Synthesis of (15E)-17 beta-hydroxyandrost-4-en-3,15-dione 15-(O-carboxymethyl)oxime, a potential hapten for testosterone immunoassays.

The key intermediate, 15-oxandrost-5-en-3 beta, 17 beta-diyl 3-acetate 17-benzoate (10), was prepared by a five-step procedure based on the addition of 4-methoxybenzyl alcohol to 3 beta-hydroxyandrosta-5, 15-dien-17-one (1). The resulting C-15 isomers were separated as acetates. In both series, the 17-ketones were reduced to 17 beta-hydroxy derivatives, and after benzoylation, the protecting methoxyphenylmethyl group at position 15 was removed with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, leaving 15 beta-hydroxyandrost-5-ene-3 beta, 17 beta-diyl 3-acetate 17-benzoate (6) and its 15 alpha-isomer 9. After Jones oxidation, both benzoates afforded the identical ketone 10. The reaction of 10 with (O-carboxymethyl)hydroxylamine in pyridine followed by diazomethane esterification gave (15E)-15-oxandrost-5-ene-3 beta, 17 beta-diyl 3-acetate 17-benzoate 15-(O-carboxymethyl)oxine methyl ester (11). Methyl ester 11 was successively submitted to acidic deacetylation, Oppenauer oxidation, potassium hydroxide treatment and reesterification with diazomethane to give (15E)-17 beta-hydroxyandrost-4-ene-3,15-dione 15-(O-carboxymethyl) oxine methyl ester (14). After purification, ester 14 was hydrolyzed with potassium hydrogen carbonate in aqueous methanol at elevated temperature into fee testosterone 15-(O-carboxymethyl)oxine (15).

Chemical synthesis of 15 beta-hydroxytestosterone and its derivatives using a (4-methoxyphenyl)methyl protecting group.

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.

Synthesis of (19E)-3 beta,17-dihydroxy-20-oxopregn-5-en-19-al 19-(O-carboxymethyl)oxime, new steroidal hapten for 17-hydroxypregnenolone.

A synthesis of (19E)-3 beta,17-dihydroxy-20-oxopregn-5-en-19-al 19-(O-carboxymethyl)oxime (15), is reported. Hydride reduction of ketone 1 gave the (20R)-hydroxy derivative 2 as the main product. Formylation of 2 followed by cleavage of the epoxide ring and mild Jones oxidation afforded aldehyde 6. Oximation with (O-carboxymethyl)hydroxylamine and subsequent methylation yielded methyl ester 8 which was selectively hydrolyzed to alcohol 9 and oxidized to ketone 10. Enolacetylation, epoxidation, and hydrolysis led to the desired 19-(O-carboxymethyl)oxime derivative of 17-hydroxypregnenolone 15.