Effects of Lactone- and Ketone-Brassinosteroids of the 28-Homobrassinolide Series on Barley Plants under Water Deficit.

The aim of this work was to study the ability of 28-homobrassinolide (HBL) and 28-homocastasterone (HCS) to increase the resistance of barley (Hordeum vulgare L.) plants to drought and to alter their endogenous brassinosteroid status. Germinated barley seeds were treated with 0.1 nM HBL or HCS solutions for two hours. A water deficit was created by stopping the watering of 7-day-old plants for the next two weeks. Plants responded to drought through growth inhibition, impaired water status, increased lipid peroxidation, differential effects on antioxidant enzymes, intense proline accumulation, altered expression of genes involved in metabolism, and decreased endogenous contents of hormones (28-homobrassinolide, B-ketones, and B-lactones). Pretreatment of plants with HBL reduced the inhibitory effect of drought on fresh and dry biomass accumulation and relative water content, whereas HCS partially reversed the negative effect of drought on fresh biomass accumulation, reduced the intensity of lipid peroxidation, and increased the osmotic potential. Compared with drought stress alone, pretreatment of plants with HCS or HBL followed by drought increased superoxide dismutase activity sevenfold or threefold and catalase activity (by 36%). The short-term action of HBL and HCS in subsequent drought conditions partially restored the endogenous B-ketone and B-lactone contents. Thus, the steroidal phytohormones HBL and HCS increased barley plant resistance to subsequent drought, showing some specificity of action.

Synthesis and biological activity of 21,22-cyclosteroids and their derivatives.

Synthesis of 21,22-cyclosteroids has been achieved starting from pregnenolone acetate. The key transformation was the Kulinkovich reaction of 17-vinyl steroids with esters. The resulting cyclopropanols were further subjected to three-membered ring-opening under various conditions including to base-, palladium or visible light-promoted isomerization and cross-coupling reaction. A number of steroidal Δ2-6-ketones and 3ß-hydroxy-Δ5-enes with functional groups at C-21 - C-23 have been synthesized via the 21,22-cyclosteroids. The antiproliferative and antihormonal activity of the obtained compounds on the cell lines of prostate (22Rv1) and breast (MCF-7) cancer was studied. The androgen receptor activity was assessed by reporter assay when the expression of signalling proteins was evaluated by immunoblotting. (20S,22R)-22-Acetoxy-21,22-cyclo-5α-cholest-5-ene with the moderate antiandrogenic potency revealed IC50 values of 18.4 ± 1.2 and 14.6 ± 1.4 µM against MCF-7 and 22Rv1 cells, respectively, and its effects on the expression of AR-V7, cyclin D1 and BCL2 were explored.

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.

Structure and Biological Activity of Ergostane-Type Steroids from Fungi.

Mushrooms are known not only for their taste but also for beneficial effects on health attributed to plethora of constituents. All mushrooms belong to the kingdom of fungi, which also includes yeasts and molds. Each year, hundreds of new metabolites of the main fungal sterol, ergosterol, are isolated from fungal sources. As a rule, further testing is carried out for their biological effects, and many of the isolated compounds exhibit one or another activity. This study aims to review recent literature (mainly over the past 10 years, selected older works are discussed for consistency purposes) on the structures and bioactivities of fungal metabolites of ergosterol. The review is not exhaustive in its coverage of structures found in fungi. Rather, it focuses solely on discussing compounds that have shown some biological activity with potential pharmacological utility.

BODIPY Conjugate of Epibrassinolide as a Novel Biologically Active Probe for In Vivo Imaging.

Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene steroids.

A photochemical approach to 18-nor-17ß-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone.

Anabolic/anticatabolic and adaptogenic effects of 24-epibrassinolide on Lupinus angustifolius: Causes and consequences.

Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N2-fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N2-fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N2-fixing activity of nitrogenase and the accumulation of poly-ß-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N2 fixation system in root nodules, as well as on protein synthesis in ripening seeds.

Biological Evaluation of a New Brassinosteroid: Antiproliferative Effects and Targeting Estrogen Receptor α Pathways.

Brassinosteroids (BS), a class of plant-specific steroid hormones, are considered as new potential anticancer agents for the treatment of tumors of different origin, including hormone-dependent cancers. Effects of a synthetic brassinosteroid BS4 ((22R,23R,24R)-22,23-dihydroxy-24-methyl-B-homo-7-oxa-5α-cholest-2-en-6-one ((3aS,7aR,7bS,9aS,10R,12aS,12bS)-10-[(2S,3R,4R,5R)-3,4-dihydroxy-5,6-dimethylheptan-2-yl]-7a,9a-dimethyl-1,3a,4,7,7a,7b,8,9,9a,10,11,12,12a,12b-tetradecahydro-3H-benzo[c]indeno[5,4-e]oxepin-3-one)) on hormone-dependent breast cancer cells and normal epithelial cells and its impact on the estrogen receptor signaling were evaluated. Cytotoxicity was assessed by MTT-test; expression of estrogen receptor α and survivin was measured by immunoblotting. Transactivation analysis of luciferase reporter gene was performed for ERα and AP-1 factors after the brassinosteroid treatment. Dock6 and Autodock Vina were used for molecular docking. BS4 revealed a significant antiproliferative effect towards the hormone-dependent breast cancer cells and was not active against normal epithelial cells. BS4 action on MCF-7 breast cancer cells was found to be complex: a decrease in ERα expression as well as in its transcription activity was accompanied by inhibition of ERα-related signaling pathways (AP-1 complex and survivin). BS4 binding mode to ERα ligand-binding domain was analyzed by molecular docking. The obtained results show that antiproliferative and antiestrogenic properties of the brassinosteroid BS4, as well as its ability to inhibit the anti-apoptotic protein survivin may be of interest for further development of anticancer agents.

Enantioselective catalytic approach to the C23-C28 subunit of 24α-methyl steroids.

Enantioselective synthesis of C23-C28 subunit of campestane steroids based on catalytic methods is reported. The synthesis was started from (S)-2-isopropyl-4-nitrobutan-1-ol, which is easily accessible by the reaction between isovaleraldehyde and nitroethylene catalyzed by only 2% of (S)-trimethylsilyldiphenylprolinol. Removal of one "extra" carbon from the nitroalcohol was achieved by Ni-catalyzed hydrodecarboxylation of the redox-active ester intermediate. The synthesized C23-C28 fragment was attached to a steroidal core by Julia-Kocienski reaction of a steroidal aldehyde with metallated C23-C28 sulfone. The obtained product of olefination was easily transformed to a precursor of campesterol and (Z)-22-dehydrocampesterol.

Molecular rearrangements of poststerone derivative steroid core with formation of unique D-homostructures of pregnane and androstane series.

20R-Hydroxy short-chain ecdysteroids were synthesized by chemo- and stereoselective reduction of poststerone acetonide with L-Selectride or LiAlH4. The same reaction with the excess of L- Selectride followed by the treatment of the reaction mixture with hydrochloric acid is accompanied by (8R)-13(14 → 8)abeo- rearrangements, which resulted in the contraction/expansion of C/D pregnane rings. The reaction of 20R-hydroxy poststerone analogs with (diethylamino)sulfur trifluoride (DAST) proceeds through intramolecular rearrangements and provides D-homo- or 13,14-seco- androstane structures.

Regio- and stereoselective C-H functionalization of brassinosteroids.

Late stage CH functionalization is a powerful tool for modification of natural compounds. Herein we report that the rhodium-catalyzed reaction of brassinosteroids with aryloxysulfonamides proceeds regio- and stereoselectively at C15 position. The derivative obtained from 24-epibrassinolide was easily transformed to the conjugate with a BODIPY dye bearing unaffected functional groups of the native brassinosteroid.

New azole derivatives of [17(20)E]-21-norpregnene: Synthesis and inhibition of prostate carcinoma cell growth.

A number of isoxazole, 1,2,3-triazole, tetrazole, and 1,2,4-oxadiazole derivatives of [17(20)E]-21-norpregnene comprising 3ß-hydroxy-5-ene and 3-oxo-4-ene fragments were prepared. Among the key steps for the synthesis of isoxazoles, 1,2,3-triazoles, and tetrazoles were (i) 1,3-dipolar cycloaddition of nitrile oxides or azides to acetylenes or nitriles and ii) dehydration of 17ß-hydroxy-17α-methylene-azoles to [17(20)E]-21-norpregnene derivatives. 1,2,4-Oxadiazoles were prepared through the formation of acetimidamides. Potency of the synthesized compounds to inhibit CYP17A1 and to suppress growth of prostate carcinoma cells was investigated. Among the new azole derivatives, four compounds were found possessing high anti-proliferative activity.

Stereoselective synthesis of α-methyl and α-alkyl ketones from esters and alkenes via cyclopropanol intermediates.

Alkenes bearing a stereocenter in the allylic position were found to undergo Kulinkovich hydroxycyclopropanation with good diastereoselectivity. For the isomerization of the resulting cyclopropanols to diastereomerically enriched α-methyl ketones, a new mild regioselective method has been developed. A sequence of stereoselective cyclopropanation and cyclopropanol ring opening was successfully employed for the construction of the C20 stereocenter in steroids.

Synthesis of ergostane-type brassinosteroids with modifications in ring A.

Herein, we present a new strategy for the preparation of a broad range of brassinosteroid biosynthetic precursors/metabolites differing by the ring A fragment. The protocol is based on the use of readily available phytohormones of this class bearing a 2α,3α-diol moiety (epibrassinolide or epicastasterone) as starting materials. The required functionalities (Δ2-, 2α,3α- and 2ß,3ß-epoxy-, 2α,3ß-, 2ß,3α-, and 2ß,3ß-dihydroxy-, 3-keto-, 3α- and 3ß-hydroxy-, 2α-hydroxy-3-keto-) were synthesized from 2α,3α-diols in a few simple steps (Corey-Winter reaction, epoxidation, oxidation, hydride reduction, etc.).

Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

Synthesis of sulfated brassinosteroids.

A number of water soluble sulfates of 24-epibrassinolide including the 2α,3α-disulfate and all possible monosulfates were synthesized. The target compounds were isolated in crystalline form as the corresponding sodium salts. Pyridine-sulfur trioxide complex was used as sulfating agent followed by transformation of the resulting pyridinium salts into the sodium sulfates by treatment with NaOH. The control of the regioselectivity was achieved by an appropriate use of acetyl and benzyl protecting groups.

Flow-cytometric analysis of reactive oxygen species in cancer cells under treatment with brassinosteroids.

To explore the underlying mechanism of cancer cell growth inhibition by brassinosteroids (BS), reactive oxygen species (ROS) generation under treatment with 28-homocastasterone and its synthetic derivatives (22S,23S)-28-homocastasterone was measured in A549 human lung adenocarcinoma cells. BS induced ROS generation in A549 cells and their growth in a time and dose-dependent manner. The maximal effect was observed for (22S,23S)-28-homocastasterone which at 30µM concentration showed a 6-fold increase of ROS generation in comparison with the control.

Effect of 24-epibrassinolide on Brassica napus alternative respiratory pathway, guard cells movements and phospholipid signaling under salt stress.

Using Brassica napus roots we observed statistically significant increase in alternative respiratory pathway in response to exogenous 24-epibrassinolide (EBL) under optimal conditions and salinity. Also we observed activation of phospholipid signaling under the same conditions in response to EBL by measuring levels of lipid second messengers - diacylglycerol (DAG) and phosphatidic acid (PA). We found that brassinosteroids cause closure of stomata in isolated leaf disks while inhibitors of alternative oxidase cancelled these effects. This study demonstrates that BRs activate total respiration rate, alternative respiratory pathway, production of PA and DAG, stimulate stomata closure and growth under optimal conditions and salinity. Also, specific inhibitor of brassinosteroids biosynthesis decreased alternative respiratory pathway and production of lipid messengers in rape plants.

C-H Acetoxylation-Based Chemical Synthesis of 17 ß-Hydroxymethyl-17 α-methyl-18-norandrost-13-ene Steroids.

Palladium-catalyzed C-H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17ß-hydroxymethyl-17α-methyl-18-nor-13-ene D-fragment. This C-H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti-doping analysis to control the abuse of this androgenic anabolic steroid.

An improved synthesis of [26-(2)H3]castasterone.

Commercially available epicastasterone has been employed as a starting material for the preparation of [26-(2)H3 ]castasterone. The chemical synthesis has been realized in 13 chemical steps and 4.6% total yield. The target compound is intended to be used as internal standard for the quantitative analysis of brassinosteroids.