Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF.

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

Liquid chromatography-mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes.

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17ß-isomers of the sulfate conjugates, but limited differentiation for 17α/17ß-isomers of glucuronide conjugates and for 3α/3ß- and 5α/5ß-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS).

16-Picolyl-androsterone derivative exhibits potent 17ß-HSD3 inhibitory activity, improved metabolic stability and cytotoxic effect on various cancer cells: Synthesis, homology modeling and docking studies.

A new androsterone derivative bearing a 16ß-picolyl group (compound 5; FCO-586-119) was synthetized in four steps from the lead compound 1 (RM-532-105). We measured its inhibitory activity on 17ß-HSD3 using microsomal fraction of rat testes as well as transfected LNCaP[17ß-HSD3] cells. We then assessed its metabolic stability as well as its cytotoxic effect against a panel of cancer cell lines. The addition of a picolyl moiety at C-16 of RM-532-105 steroid core improves the 17ß-HSD3 inhibitory activity in the microsomal fraction of rat testes, but not in whole LNCaP[17ß-HSD3] cells. Interestingly, this structural modification enhances 3-fold the metabolic stability in conjunction with a significant cytotoxic effect against pancreatic, ovarian, breast, lung, and prostate cancer cells. Because the inhibitory activity data against 17ß-HSD3 suggested that both steroid derivatives are non-competitive inhibitors, we performed docking and molecular dynamics simulations using a homology model of this membrane-associated enzyme. The results of these simulations revealed that both RM-532-105 (1) and FCO-586-119 (5) can compete for the cofactor-binding site displaying better binding energy than NADP+.

Novel, rapid, low-cost screen-printed (bio)sensors for the direct analysis of boar taint compounds androstenone and skatole in porcine adipose tissue: Comparison with a high-resolution gas chromatographic method.

This is the first report on the fabrication, characterisation and application of an electrochemical (bio)sensor system for the simultaneous measurement of skatole and androstenone. A biosensor for androstenone was fabricated using a Meldola's Blue modified SPCE (MB-SPCE) by depositing NADH and the enzyme 3α-hydroxysteroid dehydrogenase onto the MB-SPCE surface; samples of adipose tissue were analysed using the biosensors in conjunction with chronoamperometry. Cyclic voltammetry was used to investigate the electrochemical behaviour of skatole at a screen-printed carbon electrode (SPCE vs. Ag/AgCl). An oxidation peak was observed around +0.55 V (vs. Ag/AgCl) and differential pulse voltammetry was applied for quantification of skatole in adipose tissue (in-situ). Quantitative analysis was achieved using calibration plots obtained from fortified meat samples. The concentrations obtained by the electrochemical and gas chromatographic (GC) methods demonstrated a good positive correlation. The (bio)sensor system completed both measurements within 60 s, as compared to several hours for GC, and at a considerably reduced cost and complexity. Consequently, the novel (bio)sensor system should have applications for analysis of carcasses on the abattoir processing line.

Synthesis and bioevaluation of novel steroidal isatin conjugates derived from epiandrosterone/androsterone.

Steroids are classes of natural products widely distributed in nature, which have been demonstrated to exhibit broad biological functions, and have also attracted increasing interest from bioorganic and pharmaceutical researches. In order to develop novel chemical entities as potential cytotoxic agents, a series of steroidal isatin conjugations derived from epiandrosterone and androsterone were efficiently prepared and characterized, and all these obtained compounds were screened for their potential cytotoxic activities. The preliminary bioassay indicated that most of the newly synthesized compounds exhibited good cytotoxic activities against human gastric cancer (SGC-7901), melanoma (A875), and hepatocellular liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), which might be considered as promising scaffold for further development of potential anticancer agents.

Catalytic cyclometallation in steroid chemistry VI: Targeted synthesis of hybrid molecules based on steroids and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid and study of their antitumor activity.

Hybrid molecules based on a number of steroids (cholesterol, pregnenolone, androsterone) and 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid linked via mono- and diethylene glycol spacers were synthesized for the first time and studied for antitumor activity in vitro. The acid was prepared using catalytic cyclomagnesiation of oxygenated 1,2-dienes with Grignard reagent in the presence of Cp2TiCl2 as the key synthetic step. Using flow cytometry, the new molecules were shown for the first time to be efficient apoptosis inducers in the HeLa, Hek293, U937, Jurkat, and K562 cell cultures and to have dose-dependent effect on the S and G2 phases of the cell cycle.

Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6).

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17ß-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC-MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3ß-sulfate, 17α/17ß-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17ß-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17ß-estradiol-3,17-disulfate was not transported by SOAT. IN CONCLUSION: SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3' or 17' with flexibility for α- or ß- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17ß-estradiol-3,17-disulfate is not transported by SOAT.

Individual variability of human olfactory sensitivity to volatile steroids: Environmental and genetic factors.

The sensitivity to androstenone and possible factors, determining the sensitivity were investigated for the large sample of inhabitants of central Russia (n = 860). Specific anosmia was detected in 48.8% of subjects. Women were more sensitive to androstenone than men. The proportion of men, but not women perceiving the smell of androstenone as a strong one in the concentration used decreased with age. Smoking, blood group, or ethnicity had no significant effect on the expression of specific anosmia and the perception of androstenone odor intensity.

An enzymatic approach to configurationally rare trans-androsteronyl-α-glucoside and Its potential anticancer application.

Enzymatic glycosylation of sterols/steroids with glycosyltransferase HP0421 shows protein plasticity on generation of configurationally rare steryl-α-glucosides. Investigation of trans-androsteronyl-α-glucoside on tamoxifen-treated MCF-7 breast cancer cells shows dose-dependent depression of cell viability and enhanced drug effectiveness, illustrating a new avenue for the production of novel steryl-α-glucosides with useful biological activities.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis, characterization and antitumor activities of some steroidal derivatives with side chain of 17-hydrazone aromatic heterocycle.

Here a series of dehydroepiandrosterone-17-hydrazone and estrone-17-hydrazone derivatives possessing various aromatic heterocycle structures in 17-side chain of their steroidal nucleus were synthesized and their structures were evaluated. The antiproliferative activity of synthesized compounds against some cancer cells was investigated. The results have demonstrated that some dehydroepiandrosterone-17-hydrazone derivatives show distinct antiproliferative activity against some cancer cells through inducing cancer cell apoptosis, and compound 8 with a quinoline structure in 17-side chain displays excellent antiproliferative activity in vitro against SGC 7901 cancer cell (human gastric carcinoma) with an IC50 value of 1 µM. In addition, estrone-17-hydrazone derivatives having a key feature of indole group in the structure showed a special obvious cytotoxicity against HeLa cells, but almost inactive against other cells. The information obtained from the studies is valuable for the design of novel steroidal chemotherapeutic drugs.

The DHEA metabolite 7ß-hydroxy-epiandrosterone exerts anti-estrogenic effects on breast cancer cell lines.

7ß-Hydroxy-epiandrosterone (7ß-OH-EpiA), an endogenous androgenic derivative of dehydroepiandrosterone, has previously been shown to exert anti-inflammatory action in vitro and in vivo via a shift from prostaglandin E2 (PGE2) to 15-deoxy-Δ(12,14)-PGJ2 production. This modulation in prostaglandin production was obtained with low concentrations of 7ß-OH-EpiA (1-100nM) and suggested that it might act through a specific receptor. Inflammation and prostaglandin synthesis is important in the development and survival of estrogen-dependent mammary cancers. Estrogen induced PGE2 production and cell proliferation via its binding to estrogen receptors (ERs) in these tumors. Our objective was to test the effects of 7ß-OH-EpiA on the proliferation (by counting with trypan blue exclusion), cell cycle and cell apoptosis (by flow cytometry) of breast cancer cell lines MCF-7 (ERα+, ERß+, G-protein coupled receptor 30: GPR30+) and MDA-MB-231 (ERα-, ERß+, GPR30+) and to identify a potential target of this steroid in these cell lineages (by transactivations) and in the nuclear ER-negative SKBr3 cells (GPR30+) (by proliferation assays). 7ß-OH-EpiA exerted anti-estrogenic effects in MCF-7 and MDA-MB-231 cells associated with cell proliferation inhibition and cell cycle arrest. Moreover, transactivation and proliferation with ER agonists assays indicated that 7ß-OH-EpiA interacted with ERß. Data from proliferation assays on the MCF-7, MDA-MB-231 and SKBr3 cell lines suggested that 7ß-OH-EpiA may also act through the membrane GPR30 receptor. These results support that this androgenic steroid acts as an anti-estrogenic compound. Moreover, this is the first evidence that low doses of androgenic steroid exert antiproliferative effects in these mammary cancer cells. Further investigations are needed to improve understanding of the observed actions of endogenous 7ß-OH-EpiA.

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones.

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6ß,11α-dihydroxyprogesterone or 17ß-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5ß-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17ß-alcohols; activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD) has significant impact on the amount of the formed ring-D δ-lactone.

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17ß-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters.

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

7beta-Hydroxy-epiandrosterone-mediated regulation of the prostaglandin synthesis pathway in human peripheral blood monocytes.

7alpha-Hydroxy-DHEA, 7beta-hydroxy-DHEA and 7beta-hydroxy-EpiA are native metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA). Since numerous steroids are reported to interfere with inflammatory and immune processes, our objective was to test the effects of these hydroxysteroids on prostaglandin (PG) production and related enzyme gene expression. Human peripheral blood monocytes were cultured for 4 and 24 h in the presence of each of the steroids (1-100 nM), with and without addition of TNF-alpha (10 ng/mL). Levels of PGE(2), PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were measured in the incubation medium, and cell content of cyclooxygenase (COX-2), and PGE and PGD synthases (m-PGES1, H-PGDS, L-PGDS), and peroxisome proliferator activated receptor (PPAR-gamma) was assessed by quantitative RT-PCR and Western blots. Addition of TNF-alpha resulted in elevated PG production and increased COX-2 and m-PGES1 levels. Among the three steroids tested, only 7beta-hydroxy-EpiA decreased COX-2, m-PGES1 and PPAR-gamma expression while markedly decreasing PGE(2) and increasing 15d-PGJ(2) production. These results suggest that 7beta-hydroxy-EpiA is a native trigger of cellular protection through simultaneous activation of 15d-PGJ(2) and depression of PGE(2) synthesis, and that these effects may be mediated by activation of a putative receptor, specific for 7beta-hydroxy-EpiA.

3Beta-alkyl-androsterones as inhibitors of type 3 17beta-hydroxysteroid dehydrogenase: inhibitory potency in intact cells, selectivity towards isoforms 1, 2, 5 and 7, binding affinity for steroid receptors, and proliferative/antiproliferative activities on AR+ and ER+ cell lines.

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is involved in the biosynthesis of the potent androgen testosterone (T), which plays an important role in androgen-sensitive diseases. In an attempt to design compounds to lower the level of T, we designed androsterone (ADT) derivatives substituted at the position 3beta as inhibitors of type 3 17beta-HSD, and then selected the eight most potent ones (compounds 1-8) for additional studies. In an intact cell assay, they inhibited efficiently the conversion of natural substrate 4-androstene-3,17-dione into T, although they were less active in intact cells (IC50 approximately 1 microM) than in homogenated cells (IC50=57-100 nM). A study of the inhibitory potency with four other 17beta-HSDs revealed they were selective, since they do not inhibit reductive types 1, 5 and 7, nor oxidative type 2. Interestingly, they did not show any binding affinity for steroid receptors (androgen, estrogen, glucocorticoid and progestin). Only two inhibitors, 3beta-phenyl-ADT (5) and 3beta-phenylmethyl-ADT (6) showed some proliferative activities on an AR+ cell line and on an ER+ cell line, but their effects were not mediated through the androgen or estrogen receptors. This study identified selective inhibitors of type 3 17beta-HSD acting through a mixed-type inhibition, and devoid of non-suitable androgenic and estrogenic proliferative activities. The more potent inhibitors were 3beta-hexyl-ADT (2), 3beta-cyclohexylethyl-ADT (4) and 3beta-phenylethyl-ADT (7).

Androsterone 3alpha-ether-3beta-substituted and androsterone 3beta-substituted derivatives as inhibitors of type 3 17beta-hydroxysteroid dehydrogenase: chemical synthesis and structure-activity relationship.

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is involved in the biosynthesis of androgen testosterone. To produce potent inhibitors of this key steroidogenic enzyme, we prepared a series of androsterone (ADT) derivatives by adding a variety of substituents at position 3. The 3beta-substituted ADT derivatives proved to be good inhibitors (IC(50) = 57-147 nM) with better inhibitory activities obtained for compounds bearing a propyl, s-butyl, cyclohexylalkyl, or phenylalkyl group. With an IC(50) value of 57 nM, the 3beta-phenylmethyl-ADT was 6-fold more potent than ADT, the lead compound, and 13-fold more potent than 4-androstene-3,17-dione, the natural enzyme substrate used itself as inhibitor. The 3alpha-ether-3beta-substituted ADT derivatives had a lower inhibitory activity compared to the 3beta-substituted ADT analogues except for the 3beta-phenylethyl-3alpha-methl-O-ADT (IC(50) = 73 nM), which proved to be a more potent inhibitor than 3beta-phenylethyl-ADT (IC(50) = 99 nM). The results of our study identified potent type 3 17beta-HSD inhibitors for potential use in the treatment of androgen-sensitive diseases.

Pitfalls in trimethylsilylation of anabolic steroids. New derivatisation approach for residue at ultra-trace level.

Mixtures such as N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), ammonium iodide and dithioerythreitol (DTE) or MSTFA, trimethyliodosilane and DTE were used for derivatisation of anabolic steroids extracted from 2 g kidney fat and present at ng kg(-1) level. They are leading to unexpected products. Their identity and mechanism of formation have been discussed. A new silylation mixture was developed to overcome these pitfalls: N,O-bis-trimethylsilyl-acetamide was used in combination of 2.5% of MSTFA/I(2) (1000:10 (v/w)). A single product consisting in ether-TMS and/or enol-TMS derivative was observed for all tested steroids with a stability demonstrated for at least 48 h. Quantitative application was proved even at the low ng kg(-1) level in a complex biological matrices, i.e. kidney fat.

Androsterone derivatives substituted at position 16: chemical synthesis, inhibition of type 3 17beta-hydroxysteroid dehydrogenase, binding affinity for steroid receptors and proliferative/antiproliferative activity on Shionogi (AR+) cells.

A series of androsterone (ADT) derivatives substituted at position 16 were efficiently synthesized in short reaction sequences; the ether analogues were also synthesized in the case of the methyl and allyl derivatives. The aim of this study was to develop inhibitors of the steroidogenic enzyme type 3 17beta-hydroxysteroid dehydrogenase and then evaluate their ability to inhibit this activity in transfected HEK-293 cells. For each compound we measured the percentage of inhibition of the transformation of 4-androstene-3,17-dione, the natural substrate of this steroidogenic enzyme, into the active androgen testosterone. The synthesized compounds proved to be weak inhibitors of this enzyme, but interestingly, these ADT derivatives do not bind to androgen, estrogen, glucocorticoid, and progestin receptors, suggesting no unsuitable receptor-mediated effects. One exception, 16alpha-(3'-bromopropyl)-5alpha-androstane-3alpha,17beta-diol, the only compound bearing a hydroxy group at position 17beta instead of a ketone, showed a strong binding affinity for the androgen receptor (70% at 1 microM) and also exhibited an antiproliferative activity on Shionogi (AR+) cells (86% at 1 microM), which was comparable to that of hydroxyflutamide, a pure antiandrogen (100% at 1 microM).