Isolation and characterisation of a C-18 neutral steroid, oestra-5(10),7-diene-3,17-diol, from pregnant mare urine and allantoic fluid. Facile oxidation to yield oestra-5(10),6,8-triene-3,17-diol (diol of Heard's ketone)

Oestradiene-3,17-diol and oestratriene-3,17-diol (or the diol of Heard's ke tone (3-hydroxy-5(10),6.8-oestratriene-17-one) have been extracted on a lar ge scale from pooled urines and allantoic fluid obtained From pregnant mare s, Initial purification was achieved using column chromatography, and furth er purification by high performance liquid chromatography or silver nitrate (argentation) thin layer chromatography. The steroids were characterised u sing gas chromatography-mass spectrometry. Positions of the double bonds in ring B of oestradienediol were deduced on the basis of results of ultravio let (UV) and nuclear magnetic resonance (NMR) spectroscopy, hydrogenation, and incubation studies with the enzyme 5-ene-3 beta -hydroxysteroid dehydro genase/steroid-4,5-isomerase. The reference steroid, 5,7-cholestadien-3 bet a -ol (7-dehydrocholesterol), with its conjugated double bond system, behav ed entirely differently to oestradienediol, consistent with the latter havi ng no conjugated system. These data, together with detailed results of NMR studies, have led us to designate the positions of the double bonds in oest radienediol as 5(10),7-. The instability of the dienediol became apparent w hen the steroid was converted to its bis-trimethylsilyl (TMS) ether. The ph enomenon was exacerbated when derivatisation was performed at elevated temp eratures or when the fraction containing the dienediol was stored at 4 degr eesC prior to being derivatised. The Facile oxidation product was shown to be 5(10),6,8-oestratriene-3,17-diol, implying that the two steroids are rel ated and, furthermore, that all the sites of unsaturation are in the B ring . Because of the facile oxidation of oestradienediol to oestratrienediol (t he diol of Heard's ketone), we propose, that this, and by implication, Hear d's ketone itself, are artefacts of the isolation procedures which were uti lised in the original studies. A possible mechanism is proposed for the bio synthesis of 5,7-oestradienediol from a ring-B unsaturated C-19 compound, i nvolving C-19 demethylation without aromatisation. (C) 2000 Elsevier Scienc e Ltd. All rights reserved.