterations in bacterial cells, and promote reactive oxygen species (ROS) accumulation, ultimately triggering bacterial apoptosis. Mechanistic studies revealed that compound <b>P16</b> down-regulated the expression of genes associated with the type VI secretion system (T6SS), thereby conferring protection against rice bacterial blight and citrus canker. These results indicate that compound <b>P16</b> holds significant potential as a lead candidate for further development as an antibacterial agent.