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Mycobacterium tuberculosis (Mtb) is wrapped in a complex cell envelope which has inner and outer layers of membranes. The inner layer is primarily made of mycolyl arabinogalactan (mAG), whereas, the outer layer is made of glycolipids, phospholipids, sulpholipids, trehalose dimycolate (TDM) and trehalose monomycolate (TMM). This protective covering provides a thick layer of resistance to the influx of antibiotics. Enzymes that play a role in synthesis of this envelope may be excellent targets for potentiating the action of anti-Mtb compounds. Tetrahydrolipstatin (THL), a known covalent inhibitor of serine esterases including Mtb Rv3802 phospholipase A, the thioesterase activity that contains Mtb polyketide synthase-13 thioesterase domain (Pks13-TE), and Mtb Antigen 85C (Ag85C) which are all implicated in cell envelope synthesis. Thus, we synthesized THL and several congeners in the in α-side chain to identify scaffolds that improve the action of compounds active against Mtb.
The synthesis of derivative of THL is accomplished in only eight steps. Starting with the addition (S)-(+)-epichlorohydrin to a Grignard reagent with the required stereochemistry to afford a chlorohydrin derivative with 80 % yield. Epoxidation of the chlorohydrin derivative with potassium hydroxide and addition of various alkynes affords alkynols in 90 % yield. The various alkynols were stereospecifically reduced to the Z- alkene using Lindlar’s catalyst. The resulting alkenes were transformed stereoselectively to epoxides by vanadium-mediated epoxidation in combination with tert-butyl hydroperoxide in 71 % yield. Coupling reactions with a Cbz-protected Leucine amino acid produced the peptidyl arm in 80% yield. The key step in this synthesis was the insertion of carbon monoxide using a bimetallic [Lewis acid][Co(CO)4]- catalyst for regioselective carbonylation results in β-lactones in 30 % yield. Deprotection of Cbz group followed by coupling with formic acid afforded the desired compound series.
We performed MIC assays on the derivatives against model strains Mtb H37Ra and M. smegmatis. The most potent compound was further evaluated in checkerboard assays to identify synergistic combinations with different antibiotics classes. Synergy was found with macrolide, sterol, carbapenem, aminocoumarin, and isonicotinic acid classes, findings consistent with increased uptake of these drugs.

Steven Sucheck

The University of Toledo

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