Oestranes

The accurate quantitation of oestranes is of great importance in clinical chemistry to provide information about the progress of a pregnancy. In the last months of pregnancy the urinary oestriol levels normally rise, and a decline in levels is indicative of a malfunction. The oestranes are usually excreted in urine as conjugates and in most analyses these are hydrolysed prior to HPLC analysis. There are two methods currently in use for hydrolysis, these being acid hydrolysis 

When normal phase HPLC techniques have been used for the separation of the oestranes the stationary phase has usually been unmodified silica, although Diol columns have also been used (as described earUer). Using a mobile phase of hexane-ethanol (9 1) and a silica stationary phase, good resolution of the more polar oestrogens including oestradiol, 6-ketooestradiol, 16-epioestriol, oestriol, 16,17-oestriol, 2-hydroxyoestriol and 6-hydroxyoestriol has been described (Lin and Heftmann, 1981). Similarly, these authors noted that for separations of the less polar oestranes a mobile phase of hexane-ethanol (97 3) may be used. 

In general, it is suggested that epimers are better resolved using normal phase chromatography, but when separations of compounds which differ by the addition of ketone, hydroxy or carbon-carbon double bond are required, reversed phase chromatography is the technique of choice (Lin and Heftmann, 1981). 

Ion-exchange chromatography has been used for the resolution of the oestranes and-while in general this technique is much less popular than reversed phase or normal phase chromatography it can neverthe- 

Two further separation techniques have been described for the resolution of the oestranes reversed phase ion-pair partition (Hermansson, 1978) and argentation reversed phase HPLC (Tscheme and Capitano, 1977). In the former technique C2 or Cg columns were coated with 1-pentanol and used with tetrapropylammonium hydroxide as an ion-pairing reagent. In the latter technique an ODS stationary phase was used with a mobile phase of methanol-5% silver nitrate (60 40) to elute oestriol, equilin, oestrone and oestradiol. 

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O.r.d. data for a highly purified sample of 17/3-hydroxy-4,4-dimethyl-5a-oestran-3-one ° differ only slightly from a previous report. 

The structures of the 9-methyl-5a,9j8,10a-oestran-3-one (1) and its 2a- and 2/8-bromo-derivatives have been studied by X-ray crystallography. Ring A in the 2/8-bromo-compound is forced into a twist-boat conformation by steric compression (2/8-Br/9/3-Me), but the other two compounds are only slightly deformed from the chair conformation. These findings are compatible with the properties (c.d. and n.m.r.) of the same compounds in solution. 3a-Acetoxy-4a,8a,14/8-trimethyl-18-nor-5a,9/3,13a-androstan-17-one (2), another steroid of unusual configuration, derived from fusidic acid, has a twisted boat conformation of ring B. ... 

Syntheses of 16,17-disubstituted oestra-l,3 5(10),14-tetraene 3-methyl ethers involved reaction of the corresponding 16,17-epoxides with various nucleo-philes and similar reactions of the 17,20-epoxides (300) gave a range of androstanes (301). A series of 16j8-alkylated androstanes and oestranes has... 

Arthropod Sterols.—The absence of sterol biosynthesis has been observed in further examples from the class ArachnidaT The conversion of /5-sitosterol back to cholesterol was demonstrated in bees and in locusts/ where it was shown that fucosterol, but not 24-methylenecholesterol, was probably a precursor of desmosterol and cholesterol. Some interconversions of pregnane, andros-tane, and oestrane steroids have been examined in the cricket.The stages in the oxidation of the side-chain of ecdysone hormones have been studied in several species. In Calliphora, the sequence of oxidation seems to be hydroxylation at C-25, C-22,and C-20 i.e. (115)— (116)— (117)— (118). The last step, conversion... 

The 7/ -fluoro-substituent in the e-homo-oestrane derivatives (69) is unusually stable, surviving aromatization of ring a, or reduction of the 3-oxo-group by lithium aluminium hydride, except in refluxing diglyme. ... 

A number of other synthetic approaches to the steroid skeleton are discussed in the following sections, which are devoted more specifically to the synthesis of oestrane, androstane, and pregnane derivatives, as well as to seco-compounds and steroidal alkaloids. 

Both isomeric 2,3-epoxides in the 5a-cholestane, 4,4-dimethyl-5a-cholestane, and 4,4-dimethyl-5a-oestrane series afford normal diaxial fluorohydrins with... 

Condensation of the isothiouronium salt, obtained from the vinyl alcohol (413), with 2-methyl-cyclopentane-1,3-dione has led to a series of A-nor-3-oxa-steroids and similar condensation of the thia-analogue (408) with 2-methyl-cyclopentane-1,3-dione afforded the thia-oestrane (410), which was reduced catalytically to a mixture of 14a- and 14/S-H-dihydro-epimers. Reduction of the remaining conjugated 8,9-unsaturation was less easy than in the carbocyclic analogue metal-ammonia afforded thiols, and catalytic reduction was slow and led to a mixture of, presumably, 8a,9a- and 8, 9jS-epimers. The former isomer (416) which has its 9 -H favourably disposed for hydride abstraction with DDQ readily dehydrogenated to the 9(ll)-olefin which, on subsequent catalytic reduction, gave 6-thia-oestrone methyl ether. Condensation of the same thia-... 

Alkylation of the protected unsaturated ketone (433) with m-methoxyphenacyl bromide and deprotection gave the bicyclic ketone (434), which could be converted to the 8)8-methyl-D-homo oestrane (436) by a variety of methods, the best of which proceeded by direct reduction to (435) and cyclisation. ... 

Hydroxy-17a-ethynyl-oestranes and -androstanes readily add nitrile oxides to give the novel 17a-isoxazoles (533 R = Ph, Me or C02Et) with reduced oestrogenic activity. 

Backbone Rearrangements.—Reductive rearrangement of androst-4-ene-3,17-dione in HF-SbFs and in the presence of a hydrogen donor was dependent on the acidity. At high acidity the major products were the 6a-methyl-14/3-oestrane-diones (154) and (155) whereas at low acidity the major product was the... 

Miscellaneous Rearrangements.—An improved non-photochemical route to 13-epi-androstanes and -oestranes involved conversion of the appropriate 17-oximino-compounds with boiling acetic anhydride-pyridine into the enamide (176) and the enimide (177) which were readily hydrolysed to the 17-oxo-... 

The pyrrolidyl enamines of 3-oxo-steroids (176) are reduced by diborane to give the saturated 3a- (177) and 3j8-pyrrolidino-steroids (178) in good total yield. The mechanism of saturation of the olefinic bond is discussed in terms of norma borane addition to give amino-borane derivatives (Scheme 4) the BHj group at C-2 is probably displaced internally by a hydride ion. The 2)5-steroidal borane derivative (179 R = H) is stable in the absence of a 10/5-methyl group (oestrane series) and can be oxidized in the usual way to give the 3a-amino-2)5-hydroxy-derivative (180). ... 

Several groups are included in this class of steroids including corticosteroids, pregnanes, androstanes and oestranes. Extensive reviews on the chromatography of these compounds have been previously published (Heftman, 1983 Heftman and Lin, 1982 Purdy et al, 1982 O Hare and Nice, 1982 Schmidt, 1982). It is not the purpose of this section to provide a comprehensive review of the Uterature but to provide the potential chromatographer with a number of alternative strategies for the separation and detection of the various steroid hormones. For ease of reference the individual classes of steroid hormones have been separated into individual sub-sections. 

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