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).