Methandienone-I. A convenient method for the separation of 17alpha-methyl-17beta-hydroxyandrosta-i,4-dien-3-one and 17alpha-methyl-17beta-hydroxyanorosta-1,4,6-trien-3-one

17 α -Methyl-17 β -hydroxyandrosta-1,4-dien-3-one and 17α-methyl-17β-hydroxyandrosta-1,4, 6-trienone are found in the mother liquor of the reaction leading to the formation of the former from 17 α -methyl-17β -hydroxyandrosta-4-ene-3-one (I). This mother liquor usually discarded as waste product in the industrial production of 17α -methyl-17β -hydroxyandrosta-1,4-dien-3-one, can now be used for obtaining the two compounds separately using sodium metabisulfite.     

Parallel solid-phase synthesis of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-one derivatives for inhibition of type 3 17beta-hydroxysteroid dehydrogenase.

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Delta(4)-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23-58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17beta-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3beta-(N-heptanoyl-L-phenylalanine-L-leucine-aminomethyl)-3alpha-hydroxy-5alpha-androstan-17-one (42) inhibited the enzyme with an IC(50) value of 227nM, which is twice as potent as the natural substrate Delta(4)-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR(+)) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 microM (less than previously reported type 3 17beta-HSD inhibitors) and, interestingly, no proliferation at 0.1 microM.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.     

A direct radioimmunoassay for 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide.

Synthesis of the 11 alpha-hemiglutaryl derivative of 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide (androstane-diol-17G) starting from androsta-4,9(11)-diene-3,17-dione through a 10-step sequence and the preparation of its bovine serum albumin conjugate is described. By using this conjugate, antiserum was raised in rabbits which proved to be very specific for androstanediol-17G. A direct radioimmunoassay using a double antibody procedure is described for the measurement of androstanediol-17G from plasma without prior chromatography.     

Synthesis of 5 alpha-androstane-3 alpha,17 beta-diol 3- and 17-glucuronides

The glucuronidation of 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) was carried out by three different methods: The Koenigs-Knorr reaction using methyl-2,3,4-tri-O-acetyl-1 alpha-bromo-1-deoxy-beta-D-glucuronate, by employing methyl-2,3,4-tri-O-acetyl-1-O-(trichloroacetimidoyl)-alpha-D-gl ucopyranuronate (imidate procedure), and by the reaction of 2,3,4-tri-O-acetyl-alpha-D-glucopyranuronate catalyzed by trimethylsilyl trifluoromethanesulfonate (triflate method). The Koenigs-Knorr method gave the beta-anomers of both the 3- and 17-glucuronides. The imidate procedure also resulted in the beta-anomers of the 3- and 17-glucuronides, but in lower yield. The triflate method, however, yielded only the alpha-anomers of the 3- and 17-glucuronides. The structural assignments of these compounds were made from NMR spectral data obtained with a 500 mHz instrument.     

The chemistry of 9 alpha-hydroxysteroids. 3. Methods for selective formation and dehydrations of 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one

Two methods to produce the 17-cyanohydrin, using potassium cyanide in acetic acid/methanol or acetone cyanohydrin with aqueous sodium hydroxide, were followed with 9 alpha-hydroxyandrost-4-ene-3,17-dione, both providing 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one. The selectivity of one of these methods, that which uses acetone cyanohydrin, is not in agreement with a comparable reaction with the 9 alpha-unsubstituted androst-4-ene-13,17-dione to give the 17 alpha-cyano-17 beta-hydroxy product, as reported in the literature and confirmed by us. The 9 alpha-hydroxy and 17 alpha-hydroxy groups were used for the regioselective introduction of 9(11)- and 16(17)-double bonds by dehydrating 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one under different conditions.     

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

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.     

The metabolism of 17beta-estradiol-3-glucosiduronate and estrone-3-glucosiduronate in the rabbit.

[6, 7-3H]-17beta-Estradiol-3-glucosiduronate, [6, 7-3H]-estrone-3-glucosiduronate or [6, 7-3H]-estrone was administered intravenously into the rabbit, and analysis and identification of the urinary metabolites were carried out. In either case, the major urinary metabolite was found to be a diconjugate. The sequential enzymic hydrolysis indicated that this diconjugate was glucosiduronate-N-acetyglucosaminide of 17alpha-estradiol. From these results, the conversion of the estrogen glucosiduronate into a diconjugate was thought a rather universal phenomenon in the rabbit.     

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.