The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3-iodo-4-methylanilino]uracil) and EMAU (6-[3-ethyl-4-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.New antibacterial agents are needed to combat the multiply resistant gram-positive bacteria endemic in modern health care facilities (15). The 6-anilinouracils (AUs) illustrated in Fig. 1 are selective inhibitors of DNA polymerase III-c2 (pol III), an enzyme product of the polC gene (2,8,12). This enzyme is essential for the replication of the chromosome in gram-positive bacteria (7,9,17) and is found in low-GϩC-content eubacteria, including staphylococci, enterococci, streptococci, Listeria species, Bacillus species, and clostridia (1, 8). Briefly, the AUs, inhibitors of the DNA pol III enzyme, act through their capacity to mimic the guanine moiety of dGTP by forming three hydrogen bonds with cytosine at one of the two active domains (6,7,14). This leaves the second active site on the inhibitor, the aryl domain, available to bind to DNA pol III, which sequesters the enzyme into a nonproductive complex with template primer DNA (19).Structure-activity relationships of these AUs have been described previously (18, 19). The prototypic AUs, which have either weak antimicrobial activities or unacceptably low aqueous solubility (3-6, 13, 17), have now been substituted in their N3 positions and aryl rings to produce a series of more potent and more soluble molecules (13,16,19). The latest generation of these soluble forms (19) includes the N 3 -hydroxybutyl (HB) and N 3 -methoxybutyl (MB) derivatives of 6-[3Ј-ethyl-4Ј-methylanilino]uracil (EMAU) and 6-[3Ј-iodo-4Ј-methylanilino]uracil (IMAU) shown in Fig. 1. In this study, we describe the in vitro activities of HB-IMAU, HB-EMAU, MB-IMAU, and MB-EMAU against staphylococci and enterococci, bacteria that are pathogenic in humans and are difficult to treat with currently available and investigational antimicrobial agents.(This work was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 1999 [J. S. Daly, T. Giehl, N. C. Brown, C.Zhi, G. E. Wright, and R. T. Ellison III, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1808, 1999].) Materials and methods. Bacterial strains used in this study were un...