Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation, We describe the preparation, purification and characterization of 43 oxygenated sterols,. including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto. and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F-7) and 26,26,26,27,27,27-hexadeuterio (d(6)) substitution. The 7 alpha-hydroxy, 7 beta-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3 beta,26-diol (1d) and their 16,16-dideuterio analogs were also prepared. These d(2)-26-hydroxysterols and [16,16-H-2(2)]-(25R)-cholest-5-ene-3 beta;26-diol (1e) were synthesized from [16,16-H-2(2)]-(25R)-cholest-5-ene-3 beta,26-diol diacetate (2e), which can be prepared from diosgenin. The highly specific deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholesterol samples by isotope dilution methods. The Delta(5)-3 beta,7 alpha,26- and Delta(5)-3 beta,7 beta,26-triols were regioselectively oxidized/isomerited to the corresponding Delta(4)-3-ketosteroids with cholesterol oxidase. Also described are 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, its 5 beta,6 beta-isomer, cholestane-3 beta,5 alpha,6 beta-triol, their F-7 and d(6) derivatives, and d(3)-25-hydroxycholesterol, which was prepared from 3 beta-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods, including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assignments are presented, and H-1 NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side-chain protons of 30. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.