New InhA Inhibitors Based on Expanded Triclosan and Di-Triclosan Analogues to Develop a New Treatment for Tuberculosis

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new ‘direct’ InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expanding on the triclosan structure. Low Minimum Inhibitory Concentrations (MICs) were obtained for benzylphenyl compounds (12, 43 and 44) and di-triclosan derivative (39), against Mycobacterium bovis BCG although these may also be inhibiting other enzymes. The ether linked di-triclosan derivative (38) displayed excellent in vitro isolated enzyme inhibition results comparable with triclosan, but at a higher MIC (125 µg mL−1). These compounds offer good opportunities as leads for further optimisation.


Interaction with the active site residues
For the benzylphenyl ether/aniline target molecules, all the compounds displayed the correct orientation within the active site with the exception of 2 which had an inverted orientation.
Compound 2 made an H-bond, in this case the lone pairs on the oxygen act as a HBA ( Figure S2).
This compound had an inverse orientation to what was expected.
Please note that the docked structures below include the lone-pairs on the hetero atoms of ligands. Figure S2 Interactions with the active site with benzylphenyl derivative 2.
Compound 8 had the best docking and inhibition data from the benzyl phenyl ether and benzyl phenyl aniline analogue series. It displayed 63 % inhibition in the isolated enzyme assay.
Inspection of the docked structures, revealed that compound 8 ( Figure 5) had an orientation similar to TCS, with a hydrogen bond occurring between the amine linker and the 2'-OH of the cofactor, where the amine acts as an H-bond donor. Beside H-bond interactions, a л-stacking interaction with NAD + and a van der Waals interaction with Phe149 (cation-л interaction) was also observed. Mol Fitness score Tyr158 a Phe149 b NAD +a NAD +c Additional Overall similar fitness scores were displayed for the triazole linked series of compounds. The compound that had the best docking results was 23 ( Figure S3). The active site residues (blue), NAD + (magenta) and ligand.
A summary of the docking results is shown in Table S3.  Overall similar fitness scores were displayed for the bi-triclosan series of compounds. Compound 31 had the best fitness score ( Figure S4). A summary of the docking results is shown in Table S4 Mol Fitness score

tert-Butyl (4-chloro-2-methoxyphenyl)carbamate (6)
A literature procedure (3,4) was modified to synthesise compound 6. Under a nitrogen atmosphere K2CO3 (4.08 g, 29.60 mol) and iodomethane (7.37 g, 51.10 mmol) were added to a solution of the Boc protected aniline 5 (1.44 g, 5.90 mmol) in anhydrous acetone (25 mL). The suspension was heated to reflux for 6.5 h then allowed to cool to room temperature, followed by the addition of saturated NH4Cl(aq) (20 mL). The solvent was removed by evaporation and the mixture was
The solution was allowed to warm to room temperature and was stirred for 5.
The solution was allowed to stir overnight at room temperature then filtered through a bed of celite.
The solvent was removed by evaporation and the residue was dissolved in chloroform (10 mL) and washed with 5 % aqueous ammonia (3 x 10 mL) and brine (3 x 10 mL). The organic layer was dried over MgSO4, before the solvent was removed in vacuo to give an orange oil (

4-(4-Chloro-2-hydroxyphenoxy)benzaldehyde (33)
Compound 33 (8.00 g. 30.50 mmol) was suspended in AcOH (30 ml, 0.52 mol) followed by the addition of 47 % HBr (aq) (12 mL, 0.10 mol). The reaction mixture was then heated to 110 °C and stirred for 18 h. The reaction mixture was allowed to cool to room temperature before being concentrated in vacuo, the mixture was then neutralised by careful addition of NaHCO3 before

Di-1,1'(4-Iodo-phenyl)-dimethylether (40)
A literature procedure was modified to synthesise compound 40 (17). Under a nitrogen atmosphere, to a suspension of NaH (60 % dispersion in mineral oil, 0.31 g, 17.67 mmol) in anhydrous THF (13 mL), a solution of (4-iodophenyl)methanol (0.20 g, 0.86 mmol) in anhydrous THF (16 mL) was added at 0 °C and stirred for 30 min at room temperature. A solution of 4-iodobenzylbromide (0.50 g, 1.71 mmol) in dry THF (4 mL) was added to the solution at 0 °C. The solution was heated to reflux overnight and cooled to room temperature, followed by dropwise addition of water (13 mL), and extraction with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO4, before the solvent was removed in vacuo. The crude product was purified by flash column chromatography EtOAc/Petrol (1:9 v/v) to give a white powder (0.28 g, 71 %);