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A successful approach to the synthesis of 3 beta,15 beta-dihydroxy-5-androsten-17-one (14d) has been developed using trichloroethoxy ethers as intermediates in the synthesis of the corresponding alcohols. 3 beta-Methoxymethoxy-5,15-androstadien-17-one (10c) was prepared by a selenation/dehydroselenation strategy from 3 beta-methoxymethoxy-5-androsten-17-one (14c). Base-catalyzed reaction of trichloroethanol with 10c gave 3 beta-methoxymethoxy-15 beta-trichloroethoxy-5-androsten-17-one (14g). Under the same conditions, 3 beta-acetoxy-5,15-androstadien-17-one (10b) gave 3 beta-hydroxy-15 beta-trichloroethoxy-5-androsten-17-one (14f) which was characterized after conversion to 14g. Cleavage of the trichloroethoxy group in 14f with zinc or zinc/copper couple gave 14d. The acid-catalyzed hydrolysis of 17,17-ethylenedioxy-5,15-androstadien-3 beta-ol (15) gave 3 beta-hydroxy-5,15-androstadien-17-one (10a) as the major product along with 14d. However, addition of water to 10a in the presence of acid gave the desired product 14d in poor yield (15%).
Inhibitors of aromatase, the cytochrome P-450 enzyme complex responsible for the biosynthesis of estrogens, may be useful as therapeutic agents for the treatment of estrogen-dependent disease states such as breast and endometrial cancer. Several 7 alpha-thio-substituted androstenediones have proven to be potent inhibitors of aromatase in vitro and in vivo. Recent research efforts have focused on designing aromatase inhibitors with both substitution at C-7 and extended linear conjugation in rings A and B of the steroid nucleus. The targeted compounds, 7-substituted 4,6-androstadiene-3,17-diones 4-10, were prepared by the addition of either Grignard or lithium reagents to 3,3:17,17-bis(ethylenedioxy)-5-androsten-7-one (3). Inhibitory activities of the compounds were evaluated in vitro by enzyme kinetic studies employing the microsomal fraction isolated from human term placenta. 7-Benzyl- and 7-phenethyl-4,6-androstadiene-3,17-dione analogues are effective inhibitors with apparent Ki's of 60.9-174 nM, while the 7-phenyl analogue exhibited an apparent Ki of 1.424 microM. Thus, several 7-substituted 4,6-androstadiene-3,17-diones were prepared and exhibited good competitive inhibition of aromatase in vitro in human placental microsomes.
Isolation of 3 beta-hydroxy-5,7-androstadien-17-one, as a major component of steroids extracted from vein blood of the fetal gonads of the horse, supports the proposed role for the compound as a precursor for equilin formation in the placenta of the mare. The 5,7-diene was extracted from blood collected from gonadal veins of fetal ovaries and testes in situ, and from a fetal testis connected to an artery in the neck region of the mare. Perfusion of fetal gonads in the laboratory was carried out to allow longer periods of collection. In addition, isolated cell preparations from a fetal testis were incubated for 4-8 h in tissue culture to investigate steroid secretion in vitro. Final purification of neutral steroids in the extracts was carried out by high performance liquid chromatography, and identification was made by u.v. and mass spectrometry. The presence of 3 beta-hydroxy-5,7-androstadien-17-one in extracts from all sources provided evidence for its secretion in vivo and in vitro. Other 5,7-dienes, which were less polar than the C19 compound, were noted in extracts of media but not identified. These data support the view that a 5,7-diene pathway is involved in the biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one in the fetal horse gonad.
A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.
[16 alpha-2H]Pregnenolone was synthesized by catalytic deuteriation of 3 beta-hydroxy-5,16-pregnadien-20-one followed by base-catalyzed back exchange of the 17 alpha-2H atom, and [16 beta-2H]pregnenolone by catalytic hydrogenation of 3 beta-hydroxy-5,16-[16-2H]pregnadien-20-one, which had been synthesized from [16,16-2H]dehydroepiandrosterone. The labelled pregnenolones were incubated separately with the microsomal fraction of boar testis. The metabolites were analyzed by gas chromatography-mass spectrometry, and the isotope compositions of the following six metabolites were determined: 17-hydroxypregnenolone, dehydroepiandrosterone, 5-androstene-3 beta,17 alpha-diol, 5-androstene-3 beta,17 beta-diol,16 alpha-hydroxypregnenolone and 5,16-androstadien-3 beta-ol. The first four metabolites derived either from [16 alpha-2H]- or from [16 beta-2H]pregnenolone showed essentially the same isotope compositions as those of their respective precursors. The 16 alpha-hydroxypregnenolone and the 5,16-androstadien-3 beta-ol biosynthesized from [16 alpha-2H]pregnenolone lost the 2H label, while the same metabolites biosynthesized from [16 beta-2H]pregnenolone retained the albel. The result shows that the 16 alpha-hydrogen is stereospecifically removed with the retention of the 16 beta-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol.
Horse fetal gonadal tissue was incubated with 3 beta-hydroxy-5,7-pregnadien-20-one and 5,7-cholestadien-3 beta-ol and it was shown that both substrates were converted to 3 beta-hydroxy-5,7-androstadien-17-one. These findings support the proposal that in this tissue there is a 5,7-diene pathway producing 3 beta-hydroxy-5,7-androstadien-17-one, the putative precursor of equilin in the placenta.
A series of 2-carboxy-1, 4-androstadien-3-one derivatives and their alkyl esters, were prepared by high-yield syntheses. The compounds were structurally identified by physical methods. All these steroids are characterized by a marked antiglucocorticoid activity that proved long-acting in the case of the ester derivatives. 2-Carboxy-11 beta, 17 beta-dihydroxy-17-methyl-1, 4-androstadien-3-one or roxibolone, and its n-decylester or decylroxibolone, are the most promising derivatives in consideration of their pharmacological properties.
Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione, which are intermediates of microbial conversion of cholesterol, by cholesterol-degrading bacteria (12 strains of the genus Rhodococcus isolated from food of animal origin and 12 culture collection strains) were examined. All strains had the ability to degrade 4-cholesten-3-one without necessarily being able to degrade cholesterol. On the other hand, the bacteria were divided into three groups with little or no (0-10%), intermediate (10-70%) and high (70-100%) degradation abilities for 1,4-androstadiene-3,17-dione.
The 5,7-dienes, 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography-mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-diene pathway biosynthesizing 3 beta-hydroxy-5,7-androstadien-17-one, which is the proposed precursor for equilin in the placenta.
The conversion of pregnenolone to 5,16,androstadien-3 beta-ol, the first intermediate in the biosynthetic pathway of the androst-16-ene steroids, is catalysed by a microsomal enzyme system in the testes of the pig. This reaction is analogous to the conversion of pregnenolone to dehydroepiandrosterone in the biosynthesis of the androgens, since both systems involve the conversion of C21 steroids to C19 steroids by removal of the 2-carbon side chain. Cytochrome P-450SCCII catalyses the formation of the first C19 androgen intermediates, while the enzyme system that catalyzes the formation of the first androst-16-ene intermediates, so called andien-beta synthase, has not previously been well characterized. Andien-beta synthase and cytochrome P-450SCCII activities have been measured in an in vitro assay system with boar testes microsomes using [14C]pregnenolone as substrate. Both enzyme systems require NADPH and oxygen for maximal activity and are inhibited by carbon monoxide when oxygen levels are low. Classical inhibitors of cytochrome P-450 including SKF-525A, metyrapone and alpha-naphthoflavone inhibited both enzyme systems to a similar extent. In addition, inhibitory antibodies against NADPH cytochrome P-450 reductase also inhibited both enzyme activities in testes microsomes. It is concluded that the formation of 5,16,androstadien-3 beta-ol from pregnenolone in pig testes microsomes is catalyzed by cytochrome P-450.
In previous reports we described the early time sequence in in vitro [4-14C] pregnenolone metabolism in human and rat testicular homogenates and, apart from a difference in the preferred route of the conversion of pregnenolone to testosterone, we demonstrated the presence of delta 16-synthetase activity in human but not in rat testes. In the study of testicular function higher monkeys are increasingly used as a model for human reproduction. The availability of testes from 2 different species of macaques (rhesus and crab eating monkeys) enabled us to compare the in vitro metabolism of pregnenolone in these testes with human testes. The pattern obtained in both monkey species were very similar, but completely different from those found in man. The delta 4 pathway was the preferred route for the conversion of pregnenolone to testosterone in the monkeys tested, the delta 5 pathway in the humans. delta 16-Synthetase activity, a prerequisite for the synthesis of the sex pheromone precursors 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one, was clearly measurable in the human but not in the monkey testicular homogenates. So far, man and boar are the only species harbouring delta 16-synthetase activity in their testes. These in vitro data indicate that the nonhuman primates studied are not suitable models for the study of human testicular function.
Mass spectrometric identification and characterization of steroids using electrospray ionization and tandem mass spectrometry has advantages in drug testing and doping control analysis attributable to limitations of gas chromatography followed by electron ionization mass spectrometry. Steroids with an androstadiene-17beta-ol-3-one nucleus and double bonds located either at C-1 and C-4, C-4 and C-9, or C-4 and C-6 were used to determine characteristic fragmentation pathways. Diagnostic dissociation routes are proposed using deuterium labeling, MS3 experiments, and analyses of structurally closely related compounds. Steroids such as boldenone (androst-1,4-diene-17beta-ol-3-one) produced characteristic product ions at m/z 121, 135, and 147. Compounds with double bonds at C-4 and C-9 generated abundant product ions at m/z 145 and 147. Conjugated double bonds at C-4 and C-6 gave rise to an intense and characteristic signal at m/z 133. Stereochemical differentiation between 5alpha- and 5beta-isomers of androstan-17beta-ol-3-ones was possible because of significant differences in relative abundance of product ions generated by elimination of acetone from alpha,beta-saturated 3-keto steroids.
Research on human putative pheromones has recently focused on the effects of exposure to 4,16-androstadien-3-one (androstadienone). This steroid has been observed in the skin, axillary hair, and blood plasma, primarily in males. In addition to effects of the steroid on measures of physiological arousal and brain blood flow, positive mood effects have also been reported. The current study further investigated mood effects of androstadienone exposure (250 microM) in women in two experiments. Through psychophysical testing of each individual we controlled for whether any observed mood effects could be related to sensory detection of the steroid. In both experiments, we observed positive changes of women's feeling of being focused, which could not be related to sensory detection of the steroid. Overall, the patterns of results were significantly correlated between the two experiments. In conclusion, this study corroborates earlier findings suggesting that androstadienone exposure yields effects on women's mood; the feeling of being focused. The mood effects were not dependent on menstrual cycle phase. Further, these effects are replicable and occur also when androstadienone detection is rigorously controlled for across variation in menstrual cycle.
The enzyme 3-oxo-steroid: NADP+ 4-oxidoreductase (EC 1.3.1.22; 5alpha-reductase) was assayed in testicular microsomes of pigs of 3, 20 and 24 weeks of age. The activity was very low in 3-week-old animals and approximately 10-fold higher in 5- and 6-month-old pigs. The pH optimum was 6.3 in 6-month-old animals, 5.7 in 5-month-old animals, but could not be reliably determined in 3-week-old animals. The kinetic parameters for 5alpha-reductase in testis microsomes from 6-month-old animals were; K((m)(app)), 8.0 micromol/l, V((max)(app)), 6.7 nmoles/90 min/mg protein. Progesterone was a competitive inhibitor of testosterone 5alpha-reduction with an apparent K((i)(app)) of 0.86 micromol/l. However, 4,16-androstadien-3-one (dienone), which undergoes 5alpha-reduction in the biosynthesis of the pheromonally active 16-androstenes, was a comparatively poor inhibitor with a K((i)(app)) of 4.9 micromol/l. Similarly, MK434, which is a selective inhibitor of the human type 2 5alpha-reductase, but which inhibits both types 1 and 2 in the rat, was also a poor competitive inhibitor of testosterone 5alpha-reductase in the pig testis (K((i)(app)), 3.1 micromol/l). It would appear from these studies that the pig testis microsomal 5alpha-reductase corresponds to a type 1 isozyme that is not capable of reducing dienone other than under conditions where the dienone concentration would be in considerable excess of testosterone. It is, therefore, probable that substrate-specific 5alpha-reductases exist in the pig testis for the 5alpha-reduction of testosterone and dienone.
The metabolism of 17 alpha-methyl-17 beta-hydroxy-1,4-androstadien-3-one (dianabol) in human adults has been studied in detail by computer aided capillary gas chromatography mass spectrometry. After oral administration to man six metabolites were determined in the free fraction of the urine samples, the structures of which have been identified as 17-epidianabol, three isomers of 6-hydroxydianabol, 17 alpha-methyl-17 beta-hydroxy-1,4,6-androstatrien-3-one (delta 6-dianabol) and 18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one, respectively. In agreement with previous observations no measurable amounts of the administered drug itself could be detected in any of the urine samples investigated. The mass spectra of all metabolites and the main fragmentation processes are discussed in detail.
In addition to the histology of epithelial and stromal elements of prostates from intact dogs (group 0) and castrated dogs (group I), the latter of which were treated with androstenedione (group II), androstenedione plus the aromatase inhibitor 1-methyl-1,4-androstadiene-3,17-dione (group III), or androstenedione plus aromatase inhibitor and cyproterone acetate (group IV) (Habenicht and El Etreby: The Prostate 11:133-143, 1987) it was of interest to study the influence of such in vivo treatment on the prostatic 5 alpha-reductase, which is responsible for the cellular conversion of testosterone to 5 alpha-dihydrotestosterone. Michaelis constants (KM) and maximal activities (Vmax) of 5 alpha-reductase were determined under optimized incubation conditions in mechanically separated epithelium and stroma. The metabolites were separated by high-performance liquid chromatography and determined radiometrically. The main results were: 1) The mean KM (nM +/- SEM) was significantly (P less than .001) higher in epithelium (892 +/- 132) than stroma (70 +/- 11). The same was true concerning the Vmax (pmol.mg protein-1.h-1 +/- SEM) in epithelium (54.6 +/- 5.8) as compared to stroma (13.0 +/- 2.0). 2) No specific in vivo or in vitro effect of the aromatase inhibitor on the KM and Vmax data was found. 3) In prostates of intact dogs and dogs of group II the proportion of epithelial 5 alpha-reductase exceeded distinctly that of stromal 5 alpha-reductase. 4) In groups I, III, and IV the proportion of epithelial 5 alpha-reductase was rather low. These data were discussed in the light of the histological findings.