In vitro metabolic transformations of 2,4- dipyrrolidinylp yrimidine : a chemical probe for P450-mediated oxidation of tirilazad mesylate

Abstract

1. We have determined that 2,4-dipyrrolidinylp yrimidine (2,4-DPP), used as a model for studies of the metabolismof therapeutic agents containing this moiety, undergoes three characteristic hydroxylations when incubated with male rat liver microsomes. Analysis of microsomal incubates of stable isotope labelled analogues of 2,4-DPP by particle beamliquid chromatography- mass spectrometry (LC-PB-MS) has shown that the three metabolites are 4-(3-hydroxypyrrolid inyl)-2-(pyrrolidinyl)- pyrimidine (M1), 4-(2- hydroxypyrrolid inyl)-2-(pyrrolidinyl)- pyrimidine(M2) and 2-(2-hydroxypyrrolid inyl)-4- (pyrrolidinyl)- pyrimidine (M3). 2. We determined that enzymes of the cytochrome P450 family are responsible for the in vitro hydroxylations of 2,4-DPP. 3. We observed that in microsomal incubations carried out in the presence of cyanide, a single cyanide adduct is formed implicatingan iminium ion intermediate in the oxidation of the 2-pyrrolidine ring. 4. We also determined the intermolecular deuterium isotope effects for the formation of each of the three products. For M1, k_H/k_D = 14.55 ± 0.54; for M2, k_H/k_D = 6.01 ± 0.65; and for M3, k_H/k_D = 5.35 ± 1.18. 5. We interpret these data as suggesting that M2 and M3 are formed by the same mechanism,probably includingthe formation of an iminiumion, and that M1 is formed by direct hydrogen abstraction.