A fragment merging approach towards the development of small molecule inhibitors of Mycobacterium tuberculosis EthR for use as ethionamide boosters

With the ever-increasing instances of resistance to frontline TB drugs there is the need to develop novel strategies to fight the worldwide TB epidemic.


Table of Contents
Crystal structure of 28 bound to EthR (the two different binding modes for this ligand are super-imposed). Although the conformations of the side chains of residues Phe184 and Trp138 preclude the formation of sub-pockets III and IV in the sense of Figure S3, compound 28 is still capable of binding to the protein by altering the shape of the EthR hydrophobic cavity and moulding it around its own scaffold. (PDB code 5F0H)

Biophysical assays Differential scanning fluorimetry (DSF)
DSF measurements were carried out using a Bio-Rad CFX Connect machine with a 96-well reaction module. Samples (50 μl each) containing EthR (20 μM), NaCl (150 mM), Tris.HCl (20 mM, pH = 8.5), SYPRO orange (2.5x) and test compound (100 µM) in water were prepared in 96-well plates. The 96-well plates were heated linearly from 25 °C to 95 °C using a temperature increment of 0.5 °C every 30 seconds. Melting curves represent plots of the fluorescence emission intensity at λ max 490/575 nm of each sample against temperature.
Melting temperatures (T m ) were determined as the temperatures at which the minima on the negative first derivatives of the melting curves occurred.

Isothermal titration calorimetry (ITC)
An aqueous solution of the fragment to be tested (1 mM) was prepared containing NaCl (300 mM), Tris.HCl (20 mM, pH = 8.0), d 6 -DMSO (10% v/v) and glycerol (to match the 10% v/v glycerol content in the EthR stock solution). A separate aqueous solution of EthR (50 μM) containing NaCl (300 mM), Tris.HCl (20 mM, pH = 8.0) and DMSO-d 6 (10% v/v) was prepared and placed in the sample cell of a MicroCal iTC 200 microcalorimeter (GE Healthcare). The fragment solution was then titrated to the EthR solution over 39 injections (first injection of 0.4 μl and subsequent injections of 1.0 μl). Data was fitted to a one site binding model using Origin software.

Surface plasmon resonance (SPR)
The SPR assay was carried out using a BIAcore T100 instrument as described previously. 2 The assay was designed to measure the interaction of EthR with the ethA promoter DNA sequence (106 bp -experimental DNA), immobilised via biotin-streptavidin linkage onto an SA Series S Sensor Chip (BIAcore). DNA from pUC19 (113 bp) was used as the control against non-specific binding. The experimental (106 bp) and control (113 bp) DNA fragments were produced as described previously. 15,16 Biotinylated control and experimental DNA were immobilized to the chip surface to achieve stable fixation levels of 247 and 252 resonance units (RU) respectively.
For screening, EthR/ fragment solution (1-2 μM EthR and required concentration of fragment made up in running buffer (2 mM MgCl 2 , 10 mM Tris-Cl pH 7.5, 0.1 mM EDTA, 200 mM NaCl, 2% (v/v) DMSO) was flowed over the chip at 20 μL/min for 180 s. The dissociation time was 180 s. To determine binding levels, the response of the control channel at steady state was subtracted from that of the experiment channel. The chip was regenerated between samples using 0.03% w/v SDS in running buffer (passed at a flow rate of 20 μL/ min for 60 s).
For IC 50 calculations, the SPR response of EthR binding to the immobilized DNA was measured at various concentrations of compound. The resulting RUs were used to fit the data using nonlinear regression with variable slope dose-response inhibition constrains on GrapfPad PRISM 5.00. IC 50 values were calculated as the compound concentrations necessary to inhibit 50% of the maximal interaction between EthR and its DNA operator sequence.

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. For molecules, in which restricted amide rotation gives rise to multiple signals per nucleus, all signals observed are reported in the 1 H NMR and 13 C NMR spectra and the appropriate ratios of peaks in the 1 H NMR spectra reflect the ratios of different rotamers, Compounds 3, 11, 12 and 13 were shown to be rotamers in the 1H NMR.
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 procedure A 17
Amine (1 eq.), carboxylic acid (1 eq.) and diisopropylethylamine (5 eq.) were dissolved in anhydrous DCM (2 mL). COMU (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 flash column chromatography.
General procedure B (TFA deprotection) 18 The Boc-protected amine was dissolved in DCM (6 mL). TFA (3 mL) was added and the reaction mixture was stirred at room temperature for 1 -5 h. The solvent was evaporated in vacuo. The crude material was dissolved in DCM (10 mL) and washed with saturated aqueous sodium bicarbonate. The organic extracts were dried using a hydrophobic frit and evaporated in vacuo.
The reaction mixture was then allowed to warm up and was stirred at room temperature for 2 h. Ice (3.00 g) was then added and the quenched reaction mixture was stirred for 10 minutes.
The solvent was evaporated in vacuo and the residual aqueous was extracted with dichloromethane (2 x 15 mL). The organic layer was separated and dried by passing through a hydrophobic frit. The solvent was removed in vacuo and the product was purified by automated flash chromatography (0-10% MeOH in DCM) to give tert-butyl tetrahydropyrimidine-1(2H)-carboxylate (10)
After cooling to room temperature, the catalyst was removed by filtration through Celite TM and the solvent was removed in vacuo using a freeze-drier to afford 3-(piperidin-4yl)propanoic acid (32) as an off-white solid (1.06g, > 99%). 1  found, 311.2327.

Ethyl 4-(4-(aminomethyl)piperidin-1-yl)benzoate (39)
Compound 37 (175 mg, 0.48 mmol) was dissolved in DCM (6.6 mL) and TFA (2.0 mL) was added. The reaction mixture was stirred at room temperature for 2 h, after which it was concentrated in vacuo. Water (10 mL) was added to the residue and the pH was adjusted to pH 14 using 10% aqueous NaOH. The aqueous was extracted with EtOAc (2 x 30 mL) and the organic phase was dried by passing through a hydrophobic frit.