Testosterone isomers

Hamada, H., Konishi, H., Williams, H. and Scott, A. (1991) Biotransformation of testosterone isomers by a green ceU suspension cultures of Marchantia polymorpha. Phytochemistry, 30, 2269-70. 

During the enzyme reaction of a substrate, multiple metabolite isomers may be formed. Thus, a baseline LC separation of the metabolites is desired. For example, more than five hydroxylated testosterone isomers (2-, 6-, 15-, and 16-positions) were seen after an incubation of testosterone with HLM. BDS Hypersil C8 was successfuly used for a separation of the five isomers (Table 16.1). Majority of the metabolites (>82%) was due to the 6 -OH-testosterone. Similarly, two hydroxylated isomers of midazolam at 1 and 4 -positions were also isolated with the given HPLC condition. 

A solution of testosterone (54 10 g) in 300 ml methanol is cooled to 0° and treated successively with 60 ml of cold 30 % hydrogen peroxide and 20 ml of cold aqueous 10% sodium hydroxide. The reaction mixture is stored for 48 hr at 0° and then poured into ice water. The resultant oil is extracted with methylene dichloride (or ether) and the extract is dried (MgS04). Removal of the solvent affords the crude product, a mixture of 4a,5a- and 4, 5 -isomers, which is purified by chromatography. A sample of pure 17j5-hydroxy-4/3,5 -oxidoandrostan-3-one, mp 157-158°, crystallizes after trituration of the crude product with ether. 

Photoketone (118) has served as the key intermediate in a relatively simple transformation of 3-keto-10/ -steroids to 3-keto-lOa-isomers without the assistance of other functional groups suitably situated next to the ring junction—a task that appears difficult to attempt by other methods. Optimal yields of (118) are achieved by catalytic hydrogenation of the unsaturated ketone (175), the photoisomer of 1-dehydrotestosterone acetate (see section III-C). In this way, a 6-step conversion of 1-dehydrotestosterone acetate (174) to IOa-testosterone acetate (127 acetate) is achieved in good yield. ° ... 

To a solution of 1 g of the mixture of 3-ketal-isomers of compound (II) in 10 cc of acetic anhydride is added a solution of 700 mg of p-toluenesulfonic acid in 7 cc of ecetic anhydride. The reaction mixture is kept at room temperature and under stirring for 5 hours. After some time e crystalline product begins to precipitate and the precipitation is complete by diluting with water. The precipitate is filtered and crystallized from methanol to give 17a-ethynvl-19-nor-testosterone 3,17-diecetate (III), melting point 175°C to 17B°C. 

Formal isomerization of the double bond of testosterone to the 1-position and methylation at the 2-position provides yet another anabolic/androgenic agent. Mannich condensation of the fully saturated androstane derivative 93 with formaldehyde and di-methylamine gives aminoketone 94. A/B-trans steroids normally enolize preferentially toward the 2-position, explaining the regiospecificity of this reaction. Catalytic reduction at elevated temperature affords the 2a-methyl isomer 95. It is not at all unlikely that the reaction proceeds via the 2-methylene intermediate. The observed stereochemistry is no doubt attributable to the fact that the product represents the more stable equatorial isomer. The initial product would be expected to be the p-isomer but this would experience a severe 1,3-diaxial non-bonded interaction and epimerize via the enol. Bromination of the ketone proceeds largely at the tertiary carbon adjacent to the carbonyl (96). Dehydrohalogenation... 

In the case of analytes with identical elemental formulas ( true isobars ) as positional or geometrical isomers (e.g. 11-hydroxycortisol and 21-hydroxycortisol or testosterone and epi-testosterone)—a discrimination of analyte and interfering compound is not possible with any mass analyzer—even if enabling highest mass resolution. Potentially, the disintegration pattern of isomers may be different, allowing analytical discrimination but in most cases chromatographic baseline separation of analyte and isomer prior to their MS/MS detection is required for unequivocal quantitative measurement. 

Another example is illustrated in Fig. 1.3. Testosterone and epitestosterone are eluted as one peak using SE-30 as the stationary phase. By conversion of the hydroxyl group into a more bulky substituent, the slight difference in the structure is enhanced and the two epimers can be resolved. The same approach can be used for the separation of 16-and 15-keto isomers of androstan-30-ol. The initial ketones are not separated on SE-30, but after conversion into the corresponding N,N-dimethylhydrazones their separation is possible as these derivatives are eluted much more slowly than the initial compounds [3]. 

The natural androgen testosterone and a large number of other androgens and anabolic agents possess the 4,5-double bond, while the majority of metabolic products lack this unsaturation. The hypothesis was put forward that the reduction of the 4,5-double bond in the liver may represent the rate-limiting step in the inactivation of these hormones [168,221]. Upon hydrogenation of the double bond, the 5-carbon becomes asymmetric and therefore two possible isomers could result, the 5a-isomer (trans... 

Androsterone and 5)3-androsterone, which are the major metabolites, were thought to be uniquely derived from the plasma androstenedione pool. Korenman et al. [170,388] demonstrated by the double isotope tracer method using carbon-14-labeled testosterone and tritium-labeled androstenedione that neither androsterone nor 5 -androsterone is a unique metabolite of a plasma androstenedione pool. A unique steroid metabolite has been defined by Dorfman [326] as a steroid persisting or formed during metabolism which can be related to one and only one tissue steroid. Korenman et al. obtained different tritium/carbon-14 ratios for androsterone and 5/3-androsterone, suggesting that other pathways of testosterone and androstenedione metabolism also exist. These other pathways, possibly metabolism of testosterone and androstenedione by peripheral tissue, may be responsible for the relative enrichment of either the 5a- or the 5/8-isomer of the urinary metabolites [305]. 

Table 23-6 illustrates some. structure-activity effects of the androgens, such as the greatly decreased activity of the l7 -ol isomer of testosterone (epitcstustcronc). Hundreds... 

Mixtures of the latter isomers which are not normally encountered in the urine of women, except in cases of ovarian tumors, could be studied. This was done by demonstrating uniquely dehydroepiandrosterone by the study of the spontaneous loss of water which is not observed in practice during the unimolecular decomposition of testosterone. 

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