Structure-based design was used to develop a focused library of A-ring-modified diphenyl ether InhA inhibitors. From this library of analogs, two high-affinity alkyl-substituted diphenyl ethers, 6PP and 8PP, were selected for advanced study into their in vitro activity against Mycobacterium tuberculosis clinical isolates, their in vivo properties, and their signature response mode of action. 6PP and 8PP demonstrated enhanced activity against whole bacteria and showed activity in a rapid macrophage model of infection. In addition, transcriptional profiling revealed that the A-ring modifications of 6PP and 8PP increased the specificity of each analog for InhA. Both analogs had substantially longer half-lives in serum than did the parent compound, exhibited a fivefold reduction in cytotoxicity compared to the parent compound, and were well tolerated when administered orally at 300 mg/kg of body weight in animal models. Thus, the A-ring modifications increased the affinity and whole-cell specificity of the compounds for InhA and increased their bioavailability. The next step in optimization of the pharmacophore for preclinical evaluation is modification of the B ring to increase the bioavailability to that required for oral delivery.