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A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.

Although 7 beta-hydroxytestosterone is a known product of hepatic androgen metabolism, there are no published methods for its chemical synthesis except from the equally difficult to obtain 7 beta-hydroxy-4-androstene-3,17-dione. We found that several seemingly straightforward routes for its synthesis failed. Consequently, we tried to produce 7 beta-hydroxytestosterone by enzymatic oxidation of 5-androstene-3 beta, 7 beta, 17 beta-triol with cholesterol oxidase (Brevibacterium sp.), a procedure previously used to synthesize 7 beta-hydroxy-4-cholesten-3-one from 3 beta, 7 beta-dihydroxycholesterol (Alexander and Fisher 1995). However, 5-androstene-3 beta, 7 beta, 17 beta-triol was, at best, a very poor substrate for the enzyme leading to the production of 7 beta-hydroxytestosterone in only trace amounts. Thus, we explored a strategy for the enzymatic synthesis in which a C8-ester at C-17 (5-androstene-3 beta, 7 beta, 17 beta-triol 17-caprylate) would serve to mimic the bulky and hydrophobic side chain of cholesterol and thus allow the C19-steroid to act as an effective substrate. When this ester was incubated with cholesterol oxidase, it was converted efficiently to 7 beta-hydroxytestosterone-17-caprylate. Attempts to remove the ester group by several mild hydrolytic procedures caused elimination of the 7 beta-hydroxyl group; we, therefore, obtained 7 beta-hydroxytestosterone by incubation of the intermediate ester with porcine lipase.

Reaction of 3 beta-hydroxyandrosta-5,15-dien-17-one with 4-methoxybenzyl alcohol followed by acetylation gave mainly 15 beta-[(4-methoxyphenyl)methoxy]-17-oxoandrost-5-en-3 beta-yl acetate. This product was transformed by borohydride reduction and organosilyl derivatization into the orthogonally protected 17 beta-(dimethylthexylsiloxy)-15 beta-[(4-methoxyphenyl)methoxy]androst-5-en-3 beta-yl acetate and 17 beta-(dimethylisopropylsiloxy)-15 beta-[4-methoxyphenyl)methoxy]androst-5-en-3 beta-yl acetate. After deacetylation, these intermediates were submitted to Oppenauer oxidation and both yielded testosterone derivatives 17 beta-(dimethylthexylsiloxy)-15 beta-[(4-methoxyphenyl)methoxy]androst-4- en-3-one and 17 beta-(dimethylisopropylsiloxy)-15 beta-[(4-methoxyphenyl)- methoxy]androst-4-en-3-one. Removal of the (4-methoxyphenyl)methyl group from position 15 by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone treatment gave the partially protected derivatives 17 beta-(dimethylthexylsiloxy)-15 beta-hydroxyandrost-4-en-3-one and 17 beta-(dimethylisopropylsiloxy)-15 beta-hydroxyandrost-4-en-3-one. After acidic deprotection, the dimethylthexylsilyl derivative yielded 15 beta-hydroxytestosterone (15 beta,17 beta-dihydroxyandrost-4-en-3-one). Dimethylisopropylsilyl derivative was converted to the corresponding 15-hemisuccinate and 15-hemiglutarate (17 beta-hydroxy-3-oxoandrost-4-en-15 beta-yl 15-hemisuccinate and 15-hemiglutarate, respectively), which were designed as model haptens for immunoassay studies.

Hydroxylation at position 6 beta testosterone I (17 beta-hydroxyandrost-4-en-3-one) and the anabolic steroids 17 alpha-methyltestosterone II (17 beta-hydroxy-17 alpha-methylandrost-4-en-3-one), metandienone III (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone IV (4-chloro-17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), and fluoxymesterone V (9-fluoro-11 beta, 17 beta-dihydroxy-17 alpha-methylandrost-4-en-3-one) was achieved via light-induced autooxidation of the corresponding trimethysilyl 3,5-dienol ethers dissolved in isopropanol or ethanol. The reaction further yielded the 6 alpha-hydroxy isomer in low amounts. The 6 beta-hydroxy isomer of I-V and the 6 alpha-hydroxy isomers of I, III, and IV were isolated and characterized by 1H and 13C NMR, high-performance liquid chromatography, gas chromatography, and mass spectrometry. Human excretion studies with single administered doses of boldenone (17 beta-hydroxyandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, metandienone, 17 alpha-methyltestosterone, and [16,16,17-2H3] testosterone showed that 6 beta-hydroxylation is the major metabolic pathway in the metabolism of 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, and metandienone, whereas for boldenone, 17 alpha-methyltestosterone, and testosterone, 6 beta-hydroxylation is negligible.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.