Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

Abstract

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

Figure 1

Structures of (a) cephalosporins 1–3 selectively active on nonreplicating M. tuberculosis, (b) for an inactive analogue, the clinically used antibiotic cephalexin, and (c) the C-2 oxadiazole cephalosporin 5.

Figure 2

Cell-free stability of primary screening hits. Molecules were incubated at 37 °C in PBS (blue) or nonreplicating medium without (orange) or with (red) NaNO₂. Data are averages of replicate samples ± standard deviation.

Figure 3

Cell-free stability of 5. Compound 5 was incubated at 37 °C in PBS (blue) or nonreplicating medium without (orange) or with (red) NaNO₂. Data are averages of replicate samples ± standard deviation.

Scheme 1. Synthetic Route for the Preparation of Ester and Oxadiazole Analogues

Figure 4

Stability of compounds 1 and 5 in plasma. Compounds 1, 5, and cephalexin (4) were tested for stability in mouse (a) and human (b) plasma at the indicated time points. Stability was inferred by monitoring the parent ion. One of two similar experiments. Compound 1 was tested once in human plasma.

Figure 5

Bactericidal activity of compounds 1 and 5 for nonreplicating M. tuberculosis. Nonreplicating wild-type M. tuberculosis at an OD₅₈₀ of 0.01 was exposed to compounds for 7 days, and surviving bacilli were enumerated on 7H11-OADC agar plates. The inoculum is shown in yellow. The limit of detection was 1 colony arising from 10 μL of undiluted sample. Error bars represent standard deviations of triplicates. One of two similar experiments.

Figure 6

Potentiation of activity of cephalosporins against nonreplicating M. tuberculosis by reactive nitrogen species. Wild-type M. tuberculosis was resuspended at an OD₅₈₀ of 0.1 in nonreplicating medium containing indicated concentrations of NaNO₂ (0–1 mM) and dispensed into separate microtiter plates for each NaNO₂ concentration. Cells were then exposed to (a) 1 or (b) rifampicin for 7 days, after which a standard outgrowth assay was initiated to estimate the number of surviving cells. In a separate experiment, nonreplicating M. tuberculosis at a standard OD₅₈₀ of (c) 0.1 or lower inoculum of OD₅₈₀ of (d) 0.01 were treated with either 1 (red) or 5 (blue) in the presence or absence of 0.5 mM NaNO₂ for 7 days. CARA fluorescence provides an estimate of mycobacterial viability; complete loss of fluorescence is associated with ≥2–3 log₁₀ CFU reduction.

Figure 7

Bactericidal activity of (a) 1 and (b) 5 against intracellular M. tuberculosis. Mouse bone marrow-derived macrophages activated or not with 50 ng/mL of IFNγ were infected with wild-type M. tuberculosis. After a 4 h period for bacterial uptake, macrophages were washed and treated with 100 μg/mL of 1 or 5 for (a) 4 or (b) 3 days. Morphology of the macrophages was not affected by addition of 1 or 5 at the concentrations shown. One of five similar experiments.