Metabolism of 17α-methyl-5β-dihydrotestosterone in the rabbit

17α-Methy1-5β-androstane-3α, 17β-diol together with the hydroxylated metabolites 17α-methyl-5β-androstane-1β, 3α, 17β-triol, 17α-methyl-5β-androstane-3α, 12β, 17β-triol, 17α-methyl-5β-androstane-3α, 16α, 17β-triol and 17α-methyl-5β-androstane-3α, 16β, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5β-dihydrotestosterone. Biotransformations differ from the 5α-series where hydroxylation occurred at C-6 and C-15. In both series, the C-3 equatorial epimer was the major urinary excretion product among the non-hydroxylated metabolites. The 5β-compound was more resistant to metabolic hydroxylation than the 5α-compound. No evidence for epimerization at the C-17 position was observed.     

Proton magnetic resonance spectra of 17ξ-Hydroxy-17ξ-methyl-5ξ-androstane C-3 ketone and c-3ξ alcohol isomers in chloroform-d and pyridine-d5

17α-Hydroxy-17β-methyl-5β-androstan-3-one, 17μ-methyl-5α-androstane-3α, 17α-diol, 17β-methyl-5α-androstane-3β, 17α-diol, 17α-methyl-5β-androstane-3β, 17β-diol, 17β-methyl-5β-androstane-3α, 17α-diol and 17β-methy1–5β-androstane-3β, 17α-diol were synthesized for the first time. ¹H NMR spectra of all four 17ξ-hydroxy/17ξ-methyl C-3 ketones and all eight C-3 alcohols were recorded in chloroform-d and pyridine-d₅. Pyridine-induced chemical shifts are discussed. Thin-layer Chromatographic data are given.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

The addition of hypobromous acid to 3β,17β-diacetoxy-4-estrene and an equilibration study involving neighboring group participation by the a-ring acetoxy group

The reaction of 3β,17β-diacetoxy-4-estrene with N-bromoacetamide in a two phase ether/water solvent mixture gave 5-bromo-4β,17β-diacetoxy-5α-estran-3β-ol as the major product (75%). Four minor products were also isolated and identified. These were: 4α-bromo-3β, 17β-diacetoxy-5α-estran-5-ol (5%), 5-bromo-3g,17β-diacetoxy-5α-estran-4β-ol (6%), 5-bromo-4α,17β-diacetoxy-5αt-estran-3β-ol (3%), and 4β-bromo-3β,17β-diacetoxy-5α-estran-5-ol (4%). The 5-bromo-4β, 17β-diacetoxy-5α-estran-3β-ol was equilibrated by heating with oxalic acid in refluxing benzene for $\text{ca}$. 16h to give a mixture of it and 5-broino-3β,17β-diacetoxy-5α-estran-4β-ol in the ratio of 16:84 respectively. A similar equilibration mixture (14:86) was obtained under identical conditions when 5-bromo-3β,17β-diacetoxy-5α-estran-4β-ol was the starting material.     

Changes in the Mutagenic and Estrogenic Activities of 17β-Estradiol after Treatment with Nitrite

We determined the changes in the mutagenic and estrogenic activities of 17β-estradiol after a nitrite treatment. Nitrite-treated 17β-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17β-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17β-estradiol to have been formed in the reaction mixture. 2-Nitro-17β-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17β-estradiol was found to be lower than that of 17β-estradiol. These data suggest that a daily oral intake of 17β-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine

The biotransformation of dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy,17α-methylandrosta-1,4-dien-3-one) in man was studied with the aim to discover long-term metabolites valuable for the antidoping analysis. Having applied a high performance liquid chromatography for the fractionation of urinary extract obtained from the pool of several DHCMT positive urines, about 50 metabolites were found. Most of these metabolites were included in the GC-MS/MS screening method, which was subsequently applied to analyze the post-administration and routine doping control samples. As a result of this study, 6 new long-term metabolites were identified tentatively characterized using GC-MS and GC-MS/MS as 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol (M3), its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methyl-5β-androst-13-en-3α-ol, 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) and its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methylandrosta-4,13-dien-3α-ol. The most long-term metabolite M3 was shown to be superior in the majority of cases to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-1,4,13-trien-3-one and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one.     

Efficient syntheses of 17-β-amino steroids

17β-Amino steroids such as 17β-amino-1,3,5(10)-estratrien-3-ol (1), 17β-amino-5α-androstan-3β-ol (2) and, 17β-amino-3β-hydroxyandrost-5-ene (3) have been widely used as a key intermediates in the synthesis of a variety of biologically active steroid derivatives though concise, high yielding syntheses of these compounds has yet to be reported. 17β-Amino-1,3,5(10)-estratrien-3-ol (1) and 17β-amino-5α-androstan-3β-ol (2) were prepared in high yield by reductive amination of estrone and epiandrosterone using benzylamine and sodium triacetoxyborohydride followed by catalytic hydrogenolysis of the resulting 17β-benzylamino derivatives. Attempts to prepare 17β-amino-3β-hydroxyandrost-5-ene (3) from dehydroepiandosterone using a similar approach resulted in partial reduction of the double bond. 17β-Amino-3β-hydroxyandrost-5-ene (3) was ultimately obtained in high yield by reductive amination of dehydroepiandosterone using allylamine and sodium triacetoxyborohydride followed by removal of the allyl group from the resulting 17β-allylamino derivative with dimethylbarbituric acid and Pd(PPh(3))(4) as catalyst.     

17beta-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

17β-Hydroxysteroid dehydrogenase (17βHSD) type 13 is identified as a new lipid droplet-associated protein. 17βHSD type 13 has an N-terminal sequence similar to that of 17βHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17βHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17βHSD type 11, however, expression of 17βHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor α and its ligand. Instead the expression level of 17βHSD type 13 in the receptor-null mice was increased several-fold. 17βHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

5α-Estrane-3β,17β-diol and 5β-estrane-3α,17β-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.     

Polar corticosteroids in human neonatal urine; Synthesis and gas chromatography-mass spectrometry of ring a reduced 6-hydroxylate corticosteroids

This report describes the synthesis of 3α, 6β, 11β, 17α, 21-pentahydroxy-5β-pregnane-20-one, 3α, 6β, 11β, 17α, 21-pentahydroxy-5α-pregnane-20-one, 3α, 6α, 11β, 17α, 21-pentahydroxy-5β-pregnane-20-one, 3α, 6α, 11β, 17α, 21-pentahydroxy-5α-pregnane-20-one, 3α, 6β, 17α, 21-tetrahydroxy-5β-pregnane-11, 20-dione, 3α, 6β, 17α, 21-tetrahydroxy-5α-pregnane-11, 20-dione, 3α, 6α, 17α, 21-tetrahydroxy-5β-pregnane-1 1, 20-dione and 3α, 6α, 17α, 21-tetrahydroxy-5α-pregnane-11, 20-dione.The gas chromatographic-mass spectrometric properties of these compounds are given. Proof of structure was accomplished using gas chromatography-mass spectrometry, microchemical reactions, optical rotatory dispersion and nuclear magnetic resonance spectroscopy.     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

Steroids in newborns and infants identification by combined gas chromatography-mass spectrometry of the major steroids excreted by a newborn chimpanzee (Pan troglodytes)

The following steroids have been identified by combined gas chromatography-mass spectrometry in a urine specimen collected from a newborn chimpanzee; 5-androstene-3β, 17α-diol, 3β,16α (and 16β)-dihydroxy-5-androsten-17-one, 5-androstene-3β, 16α, 17β-triol, 5-androstene-3β, 16β, 17α-triol, 5-pregnene-3β, 20α-diol, 5-pregnene-3β, 20α, 21-triol, 3β,21-dihydroxy-5-pregnen-20-one, 3β, 16α-dihydroxy-5-pregnen-20-one, 5-Piegnene-3β, 16α,20α, 21-tetrol, 5-pregnene-3β,17α, 20ξ, 21-tetrol androstenetriolones and androstenetetrols.     

恶臭假单胞菌SJTE-1中氧化17β-雌二醇的17β-羟甾类脱氢酶2及其转录调控因子AraC的鉴定

[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但其代谢机制尚不清楚。本文鉴定和表征了该菌株中参与雌二醇降解与调控过程的17β-羟甾类脱氢酶2(17β-HSD2)和转录调控因子AraC。[方法]我们通过荧光定量PCR分析了17β-hsd2和araC的转录水平;我们在大肠杆菌BL21(DE3)菌株中异源表达了17β-HSD2和AraC基因,并利用金属离子亲和层析法纯化获得了重组蛋白;我们体外表征了17β-HSD2的酶学性质,利用高效液相色谱鉴定了其产物;通过电泳迁移转移法和DNase酶I足迹试验,我们鉴定了重组蛋白AraC的结合能力与结合位点。[结果]17β-HSD2和AraC可被17β-雌二醇诱导表达;蛋白序列比对结果表明17β-HSD2含有短链脱氢酶/还原酶(SDR)和β-羟甾类脱氢酶的保守结构与残基。该酶以NAD+为辅助因子,在C17位点氧化17β-雌二醇,其Km值为0.082 mmol/L,Vmax值为56.26±0.02μmol/(min·mg);5 min内可转化97.4%以上的雌二醇。转录调控因子AraC可直接结合17β-hsd2基因启动子区的特异位点;雌二醇与雌酮可解除这一结合,启动17β-hsd2基因转录;过表达AraC蛋白可抑制17β-hsd2的转录。[结论]假单胞菌SJTE-1的17β-羟甾类脱氢酶2可高效催化17β-雌二醇转化,并受到转录因子AraC的直接调控。本工作可推进细菌的雌激素降解酶学机制与调控网络研究。     

Synthesis of new steroid haptens for radioimmunoassay. Part III. 15β-carboxyethylmercaptosteroid-bovine serum albumin conjugates. Specific antisera for radioimmunoassay of 5α-dihydrotestosterone, 5α-androstane-3β, 17β-diol and 5α-androstane-3α, 17β-diol

The syntheses of 15β-carboxyethylmercapto-5α-dihydrotestosterone, 15β-carboxy-ethylmercapto-5α-androstane-3β, 17β-diol and 15β-carboxyethylmercapto-5α-androstane-3α, 17β-diol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3β, 17β-diol (3β3-diol) and 5α-androstane-3α, 17β-diol (3α-diol).     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

New steroidal lactones as 5α-reductase inhibitors and antagonists for the androgen receptor

This study reports the synthesis of several new steroidal lactones: 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-3'-oxapentanoate (11), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-propanoate (12), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-butanoate (13), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-pentanoate (14), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-hexanoate (15), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-3'-oxapentanoate (16), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-propanoate (17), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-butanoate (18), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-pentanoate (19) and 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-hexanoate (20) with a therapeutic potential as antiandrogens. The biological effect of these steroids was demonstrated in in vivo as well as in vitro experiments. In the in vivo experiments, we measured the activity of ten new steroidal derivatives on the weight of the prostate and seminal vesicle glands of gonadectomized hamsters treated with testosterone. For the in vitro studies, we determined the IC(50) values by measuring the concentration of the steroidal derivatives that inhibits 50% of the activity of the 5α-reductase enzyme present in human prostate and also its binding capacity to the androgen receptors (AR) obtained from rat's prostate cytosol. The results from these experiments indicated that compounds 11-20, significantly decreased the weight of the prostate and seminal vesicles as compared to testosterone treated animals; this reduction of the weight of these glands was comparable to that produced by Finasteride. On the other hand, compounds 11-20 inhibited the enzyme 5α-reductase, with compounds 14-19 (IC(50) values of 4.2 ± 0.95, 0.025 ± 0.003, 1.2 ± 0.45, 1.2 ± 0.1, 0.028 ± 0.003, and 0.069 ± 0.005 nM, respectively) showing the highest inhibitory activity. The results from the in vitro experiments indicated that only 15-17 bind to the AR.     

Identification and functional characterization of a putative 17��-hydroxysteroid dehydrogenase 12 in abalone (Haliotis diversicolor supertexta)

The 17β-hydroxysteroid dehydrogenases (17β-HSDs) are key enzymes in the downstream process of steroid hormone biosynthesis. To date, relatively little is known about the role of 17β-HSDs in marine gastropods. In the present study, a putative cDNA sequence encoding type 12 17β-HSD (17β-HSD-12) was identified in abalone (Haliotis diversicolor supertexta). The full-length cDNA was 1,978 bp, including an open reading frame (ORF) of 963 bp that encoded a protein of 321 amino acids. Comparative structural analysis revealed that abalone 17β-HSD-12 shared 39.8-42.8% amino acid identity with other 17β-HSD-12 homologues and that the functional domains were well conserved. Phylogenetic analysis revealed that abalone 17β-HSD-12 belonged to the short-chain dehydrogenases/reductases (SDRs) family. Functional analysis following transient transfection of the ORF in human embryonic kidney-293 (HEK-293) cells indicated that abalone 17β-HSD-12 had the ability to convert estrone (E1) into estradiol (E2). Expression analysis in vivo demonstrated that abalone 17β-HSD-12 was differentially expressed during the three reproductive stages (pre-spawning, spawning, and post-spawning). These results indicate that abalone 17β-HSD-12 is an SDR family member with a key role in steroidogenesis during the reproductive period.     

Discovery of highly potent, nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 inhibitors by virtual high-throughput screening

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the formation of the potent proliferation-stimulating hormone estradiol, and it is thus involved in the development of hormone-dependent breast cancer. Due to its high substrate specificity and the known relationships between its overexpression and disease incidence, 17β-HSD1 is considered an attractive target for drug development. Here, we have used structure-based virtual high-throughput screening to successfully identify potent nonsteroidal 17β-HSD1 inhibitors. Computational screening of a drug-like database containing 13 million compounds identified hits with a 2-benzylidenebenzofuran-3(2H)-one scaffold that we show to be highly potent 17β-HSD1 inhibitors. The most potent in the series, compound 1, showed an IC(50) of 45nM in our 17β-HSD1 inhibition assay, and also showed good selectivity for 17β-HSD1 over 17β-HSD2.     

雌二醇对大鼠颌下腺EGF、NGF生成的影响

为了研究雌二醇（estradiol-17β,E2）对颌下腺表皮生长因子（epidermal growth factor,EGF)和神经生长因子（nerve growth factor,NGF)生成的影响,研究采用免疫组织化学方法,对外源性投予雌二醇后的大鼠颌下腺进行了观察。结果证实E2明显促进颌下腺EGF和NGF的生成。提示雌二醇可能对EGF和NGF的合成起重要的调节作用。