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 4β-hydroxy, 7α-hydroxy, 7β-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 7α-hydroxy, 7β-hydroxy, and 7-keto derivatives of (25 R)-cholest-5-ene-3β,26-diol ( 1d) and their 16,16-dideuterio analogs were also prepared. These d 2-26-hydroxysterols and [16,16- 2H 2]-(25 R)-cholest-5-ene-3β,26-diol ( 1e) were synthesized from [16,16- 2H 2]-(25 R)-cholest-5-ene-3β,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 Δ 5-3β,7α,26- and Δ 5-3β,7β,26-triols were regioselectively oxidized/isomerized to the corresponding Δ 4-3-ketosteroids with cholesterol oxidase. Also described are 5,6α-epoxy-5α-cholestan-3β-ol, its 5β,6β-isomer, cholestane-3β,5α,6β-triol, their F 7 and d 6 derivatives, and d 3-25-hydroxycholesterol, which was prepared from 3β-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 1H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side chain protons of 30.