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.     

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.     

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.     

Potential limitations of recrystallization for the definitive identification of radioactive steroids

The usefulness of recrystallization in establishing the radiochemical purity of steroids is widely recognized, but the potential limitations of the technique have received little attention. The current study reports the failure of standard recrystallization procedures using methanol/water as the solvent pair to separate contaminating ¹⁴C-17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) from ³H- and ¹⁴C-labeled 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) despite ten serial crystallizations. The standard criteria of radiochemical purity were met despite gross impurity of the crystals as evidenced by thin layer chromatography. Thus, recrystallization may, under certain conditions, yield misleading results when employed as the only method for identifying radioactive steroids. These observations illustrate the importance of an optimal choice of solvent and crystallization conditions, and emphasize the need for confirmation by derivative formation and chromatography.     

Progesterone biotransformation by plant cell suspension cultures.

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid.     

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.     

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.     

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.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

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.     

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.     

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%     

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.     

Chick oviduct progesterone receptor binding of 15 beta, 17-dihydroxyprogesterone and its analogues

The structure of 16 alpha,17-epoxy-4-pregnene-3,20-dione was determined. The 20-carbonyl group eclipses the C(13)-C(17) bond. No direct correlation between the observed structure and its progestational activity could be inferred from our investigation.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.     

Determination of 11-deoxycortisol (Reichstein's compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection

A first assay based on stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) has been developed for plasma 11-deoxycortisol (Reichstein's compound S), the leading hormonal marker of 11beta-hydroxylase deficiency. A suitable internal standard being unavailable, we synthesized dideuterated 11-deoxycortisol according to a newly devised synthetic procedure. 17,21-Dihydroxy-pregna-1,4-diene-3,20-dione underwent selective deuteration using Wilkinson's catalyst. Our product [1alpha,2alpha-2H2]11-deoxycortisol was obtained in good yield (35.6%) and high isotopic purity (0.1% 2H0, 99.9% 2H2). Structural confirmation was done by MS and NMR. Our plasma work up consisted of equilibration of plasma with internal standard ([1alpha,2alpha-2H2]11-deoxycortisol), solid phase extraction with Extrelut NT columns, a clean up step using Sephadex LH-20 mini columns and preparation of heptafluorobutyrates as derivatives. Quantification was achieved by selected ion monitoring of m/z 465.40 (analyte) and m/z 467.40 (internal standard). One hundred twenty picograms of 11-deoxycortisol gave a signal to noise ratio of 10. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors <3.0%. Intraassay precision CV was 4.78% and interassay precision CV was 4.56%. We succeeded in integrating our new analyte into our already existing multisteroid ID/GC-MS plasma assay, which now, in its expanded version, is capable of determining all major diagnostic steroids of androgen related disorders in a single profile: 11-deoxycortisol, 17alpha-hydroxyprogesterone, testosterone, 4-androstenedione, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, androstanediol and 5alpha-dihydrotestosterone. The diagnostic potential of our multisteroid ID/GC-MS assay, the small amounts of plasma (0.5 ml) required, the rapid and convenient sample work up, the application of benchtop GC-MS instrumentation, and highest specificity offered by mass spectrometric detection prove our assay suitable for routine clinical use, especially in pediatric endocrinology.     

Uptake and metabolism of cortisone and cortisol by the fetal rabbit lung

Lungs of rabbit fetuses at 28 days of gestation were incubated with tritium-labeled cortisone (17α,21-dihydroxy-4-pregnene-3,11,20-trione) or Cortisol (11β,17α,21-trihydroxy-4-pregnene-3,20-dione). The fetal lungs metabolized efficiently cortisone yielding cortisol as the major product (64–71% conversion). Cortisol was poorly metabolized, only 10–14% being converted to cortisone and 68–75% of the substrate being recovered unchanged. A small amount of cortisone (5–7% of tissue radioactivity) was also found in the lungs twenty minutes after injection of labeled cortisol to the fetus $\text{in utero}$. Incubation of fetal lungs with labeled cortisone at 37° resulted in specific uptake and binding of radioactivity (predominantly cortisol) to nuclear macromolecules. The amount of cortisol bound to nuclear macromolecules was similar whether the tissue was incubated with cortisol or cortisone. These results demonstrate that the lungs of the rabbit fetus have the capacity to convert the biologically inactive cortisone to the biologically active cortisol, the reverse reaction occurring only to a limited extent.     

Steroid metabolism in primates. XX. Excretion of C21-steroids in urine of women and baboons (Papio hamadryas) during pregnancy]

Urinary excretion of 10 C21 steroids was investigated in women and baboons (Papio hamadryas) in various stages of pregnancy, in comparison to controls. In pregnant women, excretion of total C21 steroids is slightly increased, whereas in pregnant baboons it is slightly decreased. In women, excretion of tetrahydrocortisol and tetrahydrocortisone is diminished, that of 20 beta-OH-F (11-beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3,20-dione) and of 11-deoxycortisol is increased. In pregnant baboons no significant alterations in corticosteroid metabolism were established.     

Various 18-substituted progesterones

In order to test the potential biological activity of 18-substituted progesterones, 3,20-dioxo-4-pregene-18-carbonitrile (ld approximately) was converted to 3,20-dioxo-4-pregnene-18-carboxylic acid (lb approximately) and methyl 3,20-dioxo-4-pregnene-18-carboxylate (ld approximately) via a sequence of reactions involving an intramolecular hydrolysis of the 18--arbonitrile. Lithium aluminum hydride reduction of the bisethylene ketal derived from la approximately furnished 18-aminomethyl-5-pregnene-3,20-dione 3,20-bisethylene ketal (8 approximately). Acetylation and hydrolysis furnished 18-acetamidomethyl-4-pregnene-3,20-dione (lf approximately) and simple hydrolysis of 8 approximately furnished 3'alpha H-5' 6'-dihydro-2',19 beta-dimethyl-3-oxo-4-goneno [13,17-c]pyridine (9 approximately). None of the compounds exhibited any activity in Clauberg or anti-Clauberg tests.     

New Aromatic Esters of Progesterone as Antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17α-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17α-(p-chlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17α-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17α-(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 5α-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114?μM (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.