Discovery of a Novel Fluoroquinolone Antibiotic Candidate WFQ-228 with Potent Antimicrobial Activity and the Potential to Overcome Major Drug Resistance.

WFQ-101 with a unique N-1 substituent, 5-amino-4-fluoro-2-(hydroxymethyl)phenyl group, was selected as a lead compound through combination screening based on antimicrobial activity and the efflux index against quinolone-resistant (QR) Pseudomonas aeruginosa (P. aeruginosa). Through structural optimization, we identified WFQ-228 as a novel fluoroquinolone antibiotic candidate. WFQ-228 had potent and superior activity in comparison to levofloxacin (LVX) and ciprofloxacin (CIP) against clinical isolates of P. aeruginosa, Escherichia coli and Acinetobacter baumannii, including QR strains. Furthermore, WFQ-228 demonstrated the potential to overcome major mechanisms of drug resistance; its antimicrobial activity was less affected by both pump-mediated efflux and mutations of the quinolone resistance-determining region in P. aeruginosa compared with LVX and CIP. These results suggest that WFQ-228 is a promising candidate for further evaluation in the treatment of infections caused by QR Gram-negative pathogens.

Discovery of WQ-3810: Design, synthesis, and evaluation of 7-(3-alkylaminoazetidin-1-yl)fluoro-quinolones as orally active antibacterial agents.

Novel 7-(3-alkylaminoazetidin-1-yl)fluoroquinolones were designed, synthesized, and evaluated for their antibacterial activities and oral absorption rates. Against Gram-negative bacteria, 10a-e, which have various alkyl groups containing different numbers of carbon atoms (C0-C3) at the C-7 alkylaminoazetidine position, showed potent and similar antibacterial activities, whereas the activity of 10f (C4, t-Bu) was significantly lower than those of 10a-e. Conversely, the oral absorption rates of 10a-e in rats increased depending on the number of carbon atoms in the alkyl groups; 10d (C3, n-Pr) and 10e (C3, i-Pr) had high oral absorption rates (>90% at 10 mg/kg). These results demonstrated that the introduction of alkyl groups onto C-7 aminoazetidine is useful for the improvement of the oral absorption rates of these drugs while maintaining their antibacterial activities. As a conclusion, from this series of fluoroquinolones, WQ-3810 (10e), having 3-isopropylaminoazetidine as the C-7 substituent, was identified as an orally active antibacterial agent with a potent in vitro activity.

In vitro activity of WQ-3810, a novel fluoroquinolone, against multidrug-resistant and fluoroquinolone-resistant pathogens.

The aim of this study was to examine the in vitro antibacterial activity of WQ-3810, a new fluoroquinolone, against clinically relevant pathogens such as Acinetobacter baumannii, Escherichia coli and Streptococcus pneumoniae, including multidrug-resistant (MDR) and fluoroquinolone-resistant (FQR) isolates, compared with those of ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin. WQ-3810 demonstrated the most potent activity against the antimicrobial-resistant pathogens tested. Against A. baumannii, including MDR isolates, the potency of WQ-3810 [minimum inhibitory concentration for 90% of the organisms (MIC(90))=1 mg/L] was more than eight-fold higher than that of ciprofloxacin (64 mg/L) and levofloxacin (8 mg/L). Against E. coli and S. pneumoniae, including FQR isolates, WQ-3810 (MIC(90)=4 mg/L and 0.06 mg/L, respectively) was also more active than ciprofloxacin (64 mg/L and 2 mg/L) and levofloxacin (32 mg/L and 2 mg/L). Furthermore, WQ-3810 was the most potent among the fluoroquinolones tested against meticillin-resistant Staphylococcus aureus (MRSA) and Neisseria gonorrhoeae, including FQR isolates. In particular, WQ-3810 demonstrated highly potent activity against FQR isolates of A. baumannii, E. coli and S. pneumoniae with amino acid mutation(s) in the quinolone resistance-determining region of DNA gyrase and/or topoisomerase IV, which are the target enzymes of fluoroquinolones. An enzyme inhibition study performed using FQR E. coli DNA gyrase suggested that the potent antibacterial activity of WQ-3810 against drug-resistant isolates partly results from the strong inhibition of the target enzymes. In conclusion, this study demonstrated that WQ-3810 exhibits extremely potent antibacterial activity over the existing fluoroquinolones, particularly against MDR and FQR pathogens.

Simple enrichment of thiol-containing biomolecules by using zinc(II)-cyclen-functionalized magnetic beads.

A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.

[Increase in lipophilicity of enalaprilat by complexation with copper(II) or zinc(II) Ions].

Enalaprilat (H2L), which is the active metabolite of the pro-drug enalapril, is an angiotensin-converting enzyme inhibitor. Some side effects such as neurodegeneration and taste disorder can be related to copper or zinc deficiency, which would be caused by the metal complex formation of dianionic elalaprilat (L(2-)). For a better understanding of this phenomenon, we investigated the solution species of enalaprilat in the presence of copper(II) or zinc(II) ions by pH titration analysis with I=0.10 M (NaCl) at 25℃. The 1:1 complex formation constants (KML=[ML]/[M(2+)][L(2-)] M(-1)) of 10(7.4) for CuL and 10(4.4) for ZnL complexes were evaluated, indicating the presence of those complexes at a physiological pH. Furthermore, partition experiments with a two-phase system of 1-butanol/water at 25℃ disclosed that copper(II) and zinc(II) complexes of enalaprilat were partially extracted into the organic layer. In the absence of those metal ions, enalaprilat was not soluble in the 1-butanol phase. The increase in lipophilicity of enalaprilat by metal complexation suggests that the long-term administration of enalapril could be a possible risk factor for the disrupted distribution of those metal ions in biological systems.

A novel antibacterial 8-chloroquinolone with a distorted orientation of the N1-(5-amino-2,4-difluorophenyl) group.

Fluoroquinolones represent a major class of antibacterial agents with great therapeutic potential. In this study, we designed m-aminophenyl groups as novel N-1 substituents of naphthyridones and quinolones. Among newly synthesized compounds, 7-(3-aminoazetidin-1-yl)-1-(5-amino-2,4-difluorophenyl)-8-chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4) has extremely potent antibacterial activities against Gram (+) as well as Gram (-) bacteria. This compound is significantly more potent than trovafloxacin against clinical isolates: 30 times against Streptococcus pneumoniae and 128 times against methicillin resistant Staphylococcus aureus. The structure-activity relationship (SAR) study revealed that a limited combination of 1-(5-amino-2,4-difluorophenyl) group, 7-(azetidin-1-yl) group, and 8-Cl atom (or Br atom or Me group) gave potent antibacterial activity. An X-ray crystallographic study of a 7-(3-ethylaminoazetidin-1-yl)-8-chloro derivative demonstrated that the N-1 aromatic group was remarkably distorted out of the core quinolone plane by steric repulsion between the C-8 Cl atom and the N-1 substituent. Furthermore, a molecular modeling study of 4 and its analogues demonstrated that a highly distorted orientation was induced by a steric hindrance of the C-8 substituent, such as Cl, Br, or a methyl group. Thus, their highly strained conformation should be a key factor for the potent antibacterial activity.