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This paper describes the design and characterization of novel inhibitors of IleRS, whose binding affinity approaches the tightest reported for noncovalent inhibition. Compounds were designed from a binding model for the natural product pseudomonic acid-A (PS-A) together with a detailed understanding of the reaction cycle of IleRS and characterization of the mode of binding of the reaction intermediate IleAMP. The interactions of the compounds with IleRS were characterized by inhibition of aminoacylation of tRNA or PP(i)/ATP exchange at supersaturating substrate concentration and by transient kinetics and calorimetry methods. A detailed understanding of the interaction of a comprehensive series of compounds with IleRS allowed the identification of key features and hence the design of exquisitely potent inhibitors. Predictions based on these results have been recently supported by a docking model based on the crystal structure of IleRS with PS-A [Silvian, L. F., Wang J. M., and Steitz T. A. (1999) Science 285 1074-1077].

A series of beta-diketone acrylate bioisosteres 4 of pseudomonic acid A 1 have been synthesized and evaluated for their ability to inhibit bacterial isoleucyl-tRNA synthetase and act as antibacterial agents. A number of analogues have excellent antibacterial activity. Selected examples were shown to afford good blood levels and to be effective in a murine infection model.

The electronic requirements around the C1-C3 region of pseudomonic acid analogues were investigated. Synthetic routes were developed to access a range of compounds where the alpha, beta-unsaturated ester moiety had been replaced by a 5-membered ring heterocycle. The inhibition of isoleucyl tRNA synthetase from Staphylococcus aureus NCTC 6571 was determined as was the minimum inhibitory concentration (MIC) of the test compounds against that organism. Compounds possessing a region of electrostatic potential corresponding to that of the carbonyl group in the alpha, beta-unsaturated ester, and a low-energy unoccupied molecular orbital in the region corresponding to the double bond, were found to have IC50 values of 0.7-5.3 ng mL-1. However the MIC values of these compounds were in the range 2.0-8.0 micrograms mL-1, reflecting their poorer penetration into the bacterial cell.

A series of C-1 oxazole isosteres of pseudomonic acid A (mupirocin) bearing a nitroheterocycle have been synthesized, and significant differences in both spectrum of activity and potency were found between these derivatives and mupirocin. Additionally, the antibacterial potency of two members of this class of compounds against mupirocin-resistant staphylococci could not be accounted for solely by inhibition of the target enzyme isoleucyl-tRNA synthetase (IRS), indicating an additional mode of action. The most potent compound, the nitrofuran 3f (SB 205952), was the most electron affinic derivative prepared and was transformed by NAD(P)H-dependent bacterial reductases at a rate similar to that for nitrofurantoin. The second mode of action of this compound may therefore arise from its reduction to a species with cellular targets other than IRS. In in vivo studies, 3f was shown to be a very effective agent by both the subcutaneous and oral routes of administration.

The synthesis, antibacterial activities, murine pharmacokinetic and infection model data for a range of aryl and heteroaryl ketone derivatives of monic acid (2a) are reported. The best results were found for the 3-furyl and 2-methoxy thiazol-5-yl analogues.

The synthesis of a range of 5-alkyl, 5-alkenyl, and 5-heterosubstituted 2-(1-normon-2-yl) oxazoles is described. The antibacterial activity was determined as the minimum inhibitory concentration against a range of Gram-positive and Gram-negative organisms using a standard Agar dilution procedure. Compounds possessing an acid functionality directly on, or close to, the ring were found to be of greatly decreased potency, while increasing lipophilicity with greater chain length led to increased potency of these derivatives.

Semisynthetic analogues of pseudomonic acid A have been prepared containing a heterocyclyl substituted oxazole. Derivatives in which the heterocycle was thiophene, furan, pyridine, or isoxazole showed good antibacterial potency and were further evaluated in vivo. Both pharmacokinetic parameters and oral activity against an experimental intraperitoneal sepsis were superior to results obtained from previously described pseudomonic acid A derivatives.