Mass spectrometric and NMR characterization of metabolites of roxifiban, apotent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methyl ester prodrug roxifiban is an orally active, potent and selec tive antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is bei ng del eloped for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog . XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited or al absorption with the majority of the radiolabel being excreted ill faeces . After i.v. doses of C-14- roxifiban, most of the radioactivity was recove red in the urine of rat whereas the dog excreted significant amounts of rad ioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the iso xazoline ring. These hydroxylated metabolites were not detected in the urin e and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the r at with an equimolar mixture of d(o):d(4) roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these met abolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of t he correct stereochemistry of the substituted hydroxyl group on the isoxazo line ring.