Stereospecific removal of the 16 alpha-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol 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.     

Synthesis of specifically deuterium-labelled pregnanolone and pregnanediol sulphates for metabolic studies in humans.

A synthesis is reported of 3beta-hydroxy-5alpha-pregnan-20-one sulphate and the disulphate and 3-monosulphate of 5alpha-pregnane-3beta,20alpha-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3beta-hydroxy-5alpha-25R-spirostan-12-one (hemcogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 3beta-hydroxy-5alpha-[11,11-2H2]pregn-16-en-20-one (VII). Further deuterium atoms were introduced at the 3alpha and 20beta positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3beta-hydroxy-5alpha-[3alpha,11,11-2H3]pregnan-20-one (X; isotopic purity 87.2%) and 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly form the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol 20-acetate (XVII).     

Pregnane glycosides from fruits of Balanites aegyptiaca

From the mesocarp of Balanites aegyptiaca fruits, two pregnane glycosides were isolated. One is new and identified as pregn-5-ene-3β,16β,20(R)-triol 3-O-(2,6-di-O--l-rhamnopyranosyl)-β-d-glucopyranoside (balagyptin), while the other is known and assigned as pregn-5-ene-3β,16β,20(R)-triol 3-O-β-d-glucopyranoside.     

Pregnanes and pregnane glycosides from Marsdenia roylei

Two pregnanes namely desacylkondurangogenin C (1) and deniagenin (3, new) and two new pregnane glycosides designated as denin (5) and marsin (12) have been isolated from chloroform soluble extract of dried stem of Marsdenia roylei. Chemical and spectroscopic evidences are consistent with the structures of deniagenin, denin and marsin as 3beta, 11alpha, l2beta, 14beta, 17beta, 20-hexahydroxy pregn-5-ene; desacylkondurangogenin C-3-O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-L-fucopyranoside and ketocalogenin-3-O-alpha-L-fucopyranoside, respectively.     

Microbial transformation of 3β-acetoxypregna-5,16-diene-20-one by Penicillium citrinum

The biotransformation of 3β-acetoxypregna-5,16-diene-20-one (1) by using a filamentous fungus Penicillium citrinum resulted in the production of four metabolites 2–5. The structures of these compounds were elucidated by different spectroscopic analysis (1D- and 2D-NMR) and HR-ESI-MS as 3β,7β-dihydroxy-pregn-5,16(17)-dien-20-one (2), 3β-hydroxy-7α-methoxy-pregn-5,16(17)-dien-20-one (3), 3β,7β,11α-trihydroxy-pregn-5,16(17)-dien-20-one (4), and a known 3β,7α-dihydroxy-pregn-5,16(17)-dien-20-one (5). The 7-O-methylation is a novel reaction in the field of microbial transformation of pregnane steroids.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Utilization and metabolic effects of acetaldehyde and ethanol in the perfused rat liver

1. The formation of the two 16-unsaturated alcohols 5alpha-androst-16-en-3alpha-ol and 5alpha-androst-16-en-3beta-ol from [5alpha-(3)H]5alpha-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3alpha-alcohol was obtained in the presence of NADPH, whereas that for the 3beta-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at -20 degrees C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-(14)C]progesterone. 5. The production of 5alpha-androst-16-en-3-one and the two alcohols from [7alpha-(3)H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5alpha-(3)H]5alpha-androst-16-en-3-one was unaffected by storage of testis at -20 degrees C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3beta-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5alpha-androst-16-en-3alpha- and 3beta-ols, with the C(19) 3-oxo steroids as intermediates, are presented.     

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.     

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.     

New pregnane steroids from Turraea pubescens

Three new pregnane steroids, 2beta,3beta,5beta-trihydroxy-pregn-20-en-6-one (1), 3beta-hydroxy-5alpha-pregn-7,20-dien-6-one (2), and 3beta-acetoxy-5alpha-pregn-7,20-dien-6-one (3) were isolated from the twigs and leaves of Turraea pubescens, and were structurally elucidated on the basis of spectroscopic data and chemical method.     

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.     

New pregnane glycosides from Stelmatocrypton khasianum

Four new pregnane glycosides, stelmatocryptonoside A, B, C, and D (1-4), were isolated from the stems of Stelmatocrypton khasianum. On the basis of chemical and spectral data, the structures of 1-4 were established as 3beta, 16alpha-dihydroxy-pregn-5-en-20-one-16-O-beta-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside; 3beta, 20-dihydroxy-pregn-5-en-20-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->2)-beta-D-digitalopyranoside; 3beta, 16alpha-dihydroxy-pregn-5-en-20-one-16-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside; and 3beta, 16alpha-dihydroxy-pregn-5-en-20-one-16-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. This is the first report of pregnane glycosides with sugar chains linked at C-16 of the aglycone.     

Gas chromatographic-mass spectrometric study of metabolites of C21 and C19 steroids in neonatal porcine testicular microsomes.

Microsomal fractions obtained from testes of 3-week-old piglets have been incubated, separately, with progesterone, 17-hydroxyprogesterone, 5-pregnene-3 beta,20 beta-diol, 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and dehydro-epiandrosterone. The metabolites, after derivatization, have been separated by capillary gas chromatography and identified by mass spectrometry. Quantification was by selected ion monitoring. Progesterone was shown to be 17-hydroxylated and also converted into 4,16-androstadien-3-one (androstadienone). The major metabolite of 17-hydroxyprogesterone was 4-androstene-3,17-dione (4-androstenedione), but little, if any, androstadienone was formed, indicating that this particular biosynthesis did not require 17-hydroxylation. The metabolites of 5-pregnene-3 beta, 20 beta-diol were found to be 17-hydroxypregnenolone, 3 beta-hydroxy-5,16-pregnadien-20-one (16-dehydropregnenolone) and 5,16-androstadien-3 beta-ol. Dehydroepiandrosterone and 5-androstene-3 beta,17 alpha-diol were interconvertible but neither steroid acted as a substrate for 16-androstene formation. However, dehydroepiandrosterone was metabolized to a small quantity of 4-androstenedione. Under the conditions used, no metabolites of 16 alpha-hydroxypregnenolone could be detected. The present results, together with those obtained earlier, indicate that the neonatal porcine testis has the capacity to synthesize weak androgens, mainly by the 4-en-3-oxo steroid pathway. Although 16-androstenes cannot be formed from C19 steroids, progesterone served as a substrate and may be converted directly to androstadienone, without being 17-hydroxylated first. The pathway to 5,16-androstadien-3 beta-ol, however, involves 17-hydroxypregnenolone and 16-dehydropregnenolone as intermediates.     

Microbial transformation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one by Trichoderma viride

Fermentation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one (4) with Trichoderma viride under aerobic condition yielded 3beta,5alpha,6beta-trihydroxy-16-pregnen-20-one (5) and 3beta,5alpha,6beta,15beta-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by spectroscopic methods. Compounds 6 and 3beta,5alpha,15beta-trihydroxy-16-pregnen-6,20-dione (7) are reported for the first time.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

Regioselective reactions of 1,2-dehydroprogesterone: syntheses of pregnane derivatives as possible contragestational agents

A few pregnane derivatives were synthesized from 1,2-dehydroprogesterone (1). Ring A of 1,2-dehydroprogesterone was aromatized without affecting C-20, and the resulting acetoxy compound (2) after hydrolysis yielded 1-hydroxy-4-methyl-19-norpregna-1,3,5(10)-trien-20-one (3). Reactions of the phenol (3) with alkyl halides yielded the ethers 6a-6b and 7. Opening of the oxirane ring in 7 with secondary amines furnished the aminoalcohols 8a-8b. Friedelcraft's reaction of 3 with maleic anhydride and chloracetyl chloride led to the formation of 9 and 10, respectively. Base-catalyzed ring closure of 10 yielded 1-acetyl-12a-methyl-8-oxo-5[H]-1,2,3,3a,3b,4,8,9,10b,11,12, 12a-dodecahydrocyclopenta (7,8)-phenanthro (3,4-b) furan (11), which reacted with aromatic aldehydes regioselectively to furnish 12a-12b. Reaction of 1 with triethylorthoformate in the presence of boron trifluoride etherate involved the participation of C-21, and the carbonyl at C-3 remained unaffected. The product 13 was identified as 21-[2-hydroxyvinyl]-21-norpregna-1,4-diene-3,20-dione. Reductive amination with sodium cyanoborohydride in the presence of ammonium acetate did not attack ring A and smoothly furnished the amine 14 which, on reaction with succinic anhydride, gave 20-succinamylpregna-1,4-dien-3-one (15).     

Digitaloid pregnanes promote potassium-sparing diuresis in the guinea pig.

The synthesis of 17 alpha-acetoxy-3 beta-[(beta-D-glucopyranosyl)oxy]- 6 alpha-methylpregn-4-en-20-one, the glucoside of medroxyprogesterone acetate (MPA-glu), is described. MPA-glu and 14-amino-20 beta-hydroxy-3 beta-[(alpha-L-rhamnopyranosyl)oxy]-5 beta, 14 beta-pregnane (LND 623), pregnane glycosides that bind to the digitalis receptor, and digoxin, a cardiac glycoside, were infused intravenously into the anesthetized guinea pig. Each of the three steroids significantly enhanced urinary volume and sodium excretion without affecting blood pressure and creatinine clearance. Potassium excretion was markedly enhanced by digoxin but unaffected by MPA-glu or LND 623. These observations conform to previous work that demonstrated, in the rat, potassium-sparing diuresis by the glucoside of 14 beta-hydroxyprogesterone, a cardiotonic pregnane. There is a dissociation between potency to inhibit [3H]ouabain binding and the extra ATPase actions of the digitaloid pregnanes.     

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.     

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.     

Configurational analysis and relative binding affinities of 16-methyl-5alpha-androstane derivatives

The four possible isomers 16beta-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 1, 16alpha-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 2, 16beta-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 3 and 16alpha-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 4 with proven configuration were converted into the corresponding 16beta-methyl-5alpha-androstane-3beta,17beta-diol 5, 16alpha-methyl-5alpha-androstane-3beta,17beta-diol 6, 16beta-methyl-5alpha-androstane-3beta,17alpha-diol 7, 16alpha-methyl-5alpha-androstane-3beta,17alpha-diol 8, furthermore into the 16beta-methyl-17beta-hydroxy-5alpha-androstane-3-one 13, 16alpha-methyl-17beta-hydroxy-5alpha-androstan-3-one 14, 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3-one 15 and 16alpha-methyl-17alpha-hydroxy-5alpha-androstan-3-one 16. The steric structures of the resulting epimers were determined by means of 1H-, and 13C-NMR spectroscopy. In this way, comparison was possible with the C-16 epimers 5, 6 and 13, 14 prepared earlier by a different route, and the series of isomers could be completed with the steric structures of 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3beta-ol 7 and 16alpha-methyl-17alpha-hydroxy-5alpha 8 and with their 3-keto derivatives 15 and 16. The relative binding affinities of the 16-methyl-5alpha-androstane-3beta,17-diols 5, 6, 7, 8 and 17-hydroxy-16-methyl-5alpha-androstan-3-ones 13, 14, 15, 16 were studied. The introduction of a 16-methyl substituent into 5alpha-androstane molecules substantially decreases the binding affinity to the androgen receptor and 16alpha-methyl derivatives were always bound more weakly than the 16beta-methyl isomers.