Structural investigations of 13-O-demethyl-FK506 and its isomers generated by in vitro metabolism of FK506 using human-liver microsomes
W. Schüler, U. Christians, P. Schmieder, H.M. Schiebel, I. Holze, K.-F. Sewing, H. Kessler
Helv. Chim. Acta (1993) 76, 2288-2302
FK506 is currently under investigation as immunosuppressant after organ transplantation and in immune diseases. The structure of a demethylated metabolite of FK506 isolated after in vitro metabolisation by human-liver microsomes was established to be 13-O-demethyl-FK506 using two-dimensional homo- and heteronuclear NMR experiments The demethylation position was found by using HMBC spectra. In contrast to FK506, 7 different isomers of 13-O-demethyl-FK506 could be differentiated in COSY, HMBC, and HMQC spectra. The intensity of their signals was 50:18:11:9:6:6 (one isomer could not be quantified). This isomerization may be explained by epimerization at C(10) or 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. The structural variation is made possible by participation of the OH group at C(13) formed after demethylation and could be derived from HMBC spectra. Chemical exchange evidenced by ROESY spectra proved the rotational isomerism. NMR investigation also revealed at least seven isomers.