7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity.

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N(2)-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N(2)-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram- bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10µg/ml), and were inactive against the Gram- organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.

Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound.

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ "hybrids" offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, "251D," were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of Staphylococcus aureus resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens.

Hybrid antibacterials. DNA polymerase-topoisomerase inhibitors.

Novel Gram-positive (Gram+) antibacterial compounds consisting of a DNA polymerase IIIC (pol IIIC) inhibitor covalently connected to a topoisomerase/gyrase inhibitor are described. Specifically, 3-substituted 6-(3-ethyl-4-methylanilino)uracils (EMAUs) in which the 3-substituent is a fluoroquinolone moiety (FQ) connected by various linkers were synthesized. The resulting "AU-FQ" hybrid compounds were significantly more potent than the parent EMAU compounds as inhibitors of pol IIIC and were up to 64-fold more potent as antibacterials in vitro against Gram+ bacteria. The hybrids inhibited the FQ targets, topoisomerase IV and gyrase, with potencies similar to norfloxacin but 10-fold lower than newer agents, for example, ciprofloxacin and sparfloxacin. Representative hybrids protected mice from lethal Staphylococcus aureus infection after intravenous dosing, and one compound showed protective effect against several antibiotic-sensitive and -resistant Gram+ infections in mice. The AU-FQ hybrids are a promising new family of antibacterials for treatment of antibiotic-resistant Gram+ infections.

Synthesis and antibacterial activity of 3-substituted-6-(3-ethyl-4-methylanilino)uracils.

Numerous 3-substituted-6-(3-ethyl-4-methylanilino)uracils (EMAU) have been synthesized and screened for their capacity to inhibit the replication-specific bacterial DNA polymerase IIIC (pol IIIC) and the growth of Gram+ bacteria in culture. Direct alkylation of 2-methoxy-6-amino-4-pyrimidone produced the N3-substituted derivatives, which were separated from the byproduct 4-alkoxy analogues. The N3-substituted derivatives were heated with a mixture of 3-ethyl-4-methylaniline and its hydrochloride to effect displacement of the 6-amino group and simultaneous demethylation of the 2-methoxy group to yield target compounds in good yields. Certain intermediates, e.g. the 3-(iodoalkyl) compounds, were converted to a variety of (3-substituted-alkyl)-EMAUs by displacement. Most compounds were potent competitive inhibitors of pol IIIC (K(i)s 0.02-0.5 microM), and those with neutral, moderately polar 3-substituents had potent antibacterial activity against Gram+ organisms in culture (MICs 0.125-10 microg/mL). Several compounds protected mice from lethal intraperitoneal (ip) infections with S. aureus (Smith) when given by the ip route. A water soluble derivative, 3-(4-morpholinylbutyl)-EMAU hydrochloride, given subcutaneously, prolonged the life of infected mice in a dose dependent manner.

Active site directed inhibitors of replication-specific bacterial DNA polymerases.

7-Substituted-N(2)-(3,4-dichlorobenzyl)guanines potently and competitively inhibit DNA polymerases IIIC and IIIE from Gram(+) bacteria. Certain derivatives are also competitive inhibitors of DNA polymerase IIIE from Gram(-) bacteria.

Synthesis of substituted 6-anilinouracils and their inhibition of DNA polymerase IIIC and Gram-positive bacterial growth.

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.