Phenolic Steroids Research Articles

The diversity of sex steroid action: novel functions of hydroxysteroid (17β) dehydrogenases as revealed by genetically modified mouse models

Disturbed action of sex steroid hormones, i.e. androgens and estrogens, is involved in the pathogenesis of various severe diseases in humans. Interestingly, recent studies have provided data further supporting the hypothesis that the circulating hormone concentrations do not explain all physiological and pathological processes observed in hormone-dependent tissues, while the intratissue sex steroid concentrations are determined by the expression of steroid metabolising enzymes in the neighbouring cells (paracrine action) and/or by target cells themselves (intracrine action). This local sex steroid production is also a valuable treatment option for developing novel therapies against hormonal diseases. Hydroxysteroid (17β) dehydrogenases (HSD17Bs) compose a family of 14 enzymes that catalyse the conversion between the low-active 17-keto steroids and the highly active 17β-hydroxy steroids. The enzymes frequently expressed in sex steroid target tissues are, thus, potential drug targets in order to lower the local sex steroid concentrations. The present review summarises the recent data obtained for the role of HSD17B1, HSD17B2, HSD17B7 and HSD17B12 enzymes in various metabolic pathways and their physiological and pathophysiological roles as revealed by the recently generated genetically modified mouse models. Our data, together with that provided by others, show that, in addition to having a role in sex steroid metabolism, several of these HSD17B enzymes possess key roles in other metabolic processes: for example, HD17B7 is essential for cholesterol biosynthesis and HSD17B12 is involved in elongation of fatty acids. Additional studies in vitro and in vivo are to be carried out in order to fully define the metabolic role of the HSD17B enzymes and to evaluate their value as drug targets.

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1.

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

KORELASI KANDUNGAN FENOLIK DAN AKTIVITAS ANTIOKSIDAN DAUN JAMBU METE

The ethanolic extract of Anacardium occidentale L. leaves contains phenolic compounds,  avonoids, alkaloids, saponins, tannins, and steroids that could have antioxidant activity. The aim of this research was to determine the correlation between the antioxidant activity and the phenolic compounds of the ethanol extract of Anacardium occidentale L. leaf. The DPPH methode was used to determine the antioxidant activity. The total phenolic determined using Folin-Ciocalteu reagent. There was a positive correlation between antioxidant activity and total phenolic content in the ethanolic extract of cashew leaf with correlation coef cient R2 = 0.5888.Keywords: Anacardium occidentale L., antioxidant, phenolic compounds

A Mild One-Pot Method for Conversion of Various Steroidal Secondary Alcohols into the Corresponding Olefins

The addition of Tf 2 O to some hydroxy steroids in the presence of excess base directly leads to steroidal olefins. This methodology is useful for the one-pot synthesis of Δ 2 - or Δ 3 -steroids under mild conditions from the corresponding alcohols.

A spectrophotometic determination of traces of phenolic steroids in 3-ketosteroids.

Abstract As little as 0ṁ02 per cent of phenolic steroid may be determined in the presence of an excess of 3-ketosteroid. The interfering ketonic absorption is eliminated by selective reduction of ketone by potassium borohydride. The slight absorption of the reduction products may be corrected graphically.

Exploring Electron Density as a Theoretical Basis for the Biotransformation of Phenolic Steroids

Exploring Electron Density as a Theoretical Basis for the Biotransformation of Phenolic Steroids

ChemInform Abstract: Stereochemical Assignment at C-24 and C-25 of Marine 24-Ethyl-26- hydroxy Steroids Through Comparison with Synthetic (24S,25S)-, (24S, 25R)-, (24R,25R)- and (24R,25S)-Models.

ChemInform Abstract: Stereochemical Assignment at C-24 and C-25 of Marine 24-Ethyl-26- hydroxy Steroids Through Comparison with Synthetic (24S,25S)-, (24S, 25R)-, (24R,25R)- and (24R,25S)-Models.

ChemInform Abstract: Marine Natural Products as Antituberculosis Agents.

Abstract In an attempt to characterize additional structural classes that could serve as lead antituberculosis agents, 48 structurally diverse marine-derived natural and semisynthetic compounds were examined for in vitro activity against Mycobacterium tuberculosis . Three new classes of compounds including C-19 hydroxy steroids, scalarin sesquiterpenoids and tetrabromo spirocyclohexadienylisoxazolines have been identified as having potential as leads for continued investigations as new antituberculosis agents. New additions to the established antituberculosis structural classes quinone-methide and peptide are also reported.

Quantification of 19‐nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry

Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable.

An efficient C-H oxidation protocol for alpha-hydroxylation of cyclic steroidal ethers.

[reaction: see text] Various C-16 hydroxy steroids have been prepared with the aid of CrO(3)/Bu(4)NIO(4). Out of the two possible reaction courses, transition state B is favored because of less steric interference between substrate and CrO(4). Thus, C-H bonds at C-16 are oxidized selectively.

Efficient Synthesis of New Steroids Possessing an Aromatic A-Ring with a 2-Hydroxy or a 2-Fluoro Substituent

5-Fluoro-1-iodobenzocyclobutene (7) and 5-tert-butoxy-1-iodobenzocyclobutene (8) have been used for the synthesis of the title compounds. This strategy involves the use of an intramolecular Diels−Alder cycloaddition of o-xylylenes as the key step. We have shown that the introduction of a tert-butyl ether as a protecting group at the beginning of the synthesis, in place of the methyl ether used previously, is a judicious choice by which to obtain steroids possessing an aromatic A-ring with a 2-OH substituent. Finally, the vinyl groups of the synthesized fluoro and hydroxy steroids have been oxidized by the Wacker process in good yields. An X-ray crystal structure of the fluoro steroid 14b, the trans-anti-trans ring configuration of which matches those in natural products, is reported.

Determination of endocrine-disrupting phenolic compounds and estrogens in surface and drinking water by HRGC-(NCI)-MS in the picogram per liter range.

A method for the analysis of phenolic estrogenic active compounds in surface and drinking water in the picogram per liter range is described. Besides the widely used monomer bisphenol A, 4-tert-octylphenol [4-(1,1,3,3-tetramethylbutyl)phenol] and the technical isomer mixture of 4-nonylphenol; phenolic steroid hormones such as the endogenous estrogens estrone, 17alpha-estradiol, and 17beta-estradiol; and the exogenous estrogen 17alpha-ethinylestradiol were determined in water at the 20-200 pg/L level. Water samples from 1 to 5 L were extracted by solid-phase extraction (SPE) on a cartridge system containing LiChrolut EN as sorbent. The phenols and steroids were converted into their pentafluorobenzoylate esters in an extractive derivatization reaction. The derivatives were then determined by high-resolution gas chromatography with negative chemical ionization mass spectrometric detection (HRGC-(NCI)-MS) in the selected ion mode (SIM). All results were also confirmed by HRGC with electron capture detection (ECD). This highly sensitive and specific method gives a limit of detection (LOD) of 20 pg/L for bisphenol A and 4-tert-octylphenol in drinking water samples and 50 pg/L in STW effluent, respectively. The LODs for technical 4-nonylphenol, 17alpha-ethinylestradiol, and other estrogens are in the range of 50 pg/L in drinking water to 200 pg/L in STW effluent, respectively. In all river water samples in southern Germany, bisphenol A was found in concentrations ranging from 500 pg/L up to 16 ng/L, 4-nonylphenol was from 6 up to 135 ng/L, and the steroids were from 200 pg/L up to 5 ng/L. In drinking water, bisphenol A was found in concentrations ranging from 300 pg/L to 2 ng/L, 4-nonylphenol was from 2 to 15 ng/L, 4-tert-octylphenol was from 150 pg/L to 5 ng/L, and the steroids were from 100 pg/L to 2 ng/L. Mean recoveries over the whole analytical protocol, measured in bidistilled water, generally exceeded 70%. These results indicate that environmental endocrine-disrupting estrogens are not completely removed in the process of sewage treatment but are carried over into the general aquatic environment. After ground passage, they can eventually be found in drinking water.

Solid-phase synthesis of hydroxysteroid derivatives using the diethylsilyloxy linker.

Four different types of hydroxysteroids (primary alcohol, secondary alcohols, and phenol), bearing either an oxirane or an azide as a precursor of molecular diversity, were linked in good yields to solid support using the butyldiethylsilane polystyrene (PS-DES) resin. These molecules were then used as scaffolds to generate hydroxysteroid derivatives containing two levels of diversity. The proposed libraries were tested by running steroidal alcohols through a model sequence of reactions (solid-phase coupling, aminolysis of oxirane or reduction of azide, amidation, and final cleavage). As a result, two linked secondary alcohols (17beta-hydroxy-spiro-3(R)-oxirane-5alpha-androstane and 3beta-hydroxy-spiro- 17(S)-oxirane-5alpha-androstane) and a primary alcohol (spiro-17(S)-oxirane-3-(hydroxymethyl)-1,3,5(10)-estratriene) afforded good overall yields (>45%) and high HPLC purities (>90%) of hydroxysteroids derivatized as alkylamides without purification. One limitation was noted for the fourth library: the phenolic steroid linked by the diethylsilyloxy linker gave a poor overall yield of 8% of the desired model compound. Finally, the diethylsilyloxy linker was used successfully for a rapid solid-phase synthesis of a model library of twenty C19-steroid derivatives (3beta-amido-3alpha-hydroxy-5alpha-androstane-17-ones), with an average yield of 53% and average HPLC purity of 97% without purification steps.

Marine Natural Products as Antituberculosis Agents

In an attempt to characterize additional structural classes that could serve as lead antituberculosis agents, 48 structurally diverse marine-derived natural and semisynthetic compounds were examined for in vitro activity against Mycobacterium tuberculosis. Three new classes of compounds including C-19 hydroxy steroids, scalarin sesquiterpenoids and tetrabromo spirocyclohexadienylisoxazolines have been identified as having potential as leads for continued investigations as new antituberculosis agents. New additions to the established antituberculosis structural classes quinone-methide and peptide are also reported.

Ultra trace detection of a wide range of anabolic steroids in meat by gas chromatography coupled to mass spectrometry

The control on use of anabolic agents in meat producing animals is generally based on urine, faeces or hair analysis. This exercise, which is usually performed in slaughterhouses or on farms, is not relevant to imported carcasses or retail meat. A single sensitive method for a wide range of anabolic steroids was developed. After extraction of the lyophilised meat, enzymatic hydrolysis was used for deconjugation. Solid-phase extraction on a polymeric stationary phase was performed prior to hydrolysis of ester residues under alkaline conditions. Liquid–liquid partitioning was used to separate the analytes into two main categories: phenol containing molecules, such as phenolic steroids, resorcylic acid lactones and stilbenes, and Δ4-3-one containing molecules, such as most androgens and progestagens. Solid-phase extraction on silica columns was performed before applying a specific derivatisation for each compound sub-group. The combination of high-resolution chromatography with a quadrupole mass spectrometer permitted detection of 23 steroids in the 5–100 ng/kg range. Ion chromatograms for residue positive samples are shown and discussed.

Solid-phase synthesis of phenolic steroids: from optimization studies to a convenient procedure for combinatorial synthesis of biologically relevant estradiol derivatives.

During the course of our studies on therapeutic agents for the treatment of breast cancer, we became interested in the solid-phase combinatorial synthesis of estradiol derivatives that contain a functionalized side chain at either position 16 beta or 7 alpha. Both types of compounds have already demonstrated inhibitory activity toward both biosynthesis and action of estradiol. As a first step, two versatile precursors bearing an azidoalkyl side chain at either position 16 beta or 7 alpha of estradiol were synthesized using standard solution-phase methods. Afterward, the effectiveness of five linkers to attach the phenolic function of these estradiol derivatives to a polystyrene resin was investigated; they were benzylic ether (Merrifield), 4-alkoxy-benzylic ethers (Wang, Sheppard), tetrahydropyranyl ether (Ellman), benzoic ester, and o-nitrobenzyl ether. To test the linker in a synthetic context, a short sequence of reactions, including reduction of the azide and acylation of the corresponding amine, was performed on the polymer-bound estradiol derivative. While all of the tested linkers proved effective in attaching the phenol functionality of the precursor, only the o-nitrobenzyl ether photolabile linker enabled the release of the final products in acceptable purities. Consequently, this linker was used to perform successfully the solid-phase synthesis of four different classes of estradiol derivatives in acceptable yields and excellent purities. This study was preliminary to the combinatorial synthesis of larger libraries of biologically relevant estradiol derivatives.

Profiles of steroid hormones in beef from steers implanted with Synovex-S (estradiol benzoate and progesterone) in comparison to control steers

Profiles of steroid hormones (androgens, estrogens, progestogens and corticoids), their precursors and metabolites were analyzed in nine beef samples obtained from steers which had received the anabolic implant Synovex-S (200 mg progesterone plus 20 mg estradiol benzoate) and in nine samples from control steers. Analysis of phenolic steroids was performed by enzyme immunoassay after separation by HPLC. Neutral steroids were determined by GC-MS. Concentrations of the hormones progesterone and 17β-estradiol, of their precursors and metabolites (pregnenolone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, epitestosterone, α-androsterone, 17α-estradiol, and estrone) and of the corticosteroids cortisone and hydrocortisone did not differ significantly between treatments (P>0.05) but the ratio of 17β-estradiol to its metabolites and the cortisone/hydrocortisone ratio were significantly higher in beef from treated steers (P<0.01). Concentrations of testosterone and 5α-dihydrotestosterone were below the determination limits (10 ng/kg and 20 ng/kg, respectively) in both treated and control steers.

Solid-phase synthesis of phenolic steroids: Towards combinatorial libraries of estradiol derivatives

The 16β-(azidopropyl) derivative of estradiol ( 7) was synthesized and coupled to aminomethyl resin via a photolabile o-nitrobenzyl linker. Condensation of the corresponding iminophosphorane with activated acids successfully gave amides. Photocleavage resulted in good yield recovery of estradiol derivatives 15 and 16 in acceptable purities for biological screening.

Substituent effects on the binding of phenols to the D38N mutant of 3-oxo-delta5-steroid isomerase. A probe for the nature of hydrogen bonding to the intermediate.

The nature of hydrogen bonding to the intermediate of the reaction catalyzed by 3-oxo-Delta5-steroid isomerase (KSI) was investigated. Substituted phenols bind tightly to the active site of the D38N mutant of KSI, and are analogs of the intermediate dienol. These D38N-phenol complexes exhibit fluorescence, NMR, and UV spectral characteristics similar to D38N complexed with phenolic steroids. The binding of phenols to D38N is satisfactorily described by the modified Bronsted equation: log KD = 0.85(pKa) - 0.63 pi - 6.3 (n = 10, r = 0.967), where KD is the dissociation constant of the complex and pi is the hydrophobicity parameter for the phenol substituent. The high value of the Bronsted alpha (0.85 +/- 0.08) indicates that the negative charge in the D38N-phenol complex, and by implication in the KSI-intermediate complex, is localized almost exclusively on the bound ligand. It is concluded that stabilization of the anionic (dienolate) intermediate is provided by ordinary hydrogen bonds from the enzyme acids Tyr-14 and Asp-99, rather than low-barrier hydrogen bonds.

Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry.

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of allantoic and chorionic epithelia, contained no significant contamination of maternal tissues. The maternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homogenates and subcellular fractions were incubated with testosterone together with [4-(14)C] and [(2)H5 or (2)H3] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experiments (without additional cofactor) were also performed. After extraction of the tissue homogenates, neutral and phenolic (oestrogen) unconjugated steroids were separated by column chromatography. Radiolabelled studies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fractionation indicating that oestrogen production was largely confined to the microsomal fraction and time-course studies showing that the rate of formation appeared to be linear up to 90 min. In contrast, only 5-25% conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oestrogen production was detected in control incubations. These radiolabelled studies demonstrate that aromatase activity is located on the fetal allantochorionic surface and, together with the histochemical data, further delineate this activity to the chorion in mature equine placenta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the phenolic extracts from allantochorionic tissue homogenate incubations indicated the presence of substrate-derived oestradiol-17beta (E2), 6-oxo-oestradiol-17beta (6-oxo-E2) and 6beta-hydroxyoestradiol-17beta (6beta-OH-E2). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantochorionic tissue homogenates and post-mitochondrial suspensions (PMS), only E2 was identified from incubations performed using microsomal fractions prepared from this tissue. We conclude that both the microsomal and cytosol fractions are required for the conversion of E2 to the 6-oxygenated species in vitro. Using stable isotope-labelled substrates and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysis of the neutral extracts from allantochorionic tissue homogenate incubations confirmed the presence of small quantities of substrate-derived 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in the absence of cytosol. These data suggest that demethylation of C19 steroids to produce C18 neutral steroids may require the synergistic action of enzymic activities that appear to reside both in the microsomal and cytosolic fractions of equine allantochorionic tissues.