IN-VITRO METABOLIC TRANSFORMATIONS OF 2,4-DIPYRROLIDINYLPYRIMIDINE - A CHEMICAL PROBE FOR P450-MEDIATED OXIDATION OF TIRILAZAD MESYLATE

1. We have determined that 2,4-dipyrrolidinylpyrimidine (2,4-DPP), use d as a model for studies of the metabolism of therapeutic agents conta ining this moiety, undergoes three characteristic hydroxylations when incubated with male rat liver microsomes. Analysis of microsomal incub ates of stable isotope labelled analogues of 2,4-DPP by particle beam- liquid chromatography-mass spectrometry (LC-PB-MS) has shown that the three metabolites are 3-hydroxypyrrolidinyl)-2-(pyrrolidinyl)-pyrimidi ne (M1), 4-(2-hydroxy pyrrolidinyl)-2-(pyrrolidinyl)-pyrimidine (M2) a nd 2-hydroxypyrrolidinyl)-4-(pyrrolidinyl)-pyrimidine (M3). 2. We dete rmined that enzymes of the cytochrome P450 family are responsible for the in vitro hydroxylations of 2,4-DPP. 3. We observed that in microso mal incubations carried out in the presence of cyanide, a single cyani de adduct is formed implicating an iminium ion intermediate in the oxi dation of the 2-pyrrolidine ring. 4. We also determined the intermolec ular 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, pr obably including the formation of an iminium ion, and that M1 is forme d by direct hydrogen abstraction.