Highly Oxidized Ecdysteroids from a Commercial Cyanotis arachnoidea Root Extract as Potent Blood-Brain Barrier Protective Agents.

Ecdysteroid-containing herbal extracts, commonly prepared from the roots of Cyanotis arachnoidea, are marketed worldwide as a "green" anabolic food supplement. Herein are reported the isolation and complete 1H and 13C NMR signal assignments of three new minor ecdysteroids (compounds 2-4) from this extract. Compound 4 was identified as a possible artifact that gradually forms through the autoxidation of calonysterone. The compounds tested demonstrated a significant protective effect on the blood-brain barrier endothelial cells against oxidative stress or inflammation at a concentration of 1 µM. Based on these results, minor ecdysteroids present in food supplements may offer health benefits in various neurodegenerative disease states.

Diversity-Oriented Synthesis Catalyzed by Diethylaminosulfur-Trifluoride-Preparation of New Antitumor Ecdysteroid Derivatives.

Fluorine represents a privileged building block in pharmaceutical chemistry. Diethylaminosulfur-trifluoride (DAST) is a reagent commonly used for replacement of alcoholic hydroxyl groups with fluorine and is also known to catalyze water elimination and cyclic Beckmann-rearrangement type reactions. In this work we aimed to use DAST for diversity-oriented semisynthetic transformation of natural products bearing multiple hydroxyl groups to prepare new bioactive compounds. Four ecdysteroids, including a new constituent of Cyanotis arachnoidea, were selected as starting materials for DAST-catalyzed transformations. The newly prepared compounds represented combinations of various structural changes DAST was known to catalyze, and a unique cyclopropane ring closure that was found for the first time. Several compounds demonstrated in vitro antitumor properties. A new 17-N-acetylecdysteroid (13) exerted potent antiproliferative activity and no cytotoxicity on drug susceptible and multi-drug resistant mouse T-cell lymphoma cells. Further, compound 13 acted in significant synergism with doxorubicin without detectable direct ABCB1 inhibition. Our results demonstrate that DAST is a versatile tool for diversity-oriented synthesis to expand chemical space towards new bioactive compounds.

Ecdysteroids from the Aerial Parts of Paris verticillata.

Two new ecdysteroids 14-epi-polypodine B (1) and 22-oxo-hydroxyecdysterone (2), along with nine known compounds, polypodine B (3), viticosterone E (4), 20-hdroxyecdysone-2-acetate (5), 22-oxo-20-hydroxyecdysone (6), 5-hydroxyecdysone (7), pinnatasterone (8), 3-epi-20-hydroxyecdysone (9), ecdysterone (10) and stachysterone B (11), were isolated from the aerial parts of Paris verticillata. The structures of all compounds were elucidated by extensive spectroscopic analysis, quantum chemical calculations and ANN-PRA/DP4+ probability analysis. Among them, the absolute configuration of compound 1 and 2 was unambiguous determined by ECD. Also, the isolated compounds were assessed for their cytotoxic activities. Compounds 2, 3 and 7 exhibited significant cytotoxic activities against PC12, LN299 and SMCC7721 cells.

A Commercial Extract of Cyanotis arachnoidea Roots as a Source of Unusual Ecdysteroid Derivatives with Insect Hormone Receptor Binding Activity.

Ecdysteroids act as molting hormones in insects and as nonhormonal anabolic agents and adaptogens in mammals. A wide range of ecdysteroid-containing herbal extracts are available worldwide as food supplements. The aim of this work was to study such an extract as a possible industrial source of new bioactive ecdysteroids. A large-scale chromatographic isolation was performed from an extract of Cyanotis arachnoidea roots. Ten ecdysteroids (1-10) including eight new compounds were isolated and characterized by extensive nuclear magnetic resonance studies. Highly unusual structures were identified, including a H-14ß (1, 2, 4, and 10) moiety, among which a 14ß(H)17ß(H) phytosteroid (1) is reported for the first time. Compounds with an intact side chain (4-10) and 11 other natural or semisynthetic ecdysteroids (11-21) were tested for insect ecdysteroid receptor (EcR) binding activity. Two new compounds, i.e., 14-deoxydacryhainansterone (5) and 22-oxodacryhainansterone (6), showed strong EcR binding activity (IC50 = 41.7 and 380 nM, respectively). Six compounds were identified as EcR agonists and another two as antagonists using a transgenic ecdysteroid reporter gene assay. The present results demonstrate that commercial C. arachnoidea extracts are rich in new, unusual bioactive ecdysteroids. Because of the lack of an authentic plant material, the truly biosynthetic or artifactual nature of these compounds cannot be confirmed.

Diversity-oriented synthesis through gamma radiolysis: Preparation of unusual ecdysteroid derivatives activating Akt and AMPK in skeletal muscle cells.

Gamma-ray radiation is a unique way to induce chemical transformations of bioactive compounds. In the present study, we pursued this approach to the diversity-oriented synthesis of analogs of 20-hydroxyecdysone (20E), an abundant ecdysteroid with a range of beneficial, non-hormonal bioactivities in mammals including humans. Gamma irradiations of aqueous solutions of 20E were conducted either in N2- or N2O-saturated solutions. Centrifugal partition chromatography was used to fractionate crude resulting irradiated materials using a biphasic solvent system composed of tert-butyl alcohol - ethyl acetate - water (0.45:0.9:1, v/v/v) in ascending mode. Subsequently, the products were purified by RP-HPLC. Fourteen ecdysteroids, including five new compounds, were isolated, and their structure were elucidated by 1D and 2D NMR and HRMS. Compounds 2-4, 7, 9, 12 and 15 were tested for their capacity to increase the Akt- and AMPK-phosphorylation of C2C12 murine skeletal myotubes in vitro. The compounds were similarly active on Akt as their parent compound. Stachysterone B (7) and a new ring-rearranged compound (12) were more potent than 20E in activating AMPK, indicating a stronger cytoprotective effect. Our results demonstrate the use of gamma irradiation in expanding the chemical diversity of ecdysteroids to obtain new, unusual bioactive metabolites.

Naturally Occurring Ecdysteroids in Triticum aestivum L. and Evaluation of Fenarimol as a Potential Inhibitor of Their Biosynthesis in Plants.

Ecdysteroids (ECs) are steroid hormones originally found in the animal kingdom where they function as insect molting hormones. Interestingly, a relatively high number of these substances can also be formed in plant cells. Moreover, ECs have certain regulatory effects on plant physiology, but their role in plants still requires further study. One of the main aims of the present study was to verify a hypothesis that fenarimol, an inhibitor of the biosynthesis of ECs in the animal kingdom, also affects the content of endogenous ECs in plants using winter wheat Triticum aestivum L. as a model plant. The levels of endogenous ECs in winter wheat, including the estimation of their changes during a course of different temperature treatments, have been determined using a sensitive analytical method based on UHPLC-MS/MS. Under our experimental conditions, four substances of EC character were detected in the tissue of interest in amounts ranging from less than 1 to over 200 pg·g-1 FW: 20-hydroxyecdysone, polypodine B, turkesterone, and isovitexirone. Among them, turkesterone was observed to be the most abundant EC and accumulated mainly in the crowns and leaves of wheat. Importantly, the level of ECs was observed to be dependent on the age of the plants, as well as on growth conditions (especially temperature). Fenarimol, an inhibitor of a cytochrome P450 monooxygenase, was shown to significantly decrease the level of naturally occurring ECs in experimental plants, which may indicate its potential use in studies related to the biosynthesis and physiological function of these substances in plants.

Bufadienolides and ecdysteroids from the whole plants of Helleborus niger and their cytotoxicity.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5-8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1-4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1-6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

Retinoid X receptor modulates vitellogenin gene expression in black tiger shrimp, Penaeus monodon.

Effective inducing of ovarian maturation in female shrimp broodstock is important for successful breeding programs. Vitellogenesis is a biochemical process during which a yolk protein precursor vitellogenin (Vg) is synthesized and thus, can be used to indicate ovarian maturation stage. In this study, transcriptional regulation of Vg synthesis in the black tiger shrimp, Penaeus monodon was investigated. Genome walking on 5' upstream sequence of Vg gene revealed several putative binding sites of lipophilic retinoic acid response elements (RARE), and nuclear hormone responsive elements. Deletion of RARE significantly reduced the promoter activity to drive the expression of luciferase reporter gene in Sf-9 cells. To validate the trans-factor that potentially controls Vg expression through RARE, a cDNA encoding retinoid X receptor (PmRXR), one of the RARE-bound transcription factors was cloned from P. monodon's ovary. PmRXR expression was detected in various shrimp tissues, and was up-regulated during ovary development in a similar way to Vg expression. The DNA-binding domain of PmRXR protein showed specific binding to RARE-containing region on Vg 5' upstream sequence as determined by Electrophoretic Mobility Shift Assay (EMSA). Furthermore, dsRNA-mediated PmRXR silencing in previtellogenic and vitellogenic shrimp revealed that suppression of PmRXR could reduce Vg transcript in both stages. Taken together, the results presented in this study indicate that RXR is possibly an activator protein that modulates Vg expression in shrimp ovary through the binding to RARE.

Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance.

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14) were able to resensitize CEMVbl100 and LoVoDoxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 µM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

Ecdysteroids are present in the blood of wild passerine birds.

Ecdysteroids (arthropod molting hormones) play an important role in the development and sexual maturation of arthropods, and they have been shown to have anabolic and "energizing" effect in higher vertebrates. The aim of this study was to assess ecdysteroid diversity, levels according to bird species and months, as well as to observe the molting status of hard ticks (Acari: Ixodidae) infesting the birds. Therefore, blood samples and ticks were collected from 245 birds (244 songbirds and a quail). Mass spectrometric analyses showed that 15 ecdysteroids were regularly present in the blood samples. Molting hormones biologically most active in insects (including 20-hydroxyecdysone [20E], 2deoxy-20E, ajugasterone C and dacryhainansterone) reached different levels of concentration according to bird species and season. Similarly to ecdysteroids, the seasonal presence of affected, apolytic ticks peaked in July and August. In conclusion, this study demonstrates the presence of a broad range and high concentrations of ecdysteroids in the blood stream of wild-living passerine birds. These biologically active, anabolic compounds might possibly contribute to the known high metabolic rate of songbirds.

Side-chain cleaved phytoecdysteroid metabolites as activators of protein kinase B.

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete 1H and 13C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures.

Ecdysonelactones, Ecdysteroids from the Tropical Eastern Pacific Zoantharian Antipathozoanthus hickmani.

Despite a large occurrence, especially over the Pacific Ocean, the chemical diversity of marine invertebrates belonging to the order Zoantharia is largely underexplored. For the two species of the genus Antipathozoanthus no chemical study has been reported so far. The first chemical investigation of Antipathozoanthus hickmani collected at the Marine Protected Area "El Pelado", Santa Elena, Ecuador, led to the isolation of four new ecdysteroid derivatives named ecdysonelactones. The structures of ecdysonelactones A-D (1-4) were determined based on their spectroscopy data, including 1D and 2D NMR and HRMS. The four compounds of this family of ecdysteroids feature an unprecedented γ-lactone fused at the C-2/C-3 position of ring A. These derivatives exhibited neither antimicrobial nor cytotoxic activities.

Nitrogen-containing ecdysteroid derivatives vs. multi-drug resistance in cancer: Preparation and antitumor activity of oximes, oxime ethers and a lactam.

Multidrug resistance is a widespread problem among various diseases and cancer is no exception. We had previously described the chemo-sensitizing activity of ecdysteroid derivatives with low polarity on drug susceptible and multi-drug resistant (MDR) cancer cells. We have also shown that these molecules have a marked selectivity towards the MDR cells. Recent studies on the oximation of various steroid derivatives indicated remarkable increase in their antitumor activity, but there is no related bioactivity data on ecdysteroid oximes. In our present study, 13 novel ecdysteroid derivatives (oximes, oxime ethers and a lactam) and one known compound were synthesized from 20-hydroxyecdysone 2,3;20,22-diacetonide and fully characterized by comprehensive NMR techniques revealing their complete 1H and 13C signal assignments. The compounds exerted moderate to strong in vitro antiproliferative activity on HeLa, SiHa, MCF-7 and MDA-MB-231 cell lines. Oxime and particularly oxime ether formation strongly increased their inhibitory activity on the efflux of rhodamine 123 by P-glycoprotein (P-gp), while the new ecdysteroid lactam did not interfere with the efflux function. All compounds exerted potent chemo-sensitizing activity towards doxorubicin on a mouse lymphoma cell line and on its MDR counterpart, and, on the latter, the lactam was found the most active. Because of its MDR-selective chemo-sensitizing activity with no functional effect on P-gp, this lactam is of high potential interest as a new lead for further antitumor studies.

Isolation and Purification of Three Ecdysteroids from the Stems of Diploclisia glaucescens by High-Speed Countercurrent Chromatography and Their Anti-Inflammatory Activities In Vitro.

High-speed counter-current chromatography was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water (3:0.2:0.8:3, v/v). Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolicextractof D. glaucescens in one-step separation, which were identified as paristerone (I, 30.5 mg), ecdysterone (II, 7.2 mg), and capitasterone (III, 8.1 mg) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Their anti-inflammatory activities were evaluated by measuring the inhibitory ratios of ß-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds I-III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 µM, respectively.

Backstabbing P-gp: Side-Chain Cleaved Ecdysteroid 2,3-Dioxolanes Hyper-Sensitize MDR Cancer Cells to Doxorubicin without Efflux Inhibition.

P-glycoprotein (P-gp, ABCB1) over-expression, causing a multi-drug resistant (MDR) phenotype, is a major problem in cancer chemotherapy that urgently requires novel approaches. Our previous studies showed certain ecdysteroid derivatives as promising chemo-sensitizers against MDR and non-MDR cancer cell lines while also exerting mild to moderate inhibition of P-gp function. Here we report the preparation of a set of substituted 2,3-dioxolane derivatives of poststerone, a known in vivo metabolite of 20-hydroxyecdysone (20E). In contrast with previously studied ecdysteroid dioxolanes, the majority of the new compounds did not inhibit the efflux function of P-gp. Nevertheless, a strong, dose dependent sensitization to doxorubicin was observed on a P-gp transfected cancer cell line and on its susceptible counterpart. We also observed that the MDR cell line was more sensitive to the compounds' effect than the non-MDR. Our results showed for the first time that the chemo-sensitizing activity of ecdysteroids can be fully independent of functional efflux pump inhibition, and suggest these compounds as favorable leads against MDR cancer.

New ecdysteroid and ecdysteroid glycosides from the roots of Serratula chinensis.

Three new ecdysteroid glycosides (1-3) and one new ecdysteroid (4), were isolated from the roots of Serratula chinensis. Their structures were established on the basis of extensive spectroscopic analysis and chemical methods.

BBB penetration-targeting physicochemical lead selection: Ecdysteroids as chemo-sensitizers against CNS tumors.

The anticancer potential of ecdysteroids, especially their chemo-sensitizing activity has recently gained a substantial scientific interest. A comprehensive physicochemical profiling was performed for a set of natural or semi-synthetic ecdysteroids (N=37) to identify a lead compound against central nervous system (CNS) tumors. Calculated properties, such as lipophilicity (clogP), topological polar surface area (TPSA), brain-to-plasma ratio (clogBB) along with the measured blood-brain barrier specific in vitro permeability (logPe) were evaluated in parallel. Compounds with the highest CNS-availability predicted (clogBB>0.0 and logPe>-6.0) showed moderate to high lipophilicity (clogP=3.89-5.25), relatively low TPSA (94.45Å2), and shared a common apolar 2,3- and 20,22-diacetonide motif (25, 30-33). These ecdysteroids were selected for testing their capacity to sensitize SH-SY5Y neuroblastoma cells to vincristine. All of the five tested compounds exerted a remarkably strong, dose dependent chemo-sensitizing activity: at 2.5-10.0µM ecdysteroids increased the cytotoxic activity of vincristine one to three orders of magnitude in (e.g., from IC50=39.5±2.9nM to as low as 0.056±0.03nM). Moreover, analysis of the combination index (CI) revealed outstanding synergism between ecdysteroids and vincristine (CI50=0.072-0.444). Thus, based on drug-likeness, physchem character and in vitro CNS activity, compound 25 was proposed as a lead for further in vivo studies.

A new phytoecdysteroid from the roots of Achyranthes bidentata Bl.

A new phytoecdysteroid compound, named Niuxixinsterone D (1), together with two known phytoecdysteroid compounds (2 and 3) were isolated from Achyranthes bidentata Bl.. The structure of the new compound was elucidated by extensive spectral analysis, including HR-ESI-MS, 1D and 2D NMR methods. Compounds 1-3 were tested for their inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages, and compound 1 and 3 exhibited anti-neuroinflammatory activity with inhibited 29.7 and 26.0% NO production.

Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki.

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2-9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 µg/mL), which is better than that of the positive control, 2'CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS-NP computational analysis. The ChemGPS-NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space.