Fragment-Sized EthR Inhibitors Exhibit Exceptionally Strong Ethionamide Boosting Effect in Whole-Cell Mycobacterium tuberculosis Assays

Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays.


Determination of minimum effective concentration (MEC) values for ethionamide boosting of EthR fragments
Regular 7H9-based medium was prepared by adding 4.7 g Middlebrook 7H9 broth to double distilled water (900 mL) containing glycerol (2 mL, added via sterile syringe) and 0.5 mL Tween 80 (1 mL, added via sterile syringe). The mixture was stirred until all solids dissolved and passed through a 0.2 µM filter. ADC (100 mL) was aseptically added to the above 7H9based medium. [ADC was prepared by dissolving BSA fraction V (50 g), glucose (20 g) and sodium chloride (8.1 g) in water to make up a total volume of 1L. The ADC solution was filter sterilised and stored at 4 °C.] 1) Isolated M. tuberculosis cells (ATCC 27294) were grown to an OD 0.2 -0.3 in 7H9/ADC/Tween and diluted by a factor of 1000 in the required medium. 2) 50 µL of required medium was added to all wells of a 96-well clear round bottom plate, except the first row. 3) In the first row add 100 µL of ethionamide booster compound diluted in medium at twice the initial desired concentration (initial desired concentration is 50 µM). Using multichannel pipettor, transfer 50 L to each next row starting with row 1 and ending with row 12, discarding 50 µL after row 12. 4) Isoniazid and DMSO only were used as positive and negative controls respectively. 5) To each well add 50 L of the 1:1000 culture dilution (prepared in step 1 above, i.e. approximating 10,000 bacteria per well). For ethionamide synergy, add 50 µL of cell dilution (prepared in step 1 above) containing 0.2ug/mL ethionamide. 6) Plates were incubated for a total of two weeks at 37 °C in zip-lock bags. 7) Plates were read after 1 and 2 weeks with inverted enlarging mirror plate reader and graded as either growth or no growth. MEC is the lowest concentration of EthR ligand that completely inhibits growth. Photos are taken of the plates at both time points.

Macrophage (intracellular) assay
Raw264.7 macrophages (10 8 cells) were infected with H37Rv-GFP suspension at a multiplicity of infection (MOI) of 1:1 in 300 mL for 2 h at 37 °C with shaking (100 rpm). After two washes by centrifugation at 1100 rpm for 5 min, the remaining extracellular bacilli from the infected cells suspension were killed by a 1 h amikacin (20 µM, Sigma) treatment. After a final centrifugation step, 40 µL of M. tuberculosis H37Rv-GFP colonised macrophages were dispensed with the Wellmate (Matrix) into 384-well Evotec plates preplated with 10 µL of compound mixture diluted in cell medium and incubated for 5 days at 37 °C, 5% CO 2 . Macrophages were then stained with SYTO 60 (Invitrogen, S11342) for 1 h followed by plate sealing. Confocal images were recorded on an automated fluorescent ultrahigh-throughput microscope Opera (Evotec). This microscope is based on an inverted microscope architecture that allows imaging of cells cultivated in 96-or 384-well microplates (Evotec). Images were acquired with a 20x water immersion objective (NA 0.70). A double laser excitation (488 and 635 nm) and dedicated dichroic mirrors were used to record green fluorescence of mycobacteria and red fluorescence of the macrophages on two different cameras, respectively. A series of four pictures at the centre of each well were taken, and each image was then processed using dedicated image analysis. [17][18][19] The percent of infected cells, and the number of cells are the two parameters extracted from images analysis as previously reported. 18 Data of two replicates are average.  Percentage of inhibition is plotted against the log 10 of the compounds concentration, determined in the absence or in the presence of ethionamide at 1/10 of its MIC for the macrophage assay (0.033 µg/mL). Fitting was performed by Prism software using the sigmoidal dose-response (variable slope) model.

Chemistry
General Information 1 H NMR and 13 C NMR spectra were recorded using Bruker DPX-400 or Bruker DPX-500 NMR spectrometers. Chemical shifts are given in parts per million (ppm). All 13 C NMR spectra are proton decoupled. Coupling constants are reported in Hz where interpretable and the conventional abbreviations for assigning peak multiplicity are used as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad.
High resolution mass spectrometry (HRMS) was performed using a Waters LCT Premier high-resolution spectrometer in electrospray ionisation (ESI) mode.
LCMS spectra were recorded using a Waters HClass UPLC system coupled to a Waters single quad detector eluting at a constant flow rate of 0.8 mL/ min using a constant gradient of 5 -100% acetonitrile in 0.1% v/v aqueous formic acid.
Infrared spectrometry was performed using a Perkin-Elmer One FTIR Spectrometer with attenuated transmittance reflectance (ATR). The abbreviations (w) and (br) have been used to describe weak and broad IR absorbances respectively.
All commercially available reagents were used as purchased without further purification. All organic solvents used were either freshly distilled or purchased as anhydrous. Purification of intermediates and final compounds was carried out by automated flash column chromatography using Biotage SNAP Kp-Sil pre-packed columns run on either Biotage Isolera One or Biotage Isolera Four instruments.
Microwave reactions were performed using a Biotage Initiator system under reaction conditions as indicated for each individual reaction.
Following aqueous work-up, organic solutions of intermediates and final compounds were dried using Isolute ® phase separators from Biotage (referred to as hydrophobic frits).
The purity of the compounds was measured by LC-MS with UV-Vis detection and all compounds were of a purity of > 95% unless otherwise stated.

General method A 20
Amine (1 equivalent), carboxylic acid (1 equivalent) and diisopropylethylamine (5 equivalents) were dissolved in anhydrous DCM (2 mL). COMU 20 (1.1 eq.) was added and the reaction mixture was stirred at room temperature for 16 -24 h. The solvent was evaporated in vacuo. The residue was dissolved in EtOAc (10 mL) and washed with water (2 x 10 mL).
The organic layer was concentrated in vacuo and the crude material was purified by automated flash chromatography.

General method B 21
To a suspension of 1, 1'-carbonyldiimidazole (1.1 eq.) in anhydrous THF (10 mL/g of alcohol) was added slowly the alcohol (1 eq.) and the reaction mixture was left stirring at 22 °C overnight. Pyrrolidine (1 eq.) was added and the reaction mixture was stirred 22 °C for five hours. The volatile organics were removed in vacuo and the crude residue was purified by flash chromatography eluting with a gradient of 0-33% of ethyl acetate in petroleum ether (40-60).

2-Cyclopentylethan-1-ol (32)
The solids formed were removed by filtration and washed with THF (3 x 30 mL). The combined filtrate was concentrated in vacuo and re-dissolved in diethyl ether (50 mL). The bottom aqueous layer was separated and the organic layer was dried with anhydrous Na 2 SO 4 .
The drying agent was removed by gravity filtration and the solvent was evaporated in vacuo to give the alcohol 32 as a colourless liquid (1.7 g, 75%  The reaction mixture was stirred and cooled in ice/ water. N-Bromosuccinimide (1.56 g, 8.8 mmol) was added slowly and portion-wise (caution: effervescence). After the addition was complete, the ice/ water bath was removed and the reaction mixture was stirred at 22 °C overnight. The solvent was evaporated in vacuo and the desired bromide 33 was purified by flash chromatography as a pale yellow liquid (0.56 g, 36% Cyclopentylmethanol (1.0 g, 10 mmol) and triphenylphosphine (2.8 g, 10.7 mmol) were dissolved in DMF (10 mL) and cooled in an ice-water bath. Bromine (0.51 mL, 10.7 mmol) was added over 10 minutes. After the addition was complete, the reaction mixture was allowed to warm up and was stirred at ambient temperature overnight.