Discovery of novel antitubercular drugs is an effective strategy against drug-resistant tuberculosis (TB). Our previous study has identified LPX-16jas a novel antitubercular compound. Herein, we perform a comprehensive structure–activity relationship (SAR) based on LPX-16j, indicating that the central pyrimidine ring moiety was crucial for the antitubercular activities of its derivatives, and replacing the naphthyl group with hydrophobic substitutes was well tolerated. The representative derivative 5aexhibited potent activity against H37Ra, H37Rv, and clinical drug-resistant TB with minimum inhibitory concentration (MIC) values of 0.5–1.0 μg/mL. Meanwhile, 5ashowed an acceptable safety in vivoand displayed a favorable oral bioavailability with a value of 40.7%. The differential scanning fluorescence, isothermal titration calorimetry, and molecular docking assays indicated that PknB could be one of the targets of compound 5a. Overall, this study identified 5aas a novel promising lead compound with the potential to develop candidates for the treatment of drug-resistant TB.