Figure 1.
Chemical structures of 1 and 2.
Absolute configuration of 1 was determined by circular dichroism (Protocol S3). Racemic mixture was separated by high performance liquid chromatography.
Table 1.
In vitro profile of compounds 1 and 2.
Figure 2.
M. tuberculosis killing kinetics of 1 and 2 at 20x MIC.
The number of cfus were quantified after incubation with the different compounds at different times in 10 ml of 7H9 10%ADC 0,05% Tween 80 medium containing 5 µM of 1, 7.5 µM of 2, 9.4 µg/ml of Linezolid, 1.2 µg/ml of Moxifloxacine and also for internal growth control. The mean and the standard deviations of at triplicate cultures of each point are shown.
Table 2.
Activity of 1 and 2 in clinical M. tuberculosis isolates.
Table 3.
Genotypic characterization of spontaneous M. tuberculosis mutants isolated with 1 or 2.
Table 4.
Cross-Resistance study between 1 and 2.
Figure 3.
2D-TLC analysis of [14C]-labelled lipids from M. bovis BCG grown in the presence of MmpL3 inhibitors.
Cultures were grown in the absence or presence of inhibitor (3× MIC) for 8 hours, and then and labelled using [14C]acetate for 8 hours. Chloroform-methanol extracts (polar lipids) were separated using (i) System D: chloroform:methanol:water (100∶14∶0.8) in direction 1 and chloroform:acetone:methanol:water (50∶60∶2.5∶3) in direction 2 with the position of TMM is indicated by the solid arrows; (ii) System E: chloroform:methanol:water (60∶30∶6) in direction 1 and chloroform:acetic acid:methanol:water (40∶25∶3∶6) in direction 2 with the position of phosphatidylinositol (PI) and phosphatidylinositol mannosides (PIMs) indicated. Lipids were visualized by 48 h exposure on X-ray films by autoradiography (Kodak Biomax MR film).
Figure 4.
Profile of lead compounds 3 and 4.
Structure, in vitro antimycobacterial activity against M. tuberculosis H37Rv, intracellular activity against M. tuberculosis H37Rv (RAW264.7 macrophages), cytotoxicity in HepG2 cells, ClogP, clearance in mouse microsomes, % PPB and areas under the curve versus time (AUC) after oral administration (po 50 mg/kg) of compounds 3 and 4.
Figure 5.
Whole blood pharmacokinetic profile and main parameters of compounds 3 and 4.
Compounds were given orally at 50 mg/Kg suspension in 1% aqueous methylcellulose. Main pharmacokinetic parameters were established after non-compartimental analysis. AUC: Area Under the Curve; Cmax: Maximum concentration observed in whole blood; %F: percentage bioavailability.
Figure 6.
Therapeutic efficacy of Compound 3 (A) and Compound 4 (B) against H37Rv in vivo.
B6 mice were infected by intratracheal instillation with 105 CFU H37Rv per mouse. The mice were treated orally once a day from day 1 to day 8 and sacrificed on day 9. Every point represents data from lungs of one mouse. Moxifloxacin (30 mg/kg) was used as a quality control of the assay. DL: Limit of detection.