6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs as antimicrobial agents, a structure-activity relationship was developed involving substitutions of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methylanilino)uracil (EMAU). Series of N3-alkyl derivatives of both 6-AUs were synthesized and tested for their ability to inhibit purified B. subtilis DNA polymerase III and the growth of B. subtilis in culture. Alkyl groups ranging in size from ethyl to hexyl enhanced the capacity of both platforms to bind to the polymerase, and with the exception of hexyl, they also significantly enhanced their antimicrobial potency. N3 substitution of the EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold. In sum, the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents of considerable size and structural variety and, thus, can be manipulated to significantly enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.

Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial DNA polymerase IIIC (pol IIIC). In addition, analogues with 3-substituents in the uracil ring have potent antibacterial activity against Gram+ organisms in culture. In an attempt to find optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in culture. The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns maximized pol IIIC inhibition potency. Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased antibacterial activity. The most potent antibacterials were simple hydroxybutyl and methoxybutyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives.