The metabolism of chlorambucil

After injection of chlorambucil into rats, metabolites have been isolated from blood and identified by g.l.c.-mass spectrometry as (E)-4-4N,N-bis(2-chloroethyl)aminophenyl] 3-butenoic acid (3,4-dehydrochlorambucil) and 2-4-N,N-bis(2-chloroethyl)aminophenyl] acetic acid (phenyl acetic acid mustard). Analysis of urine 24 hr after administration indicates the absence of chlorambucil, 3,4-dehydrochlorambucil and phenyl acetic acid mustard but the presence of 2-4-N(2-chloroethyl) ami-nophenyl] acetic acid as the major metabolite. All metabolites isolated have been independently synthesized, allowing confirmation of structures proposed by g.l.c.-mass spectrometry. It has been shown that 3,4-dehydrochlorambucil is an intermediate in the β-oxidation of chlorambucil by injecting an authentic sample into rats and observing the production of phenyl acetic acid mustard in the blood. The contribution that metabolism makes to the anti-tumor activity of chlorambucil is discussed.