Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Progesterone fate in rabbit cornea

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one by fetal rhesus monkey testes.

Homogenates prepared from fetal rhesus monkey testes were incubated with progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one. The major progesterone metabolite was 17-hydroxy-4-pregnene-3,20-dione. Testosterone also accumulated in the progesterone incubations. 4-Androstene-3,17-dione was converted chiefly to testosterone. Testosterone was not actively metabolized by the fetal monkey testis. 17 beta-Hydroxy-5 alpha-androstan-3-one was actively converted primarily to 5 alpha-androstane-3 beta,17 beta-diol.     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.     

Modulation of the GABAA receptor by progesterone metabolites

The naturally occurring progesterone metabolites 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione reversibly enhance membrane currents elicited by locally applied GABA in bovine adrenomedullary chromaffin cells. Such potentiation was not influenced by the benzodiazepine antagonist Ro 15-1788. At concentrations in excess of those necessary to evoke potentiation of GABA currents, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregane-3,20-dione directly activated a membrane conductance. The resulting currents were potentiated by phenobarbitone and diazepam, and abolished by the GABAA-receptor antagonist, bicuculline. On outside-out membrane patches, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione activated single channel currents of similar amplitude to those evoked by GABA. The results suggest that certain naturally occurring steroids potentiate the actions of GABA and, additionally, directly activate the GABAA receptor.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Steroid transformations by species of Cephalosporium and other fungi

A total of 58 cultures, tentatively identified as species of the genus Cephalosporium, were screened in flask fermentations for their ability to effect conversions of progesterone (Delta(4)-pregnene-3,20-dione) and Reichstein's Substance S (Delta(4)-pregnene-17alpha,21-diol-3,20-dione). A large number of transformations were observed by means of a series of five paper chromatography systems rated for analysis of steroid compounds ranging in polarity from progesterone to polyhydroxylated steroids. Five different transformation products were selected for isolation and identification. For purposes of recovery, conversions were conducted under submerged conditions in either 4- or 200-liter fermentors in which the broth was agitated and aerated. The steroid substrate was dissolved in acetone and added aseptically to the growing culture in a final concentration of 0.025%. After the conversions were effected, the whole broth was extracted with chloroform, and the transformation products were recovered, either by direct crystallization from solvents or through the use of silica gel columns. It was determined that C. ciferrii 21C converted progesterone to Delta(4)-androstene-3,17-dione. Kendall's Compound F (Delta(4)-pregnene-11beta,17alpha,21-triol-3,20-dione) was converted to its 20beta-ol analogue by Geotrichum sp. 51C (during these studies, a number of cultures were taxonomically reclassified). Cephalosporium sp. 27C formed the Delta(1)-analogue of Reichstein's Substance S, and Cephalosporium sclerotigenum 31C and Verticillium aphidum both converted Substance S to the 6beta-hydroxy derivative. Paecilomyces persicinus 22C converted Substance S to a product believed to be a dihydroxylated derivative.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

Steroidogenic correlates of pregnancy in the rock hyrax (Procavia capensis)

In pregnant rock hyraxes isolated leucocytes metabolise both [3H]pregnenolone and [3H]progesterone while whole blood, erythrocytes and an erythrocyte/leucocyte mixture only metabolised [3H]progesterone. Plasma displayed no tendency to metabolically convert any one of these two steroids. In whole blood [3H]progesterone appears to be converted to 5alpha-pregnane-3,20-dione and a compound with chromatographic properties similar to that of 5alpha-pregnan-3alpha-ol-20-one. 5Alpha-pregnane-3,20-dione exhibited a high relative binding affinity for the uterine progesterone eceptor (94%), but 5alpha-pregnan-3alpha-ol-20-one displayed very little affinity for the same receptor (0.4%). 5Alpha-pregnane-3,20-dione may therefore aid in the maintenance of pregnancy. Corpora lutea metabolised progesterone to 17alpha-hydroxyprogesterone, a compound exhibiting no progestational function because of its low relative binding affinity for the uterine progesterone receptor (2%). Progesterone appears to be the main product of the corpus luteum. However, 5alpha-pregnane-3,20-dione circulated at concentrations approximately 8.5 times higher than progesterone, probably due to the metabolic conversion of progesterone to 5alpha-pregnane-3,20-dione by the blood. We conclude that in the hyrax progesterone, produced by the corpora lutea, enters the circulation, where it is reduced to 5alpha-pregnanes. 5Alpha-pregane-3,20-dione may then be transported to the uterus where it binds to the progesterone receptor to assist in the maintenance of pregnancy. This mechanism appears to be analogous to that of the African elephant which is phylogenetically related to the hyrax, except that in the elephant the 5alpha-reduced metabolites are produced by luteal tissue and not the blood.     

Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Recently several allylic steroids have been found in gonadal and breast tissues. In order to establish their presence and identity in tissues and determine the possible biological properties, a method for the synthesis of 4-pregnene-3 alpha,20 alpha-diol, 4-pregnene-3 alpha, 20 beta-diol, 4-pregnene-3 beta,20 alpha-diol, and 4-pregnene-3 beta,20 beta-diol was developed using 4-pregnene-3,20-dione (progesterone) as substrate and freshly-prepared aluminum isopropoxide in isopropyl alcohol as reducing agent. The yields were about 19%, 30%, 13%, and 38% for the 3 alpha,20 alpha-, 3 alpha,20 beta-, 3 beta,20 alpha-, and 3 beta,20 beta-diols, respectively. The structures and stereochemistry of these diols were established using proton and carbon NMR spectroscopy and infrared and mass spectrometry.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.     

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-α-cyclodextrin complex

Newborn rat adrenocortical cells were successfully cultured in a serum free carrier protein free medium (SPFM) by using -cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol--cyclodextrin with a molar ratio of almost 1 was obtained for a 5 × 10^–5 mol/1 -cyclodextrin concentration. Cell cultures incubated with [4-¹⁴C] cholesterol--cyclodextrin in SPFM produced, under ACTH stimulation, various ¹⁴C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone. As measured by gas chromatography and mass spectrometry, the ratio between corticosteroids (21-hydroxylated steroids) and 20-reduced steroids produced in SPFM with cholesterol--cyclodextrin was equal to 1.8. This corresponds to a value of 3.6 times higher than that found in the serum free medium with cholesterol-albumin. Consequently, the chemically defined SPFM with cholesterol--cyclodextrin used in this study is more suitable for corticosteroidogenesis by adrenal cells in culture than a serum free medium with cholesterol-albumin.Abbreviations -CD -cyclodextrin - ACTH adrenocorticotropic hormone - 20-dihydroprogesterone 20-hydroxy-4-pregnene-3-one - 11-hydroxy-20-dihydroprogesterone 11, 20-dihydroxy-4-pregnene-3-one - 11-hydroxyprogesterone 11-hydroxy-4-pregnene-3,20-dione - C--CD cholesterol--cyclodextrin complex - corticosterone 11,21-dihydroxy-4-pregnene-3,20-dione - deoxycorticosterone 21-hydroxy-4-pregnene-3,20-dione - 18-hydroxy-11-deoxycorticosterone 18,21-dihydroxy-4-pregnene-3,20-dione - 18-hydroxy-20-dihydroprogesterone 18-20-dihydroxy-4-pregnene-3-one - 18-hydroxyprogesterone 18-hydroxy4-pregnene-3,20-dione - progesterone 4-pregnene-3,20-dione - SFM-S serum-free medium - SPFM serum-free protein-free medium - SSM serum supplemented medium     

Stereospecific 1,4-addition to an alpha,beta-unsaturated steroidal epoxide: syntheses of new 15-oxygenated sterols

3 beta-Benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (1) is a key intermediate in the synthesis of C-7 and C-15 oxygenated sterols. Treatment of 1 with benzoyl chloride resulted in the formation of 3 beta,15 alpha-bis-benzoyloxy-7 alpha-chloro-5 alpha-cholest-8(14)-ene (2). Reaction of 2 with LiAlH4 or LiAlD4 resulted in the formation of 5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3a) or [14 alpha-2H]5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3b). Diol 3b was selectively oxidized by Ag2CO3/celite to [14 alpha-2H]5 alpha-cholest-7-en-15 alpha-ol-3-one (4). Treatment of 1 with MeMgI/CuI gave 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 alpha-diol (5). Selective oxidation of 5 with pyridinium chlorochromate (PCC)/pyridine or oxidation with PCC resulted in the formation of 7 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one (6) and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3,15-dione, respectively. Reduction of 6 with LiAlH4 yielded 5 and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 beta-diol (6). Reaction of 1 with benzoic acid/pyridine gave 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-en-15 alpha-ol (9). Treatment of 9 with LiAlH4 or ethanolic KOH resulted in the formation of 5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol (10). Dibenzoate 9, upon brief treatment with mineral acid, gave 3 beta-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (11). Oxidation of 9 with PCC yielded 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (12). Ketone 12 was also prepared by the selective hydride reduction of 5 alpha-cholest-8(14)-en-7 alpha-ol-3,15-dione (13) to give 5 alpha-cholest-8(14)-ene-3 beta,7 alpha-diol-15-one (14), which was then treated with benzoyl chloride to produce 12.     

Effect of 5 alpha-dihydrocorticoids on enzymes of gluconeogenesis in rat liver

5 alpha-Dihydrocortisol (11 beta, 17, 21-trihydroxy-5 alpha-pregnane-3,20-dione), 5 alpha-dihydrocorticosterone (11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione) as well as cortisol (11 beta, 17, 21-trihydroxy-4-pregnene-3,20-dione) and corticosterone (11 beta, 21-dihydroxy-4-pregnene-3,20-dione) were administered for seven days to male rats. Blood glucose increased in cortisol- and corticosterone-treated rats and blood insulin decreased after 5 alpha-dihydrocorticosteroid treatment. In the liver, total protein was elevated after cortisol, corticosterone and 5 alpha-dihydrocorticosterone application. Phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in liver were significantly lowered after treatment with 5 alpha-dihydrocortisol and 5 alpha-dihydrocorticosterone.     

Synthesis of 21-amino-5-pregnene-3,20-dione bysethylene ketal

21-Amino-5-pregnene-3,20-dione bisethylene ketal was obtained in good yield by lithium aluminum hydride reduction of 21-azido-5-pregnene-3,20-dione bisethylene ketal. The azido bisethylene ketal was synthesized by the sequence: deoxycorticosterone → deoxycorticosterone 21-p-toluene-sulfonate → 21-azidoprogesterone → 21-azido-5-pregnene-3, 20-dione bisethylene ketal. The structure of the title compound was confirmed by its conversion to the known 21-acetylaminoprogesterone. 21-Amino-5-pregnene-3,20-dione bisethylene ketal is a stable aminosteroid which is a useful intermediate for the synthesis of C-21 nitrogen derivatives of progesterone.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.     

New aromatic esters of progesterone as antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17alpha-(pchlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1-4 have a weak inhibitory activity on 5alpha-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 microM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds. The binding efficiency of the synthesized steroids 1-4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1-4.