In this study, three novel series of benzoxazinone, benzothiopyranone and benzopyranone derivatives were designed through scaffold morphing from benzothiazinones to target DprE1. All compounds were evaluated for their in vitro activities against Mycobacterium tuberculosis and cytotoxicity against Vero cell line. Among these three series, the benzothiopyranone series displayed excellent antimycobacterial activity and low cytotoxicity. In particular, compound 6b exhibited potent in vitro activity against both drug-susceptible and drug-resistant tuberculosis clinical strains with MICs <0.016 μg/mL. In addition, compound 6b demonstrated excellent ADME/T and PK properties and potent in vivo efficacy with bactericidal activity in an acute mouse model of tuberculosis. The antituberculosis effect of compound 6b is most likely attributed to its excellent anti-DprE1 activity. As such, compound 6b is under evaluation as a potential clinical candidate for treatment of tuberculosis. The current study provided new insight into the structural and pharmacological requirements for DprE1 inhibitors as potent antitubercular agents.