Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase

Abstract

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol was previously shown to be an effective inhibitor of the glucosyltransferase in liver that forms glucosylceramide. Since the inhibitor consists of four isomers, it was important for further testing to determine which isomer was most effective and to devise a method for preparation of this isomer. The mixture of isomers was synthesized as described before and separated by crystallization into two diastereomers, differing in migration rate with thin-layer chromatography (TLC) and in retention time with high performance liquid chromatography (HPLC). The slower moving diastereomer, which proved to be the active inhibitor, was separated into its enantiomers by crystallization with dibenzoyltartaric acid isomers. The inhibitory activity resided in the less soluble salt formed with the D-tartaric acid compound. The optical isomers could be characterized by TLC as their (1R)-(-)-camphanate esters. Using a second synthetic route, starting with L-threo- and DL-erythro-1-phenyl-2-amino-1,3-propanediol, we tentatively established the active form of the inhibitor to be the D-threo (1S,2R) isomer. 13C NMR spectroscopy supported the threo and erythro assignments. Kinetic analysis showed that it acted uncompetitively against UDP-glucose and by mixed competition against ceramide, with Ki of 0.7 microM. The DL-erythro and DL-threo compounds inhibited brain galactosylceramide synthetase to a small extent. Glucosylceramide glucosidase activity of liver was unaffected by the DL-threo mixture.