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The total synthesis of (+)-03219A, a rare Δ8,9-pregnene isolated from the marine-derived Streptomyces sp. SCSIO 03219, is described that is based on a series of transformations that enable progression from epichlorohydrin to an ent-estrane, then conversion to a nat-androstane, and finally establishment of the natural product target. Key to the success of these studies was implementation of two rearrangement processes to formally invert the quaternary center at C13 and establish the C10 quaternary center.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

The IL6/JAK2/STAT3 axis dysregulation and the related downstream pathways are a major contributor to the progression of non-small-cell lung carcinoma (NSCLC) and mainly affect apoptosis. Furthermor...

Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17β-reduced derivatives thus evidencing the presence of 7α-, 7β-hydroxylase and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7β-position, while much lower 7α-hydroxylation was observed. Along with 7β-hydroxy-ADD and its corresponding 7α-isomer, their respective 17β-alcohols were produced. In this study, transformation of ADD, androst-4-en-17β-ol-3-one (testosterone, TS) and androst-5-ene-3β,17β-diol (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17β-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, 1H and 13C NMR spectroscopy data. 17β-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17β-alcohols, namely, androsta-1,4-diene-17β-ol-3-one (1-dehydro-TS), and androst-5-ene-3β,17β-diol. Production of the 7α- and 7β-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. β-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction. Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/β)- and 17β-alcohols.

Four isomeric N-alkynylaminosteroids of the androstane and pregnane series were synthesized and tested in vitro for inhibition of CYP450 17A1 heterologously expressed in transgenic yeast Yarrowia lipolytica. The highest inhibitory activity was observed in the case of steroids containing a side chain of five atoms. At equal concentrations of the substrate and the inhibitor (50 μM), the pregnenolone derivative containing the N-propynyl fragment caused a 5-fold decrease of reduced the progesterone conversion.

Withanolides are steroidal lactones widespread in Nightshade plants with often potent antiproliferative activities. Additionally, the structural diversity of this compound class holds much potential for the discovery of novel biological activity. Here, we report two newly characterised withanolides, named irinans, from Physalis peruviana with highly unusual truncated backbones that resemble mammalian androstane sex hormones. Based on biomimetic chemical reactions, we propose a model that links these compounds to withanolide biosynthesis. Irinans have potent antiproliferative activities, that are however lower than those of 4ß-hydroxywithanolide E. Our work establishes androwithanolides as a new subclass of withanolides.

Background Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood. Results High-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed. Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115. Conclusions High expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry. Electronic supplementary material The online version of this article (10.1186/s12896-019-0533-7) contains supplementary material, which is available to authorized users.

The interaction of human monooxygenases and pathogenic fungi with previously obtained esters of isomeric 7-methyl-19-nor-testosterones and a number of heteroaromatic acids – derivatives of pyridine and pyrazine, was studied. Interaction with the active center of CYP11B1 derivatives of steroids of the androstane series containing methyl group at C7 and residues of heteroaromatic acids at C17β is shown.

The induction of cytochrome P450s can result in reduced drug efficacy and lead to potential drug-drug interactions. The xenoreceptors-aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR)-play key roles in CYP induction by xenobiotics. In order to be able to rapidly screen for the induction of three enzymes (CYP1A1, CYP2B6, and CYP3A4), we generated a stable AhR-responsive HepG2 cell line, a stable CAR-responsive HepG2 cell line, and a stable PXR-responsive HepG2 cell line.To validate these stable xenoreceptor-responsive HepG2 cell lines, we evaluated the induction of the different Gaussia reporter activities, as well as the mRNA and protein expression levels of endogenous CYPs in response to different inducers.The induction of luciferase activity in the stable xenoreceptor-responsive HepG2 cell lines by specific inducers occurred in a concentration dependent manner. There was a positive correlation between the induction of luciferase activities and the induction endogenous CYP mRNA expression levels. These xenoreceptor-responsive HepG2 cell lines were further validated with known CYP1A1, CYP2B6, and CYP3A4 inducers.These stable xenoreceptor-responsive HepG2 cell lines may be used in preclinical research for the rapid and sensitive detection of AhR, CAR, and PXR ligands that induce CYP450 isoforms.

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16&alpha, brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16&alpha, of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding &beta, substituted analogues. For the 16&alpha, brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit &mu, M EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.

New steroidal D-homo androstane derivatives with 5β,6β-epoxy-3,16-dicarbonyl, 6α- and 6β-hydroxy-3,16-dicarbonyl and 3β,5α-dihydroxy-6,16-dicarbonyl moieties were synthesized and confirmed by NMR spectroscopy. Novel and starting compounds were evaluated for their potential cytotoxicity in vitro against seven human cancer cell lines (MCF-7, MDA-MB-231, PC3, HeLa, HT-29, A549 and CEM) and one human noncancerous cell line (MRC-5). The most sensitive cell line was MDA-MB-231 derived from female reproductive tissue, wherein all compounds showed moderate to strong cytotoxic activity. Also, new compound with 5β,6β-epoxy-3,16-dicarbonyl moieties showed strong cytotoxic activity against colon adenocarcinoma (HT-29). In this work, in silico ADME properties of novel compounds were assessed by comparing calculated molecular properties with Lipinski, Veber, Egan, Ghose and Muegge criteria. The synthesis and structure elucidation of new oxygen-containing androstane derivatives was reported. New derivatives were tested for their in silico ADME properties and cytotoxicity against different human cancer cell lines.

17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a mitochondrial enzyme known for its potential role in Alzheimer’s Disease (AD). 17β-HSD10, by its oxidative activity, could decrease the concentration of two important neurosteroids, allopregnanolone (ALLOP) and 17β-estradiol (E2), respectively preventing their neurogenesis and neuroprotective effects. Since the inhibition of 17β-HSD10 could lead to a new treatment for AD, we developed two biological assays using labeled ALLOP or E2 as substrates to measure the inhibitory activity of compounds against pure 17β-HSD10 protein. After the optimization of different parameters (time, concentration of enzyme, substrate and cofactor), analogs of the first reported steroidal inhibitor of 17β-HSD10 in intact cells were screened to determine their inhibitory potency for the ALLOP or the E2 oxidation. One compound, androstane derivative 5, possesses the best dual inhibition against both transformations (ALLOP, IC50 = 235 μM and E2, IC50 = 610 μM). Some compounds are dual inhibitors to a lesser extent, and others seem selective for one of the transformations in particular. By developing two reliable assays and by identifying a first generation of steroidal inhibitors of pure 17β-HSD10, this preliminary study opens the door to new and more potent inhibitors.

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating complex roles in cell and systems biology. Species differences in CAR's effector pathways remain poorly understood, including its role in regulating liver tumor promotion. We developed transgenic mouse models to assess genome-wide binding of mouse and human CAR, following receptor activation in liver with direct ligands and with phenobarbital, an indirect CAR activator. Genomic interaction profiles were integrated with transcriptional and biological pathway analyses. Newly identified CAR target genes included Gdf15 and Foxo3, important regulators of the carcinogenic process. Approximately 1000 genes exhibited differential binding interactions between mouse and human CAR, including the proto-oncogenes, Myc and Ikbke, which demonstrated preferential binding by mouse CAR as well as mouse CAR-selective transcriptional enhancement. The ChIP-exo analyses also identified distinct binding motifs for the respective mouse and human receptors. Together, the results provide new insights into the important roles that CAR contributes as a key modulator of numerous signaling pathways in mammalian organisms, presenting a genomic context that specifies species variation in biological processes under CAR's control, including liver cell proliferation and tumor promotion.

Background: Steroids play an important role in life because they can regulate a variety of biological processes and have been widely used in medicine namely as antiinflammatory, anabolic, contraceptives and anticancer drugs. In recent years, there has been an increasing interest in the introduction of the oxime group in a large variety of molecules in order to increase their biological effects. This review highlights steroidal oximes with anticancer properties and their potential mechanisms of action, as well as data on their relative potencies reported in literature in the last few years. Methods: To prepare this review, an extensive literature search was performed on three databases, PubMed, ISI Web of Knowledge and Science Direct, to generate a critical but comprehensive overview of the potential antitumor activities of steroidal oximes. The main keywords used for the search consisted of combinations of the following terms or their synonyms: steroidal oximes, anticancer activity and enzymatic inhibitory activity. The abstracts and full texts were evaluated for their clarity and scientific merit and to further help on the selection of other articles. Results: Over the last decades the introduction of oxime groups in the steroid scaffold is originating molecules with relevant antitumor activities, as well as steroid sulfatase, aromatase, 17α-hydroxylase-17,20-lyase, 5α-reductase and 17β-hydroxysteroid dehydrogenase type 1 inhibitory activities. As relevant examples, pregnenolone 20-oximes showed high activity as 17α-hydroxylase-17,20-lyase and 5α-reductase inhibitors and the introduction of an oxime group at C-6 in androstane series also led to relevant results as aromatase inhibitors. Interestingly, the introduction of this functional group frequently improves the bioactivity when compared with non-oxime analogous compounds, which can be due to extra interactions with biological targets. In addition, it has been observed that varying the position of the hydroximino group on the parent skeleton leads to remarkable changes in the antitumor activity. Conclusion: The recent advances in synthesis and in vitro bioactivity studies of steroidal oximes contributed to understand the potential interest of the introduction of this functional group in the steroidal nucleus in the development of anticancer molecules. Moreover, the cytotoxic/enzyme inhibitory activity usually depends on the position of the oxime group in different steroid scaffolds. However, despite the promising results, it is necessary to perform more in vitro and in vivo assays not only to better explore the mechanisms of action but also to confirm the potential effectiveness and safety of this interesting family of compounds in clinical practice.

d -ring–fused and d -homo lactone compounds in estratriene and androstane series were synthesized using microwave-assisted reaction conditions. Microwave-irradiated synthesis methods were convenient and effective, and provided high yields with short reaction times. Their inhibition of C17,20-lyase and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) activities were studied in in vitro enzyme assays. d -ring–fused triazolyl estrone analog 24 showed potent inhibition of NADH-complexed 17β-HSD1, with a binding affinity similar to that of the substrate estrone; its inhibition against NADPH-complexed 17β-HSD1 was markedly weaker. Compound 24 also significantly and selectively reduced proliferation of cancer cell lines of gynecological origin. This estrane triazole changed the cell cycle and induced apoptosis of HeLa, SiHa, and MDA-MB-231 cancer cells, measured by both increased subG1 fraction of cells and activation of caspase-independent signaling pathways. A third mode of anti-estrogenic action of 24 saw increased mRNA expression of the SULT1E1 gene in HeLa cells; in contrast, its 3-benzyloxy analog 23 increased mRNA expression of the HSD17B2 gene, thus showing pronounced pro-drug anti-estrogenic activity. Estradiol-derived d -ring triazole compound 24 thus acts at the enzyme, gene expression and cellular levels to decrease the production of active estrogen hormones, demonstrating its pharmacological potential.

The Schisandraceae family is reported to have a range of pharmacological activities, including anti-inflammatory effects. As with all herbal preparations, extracts of Schisandra species are mixtures composed of >50 lignans, especially schizandrins, deoxyschizandrins, and gomisins. In China, Schisandra sphenanthera extract (SSE) is often coadministered with immunosuppressant treatment of transplant recipients. In cases of coadministration, the potential for herb-drug interactions (HDIs) increases. Clinical studies have been used to assess HDI potential of SSE. Results demonstrated that chronic SSE administration reduced midazolam (MDZ) clearance by 52% in healthy volunteers. Although clinical studies are definitive and considered the "gold standard," these studies are impractical for routine HDI assessments. Alternatively, in vitro strategies can be used to reduce the need for clinical studies. Transporter-certified sandwich-cultured human hepatocytes (SCHHs) provide a fully integrated hepatic cell system that maintains drug clearance pathways (metabolism and transport) and key regulatory pathways constitutive active/androstane receptor and pregnane X receptor (CAR/PXR) necessary for quantitative assessment of HDI potential. Mechanistic studies conducted in SCHHs demonstrated that SSE and the more commonly used dietary supplement Schisandra chinensis extract (SCE) inhibited CYP3A4/5-mediated metabolism and induced CYP3A4 mRNA in a dose-dependent manner. SSE and SCE reduced MDZ clearance to 0.577- and 0.599-fold of solvent control, respectively, in chronically exposed SCHHs. These in vitro results agreed with SSE clinical findings and predicted a similar in vivo HDI effect with SCE exposure. These findings support the use of an SCHH system that maintains transport, metabolic, and regulatory functionality for routine HDI assessments to predict clinically relevant clearance interactions.

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0-7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with Km , 0.80 ± 0.06 μM; Vmax , 30.36 ± 1.97 μmol·min-1; Kcat , 607 ± 39 μmol·μM-1·min-1; Kcat /Km , 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp, one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.

Cytochromes P450 are key enzymes for steroid hormone biosynthesis in human body. They are considered as targets for the screening of novel high efficient drugs. The results of screening of bile acids and androstane derivatives toward human recombinant steroid 17α-hydroxylase/17,20-lyase (CYP17A1) are presented in this paper. A group of steroids, binding with micromolar or submicromolar affinity (in a range from 9 μM - less than 0.1 μM), was identified. Results presented here showed that these steroidal compounds are able to decrease rate of hydroxylation of essential CYP17A1 substrate - progesterone, while some compounds completely inhibited enzyme activity. Structure-activity relationship (SAR) analysis based on in vitro and in silico studies showed that high affinity of the enzyme to bile acids derivatives is correlated with side chain hydrophobicity and presence of hydroxyl or keto group at C3 position. From the other side, bile acid-derived compounds with more polar side chain or substituents at C7 and C12 positions possess higher Kd values. Among androstane-derived steroids couple of Δ5-steroids with hydroxyl group at C3 position, as well as 16,17-secosteroids, were found to be high affinity ligands of this enzyme. The data obtained could be useful for the design of novel highly efficient inhibitors of CYP17A1, since the bile acids-derived compounds are for first time recognized as effective CYP17A1 inhibitors.

Starting from 3β-acetoxy-17a-homo-17-oxa-androst-5-en-16-one (1) and in the reaction with N-bromoacetamide (NBA) and perchloric acid, the 5α-bromo-6β-hydroxy (2), 5β,6β-epoxy (3) and 5α,6β-dibromo (4) derivatives were obtained. The structure of compounds 2–4, particularly stereochemistry at C5 and C6, is established by detailed NMR and X-ray analysis. The in vitro antiproliferative activity of newly synthesized compounds 2–4 against six human tumor cell lines was evaluated. All three compounds showed a significant toxicity toward hormone-independent breast adenocarcinoma MDA-MB-231 and cervical carcinoma cells HeLa, while dibromo derivative 4 was active toward five human cancer cell lines. These new 5,6-disubstituted-D-homolactone steroidal compounds have also displayed selectivity toward cancerous cell lines against inactivity found for noncancerous control cell line. This selectivity was not found for control compound, well-known chemotherapy drug cisplatin.

The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells.

Benfluralin, an herbicide of the dinitroaniline class used in weed control, was first registered in the United States in 1970. Increased incidence of liver tumors was observed in the 2 year dietary carcinogenicity studies. A review of the toxicology database provides evidence that the mode of action (MOA) of benfluralin responsible for hepatocellular adenoma and carcinoma in rodents depends on activation of the constitutive androstane (CAR)/pregnane X (PXR) receptors, that triggers enzyme induction and altered gene expression leading to hepatocyte proliferation. After prolonged exposures at high dose levels, altered hepatic foci and liver tumors are observed. This hepatocarcinogenic MOA has been described in rodents following long-term dietary exposures to other CAR/PXR activator chemicals, such as phenobarbital, and is generally considered as non-relevant in humans due to differences between human and rodent responses. We analyzed the existing and newly acquired toxicology data to establish that the hepatocarcinogenic MOA of benfluralin in rodents includes the same key events previously described in the rodent MOA of phenobarbital. A weight of evidence approach was taken to establish temporal and dose-related concordance of the causal key events supporting the conclusion that rodent liver carcinogenicity of benfluralin is unlikely to be relevant for human cancer risk.

This paper describes the synthesis of a new A-homo lactam D-homo lactone androstane derivative from dehydroepiandrosterone. To evaluate the impact of the introduction of nitrogen in the parental scaffold on biological activity, a new androstane enamide-type lactam derivative was prepared and characterized. The new compound as well as starting compounds were screened for cytotoxic, anti-angiogenic and anti-inflammatory activities using several human cancer cell lines (MCF-7, MDA-MB-231, PC3, CEM, G-361, HeLa), endothelial (HUVEC) and non-tumour (MRC-5 and BJ) cell lines. Strong cytotoxic and anti-inflammatory activity with a broad therapeutical window was demonstrated by the A-homo lactam D-homo lactone androstane derivative. The induction of apoptosis in treated PC3 cultures was confirmed using apoptotic morphology screening and a fluorescent double-staining method. New A-homo lactam D-homo lactone androstane derivative induced apoptosis more than the tested reference compounds, Formestane and Doxorubicin. An in silico ADME analysis showed that the compounds possess drug-like properties.

Oxime ethers have attracted much interest as important precursors and intermediates for the preparation of a wide variety of drugs and natural products. They can be easily converted into important functional groups such as amino alcohols and hydroxy ketones. Therefore, the development of methodologies for the preparation of oxime ethers is of considerable interest. Various researchers have studied the interesting biological properties of oxime ether derivatives such as anticonvulsant, anti-inflammatory, antineoplastic, anti-enteroviral, antimicrobial, antitumor, and anti-Helicobacter pylori activities [1]. Since clinical use of almost all anticancer drugs has been limited by the toxicity to normal tissues, important goal of cancer chemotherapy is to amplify the selective inhibition of tumor cells while decreasing toxicity to normal tissues. In order to develop more efficient and selective antitumor agents, here we report the efficient synthesis of 17-substituted O-alkylated androstane derivatives, and investigate their antiproliferative activity (IC50 after 72 h, MTT test) against three tumor cell lines (MDA-MB-231, HeLa and HT-29) and one healthy cell line (MRC-5). In continuation of our work on nitrogen containing androstane derivatives [2-4], synthesis of respective novel compounds and their evaluation in a human carcinoma cell lines will be presented and discussed. Acknowledgements: This work was financialy supported by Ministry of Education, Science and Technological development of the Republic of Serbia (Project No. 172021). [1] K. Sharma, S. B. Mishra, A. K. Mishra, Helv. Chim. Acta 94 (2011), 2256. [2] J. Ajdukovic, E. Djurendic, E. Petri, O. Klisuric, A. Celic, M. Sakac, D. Jakimov, K. Penov Gasi, Bioorg. Med. Chem. 21 (2013), 7257. [3] E. Djurendic, J. Ajdukovic, M. Sakac, J. Csanadi, V. Kojic, G. Bogdanovic, K. Penov Gasi, Eur. J. Med. Chem. 54 (2012), 784. [4] J. J. Ajdukovic, K. M. Penov Gasi, D. S. Jakimov, O. R. Klisuric, S. S. Jovanovic-Santa, M. N. Sakac, L. D. Aleksic, E. A. Djurendic, Bioorg. Med. Chem. 23 (2015), 1557.

Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development.

1. Nuclear receptors CAR (NR1I3) and PXR (NR1I2) are major ligand-activated transcriptional regulators of xenobiotic metabolism and disposition and modulators of endobiotic metabolism. Differences in xenobiotic selectivity between the human and rodent receptors are well recognized but there is lack of such information on properties of CAR and PXR in important domestic animals. 2. The pig and bovine receptors were cloned and their ligand profiles were systematically compared to corresponding human and mouse forms utilizing a panel of xenobiotics and structural analysis. 3. Pig CAR and PXR resemble their human counterparts which can be rationalized by only modest amino acid changes between critical residues of the human ligand-binding pockets (H203Q for CAR, L210V and M243I for PXR). 4. In contrast, bovine CAR shows a blunted response to CAR agonists and inverse agonists. These changes are likely due to disruptive mutations at or near critical hydrogen bond-forming residues (N165I, Y326F). The unresponsiveness of bovine PXR to human- and mouse-selective agonists may be related to substitutions at important ligand-contacting residues R410Q and F305V, respectively. 5. Our findings have implications for regulation of drug-metabolizing enzymes and transporters and pharmacokinetics in cattle and pigs.

Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750 ppm equating to 105 mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh β transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28 days of treatment. In these dose–response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA.

Hexabromocyclododecane (HBCD) is a brominated flame retardant found in the environment and human tissues. The toxicological effects of HBCD exposure are not clearly understood. We employed whole-genome RNA-sequencing on liver samples from male and female Fischer rats exposed to 0, 250, 1250, and 5000 mg technical mixture of HBCD/kg diet for 28 days to gain further insight into HBCD toxicity. HBCD altered 428 and 250 gene transcripts in males and females, respectively, which were involved in metabolism of xenobiotics, oxidative stress, immune response, metabolism of glucose and lipids, circadian regulation, cell cycle, fibrotic activity, and hormonal balance. Signature analysis supported that HBCD operates through the constitutive androstane and pregnane X receptors. The median transcriptomic benchmark dose (BMD) for the lowest statistically significant pathway was within 1.5-fold of the BMD for increased liver weight, while the BMD for the lowest pathway with at least three modeled genes (minimum 5% of pathway) was similar to the lowest apical endpoint BMD. The results show how transcriptional analyses can inform mechanisms underlying chemical toxicity and the doses at which potentially adverse effects occur. This experiment is part of a larger study exploring the use of toxicogenomics and high-throughput screening for human health risk assessment.

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.

Tourette's syndrome (TS) is a neurodevelopmental disorder characterised by recurring motor and phonic tics. The pathogenesis of TS is considered to reflect dysregulations in the signalling of dopamine (DA) and other neurotransmitters, which lead to excitation/inhibition imbalances in cortico-striato-thalamocortical circuits. The causes of these deficits may reflect complex gene × environment × sex (G × E × S) interactions; indeed, the disorder is markedly predominant in males, with a male-to-female prevalence ratio of approximately 4 : 1. Converging lines of evidence point to neuroactive steroids as being likely molecular candidates to account for G × E × S interactions in TS. Building on these premises, our group has begun examining the possibility that alterations in the steroid biosynthetic process may be directly implicated in TS pathophysiology; in particular, our research has focused on 5α-reductase (5αR), the enzyme catalysing the key rate-limiting step in the synthesis of pregnane and androstane neurosteroids. In clinical and preclinical studies, we found that 5αR inhibitors exerted marked anti-DAergic and tic-suppressing properties, suggesting a central role for this enzyme in TS pathogenesis. Based on these data, we hypothesise that enhancements in 5αR activity in early developmental stages may lead to an inappropriate activation of the 'backdoor' pathway for androgen synthesis from adrenarche until the end of puberty. We predict that the ensuing imbalances in steroid homeostasis may impair the signalling of DA and other neurotransmitters, ultimately resulting in the facilitation of tics and other behavioural abnormalities in TS.

Pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR), members of the nuclear receptor superfamily, are two major xeno-sensing transcription factors. They can be activated by a broad range of lipophilic xenobiotics including therapeutics drugs. In addition to xenobiotics, endogenous compounds such as steroid hormones and bile acids can also activate PXR and/or CAR. These nuclear receptors regulate genes that encode enzymes and transporters that metabolize and excrete both xenobiotics and endobiotics. Sulfotransferases (SULTs) are a group of these enzymes and sulfate xenobiotics for detoxification. In general, inactivation by sulfation constitutes the mechanism to maintain homeostasis of endobiotics. Thus, deciphering the molecular mechanism by which PXR and CAR regulate SULT genes is critical for understanding the roles of SULTs in the alterations of physiological and pathophysiological processes caused by drug treatment or environmental exposures.

Five 14α-hydroxylated derivatives of androstane and pregnane steroids were obtained using mycelium of the mold fungus Curvularia lunata (VKPM F-981). The conditions for microbiological transformation of androst-4-en-3,17-dione that enabled its 14α-hydroxy analog to be obtained in yields up to 60% with substrate loading 6 g/L were determined. The 21-acetoxy analog of proligestone was synthesized from 14α-hydroxycortexolone that was formed simultaneously with hydrocortisone during hydroxylation of cortexolone by C. lunata. The resulting 14α- and 14α,21-hydroxysteroids could be used as precursors for the synthesis of new drugs.

The novel androstane-type steroidal glycoside 1 was isolated from the roots of Asparagus curillus Buch.-Ham .e x Roxb. Its structure was elucidated as (2a,3b,5a,17b)-17-(1-methoxyethoxy)-17- methylandrostane-2,3-diol 3-(b-d-digitoxopyranoside) by means of chemical and advanced spectral analysis. Introduction. - Asparagus curillus (Buch.-Ham.) ex Roxb., belonging to the family Liliaceae, is a reputed medicinal shrub distributed in the tropical and temperate climate (1000 to 2250 meter altitude) of central Himalaya and is known for its wide spectrum of medicinal utility including diabetes. Traditionally, ripe fruits from the plant are often used for abortion. The extract obtained from the plant is also used to increase the appetite and secretion of milk in lactating women (1) (2). Previous phytochemical reports on the plant have lead to the isolation of oligospirostanosides, oligofurostano- sides, spirostanol, furostanol glycosides, and other steroidal saponins (3 - 6). Recently, the plant was also screened for the concentration level of nine trace elements (Zn, Cu, Mn, Fe, Co, Na, K, Ca, and Li) by atomic absorption spectroscopy (7). In the present study, we report a novel androstane type steroidal glycoside 1 (Fig. 1), isolated for the first time from the EtOH extract of Asparagus curillus roots.

Cytochrome P450 enzymes, CYP3A and CYP1A are major drug metabolizing enzymes in the liver. CYP3A enzymes have a major role in the metabolism of 30-40% of all used drugs. CYP1A2 is a key enzyme having an important role in the metabolic clearance of 5% of currently marketed drugs. CYP1A2 participates in the metabolic activation of chemical mutagens in cooked food, therefore its activity is suspected to be on e of the possible risk factors determining the carcinoge nicity of heterocyclic amines in human beings. In a previous report, we have reported the induction of CYP3A2 and the inhibition of CYP1A2 by Fibrate (CFA) and proved CYP1A2 inhibition to be PPAR α-dependent. CYP3A2 and CYP1A2 have been reported to be induced in the liver by Phenobarbital (PB) wh ile Fibrates was reported to induce CYP3A2. However the exact mechanism of the induction of CYP3A2 by CFA and PB and induction of CYP1A2 by PB has not been clarified yet whether it is through Constituti ve Androstane Receptor (CAR) or other receptor as PPAR α or Pregnane X Receptor (PXR). We treated Wistar female rats (with normal expression of CAR protein) and Wistar femal Kyoto rats (with low expr ession of CAR protein) with PB and Clofibric Acid (CFA). PB caused a high CYP3A2 induction in Wistar female rats and a low induction in (WKY) indicating that PB induced CYP3A2 in a CAR-dependent manner. Interestingly, PB treatment induced CYP1A2 in Wistar female rats and failed to induce it in (WKY) indicating that the induction of CYP1A2 by PB to b e CAR-dependent. Moreover CFA induced CYP3A2 protein similarly in both rat strains indicating that CYP3A2 induction by Fibrates is CAR-independent and most probably to be PXR or PPAR α-dependent. For the best of our knowledge this is the first rep ort that shows a clear evidence of the CAR-dependent induction of CYP1A2 and CYP3A2 by PB and the CAR-independent induction of CYP3A2 by fibrates.

An efficient synthesis of several A- and B-modified D-homo lactone androstane derivatives from 3β-hydroxy-17-oxa-D-homoandrost-5-en-16-one (1) is reported. 17-Oxa-Dhomoandrost- 4-ene-3,16-dione (2), obtained by the Oppenauer oxidation of compound 1, was converted via the unstable intermediate 3,16-dioxo-4,17-dioxa-D-homoandrostane- 5α-carboxaldehyde (3) to 17-oxa-D-homo-3,5-seco-4-norandrostan-5-one-3-carboxylic acid (4), which was also obtained directly from compound 2. Compound 1 was acetylated to give 17-oxa-D-homoandrost-5-en-16-on-3β-yl acetate (5) which was then oxidized with chromium(VI)-oxide in 50% acetic acid or with meta-chlorperbenzoic acid and chromium(VI)-oxide to yield compounds 6-8 and 5α-hydroxy-17-oxa-D-homoandrostane- 6,16-dion-3β-yl acetate (9), respectively. The oximination of compound 9 gave a mixture of 6(E)-hydroximino-5α-hydroxy-17-oxa-D-homoandrostan-16-on-3β-yl acetate (10) and 6(Z)-hydroximino-5α-hydroxy-17-oxa-D-homoandrostan-16-on-3β-yl acetate (11), the hydrolysis of which gave 6(E)-hydroximino-3β,5α-dihydroxy-17-oxa-D-homoandrostan- 16-one (12) and 6(Z)-hydroximino-3β,5α-dihydroxy-17-oxa-D-homoandrostan-16-one (13). 6-Nitrile-17-oxa-5,6-seco-D-homoandrostane-5,16-dion-3β-yl acetate (14) was obtained under the Beckmann fragmentation of compounds 10 and 11. Only pure and stable compounds (1, 2, 4, 5, 9 and 14) were tested in vitro on six malignant cell lines (MCF-7, MDA-MB-231, PC-3, HeLa, HT-29, K562) and one non-tumor MRC-5 cell line. Significant antiproliferative activity against MDA-MB-231 cells showed compounds 1, 5 and 9, while compound 2 exhibited a strong antiproliferative activity. Only compound 14 showed weak antiproliferative activity against MCF-7 cells. All tested compounds were not toxic on MRC-5 cells, whereas Doxorubicin was highly toxic on these cells. [Projekat Ministarstva nauke Republike Srbije, br. 172021]

The majority of breast cancer cases are estrogen receptor positive (ER+). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3), 7α-allylandrost-4-ene-3,17-dione (6), 7α-allylandrost-4-en-17-one (9), 7α-allyl-3-oxoandrosta-1,4-dien-17β-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance.

The synthesis of hydroxy-androstane-1,4-diene-3,17-dione derivatives (2a-d) by biotransformations with Pseudomonas alcaliphila of bile acids (1a-d) was reported. The scale-up of addition of deoxycholic acid (1a), cholic acid (1b), and chenodeoxycholic acid (1c) (1 g/L until 10 g/L) to the culture broth of P. alcaliphila switches the degradation process to the synthesis of 12β-hydroxy-androstane-1,4-diene-3,17-dione (2a, 95%), 7α,12β-dihydroxy-androstane- 1,4-diene-3,17-dione (2b, 52%), and 7α-hydroxy-androstane-1,4-diene-3,17-dione (2c, 23%) Also hyodeoxycholic acid (1d) was transformed by P. alcaliphila to 6α-hydroxy-androstane-1,4-diene-3,17-dione (2d) with as much good yields (83%) but with lower concentration (1 g/L) in the culture broth.

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drugdrug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drugdrug interaction possibilities, the inducing properties of artemisinin metabolites should be considered. German Federal Ministry of Education and Research (BMBF) HepatosSys network [0313081B, 0313080F, 0313080I]; Deutsche Forschungsgemeinschaft (Germany) [KE 1629/1-1]; Robert Bosch Foundation, Stuttgart, Germany info:eu-repo/semantics/publishedVersion

Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility.

Nocardia sp. was isolated from an exotic soil of the northwestern Himalayas and was capable of selectively cleaving the side chain of sterols (cholesterol and phytosterol) yielding androstane steroids, in the presence of a hydrophobic metal chelating agent, after an incubation period of 24 h. Nocardia sp. was identified on the basis of morphological and biochemical characteristics accomplished with 16S rDNA sequencing. An extracellular production of 1,4-androstadiene-3,17-dione (ADD) was observed in the fermentation medium. The conversion studies were carried out with a cholesterol concentration ranging from 0.3 to 3 g/l, but the fermentation conditions in biotransformation experiments gave the maximum yields (theoretical yield was 90 %) at 0.5 g/l cholesterol concentration with pH 7.2 in the presence of Tween 80 concentration 2 g/l; in addition, th effects of the media were also studied.

New 17-picolyl and 17-picolinylidene androstane derivatives, 3 – 10 , 15 , 18 , 19 , 22 and 23 , were synthesized starting from 17α-picolyl-androst-5-en-3β,17β-diol ( 1 ) and 17(Z)-picolinylidene-androst-5-en-3β-ol ( 2 ). Reaction of 1 with m -chloroperoxybenzoic acid gives 5α,6α-epoxy N-oxide derivative 3 , or, with Jones reagent, 3,6-dione derivative 4 ; while 17α-picolyl-androst-5-en-3β,4α,17β-triol ( 5 ) or 3β,4β,17β-triol ( 6 ) derivatives are obtainable from 1 using SeO 2 in dioxane. Base-catalyzed tosyl group elimination from 7 or 9 affords AB conjugated derivatives 8 and 10 . Oppenauer oxidation of 1 and 2 yields 4-en-3-one derivatives 11 and 12 , which, with H 2 O 2 in 4 M NaOH, affords 4α,5α and 4β,5β-epoxides 13 , 14 , 16 and 17 . New 4-methoxy-3-keto derivatives 15 and 18 were obtained from 13 and 14 , or, with methanol in 4 M NaOH, from 16 and 17 . Reduction of 11 with NaBH 4 gives 22, which was then acetylated to obtain 23 . All new derivatives were screened for antitumor activity against human breast adenocarcinoma ER+, MCF-7; human breast adenocarcinoma ER-, MDA-MB-231; prostate cancer AR-, PC-3; human cervix carcinoma, HeLa; and colon cancer, HT-29 cells; as well as one human non-tumor cell line, MRC-5. Compounds 3 , 5 , 6 , 8 , 10 , 18 , 19 and 22 exhibited significant antitumor activity against MDA-MB-231 breast cancer cells; while 5 , 6 and 10 also showed strong cytotoxicity against HT-29. Only compound 19 exhibited significant activity against MCF-7 breast cancer cells. No compounds displayed cytotoxicity against non-tumor MRC-5 cells.

A series of new 16E-arylidene androstane derivatives were synthesized and characterized. The new compounds were screened for their anticancer activities against the human cancer cell lines SW480, A549, HepG2 and HeLa in vitro using the MTT assay. The results of the in vitro study showed that a number of compounds have shown IC(50) values lower than 20 μM against the four cancer cell lines.

Here, we have demonstrated that xenobiotic activation of the nuclear receptor (CAR, NR1I3) can result in arresting DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase. Huh7 cells over-expressing CAR were either treated with dimethyl sulfoxide, the CAR activator TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; androstenol, 16,(5α)-androsten-3α-OL), or repressor androstenol; these treatments were then followed by adriamycin treatment to damage DNA. FACS analysis revealed that CAR-activation by TCPOBOP increased the rate of arrested Huh7 cells at the G2/M phase (4N DNA content) after DNA damage by adriamycin. This increase correlated with the increase of cell viability in TCPOBOP-treated Huh7 cells, as determined by MTT assays. Real-time polymerase chain reaction analysis determined that, as regulated by CAR, the growth arrest and DNA damage-inducible γ (GADD45γ) and Cyclin G2 genes increased and decreased, respectively, as TCPOBOP increased the number of Huh7 cells arrested at the G2/M phase. Thus, the results suggest that CAR regulates cell cycle, increasing G2/M arrest, and delaying the death of DNA-damaged cells.

Background: Brain tumours present unique challenges to conventional therapies and pose major health problems around the world. Brain tumour stem cells (BTSCs) represent a small fraction of tumour cells that maintain growth, drug resistance and recurrence properties. Constitutive androstane receptor (CAR) is a nuclear receptor transcription factor that regulates drug metabolism and homoeostasis. In this study, we examined the effect of CAR agonist, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehydeO-(3,4-dichlorobenzyl)oxime (CITCO) on BTSCs. Methods: The expression of CAR in BTSCs was detected by quantitative RT–PCR and western blot. The antiproliferative effect of CITCO on BTSCs was determined by WST-1 and 3H thymidine uptake assays. The effect of CITCO on CD133 expression, cell cycle progression and apoptosis in BTSCs was analysed by immunostaining and flow cytometry. The in vivo effect of CITCO was studied using subcutaneous (s.c.) BTSC xenograft in nude mice. Results: We show for the first time that BTSCs express altered levels of nuclear receptors compared with glioma cells. The expression of CAR mRNA and protein was low in BTSCs and that increased following treatment with CITCO in culture. CITCO induced a dose-dependent decrease in growth and expansion of CD133+ BTSCs as gliospheres in culture. Cell cycle arrest and apoptosis in BTSCs were induced by CITCO, but not in normal astrocytes. Growth of s.c BTSC xenograft in nude mice was also inhibited by CITCO. Conclusion: These findings indicate that CITCO inhibits the growth and expansion of BTSCs, suggesting the use of CAR agonists for the treatment of brain tumour.

The effects of varying dosages of intraperitoneal (i.p.) injections of streptozotocin (STZ) on the disease state of STZ-induced diabetic rats were assessed. When administered as a single i.p. injection, 50 mg.k g-1 STZ is considered safe. However, it is necessary to set the dosage interval to approximately 7 days in the case of 2 i.p. injections of 50 mg.k g-1 STZ. The serum levels of total bile acids (TBA), which are ligands of constitutive androstane receptors, related to the regulation of mRNA, expression of cytochrome P450, conjugation enzymes, and transporters, differed between rats that received single and double STZ administrations. TBA levels in the model rats should be stable otherwise it could lead to differences in regulation of mRNA expression. When a single dose of STZ was administered, the TBA levels stabilized within 4 weeks of administration. However, when 2 doses of STZ were administered, TBA levels stabilized within 2 weeks of administration.

E-mail: yuzhifang@njau.edu.cnReceived June 9, 2014, Accepted July 5, 2014A new androstane derivative, 4β-methyl-15-oxa-14β-androstane-7-ene-4α-carboxylic acid (1) and a knownone 4β-methyl-15-oxa-14β-androstane-7-ene-4α-hydroxyl (2) were isolated from the EtOAc extract of thecultures of the fungus Marasmiellus ramealis (Bull.) Singer. Their structures were elucidated on the basis of1D and 2D NMR as well as MS spectroscopic data analysis. The inhibitory activity of two isolates againstacetylcholinesterase (AChE) revealed that compound 1 exhibited definitely inhibitory activity.Key Words : Marasmiellus ramealis (Bull.) Singer, Fermentation, Androstane derivative, AChE inhibitoryactivityIntroductionMarasmiellus ramealis (Bull.) Singer, belonging to thefamily Marasmiaceae, is a small, thin, white mushroom,usually growing on the deadwood. The fungi is an ediblefungus with a wide distribution in most parts of China,especially in Hainan, Hunan,Yunnan, and Tibet province.

The nuclear receptor constitutive active/androstane receptor (CAR) acts as a sensor of toxic byproducts derived from the endogenous metabolism and exogenous chemicals. We previously reported that CAR is responsible for exacerbating hepatic injury and fibrosis in a dietary model of non-alcoholic steatohepatitis (NASH) via upregulation of lipid peroxidation. In this study, we investigated the pathological roles of the CAR in the development of hepatocellular carcinoma in NASH model. CAR +/+ and CAR ―/― mice were fed methionine- and choline-deficient (MCD) diet after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine (DEN) at 2 weeks of age. Interestingly, the MCD diet dramatically promoted DEN-induced hepatocarcinogenesis in CAR +/+ mice. However, the deletion of CAR leads to a significantly lower tumor incidence and smaller tumor diameter. Hepatocytes of MCD-treated-CAR +/+ mice showed a significantly higher staining frequency of Ki-67, a marker of cell proliferation, and exhibited a higher expression of c-Myc and FoxM1 transcripts compared with MCD-treated CAR ―/― mice. Immunohistochemistry revealed the nuclear translocation of CAR thus suggesting that the activation of CAR signaling increased in the hepatocytes of CAR +/+ mice fed MCD diet. In addition, in vitro experiments using the CAR stably expressed cell line with TCPO-BOP have suggested that CAR activation directly leads to cell proliferation. Survival was significantly lower in the CAR +/+ mice fed the MCD diet in comparison with the CAR ―/― mice. Taken together, these results suggest that CAR may therefore play a critical role in the hepatocarcinogenesis of the murine NASH model via the upregulation of cell proliferation.

Drug-metabolizing enzymes (DMEs) and transporters play pivotal roles in the disposition and detoxification of numerous foreign and endogenous chemicals. To accommodate chemical challenges, the expression of many DMEs and transporters is up-regulated by a group of ligand-activated transcription factors namely nuclear receptors (NRs). The importance of NRs in xenobiotic metabolism and clearance is best exemplified by the most promiscuous xenobiotic receptors: pregnane X receptor (PXR, NR1I2) and constitutive androstane/activated receptor (CAR, NR1I3). Together, these two receptors govern the inductive expression of a largely overlapping array of target genes encoding phase I and II DMEs, and drug transporters. Moreover, PXR and CAR also represent two distinctive mechanisms of NR activation, whereby CAR demonstrates both constitutive and ligand-independent activation. In this review, recent advances in our understanding of PXR and CAR as xenosensors are discussed with emphasis placed on the differences rather than similarities of these two xenobiotic receptors in ligand recognition and target gene regulation.

Polychlorinated biphenyls (PCBs) are ubiquitous contaminants found as complex mixtures of coplanar and non-coplanar congeners. The hepatic temporal and dose-dependent effects of the most abundant non-dioxin-like congener, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), were examined in immature, ovariectomized C57BL/6 mice, and compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand. Animals were gavaged once with 300 mg/kg PCB153 or sesame oil vehicle and sacrificed 4, 12, 24, 72 or 168 h post dose. In the dose-response study, mice were gavaged with 1, 3, 10, 30, 100 or 300 mg/kg PCB153 or sesame oil for 24 h. Significant increases in relative liver weights were induced with 300 mg/kg PCB153 between 24 and 168 h, accompanied by slight vacuolization and hepatocellular hypertrophy. The hepatic differential expression of 186 and 177 genes was detected using Agilent 4 x 44 K microarrays in the time course (|fold change|> or =1.5, P1(t)> or =0.999) and dose-response (|fold change|> or =1.5, P1(t)> or =0.985) studies, respectively. Comparative analysis with TCDD suggests that the differential gene expression elicited by PCB153 was not mediated by the AhR. Furthermore, constitutive androstane and pregnane X receptor (CAR/PXR) regulated genes including Cyp2b10, Cyp3a11, Ces2, Insig2 and Abcc3 were dose-dependently induced by PCB153. Collectively, these results suggest that the hepatocellular effects elicited by PCB153 are qualitatively and quantitatively different from TCDD and suggestive of CAR/PXR regulation.