Conversion of androstenedione to 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-4-androsten-17-one by the testicular microsomal fraction of Sprague-Dawley rats

When androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7 alpha-hydroxyandrostenedione. The metabolites were identified as 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one catalyzed by the 3 beta-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Synthesis of some 6 beta-acetylthio hydroxysteroids

The reactions of 3 beta-hydroxy-20-oxo-5-pregnene-16 alpha-carbonitrile, 3 beta-hydroxy-5-androsten-17-one, 3 beta-hydroxy-5-pregnen-20-one, and 5-cholesten-3 beta-ol with thioacetic acid in dioxane afford mainly 6 beta-acetylthio derivatives which were characterized by IR, NMR (1H, 13C), and mass spectroscopy. A similar reaction of 17 beta-hydroxy-1,4-androstadien-3-one yields chiefly the known 1 alpha-SCOCH3 derivative.     

6-Oxygenation of 3-oxo-4-ene-steroids in high yields after 3-imine-formation

3-Imine formation between primary amines and 3-oxo-4-ene-steroids, followed by hydrolysis of the imines (either spontaneously during work up or induced by acetic acid) has been shown to cause 6-oxygenation of the steroids tested (17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione and 4-cholesten-3-one). The main products are the 6 beta-hydroxy- and the 6-oxo-derivatives of the respective steroid. These derivatives were identified by chromatographic mobilities and by gas chromatography-mass spectrometry. The formation of 6 beta-hydroperoxy-derivatives is suggested and these derivatives were tentatively identified. The highest yields of 6-oxygenated products (30-50%) were found when cadaverine and spermine were reacted with the steroids. The addition of reduced glutathione during hydrolysis of the steroid 3-imines of cadaverine, hexylamine and ethanolamine as well as addition of ascorbic acid during the hydrolysis of the steroid 3-imines of cadaverine substantially reduced the 6-oxygenation. Steroid 3-imine formation and hydrolysis which yields 6-oxygenated derivatives has also been shown to occur during work up (evaporation) of organic solvent extracts of rat liver microsomes (105,000 g sediments) to which 17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione or 4-cholesten-3-one respectively had been added. It is concluded that there is a risk that these organic reactions are mistaken for enzymatic conversions during in vitro investigations of 3-oxo-4-ene-steroids.     

A radioimmunoassay for 7 alpha-hydroxy dehydroepiandrosterone in human plasma.

A radioimmunoassay for plasma 3 beta, 7 alpha-dihydroxy-5-androsten-17-one (7 alpha-hydroxy DHA) has been developed using anti-sera raised against 3 beta, 7 alpha-dihydroxy-5-androstene-17 beta-carboxyl-bovine serum albumin conjugate and [1, 2 (n) - 3H] 7 alpha-hydroxy DHA as the radioligand. Significant cross reactivity was found with 3 beta, 7 alpha-dihydroxy-5-pregnen-20-one (44%), 3 beta, 7 beta-dihydroxy-5-androsten-17-one (6%), 3 beta, 6 beta-dihydroxy-4-androsten-17-one (2.5%), 3 beta-hydroxy-5-androsten-17-one (DHA, 2%), 3 beta, 7 beta-dihydroxy-5-pregnen-20-one (2%) and 7 alpha-hydroxy-4-androstene-3, 20-dione (1%). 7 alpha-Hydroxy DHA was extracted from plasma and separated from cross-reacting factors using alumina micro-columns. The separation of bound and free steroid was achieved using dextran-coated charcoal. The concentration of 7 alpha-hydroxy DHA in the plasma of breast cancer patients was significantly lower than the concentrations in the plasma of normal women, hospitalized women suffering from non-endocrine diseases and patients with benign breast disease. The decrease in the concentration of 7 alpha-hydroxy DHA in the plasma of pregnant women was not significant.     

Kinetic analysis of the placental microsomal 3 beta-hydroxysteroid dehydrogenase activity.

A sensitive accurate assay for the placental microsomal 3 beta-hydroxysteroid dehydrogenase (E.C.1.1.1.51) has been developed using tritiated substrates. Kinetic analysis of the enzyme with 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-5-pregnen-20-one indicates that the apparent Km values for these substrates are orders of magnitude less than previously described. Analyses were carried out with microsomal preparations from two different placentas. For placenta 1 the apparent Km value for 3 beta-hydroxy-5-androsten-17-one was 14 nM and for 3 beta-hydroxy-5-pregnen-20-one was 36 nM; for placental 2 apparent Km values were 19 nM and 42 nM respectively. The analyses were performed over wide ranges of substrate concentration (about 200 fold), both above and below the Km values and no deviation from linearity of Eadie-Hoftsee plots was observed.     

Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.     

A preovulatory rise of dehydroepiandrosterone in the mare measured by radioimmunoassay.

A radioimmunoassay procedure was developed for the measurement of dehydroepiandrosterone (DHA) in peripheral serum in nonpregnant mares. The synthesis and conjugation of 3beta-hydroxy-5-androsten-19-al-17-one 19(0-carboxymethyl) oxime is described. Antisera were developed against this antigen and characterized. The most specific antiserum was used to measure DHA. Concentrations of DHA were greatest immediately before ovulation.     

Antiprogestational agents. The synthesis of 7-alkyl steroidal ketones with anti-implantational and antidecidual activity

A series of 7α- and 7β- alkyl derivatives of steroidal 4-en- and 5-en-3-ones were prepared by 1,6-conjugate addition of organocopper reagents to various steroidal 4,6-dien-3-ones of the androstane, estrane and gonane series. Biological study of these and related compounds revealed that 17β-hydroxy-7α-methyl-5-androsten-3-one (2), 17β-hydroxy-7α-methyl-5-estren-3-one acetate and 17β-hydroxy-7α-methyl-4-estren-3-one acetate had significant anti-implantational and antidecidual activities. The contragestative effects were associated with the latter antihormonal properties, and not with the androgenicity of these compounds.     

Analysis of profiles of unconjugated steroids in rat testicular tissue by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) method for analysis of unconjugated steroids in a rat testis is described. A combined solvent-solid extraction procedure, utilizing Lipidex 1000 and Sep-Pak C18, gives a 25-fold purified extract. Steroids in this extract are fractionated by straight phase high-performance liquid chromatography (HPLC) on a LiChrosorb DIOL column in n-hexane-2-propanol, 92:8 (v/v). Four fractions are collected and the steroids are converted to tert-butyldimethylsilyl (TBDMS), 3-enol-TBDMS, and mixed TBDMS-trimethylsilyl (TMS) derivatives using TBDMS- and TMS-imidazole with sodium formate as catalyst under conditions suitable for the steroids present in the respective fractions. The derivatives are purified by reversed phase HPLC in 100% methanol and are analyzed by GC-MS, using selected ion monitoring of the major ions of high mass. For quantification, a mixture of known amounts of ten 14C-labelled steroids, [3H]estradiol and [2H3]estradiol are added to the testis homogenate. The mean concentrations (ng/g wet wt) of the twelve steroids determined were: 4-androstene-3, 17-dione, 4.0; testosterone, 127; 17 beta-hydroxy-5 alpha-androstan-3-one, 4.5; 5 alpha-androstane-3 alpha, 17 beta-diol, 5.7; 5 alpha-androstane-3 beta, 17 beta-diol, 1.5; progesterone, 5.5; 17 alpha-hydroxyprogesterone, 14.4; 3 beta-hydroxy-5-androsten-17-one, 0.07; 5-androstene-3 beta, 17 beta-diol, 0.25; 3 beta-hydroxy-5-pregnen-20-one, 10.3; 3 beta, 17 beta-dihydroxy-5-pregnen-20-one, 0.95; and estradiol, 0.025. Variations between animals were large whereas testes from the same animal in most cases had similar steroid concentrations.     

Synthesis of 7-substituted-5-androstene derivatives promoted by SmI2

The 7-substituted-5-androstene derivatives 2a-10a and 2b-10b were prepared by reaction of 3beta,17beta-di(tert-butyldimethylsilyloxy)-5-androsten-7-one 1 with different organic halides. The resulting 7alpha- and 7beta-isomers were carefully separated by column chromatography. The structural assignments of the 7alpha- and 7beta-isomers were determined by 13C-NMR.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

Expeditious and convenient synthesis of pregnanes and its glycosides as potential anti-dyslipidemic and anti-oxidant agents

A series of new pregnane derivatives and its glycosides were synthesized in order to find new 'leads' against some important targets. The 3beta-hydroxy-16alpha-(2-hydroxy ethoxy) pregn-5-en-20-one (5) was synthesized from 3beta-hydroxy-5,16-pregnadiene-20-one (2) by adopting general modified procedure using BF(3):Et(2)O as a catalyst. Reduction of 5, with sodium borohydride yielded 3beta,20beta-dihydroxy-16alpha-(2-hydroxy ethoxy) pregn-5-en (7) as the major isolable product. O-alkylation of the C-20-oxime-pregnadiene (9) with 1,5-dibromopentane yielded 20-(O-5-bromopentyl)-oximino-3beta-hydroxy-pregn-5,16-diene (11). Synthesis of C-16 substituted pregnane glycosides (20) and (21) were accomplished with the imidate method using BF(3):Et(2)O. The synthesis of 4-chlorobenzoate (3) and 2-chlorobenzoate (4), derivatives of 2 were also accomplished. These compounds were evaluated for their anti-dyslipidemic and anti-oxidant activity and amongst them compounds 3 and 7 showed more lipid lowering and anti-oxidant activity.     

Preparation of Metabolites of Spironolactone by Microbial Oxygenation

It has been postulated that 3-(3-oxo-7alpha-methylthio-4-androsten-17alpha-yl)propionic acid gamma-lactone (I) is a key intermediate in the metabolism of the anti-aldosterone agent, spironolactone (3-[3-oxo-7alpha-acetylthio-17beta-hydroxy-4-androsten-17alpha-yl] propionic acid gamma-lactone). In the present study it was found that microbial oxygenation of I by Chaetomium cochloides QM 624 gave three metabolites which retained sulfur in their molecules and were found to be identical to the human metabolites of spironolactone.     

Synthesis of 16 alpha,19-dihydroxy-4-androstene-3,17-dione and 3 beta,16 alpha,19-trihydroxy-5-androsten-17-one and their 17 beta-hydroxy-16-keto isomers

3 beta,16 alpha,19-Trihydroxy-5-androsten-17-one and 16 alpha,17-dihydroxy-4-androstene-3,17-dione were synthesized from the 5 alpha-bromo-6 beta,19-epoxy-17-ketone derivative 1, using the bromination at C-16 alpha of the 17-ketone 1 and the controlled alkaline hydrolysis of the 16 alpha-bromo-17-ketones 2 and 11 as key reactions. Zinc dust reductive cleavage of the 6 beta,19-epoxy-16 alpha-hydroxy-17-ketones 4 and 12, produced by controlled hydrolysis, gave the corresponding 19-alcohol derivatives 6 and 14, which were rearranged to the 17 beta-hydroxy-16-ketones 7 and 15 when treated with sodium hydroxide. The 3 beta,16 alpha,17 beta,19-tetrol 8 was obtained from the 16 alpha-ketol 6 by reaction with sodium borohydride.     

Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization.

The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17 alpha-carboxyethynyl-17-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17alpha-carboxyethynyl-17-hydroxy-5alpha-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxymethyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89,500 and a carbohydrate content of 17%. The association constant (M-1) at 4 degrees C was 1.1 X 10(8) and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCl dissociated the protein into subunits having a molecular weight of 28,400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.     

Syntheses and ligand-binding studies of 1 alpha- and 17 alpha-aminoalkyl dihydrotestosterone derivatives to human sex hormone-binding globulin

We report on the syntheses of 1 alpha- and 17 alpha-aminoalkyl dihydrotestosterone (DHT) derivatives and the particularly high binding affinity of the 1 alpha-aminohexyl ligand for human sex hormone-binding globulin (SHBG). The two 17 alpha-aminopropyl-17 beta-hydroxy-5 alpha-androstan-3-one (1) and 17 alpha-aminocaproylamidoethyl-17 beta-hydroxy-5 alpha-androstan-3-one (2) derivatives were synthesized via a 17beta-spirooxirane intermediate in high yields. The 1 alpha-aminohexyl-17 beta-hydroxy-5 alpha-androstan-3-one compound (3) was obtained in a seven step synthesis using a copper-catalyzed conjugate addition of a omega-silyloxyhexyl Grignard reagent to 17 beta-benzoyloxy-5 alpha-androst-1-en-3-one. All structures were elucidated based on 1H NMR spectroscopy and mass spectral analyses. The three aminosteroid derivatives were tested as ligands for SHBG by competition experiments with tritiated testosterone as tracer under equilibrium conditions. The association constants of the two 17 alpha-DHT derivatives were approximately 1 x 10(7) M(-1), whereas the 1 alpha-DHT derivative showed a remarkably high binding affinity to SHBG with an association constant of 1.40 x 10(9) M(-1).These aminoalkyl derivatives, substituted either at the D-ring or the A-ring of the steroid skeleton, can be easily coupled onto a carboxymethylated solid state surface of a biosensor. Such a device lends itself to kinetic and thermodynamic studies aimed to provide a better understanding of the biospecific interaction of steroids with SHBG.     

Microbiologic oxidation of estratrienes and estratetraenes by Streptomyces roseochromogenes ATCC 13400

Incubation of estrone (1a) with Streptomyces roseochromogenes ATCC 13400 yielded a mixture of 3,16 alpha-dihydroxyestra-1,3,5(10)-trien-17-one (3a) and 3,17 beta-dihydroxyestra-1,3,5(10)-trien-16-one (4a). Transformation of 3-methoxyestra-1,3,5(10)-trien-17-one (1b), 3-hydroxyestra-1,3,5(10),9(11)-tetraen-17-one (2a), and 3-methoxyestra-1,3,5(10),9(11)-tetraen-17-one (2b) with the same microorganism gave the corresponding mixtures of 16 alpha-hydroxy-17-ketones and 17 beta-hydroxy-16-ketones (3b and 4b, 6a and 7a, 6b and 7b, respectively). In addition, in these three last experiments, the 16 beta-17 beta-dihydroxy derivatives 5b, 8a, and 8b, respectively, were also isolated. The complete assignments of the 13C nuclear magnetic resonance spectra of these compounds are given.     

Alpha-methylene-gamma-lactone fused to steroidal ring D as potential antitumor agent

Steroidal alpha-methylene-gamma-lactone la has been synthesised from 3 beta-hydroxy-5-androsten-17-one 2 and showed to be active against HeLa cells.     

In vitro metabolism of androgens by mammalian cauda epididymal spermatozoa.

The in vitro metabolism of [3H] testosterone (17beta-hydroxy-4-androsten-3-one), [3H] androstenedione (4-androstene-3,17-dione) and [3H] dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) by cauda epididymal spermatozoa from the rat, rabbit, hamster, guinea-pig and ram, varied between species. There were differences in the androgens utilized, the extent of their conversion and the identities of the metabolites formed. Of the steroid substrates tested rat spermatozoa metabolized testosterone preferentially while spermatozoa from guinea-pig transformed [3H] dehydroepiandrosterone (DHEA) almost exclusively. Rabbit spermatozoa converted all three [3H] androgens while hamster sperm utilized [3H] testosterone and [3H] DHEA. Spermatozoa collected from rams killed at the abattoir metabolized both [3H] androstenedione and [3H] DHEA but this capacity was dramatically reduced in spermatozoa collected from rams subjected to short-term anaesthesea. The results are discussed in relation to the possible direct roles of androgens in sperm physiology.