STRUCTURAL INVESTIGATIONS OF 13-O-DEMETHYL-FK506 AND ITS ISOMERS GENERATED BY IN-VITRO METABOLISM OF FK506 USING HUMAN-LIVER MICROSOMES

FK506 is currently under investigation as immunosuppressant after orga n transplantation and in immune diseases. The structure of a demethyla ted metabolite 1 of FK506 isolated after in vitro metabolism by human- liver microsomes was established using two-dimensional homo- and heter onuclear NMR experiments. The demethylation position was found to be a t O-C(13) using HMBC spectra. In contrast to FK506, 7 different isomer s could be differentiated in COSY, HMBC, and HMQC spectra. The intensi ty of their signals was 50:18:11:9:6:6 (one isomer could not be quanti fied). This isomerization may be explained by epimerization at C(10) o r alternative formations of the hemiketal ring between C(10) and C(13) or C(9) and C(13), in addition to cis/trans-isomerism about the amide bond (see Scheme). The structural variation is possible by participat ion of the OH group at C(13) formed after demethylation and could be d erived from HMBC spectra. Chemical exchange evidenced by ROESY spectra proved the rotational isomerism. NMR investigation of the structure o f 13-O-demethyl-FK506(1) revealed at least seven isomers.