A new aza-analogue of
furamidine, 6-[5-(4-amidinophenyl)-
furan-2-yl]nicotinamidine (DB820), has potent in vitro antitrypanosomal activity; however, it suffers from poor oral activity because of its positively charged amidine groups. The dimethoxyamidine
prodrug of DB820, N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-
furan-2-yl}-nicotinamidine (
DB844), has potent oral activity in mouse models of both early-stage and CNS
African trypanosomiasis. Metabolism of
DB844 in human liver microsomes (HLM) was investigated using liquid chromatography-mass spectrometry (LC-MS/MS). The metabolism of
DB844 in HLM was
NADPH-dependent and resulted in the production of eight metabolites over a 90?min incubation. O-Demethylation and N-dehydroxylation reactions resulted in the metabolic conversion of
DB844 to its active DB820 metabolite. Chromatographic conditions used for LC-MS analysis allowed for the separation and identification of all metabolites including positional isomers. Demethylation of either the phenyl or
pyridine side of
DB844 (
DB844 m/z 366.2) resulted in the production of two metabolites (M1A, M1B), each with a molecular ion of m/z of 352.3 and MS(2) fragments of 288.1, 305.2, 321.2 and 335.2. However, the intensities of the MS(2) fragments were different among the two isomeric metabolites, and comparison to an authentic standard allowed for the structural determination of each metabolite. The isomeric metabolites M2A and M2B, resulting from
amidoxime reductions of M1A and M1B, were also chromatographically separated and had distinguishable MS(2) profiles that allowed for their structural assignments when compared to an authentic standard. The di-
amidoxime product resulting from O-demethylation of either side of
DB844 was also identified as an abundant metabolite during microsomal incubations. The active antitrypanosomal metabolite, DB820, was the last metabolite to be formed and thus provides evidence that
DB844 may effectively be metabolized to its active metabolite in vivo.