New classes of anti-tuberculosis agents are needed to combat resistance to existing agents and to shorten the duration of therapy. This revised proposal explores the potential of 1,4 benzoxazines as anti-tuberculosis agents. A screening program identified six 1,4 benzoxazines with sub-microgram/ml MICs against M. tuberculosis and no detectable mammalian cell cytotoxicity with resulting selectivity indices of >100. Several structure-activity relationships were also revealed. These compounds were also active at low microgram/ml levels against M. tuberculosis in murine macrophages. Polar surface area calculations predict good absorption when administered orally and preliminary results suggest low toxicity in mice upon oral administration. New preliminary data suggests a lack of formation of glutathione adducts, activity against drug-resistant strains and (via expression profiling) a mechanism of action not shared by existing agents. In order to identify a lead compound from this class a focused library of approximately one hundred and fifty new 1,4 benzoxazines will be synthesized using a strategy that covers substitution at all possible position with minimal synthetic effort. Both classical and parallel synthesis will be used to produce 4-dihydro-2H-1,4-benzoxazines via reaction of polycarbonyl compounds (2,4 diketo acids and tetraketones) with oaminophenols. Additional analogs will be produced by chlorination and then reaction with N, O- and S nucleophilic reagents resulting in the production of 3-vinyl-2H-1,4 benzoxazines. These compounds are predicted to be drug-like and orally available based on """"""""Lipinski rules"""""""" in addition to low polar surface area and numbers of rotatable bonds. All compounds will be assessed for activity against both logarithmic phase M. tuberculosis as well as against non-replicating M. tuberculosis using low oxygen incubation. All compounds will also be assessed for toxicity to a mammalian cell line. Potent (MIC<1 ug/ml) and selective (selectivity indices>100) compounds will be assessed for anti-TB activity against drug-resistant strains, and activity in murine macrophages. Active compounds will be assessed for stability in the presence of human microsomes, P-450 mediated cytotoxicity and protein binding. Compounds with the best in vitro profiles will be evaluated for pharmacokinetic properties in mice including oral bioavailability and lung tissue concentrations. The best candidates will be assessed for comparative passage through CACO-2 cells vs. a bovine brain epithelial cell line, as well as for spectrum of activity and frequency of resistance. Mechanism of action will be investigated by transcriptional profiling as well as identification of gene mutations in resistant mutants using microarray-based screening and sequencing. This exploratory R21 proposal is expected to determine the potential of this class to yield a clinically useful agent for tuberculosis and to provide a lead compound(s) for further optimization and pre-clinical development. ? ? ?
