Evaluation of the effect of bacterial efflux pumps on the antibacterial activity of the novel fluoroquinolone besifloxacin.

This study examined the susceptibility of a variety of wild-type strains and efflux pump mutants to besifloxacin and the comparator agents sparfloxacin, ciprofloxacin, norfloxacin, moxifloxacin, tetracycline, and ethidium bromide. Organisms tested included Staphylococcus aureus (mepA or norA), Streptococcus pneumoniae (pmrA, patB), Escherichia coli (acrAB::Tn903, tolC::Tn10), Haemophilus influenzae (acrAB) and Pseudomonas aeruginosa (mepAB-oprM, oprM::ΩHg(r) rpsL). The minimal inhibitory concentrations (MIC) of besifloxacin and comparators were also measured in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide mchlorophenyl- hydrazone, or sodium orthovanadate. Overall, very few meaningful changes (>2-fold) in besifloxacin MIC values resulted from the presence of efflux pump mutations or efflux pump inhibitors. In summary, the novel fluoroquinolone besifloxacin is no exception to the observation that newer fluoroquinolones are generally less affected by efflux pump-mediated resistance than older fluoroquinolones.

Carbon-carbon-linked (pyrazolylphenyl)oxazolidinones with antibacterial activity against multiple drug resistant gram-positive and fastidious gram-negative bacteria.

In an effort to expand the spectrum of activity of the oxazolidinone class of antibacterial agents to include Gram-negative bacteria, a series of new carbon-carbon linked pyrazolylphenyl analogues has been prepared. The alpha-N-substituted methyl pyrazole (10alpha) in the C3-linked series exhibited very good Gram-positive activity with MICs

Enols as potent antibacterial agents.

This paper describes the discovery of alpha-trifluoroketoacetamides as potent antibacterial agents against Gram-positive organisms. The initial SAR indicates that the aryl ethyl side chain is essential in maintaining antibacterial activity. The SAR observations have been utilized to design a bioisostere for the alpha-trifluoroketoacetamide with good activity against Gram-positive organisms.

3-Arylpiperidines as potentiators of existing antibacterial agents.

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

The discovery of linezolid, the first oxazolidinone antibacterial agent.

The emergence of new antibiotic-resistance in the significant Gram-positive pathogens in the last decade created a substantial medical need for new classes of antibacterial agents. Pharmacia Corporation scientists initiated a discovery research program in oxazolidinone chemistry and biology. Indanone-, tetralone-, and indoline-subunit oxazolidinones provided proof-of-concept interim improvements in antibacterial activity and safety SAR for the program. A method for enantiomeric enrichment of analogs was developed and intensive synthesis and evaluation efforts were undertaken with three oxazolidinone subclasses; the piperazine, indoline, and tropones. Members of the piperazinyl-phenyloxazolidinones possessed the most suitable chemical characteristics and biologic activity of the three subclasses. The monofluorophenyl congener eperezolid and the morpholino analog linezolid emerged as the first clinical candidates from the piperazine oxazolidinones. Linezolid was selected for continued human clinical evaluation based upon its' superior pharmacokinetic profile. Microbiologic testing revealed that linezolid compared very favorably against comparator antibiotics in vitro and in animal infection models. Linezolid possessed a unique mechanism of action in that it inhibited functional 70S initiation complex formation and did not cross-react with existing bacterial resistance. Oral bioavailability in humans was determined to be 100% and twice daily dosing in humans resulted in blood levels which even at trough values were in excess of the MIC90 for significant Gram-positive pathogens. The preclinical promise of linezolid was realized in human clinical trials where linezolid was highly efficacious in the treatment of medically significant Gram-positive infections.

Oxazolidinones: a new class of antibacterials.

The oxazolidinones represent the first truly new class of antibacterial agents to reach the marketplace in several decades. They have a unique mechanism of action involving inhibition of the initiation step of protein synthesis and are not cross-resistant to other classes of antibiotics. The first marketed member of that class, linezolid (Zyvox), shows good efficacy with an impressive antibacterial spectrum (including activity against gram-positive organisms resistant to other drugs), and a pharmacodynamic/pharmacokinetic relationship best characterized by time above the minimum inhibitory concentration. The agent is effective by both the intravenous and oral route of administration. Although technically classified as bacteriostatic against a number of pathogens in vitro, linezolid behaves in vivo like a bactericidal antibiotic.

Substituent effects on the antibacterial activity of nitrogen-carbon-linked (azolylphenyl)oxazolidinones with expanded activity against the fastidious gram-negative organisms Haemophilus influenzae and Moraxella catarrhalis.

A series of new nitrogen-carbon-linked (azolylphenyl)oxazolidinone antibacterial agents has been prepared in an effort to expand the spectrum of activity of this class of antibiotics to include Gram-negative organisms. Pyrrole, pyrazole, imidazole, triazole, and tetrazole moieties have been used to replace the morpholine ring of linezolid (2). These changes resulted in the preparation of compounds with good activity against the fastidious Gram-negative organisms Haemophilus influenzae and Moraxella catarrhalis. The unsubstituted pyrrolyl analogue 3 and the 1H-1,2,3-triazolyl analogue 6 have MICs against H. influenzae = 4 microgram/mL and M. catarrhalis = 2 microgram/mL. Various substituents were also placed on the azole moieties in order to study their effects on antibacterial activity in vitro and in vivo. Interesting differences in activity were observed for many analogues that cannot be rationalized solely on the basis of sterics and position/number of nitrogen atoms in the azole ring. Differences in activity rely strongly on subtle changes in the electronic character of the overall azole systems. Aldehyde, aldoxime, and cyano azoles generally led to dramatic improvements in activity against both Gram-positive and Gram-negative bacteria relative to unsubstituted counterparts. However, amide, ester, amino, hydroxy, alkoxy, and alkyl substituents resulted in no improvement or a loss in antibacterial activity. The placement of a cyano moiety on the azole often generates analogues with interesting antibacterial activity in vitro and in vivo. In particular, the 3-cyanopyrrole, 4-cyanopyrazole, and 4-cyano-1H-1,2,3-triazole congeners 28, 50, and 90 had S. aureus MICs

Piperazinyl oxazolidinone antibacterial agents containing a pyridine, diazene, or triazene heteroaromatic ring.

Oxazolidinones are a novel class of synthetic antibacterial agents active against gram-positive organisms including methicillin-resistant Staphylococcus aureus as well as selected anaerobic organisms. Important representatives of this class include the morpholine derivative linezolid 2, which is currently in phase III clinical trials, and the piperazine derivative eperezolid 3. As part of an investigation of the structure-activity relationships of structurally related oxazolidinones, we have prepared and evaluated the antibacterial properties of a series of piperazinyl oxazolidinones in which the distal nitrogen of the piperazinyl ring is substituted with a six-membered heteroaromatic ring. Compounds having MIC values

Ribosomes from an oxazolidinone-resistant mutant confer resistance to eperezolid in a Staphylococcus aureus cell-free transcription-translation assay.

Oxazolidinone-resistant mutants of Staphylococcus aureus, isolated with a spiral plating technique, had a 16-fold higher MIC (2 versus 32 microg/ml) of eperezolid when compared to the parental sensitive strain. Eperezolid inhibited in vitro protein translation with 50% inhibitory concentrations of 30 microM for the oxazolidinone-sensitive S30 extract and 75 microM for the resistant extract. Experiments mixing various combinations of S100 and crude ribosome preparations from oxazolidinone-sensitive and -resistant S. aureus strains in a transcription-translation assay demonstrated that the resistant determinant resided within the ribosomal fraction. Ribosomes from the oxazolidinone-resistant strain bound less drug than ribosomes from the sensitive strain, indicating that the ribosome is the site of action for the oxazolidinones. These experiments demonstrate that an alteration of the ribosome is responsible for some or all of the oxazolidinone resistance observed in the S. aureus mutant.

Synthesis and antibacterial activity of [6,5,5] and [6,6,5] tricyclic fused oxazolidinones.

A series of conformationally restricted, [6,5,5] and [6,6,5] tricyclic fused oxazolidinones were synthesized and tested for antibacterial activity. Several compounds in the trans-[6,5,5] series demonstrated potent in vitro and in vivo activity. This work provides valuable information regarding the preferred conformational orientation of the oxazolidinones at the binding site.

Serum inhibitory titers and serum bactericidal titers for human subjects receiving multiple doses of the antibacterial oxazolidinones eperezolid and linezolid.

In Phase I trials subjects received multiple doses of eperezolid (PNU-100592; formerly U-100592) and linezolid (PNU-100766; formerly U-100766), and steady-state samples were drawn at the projected peak and trough timepoints. Serum inhibitory titer and serum bactericidal titer values were determined using single strains of Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae. Serum inhibitory titer values generally correlated with drug concentration in serum and inherent organism susceptibility. Against S. aureus and E. faecalis sera from patients dosed with either drug were generally inhibitory at the peak timepoint, but at trough only linezolid exhibited a persistent effect. No bactericidal activity was seen for either drug against S. aureus or E. faecalis. The sera from patients dosed with either drug exhibited inhibition of S. pneumoniae at peak and trough. Bactericidal activity was seen against S. pneumoniae for both drugs at peak time and at trough for many of the sera for patients on the higher dose regimens. The results demonstrated that the sera from most human subjects dosed with eperezolid or linezolid were inhibitory to S. aureus and E. faecalis and S. pneumoniae and that many of the samples exhibited bactericidal activity for S. pneumoniae.

Oxazolidinones: new antibacterial agents.

The oxazolidinones are a new chemical class of synthetic antibacterial agents that are active orally or intravenously against multidrug-resistant Gram-positive bacteria. Their unique mechanism of action and activity against bacteria that pose therapeutic problems in hospital and community treatments make them promising candidates for antimicrobial agents.

Oxazolidinone antibacterial agents: development of the clinical candidates eperezolid and linezolid.

Antimicrobial resistance is a significant nosocomial problem and is of increasing importance in community-acquired infections. One approach for overcoming resistance is the discovery and development of agents with new mechanisms of action. The oxazolidinones make up a relatively new class of antimicrobial agents which possess a unique mechanism of bacterial protein synthesis inhibition. Eperezolid and linezolid are two novel analogues that have demonstrated a variety of positive attributes. These agents inhibit many clinically-significant bacterial species both in vitro and in animal models of human infection. Furthermore they have oral bioavailability, and are well tolerated in humans at doses which produce plasma concentrations in excess of the levels predicted to be necessary for efficacy. In this review, we discuss the key information from the literature that supports the Phase II development of linezolid.

In vitro activity of linezolid and eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria.

Linezolid (formerly U-100766) and eperezolid (formerly U-100592) are novel oxazolidinone antimicrobial agents that are active against multi-drug-resistant staphylococci, streptococci, enterococci, corynebacteria, and mycobacteria. Preliminary studies also demonstrated that the compounds inhibited some test strains of anaerobic bacteria. Therefore, we extended the in vitro evaluation of these agents to include a total of 54 different anaerobic species. Minimal inhibitory concentration (MIC) values were determined using a standard agar dilution method for 143 anaerobic bacterial isolates. Eperezolid and linezolid demonstrated potent activity against the anaerobic Gram-positive organisms with most MIC values in the range of 0.25-4 microg/mL. Viridans streptococci demonstrated MICs of 1-2 microg/mL; Peptostreptococcus species and Propionibacterium species were inhibited by

In vitro activities of U-100592 and U-100766, novel oxazolidinone antibacterial agents.

Oxazolidinones make up a relatively new class of antimicrobial agents which possess a unique mechanism of bacterial protein synthesis inhibition. U-100592 (S)-N-[[3-[3-fluoro-4-[4-(hydroxyacetyl)-1-piperazinyl]- phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide and U-100766 (S)-N-[[3-[3-fluoro-4-(4-morpholinyl)phenyl]- 2-oxo-5-oxazolidinyl]methyl]-acetamide are novel oxazolidinone analogs from a directed chemical modification program. MICs were determined for a variety of bacterial clinical isolates; the respective MICs of U-100592 and U-100766 at which 90% of isolates are inhibited were as follows: methicillin-susceptible Staphylococcus aureus, 4 and 4 micrograms/ml; methicillin-resistant S. aureus, 4 and 4 micrograms/ml; methicillin-susceptible Staphylococcus epidermidis, 2 and 2 micrograms/ml; methicillin-resistant S. epidermidis, 1 and 2 micrograms/ml; Enterococcus faecalis, 2 and 4 micrograms/ml; Enterococcus faecium, 2 and 4 micrograms/ml; Streptococcus pyogenes, 1 and 2 micrograms/ml; Streptococcus pneumoniae, 0.50 and 1 microgram/ml; Corynebacterium spp., 0.50 and 0.50 micrograms/ml; Moraxella catarrhalis, 4 and 4 micrograms/ml; Listeria monocytogenes, 8 and 2 micrograms/ml; and Bacteroides fragilis, 16 and 4 micrograms/ml. Most strains of Mycobacterium tuberculosis and the gram-positive anaerobes were inhibited in the range of 0.50 to 2 micrograms/ml. Enterococcal strains resistant to vancomycin (VanA, VanB, and VanC resistance phenotypes), pneumococcal strains resistant to penicillin, and M. tuberculosis strains resistant to common antitubercular agents (isoniazid, streptomycin, rifampin, ethionamide, and ethambutol) were not cross-resistant to the oxazolidinones. The presence of 10, 20, and 40% pooled human serum did not affect the antibacterial activities of the oxazolidinones. Time-kill studies demonstrated a bacteriostatic effect of the analogs against staphylococci and enterococci but a bactericidal effect against streptococci. The spontaneous mutation frequencies of S. aureus ATCC 29213 were <3.8 x 10(-10) and <8 x 10(-11) for U-100592 and U-100766, respectively. Serial transfer of three staphylococcal and two enterococcal strains on drug gradient plates produced no evidence of rapid resistance development. Thus, these new oxazolidinone analogs demonstrated in vitro antibacterial activities against a variety of clinically important human pathogens.

Synthesis and antibacterial activity of U-100592 and U-100766, two oxazolidinone antibacterial agents for the potential treatment of multidrug-resistant gram-positive bacterial infections.

Bacterial resistance development has become a very serious clinical problem for many classes of antibiotics. The 3-aryl-2-oxazolidinones are a relatively new class of synthetic antibacterial agents, having a new mechanism of action which involves very early inhibition of bacterial protein synthesis. We have prepared two potent, synthetic oxazolidinones, U-100592 and U-100766, which are currently in clinical development for the treatment of serious multidrug-resistant Gram-positive bacterial infections caused by strains of staphylococci, streptococci, and enterococci. The in vitro and in vivo (po and iv) activities of U-100592 and U-100766 against representative strains are similar to those of vancomycin. U-100592 and U-100766 demonstrate potent in vitro activity against Mycobacterium tuberculosis. A novel and practical asymmetric synthesis of (5S)-(acetamidomethyl)-2-oxazolidinones has been developed and is employed for the synthesis of U-100592 and U-100766. This involves the reaction of N-lithioarylcarbamates with (R)-glycidyl butyrate, resulting in excellent yields and high enantiomeric purity of the intermediate (R)-5-(hydroxymethyl)-2-oxazolidinones.

Identification of a novel oxazolidinone (U-100480) with potent antimycobacterial activity.

During the course of our investigations in the oxazolidinone antibacterial agent area, we have identified a subclass with especially potent in vitro activity against mycobacteria. The salient structural feature of these oxazolidinone analogues, 6 (U-100480), 7 (U-101603), and 8 (U-101244), is their appended thiomorpholine moiety. The rational design, synthesis, and evaluation of the in vitro antimycobacterial activity of these analogues is described. Potent activity against a screening strain of Mycobacterium tuberculosis was demonstrated by 6 and 7 (minimum inhibitory concentrations or MIC's < or = 0.125 micrograms/mL). Oxazolidinones 6 and 8 exhibit MIC90 values of 0.50 micrograms/mL or less against a panel of organisms consisting of five drug-sensitive and five multidrug-resistant strains of M. tuberculosis, with 6 being the most active congener. Potent in vitro activity against other mycobacterial species was also demonstrated by 6. For example, 6 exhibited excellent in vitro activity against multiple clinical isolates of Mycobacterium avium complex (MIC's = 0.5-4 micrograms/mL). Orally administered 6 displays in vivo efficacy against M. tuberculosis and M. avium similar to that of clinical comparators isoniazid and azithromycin, respectively. Consideration of these factors, along with a favorable pharmaco-kinetic and chronic toxicity profile in rats, suggests that 6 (U-100480) is a promising antimycobacterial agent.