Late-Stage Functionalization of the Rifamycin Core via Click Chemistry Toward New Antibacterial Derivatives

Antimicrobial resistance (AMR) threatens global health, particularly through the rise of multidrug-resistant tuberculosis (MDR-TB) and other critical bacterial infections such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Rifamycins remain frontline antibiotics but are increasingly undermined by resistance. Here, we introduce a click-enabled platform for the synthesis of C8-functionalized rifamycins, which can be converted in a single additional step into efficacious 3'-hydroxy-5'-aminobenzoxazinorifamycins (bxRifs) and enzymatically into 25-deacetylated rifamycins (deAcRifs), providing access to novel antibacterial scaffolds that expand beyond the scope of traditional C8 modifications. Accordingly, we establish a modular strategy for late-stage analog development of the complex natural product rifamycin S, wherein azido and alkyne functionalities are installed via tailored core chemistry and converted into 1,2,3-triazoles through copper(I)-catalyzed click chemistry. Another key feature of this work is the development of systematic HPLC purification methods, enabling the isolation of analytically pure compounds despite structural complexity. The resulting analogs exhibit distinct antibacterial profiles, notably against Gram-positive bacteria including MRSA and <i>Streptococcus mutans</i>, informing structure-activity relationships and offering a foundation for further optimization. This approach supports the rapid diversification of rifamycin scaffolds to combat the escalating threat of AMR, while also establishing a foundation for future discovery through bioorthogonal applications.