IntroductionBacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. Previous screening of a focused chemical compound library using a polyU mRNA‐directed aminoacylation/translation protein synthesis system from Escherichia coli yielded a lead series of compounds that inhibit protein synthesis with IC50's ranging from 3 to14 μM (Ribble, et. al. (2010) AAC, 54, 4648–4657). This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3‐d]pyrimidin‐4‐ol (e.g. 4H‐pyridopyrimidine). These compounds inhibited bacterial growth with minimum inhibitory concentrations (MICs) ranging from 0.25 to 32 μg/ml.ResultsOver 100 analogs of the initial compounds were designed and synthesized and assayed. In biochemical protein synthesis reactions nineteen of the compounds exhibited IC50 values less than 2.0 μM and seven less than 1.0 μM. These compounds were active against S. pneumoniae (MIC=2 μg/mL), H. influenzae (MIC=1 μg/mL), and M. catarrhalis (MIC≤0.12 μg/mL) and S. aureus (4= μg/ml). Macromolecular synthesis (MMS) assays in E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis.ConclusionOptimization of 4H‐pyridopyrimidines yields potent inhibitors of protein synthesis with good activity against respiratory pathogens.