i In this paper, the numbering of the compounds retains the numbering used in the online Open Source Antibiotics project, maintaining the connection between data in this paper and the live research. Molecules in Open Source Antibiotics are numbered according to a convention described online () and in the Supplementary Information. In the rest of this paper, the prefix "OSA_" is omitted. The chemotype was of previous interest because of a screen of a GlaxoSmithKline internal library collection for inhibitors of the transforming growth factor β1 (TGF-β1) type I receptor (ALK5), where compound 812 displayed an IC50 of 0.46 µM against ALK5 and no inhibition of p38 kinase. Optimization led to the selective inhibitor SB-431542 (IC50 94 nM against ALK5 and still no inhibition of p38 kinase, CC50 > 30 µM), which inhibits TGF-β1-induced fibronectin mRNA formation (IC50 0.05 µM) while displaying no measurable cytotoxicity in a 48 hour XTT assay (CC50 > 30 µM). The promising activity against MRSA, and the low cytotoxicity observed in the ALK5 research, stimulated us to pursue a new hit-to-lead campaign. Three regions of the main scaffold were modified: the bicyclic imidazole core (black), the "northwest" aryl substituent (blue), and the "southwest" aryl substituent (green). Diversification of these motifs, as well as their linkages, formed the basis of our synthetic studies of this chemotype. The aim was to generate potent compounds with promising physicochemical properties and improved metabolic stability while maintaining an acceptable toxicity vs. mammalian cells.