The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide.