Synthesis of 3α,7α-dihydroxy-5β-androstan-17-one

A short and efficient method for the stereospecific synthesis of 3α,7α-dihydroxy-5β-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

Synthesis and pyrogenic effect of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione

The first chemical synthesis of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione is reported. In this method, the 17β-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups : 3α-OH by a $\text{tert}$-butyldimetfaylsilyl group and 7α-OH by an acetoxy group. The capacity of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods : supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7α-hydroxyl substituted one did not.     

Synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxyandrost-5-en-17-one via microbial 7α-hydroxylation

The synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7α-hydroxylation has been accomplished. At the first stage, 3β,7α-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7α-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC_,¹H NMR, MS, UV- and IR-spectrometry.The combination of microbial and chemical steps ensured 54.6% yield of the target 3β-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.     

C-19-steroid 7α-hydroxylation by rat testes. isolation and identification of: 7α, 17β-dihydroxy-5α-androstan-3-one, 5α-androstan-3α,7α,17β-triol and 5α-androstane-3β,7α, 17β-triol

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7α-hydroxylated metabolites could be isolated and identified: 7α,17β-dihydroxy-4-androsten-3-one (7α-hydroxy-testosterone), 7α-17β-dihydroxy-5α-androstan-3-one (7α-hydroxy-Dht), 5α-androstan-3α,7α,17β-triol (7α-hydroxy-3α-A'DIOL) and 5α-androstane-3β,7α,l7β-triol (7α-hydroxy-3β-A'DIOL). To our knowledge this is the first demonstration of the formation of 5α-reduced-7α-hydroxylated metabolites of testosterone in the male gonad. These 5α-reduced-7α-hydroxylated metabolites could also be isolated after incubations of 5α-androstane-3α,17β-diol (3α-A'D10L) with testicular homogenates in the presence of a NADPH-generating system.Measured as the sum of 7α-hydroxy-testosterone, 7α-hydroxy-Dht. 7α-hydroxy-3α-A'DIOL and 7α-hydroxy-3β-A'DIOL formed using testosterone as substrate, total 7α-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3α-A'DIOL as substrate total 7α-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.     

Metabolism of 17β-hydroxy-2α-methyl-5α-androstan-3-one in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α-methyl-5α-androstan-3-one from the rabbit dosed orally were investigated. Together with oxidation-reduction of the oxygen functions at C-3 and C-17 hydroxylation occurred at C-15, C-16, and at the 2α-methyl positions of the steroid nucleus.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

Conversion of 3β,15β,16β-trihydroxy-5-androsten-17-one to 1,3,5(10)-oestratriene-3,15β,16β,17β-tetrol by placental homogenates

Incubation of 3β, 15β, 16β-trihydroxy-5-androsten-17-one with a homogenate of placental tissue resulted in the formation of an oestetrol (l, 3, 5(10)-oestratriene-3, 15β,16β,17β-tetrol), the identity of which was confirmed by gas chromatography-mass spectrometry.This finding suggests that androstenetriolones can act as precurors of oestetrols in the human foetoplacental unit.     

Synthesis of 16α-bromoacetoxy androgens and 17 β-bromoacetylamino-4-androsten-3-one: Potential affinity labels of human placental aromatase

A novel synthesis of 16α-hydroxy-4-androstene-3,17-dione ($\text{3}$), 16α-hydroxy-4-androstene-3, 6,17-trione ($\text{4}$), 17β-amino-5-androsten-3β-ol ($\text{10}$) and 17β-amino-4-androsten-3-one (14) is described. 16α-Bromoacetoxy-4-androstene-3, 17-dione ($\text{5}$), 16α-bromoacetoxy-4-androstene-3, 6,17-trione ($\text{6}$) and 17β-bromoacetylamino-4-androsten-3-one ($\text{15}$) were synthesized as potentially selective irreversible inhibitors of androgen aromatases. 16α-Bromo-4-androstene-3,17-dione ($\text{1}$) and 16α-bromo-4-androstene-3, 6,17-trione ($\text{2}$) were converted to compounds $\text{3}$ and $\text{4}$ in 80–90% yield by controlled stereospecific hydrolysis using sodium hydroxide in aqueous pyridine. Reductive amination of 3β-hydroxy-5-androsten-17-one and 3-methoxy-3,5-androstadien-17-one ($\text{11}$) using ammonium acetate and sodium cyanohydridoborate (NaBH₃CN) and a subsequent treatment with acid gave the amines $\text{10}$ and $\text{14}$ respectively, as a salt. The corresponding 17-imino compounds $\text{9}$ and $\text{13}$ were also isolated from the reaction mixtures when methanol was used as a solvent for the reaction. The 16α-hydroxyl compounds $\text{3}$ and $\text{4}$ and the 17β-amino compound $\text{14}$ were con- verted to the corresponding bromoacetyl derivatives, $\text{5}$, $\text{6}$, and $\text{15}$, with bromoacetic acid and N,N'-dicyclohexylcarbodiimide.     

Degradation of tomatine to 3β-hydroxy-5α-pregn-16-en-20-one by ripe tomatoes

Tomatine-4-¹⁴C was prepared by foliar administration of cholesterol-4-¹⁴C and silicone oil to tomato plants. Chromatographically homogeneous tomatine-4-¹⁴C in 96% ethanol, when incubated in whole, ripe tomatoes was rapidly converted to 3β-hydroxy-5α-pregn-16-en-20-one in combined form. The identity of this steroid and its acetyl derivative was established by comparing their TCL mobilities with reference materials and by recrystallizing them to constant specific activity.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

A direct radioiodination technique for the radioimmunoassay of 17α-ethynyl, 17β-hydroxy-4-estren-3-one

A new technique is described for the direct labelling of norethisterone with¹²⁵I for use in radioimmunoassay. This method allows the preparation of tracers possessing both high specific activity and unaltered immunological properties. The steroid is radioiodinated in the presence of H₂O₂ using acetic acid as the solvent, in a sealed vial heated at 100°C. The reaction mixture is then chromatographed on bidimensional t.l.c. and six major radioactive spots are separated; only one of these fractions possess immunoreactivity. When submitted to repeated t.l.c. it behaves as a single compound, possibly a monoiodinated derivative, substituted in Ring A.The new tracer was compared in RIA with both the tyrosine-iodinated and the tritiated tracers. The standard inhibition curve obtained with the new labelled ligand had the same slope as that obtained using the tritiated one; on the other hand the slope of the curve obtained with the tyrosine iodinated derivative was slightly higher than that of the directly iodinated one.The results obtained indicate that the possibility exists of radioiodinating steroids without losing their immunoreactivity.     

Conversion of 15α-hydroxy-11-oxo-progesterone to 3β,11α,15β-trihydroxy-5-pregnen-20-one

3β,11α,15β-Trihydroxy-5-pregnen-20-one, an intermediate required for the synthesis of the oogoniols, has been prepared from 15α-hydroxy-11-oxo-progesterone in an overall yield of 16%. The three isomers (at C-11, C-15) of the pregnene were also prepared.     

Isolation and identification of etiocholan-3α-ol-17-one and etiocholane-3α, 11β-diol-17-one from human blood plasma

Two hitherto unidentified 17-ketosteroids were isolated from 600 ml. of normal human male plasma. By such criteria as infrared and sulfuric acid spectra and mobility of the free, the acetylated, and the oxidized compounds in several systems of paper chromatography, these 17-ketosteroids were identified as etiocholan-3α-ol-17-one and etiocholane-3α-11β-diol-17-one.     

The formation of 3α- and 3β-methoxy-Δ4-androsten-17-one and Δ3,5-androstadien-17-one by acid-catalyzed methanolysis

— 3α- and 3β-Methoxy-Δ⁴-androsten-17-one (IVb and Vb) were produced along with a smaller amount of Δ^3,5-androstadien-17-one when the 17-ethylene ketal (I) of Δ⁴-androstene-3,17-dione was reduced with sodium borohydride and the resulting mixture of epimeric 3-alcohols was refluxed in methanol containing acetic acid and water. The predominance of the exchange reaction with methanol over elimination is explicable on the basis of steric hindrance from C-19. Proof of structure for the previously unreported epimeric methoxy compounds was obtained by a study of their chemical and physical properties. The epimer with an equatorial methoxyl group proved to be faster moving in an adsorption system of chromatography in contrast to expectation based on the known rate of movement of epimeric 3-hydroxy compounds.