The effect of DMSO on growth of <i>M. tuberculosis</i>.

<p><i>M. tuberculosis</i> growth was monitored in 96-well plates. DMSO was added as serial dilutions at a starting concentration of 40%. After 5 d of incubation at 37°C % growth was calculated by dividing RFU (triangles) or OD₅₉₀ (squares) by the average of 4 control culture wells containing no DMSO. Results are the average of 8 independent dose response curves; error bars represent standard deviation.</p

Stability of fluorescent signal in the absence of antibiotic selection.

<p><i>M. tuberculosis</i> CHEAM3 was grown in 100 µL of 7H9-Tw-OADC-H or 100 µL of 7H9-Tw-OADC in 96-well plates. Fluorescence (dotted bars) and optical density (hashed bars) were measured after 5 d of incubation at 37°C. Measurements are average all of well. Results of 2 independent experiments are shown.</p

MIC determination of imidazo[1,2-a]pyridine compounds.

<p>MICs were determined in liquid medium using two readouts of growth (OD and fluorescence). The values from both readouts were combined and results are the average and standard deviations for a minimum of two independent runs.</p

MIC of known anti-tuberculosis drugs.

<p>MICs were determined in liquid medium using both readouts of growth, OD and fluorescence (RFU). Results are the average and standard deviations of a minimum of two independent runs.</p>(1)<p>MIC values as recorded by Changsen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060531#pone.0060531-Changsen1" target="_blank">[22]</a>.</p

Correlation of fluorescence and optical density as measures of growth. A.

<p>Growth of <i>M. tuberculosis</i> CHEAM3 measured in 96-well plates. CFU – squares; OD₅₉₀ - triangles. Cells were grown in 100 µL of 7H9-Tw-OADC in 96-well plates at a theoretical starting OD₅₉₀ of 0.02. Results for OD₅₉₀ are the average of all wells in the plate +/− SD. Samples were taken from 3 wells for CFU counts; results are the average +/− SD<b>. B.</b> Correlation of OD₅₉₀ and RFU during growth in 96-well plates. Measurements were taken on days 3, 4, 5 and 6.</p

Assay reproducibility.

<p><b>A</b> Dose response curve reproducibility for rifampicin. Results are the average of 8 independent dose response curves generated from the fluorescence +/− standard deviation. The MIC curves were generated using the Gompertz model <b>B</b> MIC reproducibility for rifampicin. MICs were calculated for rifampicin over a period of 12 months using fluorescence or OD. Results are for 337 independent rifampicin dose response curves are shown in a box and whiskers plot. MIC values that are outside the 99 percentile are indicated as outliers.</p

Final assay plate layout.

<p>The plate format is shown. Column 1 contains maximum inhibition control (rifampicin) in Rows A–D and minimum inhibition control (no compound) in Rows E–H. Column 12 is not inoculated with cells. Compounds are tested across each Row A–G in a two-fold serial dilution starting at 20 µM – concentrations are indicated below. A rifampicin dilution series is included in Row H starting at 20 nM.</p

Advancement of Imidazo[1,2‑<i>a</i>]pyridines with Improved Pharmacokinetics and nM Activity vs. <i>Mycobacterium tuberculosis</i>

A set of 14 imidazo­[1,2-<i>a</i>]­pyridine-3-carboxamides was synthesized and screened against <i>Mycobacterium tuberculosis</i> H₃₇Rv. The minimum inhibitory concentrations of 12 of these agents were ≤1 μM against replicating bacteria and 5 compounds (<b>9</b>, <b>12</b>, <b>16</b>, <b>17</b>, and <b>18</b>) had MIC values ≤0.006 μM. Compounds <b>13</b> and <b>18</b> were screened against a panel of MDR and XDR drug resistant clinical <i>Mtb</i> strains with the potency of <b>18</b> surpassing that of clinical candidate <b>PA-824</b> by nearly 10-fold. The <i>in vivo</i> pharmacokinetics of compounds <b>13</b> and <b>18</b> were evaluated in male mice by oral (PO) and intravenous (IV) routes. These results indicate that readily synthesized imidazo­[1,2-<i>a</i>]­pyridine-3-carboxamides are an exciting new class of potent, selective anti-TB agents that merit additional development opportunities

The representative aminothiazole 20 possesses bactericidal activity.

<p><i>M</i>. <i>tuberculosis</i> was inoculated to a starting of OD₅₉₀ of 0.1 in medium containing compound 20. CFU/mL was enumerated at indicated time points by serial dilution onto solid medium. The limit of detection was 20. Note that the lines for 0.625, 1.25 and 2.5 overlap.</p

Chemical synthesis of C-4 ketone and carboxamide analogs.

<p>Reagents: (i) EDC.HCl, HOBt, NCH₃(OCH₃), DIPEA, CH₂Cl₂, 16 h; (ii) CH₃MgBr, THF, -78°C–r.t., 2 h; (iii) 1-adamantanoyl chloride, Et₃N, THF, r.t., 1 h.</p