By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.