Intermediates in the reduction of the antituberculosis drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, in aqueous solution.

Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
Organic & Biomolecular Chemistry (Impact Factor: 3.57). 07/2008; 6(11):1973-80. DOI: 10.1039/b801859f
Source: PubMed

ABSTRACT The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO(2) (-) species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermediate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action.

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    ABSTRACT: Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.
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    ABSTRACT: PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F(420) )-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis. PA-824 (250 µM) was incubated with the 9000 × g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis. PA-824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des-nitro product. Six of these products, but not the des-nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn-catalyzed des-nitrification and radiolytic reduction of PA-824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem-catalyzed product of imidazole ring hydration (M2). PA-824 was metabolized by des-nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA-824 was selectively bioactivated in M. tuberculosis and there was no evidence for 'cross-activation' by human enzymes.
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    ABSTRACT: In an attempt to highlight structural features required for potent antitubercular activity, five pharmacophoric features were developed for PA-824 and its analogs. The generated pharmacophore indicated importance of a nitro group, three hydrogen bond acceptor features, and a distal aromatic ring for potent activity. The model based on pharmacophore alignment has good correlation coefficient for the training set (r(2) = 0.81, SD = 0.31, F = 122.9, N = 152), which was evaluated using a test set (Q(2) = 0.77, root-mean-square error = 0.35, Pearson-R = 0.88, N = 49). Structure-activity relationship investigation further revealed that hydrophobic substitutions at the para-position of distal aromatic ring could lead to more potent analogs. The most active and inactive compounds were further studied using density functional theory at B3LYP/3-21*G level. The calculated electrostatic profile indicated that these compounds possess maximum negative potential in the vicinity of nitro group extending laterally to the imidazole ring. Furthermore, the calculated electron affinity values indicate the stability of radical anions, which could form upon one electron reduction in the biological system, thus, indicating the electron acceptor capacity of these compounds. Results of this study are expected to be useful in the design of novel potent nitroimidazoles as antitubercular agents.
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