Microbial transformation of 17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one (STS 557) and 17α-cyanomethyl-19-nortestosterone by Mycobacterium smegmatis

Microbial transformation of the new progestagen STS 557 (17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one) by $\text{Mycobacterium smegmatis}$ yielded predominantly ring A-aromatized compounds: 17α-cyanomethyl-1,3,5(10),9(11)-estratetraene-3, 17-diol, 17α-cyanomethyl-1,3,5(10)-estratriene-3, 17-diol and the corresponding 3-methyl ethers. The analogous compound without the 9(10) double bond, 17α-cyanomethyl-19-nortestosterone, was transformed mainly to 5α-hydrogenated metabolites: 17α-cyanomethyl-17-hydroxy-5α-estran-3-one, 17α-cyanomethyl-17-hydroxy-5α-1-estren-3-one, 17α-cyanomethyl-5α-estrane-3α, 17-diol, and 17α-cyanomethyl-5α-estrane-3β, 17-diol. From these results, it is concluded that 4,9-dien-3-oxo compounds are not substrates for enzymatic 5α-hydrogenation.     

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.     

In vitro metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by the rat intestine

The metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by the 800 g supernatant fraction by different parts of the gastrointestinal tract from male rats was investigated. This metabolism tended to be higher in immature than in mature animals. Administration of dexamethasone or long-acting ACTH to immature and mature rats increased testosterone metabolism to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α, 17ß-diol by ileum tissue. No such effect could be observed following administration of progesterone, estradiol, prolactin, LH or FSH in mature animals. Development of the gastrointestinal tract from the immature to themmature stage was associated with augmented metabolism of testosterone to 17ß-hydroxy-5α-androstane-3-one and 5α-androstane-3α,17ß-diol in the ileum.     

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.     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

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.     

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.     

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.     

Identification of a unique sertoli cell steroid as 3α-hydroxy-4-pregnen-20-one (3α-dihyroprogesterone: 3α-DHP)

An allylic steroid produced from progesterone by rat Sertoli cells and which does not appear to have been described previously as a product of gonadal or adrenal tissues has been isolated and identified as 3α-hydroxy-4-pregnen-20-one (3α-dihydroprogesterone; 3α-DHP). 3α-DHP appears to be a reactive molecule which is easily oxidized or dehydrated and its identification was possible by a combination of microchemical procedures, derivative formation, specific enzyme reaction, TLC, GC, HPLC, spectrophotometry and mass spectrometry. The biological functions of this Sertoli cell steroid are not known, but it is suggested that 3α-DHP is more than a metabolic waste product because (a) its production rate varies with age and is highest at the onset of meiosis, and (b) there appear to be specific receptors for it in the testis.     

The crystal and molecular structure of d1-17β-hydroxy-8α-androst-4-en-3-one (8-isotestosterone)

The molecular conformation of d1-8-isotestosterone has been determined crystallographically. Crystals of the title compound belong to the space group P2₁/c with a = 11.449(4), b = 10.962(4), c = 25.860(5) , β = 100.95(4)⁰, with two molecules in the asymmetric unit. The structure has been refined to a final R value of 0.052 for 2227 reflections. Unlike testosterone, which is a flat molecule, its 8-isomer has a folded conformation. The conformations of the ring-B in the two crystallographically independent molecules (A and B) correspond to the twist form and differ significantly from one another.     

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.     

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.     

Conversion of 20α-hydroxy-4-pregnen-3-one to progesterone by human endometrium in vitro

Human endometrial tissues during the reproductive cycle were incubated in vitro with tritium labelled 20α-hydroxy-4-pregnen-3-one. It was shown that 20α-hydroxysteroid dehydrogenase, catalyzing the oxidative conversion of 20α-hydroxy-4-pregnen-3-one to progesterone with NAD, is mainly associated with the mitochondrial fraction of endometrial cells. Histochemical studies showed that glandular cells of the endometrium have high activity while the stromal cells have only weak activity. Conversion of 20α-hydroxy-4-pregnen-3-one to progesterone in the endometrium at different phases of the menstrual cycle indicated a progressive increase from the early proliferative phase until the late secretory phase. The conversion rate in decidua was higher in the first trimester of gestation than in the latter half. In patients with primary sterility the conversion rate was very high in the endometrium. The reason for this phenomenon is unknown.