Simultaneous Determination of Selected Steroids with Neuroactive Effects in Human Serum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person's sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 µL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 µmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches.

Analytical Methods for Brassinosteroid Analysis: Recent Advances and Applications.

Brassinosteroids (BRs) are plant steroidal hormones that play crucial roles in plant growth and development. Accurate quantification of BRs in plant tissues is essential for understanding their biological functions. This study presents a comprehensive overview of the latest methods used for the quantification of BRs in plants. We discuss the principles, advantages, and limitations of various analytical techniques, including immunoassays, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) that are used for the detection and quantification of BRs from complex plant matrices. We also explore the use of isotopically labeled internal standards to improve the accuracy and reliability of BR quantification.

Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

Synthesis and neuroprotective activity of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives.

A library of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives was prepared from N-Boc-3-azetidinone employing the Horner-Wadsworth-Emmons reaction, rhodium(I)-catalyzed conjugate addition of arylboronic acids, and subsequent elaborations to obtain N-unprotected hydrochlorides, N-alkylated and N-acylated azetidine derivatives. The compounds were evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity, revealing several derivatives to possess AChE inhibition comparable to that of the AChE inhibitor rivastigmine. The binding mode of the AChE inhibitor donepezil and selected active compounds 26 and 27 within the active site of AChE was studied using molecular docking. Furthermore, the neuroprotective activity of the prepared compounds was evaluated in models associated with Parkinson's disease (salsolinol-induced) and aspects of Alzheimer's disease (glutamate-induced oxidative damage). Compound 28 showed the highest neuroprotective effect in both salsolinol- and glutamate-induced neurodegeneration models, and its protective effect in the glutamate model was revealed to be driven by a reduction in oxidative stress and caspase-3/7 activity.

Small change - big consequence: The impact of C15-C16 double bond in a D­ring of estrone on estrogen receptor activity.

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.

Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

Ring-fused 3ß-acetoxyandrost-5-enes as novel neuroprotective agents with cholinesterase inhibitory properties.

Alzheimer´s disease (AD) is an intellectual disorder caused by organic brain damage and cerebral atrophy, characterized by the loss of memory, judgment, and abstract thinking followed by declining cognitive functions, language, and the ability to perform daily living activities. Many efforts have been made to decrease the effects of the disease but also to block the neurodegenerative process. Cholinesterase inhibitors (ChEIs) are a group of medicines that act at the neurotransmission of acetylcholine, preventing its excessive breakdown and helping to improve cognitive functions in patients with AD. In this work, 16 chiral steroids, namely ring-fused 3ß-acetoxyandrost-5-ene derivatives, their precursor and two 16-dehydroprogesterone-derived dioximes, were assessed as cholinesterase inhibitors and neuroprotective agents. The results demonstrated that some of the tested steroids are cholinesterase inhibitors and the majority selective for acetylcholinesterase inhibition. Albeit, one ring-fused 3ß-acetoxyandrost-5-ene containing N-methylpiperidine ring (compound 2g) demonstrated to be a selective and potent inhibitor of the butyrylcholinesterase enzyme. (S)- 4,4a,5,6,7,8-(hexahydronaphthalen-2-one)-fused 3ß-acetoxyandrost-5-ene (compound 6) showed high neuroprotective effect, high ability to restore the mitochondrial membrane potential from glutamate intoxication, and dramatic improvement in cell morphology. The described results provided relevant structure-activity relationship data.

Synthesis and Biological Evaluation of New Isoxazolyl Steroids as Anti-Prostate Cancer Agents.

Steroids with a nitrogen-containing heterocycle in the side chain are known as effective inhibitors of androgen signaling and/or testosterone biosynthesis, thus showing beneficial effects for the treatment of prostate cancer. In this work, a series of 3ß-hydroxy-5-ene steroids, containing an isoxazole fragment in their side chain, was synthesized. The key steps included the preparation of Weinreb amide, its conversion to acetylenic ketones, and the 1,2- or 1,4-addition of hydroxylamine, depending on the solvent used. The biological activity of the obtained compounds was studied in a number of tests, including their effects on 17α-hydroxylase and 17,20-lyase activity of human CYP17A1 and the ability of selected compounds to affect the downstream androgen receptor signaling. Three derivatives diminished the transcriptional activity of androgen receptor and displayed reasonable antiproliferative activity. The candidate compound, 24j (17R)-17-((3-(2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-androst-5-en-3ß-ol, suppressed the androgen receptor signaling and decreased its protein level in two prostate cancer cell lines, LNCaP and LAPC-4. Interaction of compounds with CYP17A1 and the androgen receptor was confirmed and described by molecular docking.

Local brassinosteroid biosynthesis enables optimal root growth.

Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

Synthesis of New Brassinosteroid 24-Norcholane Type Analogs Conjugated in C-3 with Benzoate Groups.

The metabolism of brassinosteroid leads to structural modifications in the ring skeleton or the side alkyl chain. The esterification and glycosylation at C-3 are the most common metabolic pathways, and it has been suggested that conjugate brassinosteroids are less active or inactive. In this way, plants regulate the content of active brassinosteroids. In this work, the synthesis of brassinosteroid 24-norcholane type analogs conjugated at C-3 with benzoate groups, carrying electron donor and electron attractant substituents on the aromatic ring, is described. Additionally, their growth-promoting activities were evaluated using the Rice Lamina Inclination Test (RLIT) and compared with that exhibited by brassinolide (used as positive control) and non-conjugated analogs. The results indicate that at the lowest tested concentrations (10-8-10-7 M), all analogs conjugated at C-3 exhibit similar or higher activities than brassinolide, and the diasteroisomers with S configuration at C-22 are the more active ones. Increasing concentration (10-6 M) reduces the biological activities of analogs as compared to brassinolide.

Synthesis and Biological Activity of Brassinosteroid Analogues with a Nitrogen-Containing Side Chain.

Brassinosteroids are a class of plant hormones that regulate a broad range of physiological processes such as plant growth, development and immunity, including the suppression of biotic and abiotic stresses. In this paper, we report the synthesis of new brassinosteroid analogues with a nitrogen-containing side chain and their biological activity on Arabidopis thaliana. Based on molecular docking experiments, two groups of brassinosteroid analogues were prepared with short and long side chains in order to study the impact of side chain length on plants. The derivatives with a short side chain were prepared with amide, amine and ammonium functional groups. The derivatives with a long side chain were synthesized using amide and ammonium functional groups. A total of 25 new brassinosteroid analogues were prepared. All 25 compounds were tested in an Arabidopsis root sensitivity bioassay and cytotoxicity screening. The synthesized substances showed no significant inhibitory activity compared to natural 24-epibrassinolide. In contrast, in low concentration, several compounds (8a, 8b, 8e, 16e, 22a and 22e) showed interesting growth-promoting activity. The cytotoxicity assay showed no toxicity of the prepared compounds on cancer and normal cell lines.

2-Deoxyglycoside Conjugates of Lupane Triterpenoids with High Cytotoxic Activity-Synthesis, Activity, and Pharmacokinetic Profile.

A set of 41 glycosidic conjugates of pentacyclic triterpenes was synthesized in order to improve the solubility of highly cytotoxic parent compounds. Their in vitro cytotoxic activity was evaluated in 25 cancer cell lines and 2 noncancer fibroblasts. Fifteen compounds had high cytotoxicity on the T-lymphoblastic leukemia cell line CCRF-CEM and 6 of them were active in multiple cell lines of various histogenic origin and not toxic in fibroblasts. Compound 11a had IC50 of 0.64 µM in CCRF-CEM cells, 0.60 µM in K-562 cells, and 0.37 µM in PC-3 cells; compound 12a had IC50 of 0.64 µM in CCRF-CEM cells and 0.71 µM in SW620 cells; compound 17b had IC50 of 0.86 µM in HCT116 cells and 0.92 µM in PC-3 cells. Compounds 11b and 12b were slightly less active than the previously mentioned derivatives; however, their solubility was significantly better, and therefore they were selected for the in vivo evaluation of the pharmacokinetic profile in mice. In both compounds, the maximum concentration in plasma was achieved very rapidly-the highest level in plasma was found 1 h after administration (22.2, respectively, 6.4 µM). For compound 12b, the resorption was followed with fast elimination, and 12 h after administration, the compound was not detected in plasma. In contrast, compound 11b was eliminated more slowly; it was still present in plasma after 12 h, but its concentration dropped below the detection limit after 24 h. The elimination half-time determined for compound 11b was 2.4 h and for compound 12b just about 1.4 h. These values are reasonable for further drug development.

Synthesis, characterization and antiproliferative activity of seco analogues of brassinosteroids.

Synthesis and structure-activity relationship analysis of a two groups of 2,3-seco analogues of brassinosteroids (BRs) were performed to examine their antiproliferative activities. Two steroid skeletons were chosen for the preparation of seco analogues - cholestane and stigmastane. The synthetic strategy consists of multistep reactions and detailed analysis of compounds prepared. We have discovered unpublished behaviour of 2,3-seco-2,3-dihydroxy-6-ketones leading to formation of intramolecular ketal with two new steroidal rings. Their reaction intermediates were also characterized in some cases. All compounds prepared were fully characterized with NMR and MS techniques. Most of compounds were tested for in vitro cytotoxicity on three cancer cell lines (CEM, MCF7, and HeLa) and normal human fibroblasts (BJ). It was discovered that some seco analogues caused apoptosis in cancer cells. The most promising seco derivative 28 proved to have high therapeutic index.

The novel brassinosteroid analog BR4848 inhibits angiogenesis in human endothelial cells and induces apoptosis in human cancer cells in vitro.

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17ß-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G2/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G2/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERß, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.

Regulation of the membrane structure by brassinosteroids and progesterone in winter wheat seedlings exposed to low temperature.

Steroids constitute one of the most important groups of compounds of regulatory properties both in the animal and plant kingdom. In plants, steroids such as brassinosteroids or progesterone, by binding to protein receptors in cell membranes, regulate growth and initiate processes leading to increased tolerance to stress conditions. Due to their structural similarities to sterols, these steroids may also directly interact with cellular membranes. Our aim was to determine the changes of the structural parameters of lipid membranes under the influence of hydrophobic steroid compounds, i.e., 24-epibrassinolide (EBR) and its precursor-24-epicastasterone (ECS) and progesterone (PRO). Lipids were isolated from wheat seedlings with different tolerances to frost, grown at low temperatures (5 °C) for 1.5 and 3 weeks (acclimation process). Control plants were cultured continuously at 20 °C. From galactolipids and phospholipids, the main polar lipid fractions, the monolayers were formed, using a technique of Langmuir trough. EBR and ECS were introduced into monolayers, together with lipids, whereas the PRO was dissolved in the aqueous sub-phase upon which the monolayers were spread. Measurements performed at 25 °C and 10 °C showed a significant action of the tested compounds on the physicochemical properties of the monolayers. EBR and PRO increased the area per lipid molecule in monolayers, resulting in formation of more flexible surface structures while the presence of the ECS induced the opposite effect. The influence of the polarity of lipids and steroids on the interactions in the monolayer was discussed. Lipids extracted from the membranes of wheat with the most tolerance to frost were characterized by the highest fatty acid unsaturation and steroids had a relatively weak effect on the parameters of the structure of their monolayers.

Synthesis and antiproliferative properties of new hydrophilic esters of triterpenic acids.

To improve the properties of cytotoxic triterpenoid acids 1-5, a large set of hydrophilic esters was synthesized. We choose betulinic acid (1), dihydrobetulinic acid (2), 21-oxoacid 3 along with highly active des-E lupane acids 4 and 5 as a model set of compounds for esterification of which the properties needed to be improved. As ester moieties were used - methoxyethanol and 2-(2-methoxyethoxy)ethanol and glycolic unit (type a-d), pyrrolidinoethanol, piperidinoethanol and morpholinoethanol (type f-h), and monosaccharide groups (type i-l). As a result, 56 triterpenic esters (49 new compounds) were obtained and their cytotoxicity on four cancer cell lines and normal human fibroblasts was tested. All new compounds were fully soluble at all tested concentrations, which used to be a problem of the parent compounds 1 and 2. 16 compounds had IC50 < 10 µM on at least one cancer cell line, 12 compounds had cytotoxicity of <10 µM against at least three of four tested cancer cell lines. The highest activity was found for compound 3c (1.8 µM on MCF7, 2.8 µM on HeLa, and 1.6 µM on G-361 cells) which also had no toxicity on non-cancerous BJ fibroblasts at the highest tested concentration (50 µM). High selective cytotoxicity was also found in compounds 1k, 2k, 3c, and 3i that are ideal candidates for drug development. Therefore, more studies to identify the mechanism of action were performed in case of 1k, 3c, and 3g such as effects on cell cycle and apoptosis. It was found that compounds 3c and 3g can induce apoptosis via caspase-3 activation and modulation of protein Bcl-2 in G-361 cells. In conclusion, compounds 1k, 3c, and 3g show high and selective cytotoxicity, therefore they are significantly better candidates for anti-cancer drug development than the parent acids 1-5.

Synthesis of novel aryl brassinosteroids through alkene cross-metathesis and preliminary biological study.

A series of phenyl analogues of brassinosteroids was prepared via alkene cross-metathesis using commercially available styrenes and 24-nor-5α-chola-2,22-dien-6-one. All derivatives were successfully docked into the active site of BRI1 using AutoDock Vina. Plant growth promoting activity was measured using the pea inhibition biotest and Arabidopsis root sensitivity assay and then was compared with naturally occuring brassinosteroids. Differences in the production of plant hormone ethylene were also observed in etiolated pea seedlings after treatment with the new and also five known brassinosteroid phenyl analogues. Antiproliferative activity was also studied using normal human fibroblast and human cancer cell lines.

Synthesis of 3α-deuterated 7α-hydroxy-DHEA and 7-oxo-DHEA and application in LC-MS/MS plasma analysis.

7-Oxygenated metabolites of dehydroepiandrosterone (DHEA) are known for their neuroprotective and immunomodulatory properties. These neuroactive steroids are currently predominately analysed by mass spectrometry, for which the use of internal deuterated standards is necessary. The aim of this study was to synthesize the deuterated derivatives of 7α-hydroxy-DHEA and 7-oxo-DHEA and test them in liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to enhance the performance characteristics of this method. Here we report the synthesis of 3α deuterium-labelled 7α-hydroxy-DHEA and 7-oxo-DHEA. Deuterium was introduced into the 3α position by reduction of the corresponding 3-ketone with a protected 17-carbonyl group using NaBD4. Our new procedure allows the easier synthesis of deuterated steroid labelled compounds. The use of these deuterated steroids enabled us to improve the human plasma LC-MS/MS analysis of 7α-hydroxy-DHEA and 7-oxo-DHEA in terms of sensitivity, precision and recovery.

Structure activity relationship studies on cytotoxicity and the effects on steroid receptors of AB-functionalized cholestanes.

Structure-activity relationship analysis and profiling of a library of AB-functionalized cholestane derivatives closely related to brassinosteroids (BRs) were performed to examine their antiproliferative activities and activities on steroid hormone receptors. Some of the compounds were found to have strong cytotoxic activity in several human normal and cancer cell lines. The presence of a 3-hydroxy or 3-oxo group and 2,3-vicinal diol or 3,4-vicinal diol moiety were found to be necessary for optimum biological activity, as well as a six-membered B ring. According to the profiling of all steroid receptors in both agonist and antagonist mode, the majority of the cholestanes were weakly active or inactive compared to the natural ligands. Estrogenic activity was detected for two compounds, two compounds possessed antagonistic properties on estrogen receptors and seven compounds showed agonistic activity. Two active cholestane derivatives were shown to strongly influence cell viability, proliferation, cell cycle distribution, apoptosis and molecular pathways responsible for these processes in hormone-sensitive/insensitive (MCF7/MDA-MB-468) breast cancer cell lines.

Pentacyclic triterpenoids with nitrogen- and sulfur-containing heterocycles: synthesis and medicinal significance.

Triterpenoids are natural compounds with a variety of biological activities and are usually produced by plants as secondary metabolites. In recent decades, scientists focused on the properties of triterpenoids have discovered many activities, such as antitumor, antiviral, antimicrobial, anti-inflammatory and others. Thousands of new triterpenoids with various skeletal modifications have now been synthesized. One of the most important modifications is the formation of a new heterocyclic ring. The simple fact that the vast majority of currently used drugs are heterocyclic compounds has encouraged a lot of researches to synthesize analogous triterpenoid derivatives in order to find new molecules with higher activities and consequently optimize their pharmacological profile. The biological properties of triterpenoid heterocycles are very promising and many of them have been studied, especially as antitumor agents. This review is mainly focused on the synthesis of various types of nitrogen and occasionally sulfur-containing heterocyclic triterpenoids for their potential use as future drugs in medicine and in addition discusses their overall biological activities.