Regioselective hydroxylations of 2-nitrofluorene in vivo: a nuclear magnetic resonance study

The time-dependent metabolism of 2-nitrofluorene (2-NF) in vivo has been studied after ip administration to rats. After hydrolysis with beta-glucuronidase/arylsulfatase, five hydroxylated 2-nitrofluorenes were isolated by HPLC and identified by a combination of data from NMR, UV, and MS experiments. These metabolites were characterized as 6,9-dihydroxy-2-nitrofluorene (I), 9-hydroxy-2-nitrofluorene (II), 6-hydroxy-2-nitrofluorene (III), 7-hydroxy-2-nitrofluorene (IV), and 8-hydroxy-2-nitrofluorene (V). The highest amounts of metabolites were found after 24 h of injection, and although the major ones were still present after 48 h, only traces of them could be found beyond that time. A unique feature of the present study was our ability to establish the position of the different hydroxylations that occur in vivo by NMR techniques and, thus, to observe the regioselectivity of the metabolism of the 2-NF in vivo. Furthermore, two conjugated metabolites were identified by 1H-NMR and ESI-MS as 6-[(hydroxysulfonyl)oxy]-2-nitrofluorene (2-nitrofluorene 6-sulfate) (VI) and 7-[(hydroxysulfonyl)oxy]-2-nitrofluorene (2-nitrofluorene 7-sulfate) (VII).