Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene.

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.

Phosphine boranes as less hydrophobic building blocks than alkanes and silanes: Structure-property relationship and estrogen-receptor-modulating potency of 4-phosphinophenol derivatives.

Increasing structural options in medicinal chemistry is important for the development of novel and distinctive drug candidates. In this study, we focused on phosphorus-containing functionalities. We designed and synthesized a series of phosphinophenol derivatives and determined their physicochemical properties, including hydrophobicity parameter LogP, and their biological activity toward estrogen receptor (ER). Notably, the phosphine borane derivatives (9 and 14) exhibited potent ER-antagonistic activity, exceeding the potency of the corresponding alkane (15) and silane (16) derivatives, despite having a less hydrophobic nature. The determined physicochemical parameters will be helpful for the rational design of phosphorus-containing biologically active compounds. Our results indicate that phosphine boranes are a promising new chemical entry in the range of structural options for drug discovery.

Construction of benzofuranone library via a metal-free, one-pot intermolecular condensation, and their application as efficient estrogen receptor ß modulators.

Facile synthesis of benzofuranone was achieved through a metal-free, one-pot intermolecular condensation between α-hydroxy aryl ketones and resorcinol derivatives. A library of 20 compounds with moderate to good overall yields was prepared. These compounds showed strong binding toward estrogen receptors along with good selectivity for ERß (>190-fold over ERα). Anti-proliferative activity on DU-145, U-87, and MCF-7 cells gave inhibition IC50 values in the low µM range, which suggested the promising potential therapeutic applications of these new classes of benzofuranones.

Estrogen Receptor α (ERα)-targeting Compounds and Derivatives: Recent Advances in Structural Modification and Bioactivity.

Breast cancer is the most common cancer suffered by female, and the second highest cause of cancer-related death among women worldwide. At present, hormone therapy is still the main treatment route and can be divided into three main categories: selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). However, breast cancer is difficult to cure even after several rounds of anti-estrogen therapy and most drugs have serious side-effects. Here, we review the literature published over the past five years regarding the isolation and synthesis of analogs and their derivatives.

Methyl-Hispolon from Phellinus lonicerinus (Agaricomycetes) Affects Estrogen Signals in MCF-7 Breast Cancer Cells and Premature Aging in Rats.

We studied Phellinus lonicerinus to determine the cytotoxic effect and the dual estrogenic activities of methyl-hispolon and their relation to estrogen signals in vivo and in vitro. The Glide scores of methyl-hispolon-estrogen receptor α (ERα) and methyl-hispolon-ERß docked complexes were -7.29 kcal/mol and -6.68 kcal/mol in docking simulations. Methyl-hispolon had a significant antiproliferative effect for estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen, and it exhibited dual estrogen activities. Methyl-hispolon increased the serum E2 in rats with premature ovarian failure and fulfilled the estrogenic function in the uterus and ovary. Methyl-hispolon significantly inhibited the expression of Ras, API, ERα, C-myc, and cyclinDl, as well as their gene transcription in RL95-2 cells. The phosphorylation of ERK1/2 was inhibited by methyl-hispolon. Thus, methyl-hispolon has potential use in treating estrogen deficiency-related diseases, with good antitumor effects and estrogenic activity.

Food components and environmental chemicals of inhibiting human placental aromatase.

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy.

Identification of Compounds That Inhibit Estrogen-Related Receptor Alpha Signaling Using High-Throughput Screening Assays.

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.

Design and synthesis of fluorescently labeled steroidal antiestrogens.

A set of derivatives of 11ß-(4-oxyphenyl)estradiol were prepared as potential fluorescent imaging agents for the evaluation of the estrogen receptor. The compounds were designed based on the established affinity and selectivity of 11ß-[4-(dimethylethoxy)phenyl]estradiol (RU39411) as an estrogen receptor (ER) antagonist. The 5-(dimethylamino) naphathalene-1-sulfonyl (dansyl) and 7-nitrobenzo[c][1,2,5] oxadiaol-4-yl (NBD) moieties were selected based on their fluorescent and physicochemical properties. A convergent synthesis was developed that culminated in the [3 + 2] copper (I) assisted alkyne-azide cycloaddition coupling of the steroidal and fluorescent components. Good yields were obtained for the intermediates and final products, and the structural variations in the steroid component will permit evaluation of ER affinity and selectivity.

Antagonists for Constitutively Active Mutant Estrogen Receptors: Insights into the Roles of Antiestrogen-Core and Side-Chain.

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both nonpolar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.

Endocrine effects after ozonation of tamoxifen.

Ozonation is used as additional wastewater treatment option to remove recalcitrant micropollutants. It also removes the estrogenic activity found in wastewater but not always the anti-estrogenic activity. This can be explained by an incomplete removal of anti-estrogenic micropollutants or by formation of transformation products (TPs) which retain the activity. The present study investigates the degradation of the anti-estrogenic pharmaceutical tamoxifen in pure water, regarding TP formation and related anti-estrogenic effect using Arxula adeninivorans yeast estrogen screen (A-YES). In total, five transformation products were detected: three N-oxides and two further products (TP 270 and TP 388). For the transformation product TP 270 a correlation of the extent of formation with an increase of the anti-estrogenic activity was determined, demonstrating that transformation products from ozonation can be more active in a bioassay than the parent compounds. Our study shows also that the transformation of tamoxifen to N-oxides reduces the anti-estrogenic activity. The reactivity of amines towards ozone typically increases with pH, since only deprotonated amines react with ozone. Hence, removal of the endocrine activity by N-oxide formation may be disfavored at low pH.

Protective effects of psoralidin on IL­1ß­induced chondrocyte apoptosis.

Chondrocyte apoptosis serves a key role in the pathogenesis of osteoarthritis. The present study aimed to investigate the protective effects of psoralidin on interleukin (IL)­1ß­induced chondrocyte apoptosis and explore the underlying mechanisms. Chondrocytes were isolated from the articular cartilage of Sprague­Dawley rats and were treated with 10 ng/ml IL­1ß and various doses of psoralidin (5, 10 or 15 µM). The ratio of apoptosis was measured by Annexin V/propidium iodide double­labeling fluorescence­activated cell sorting (FACS) analysis. Caspase­3 and ­9 activity was determined using a quantitative colorimetric assay. Intracellular levels of reactive oxygen species (ROS) were assessed using a dichlorofluorescein diacetate­labeling FACS analysis, and the release of nitric oxide (NO) was measured using the Griess reaction method. In addition, protein expression levels were detected by western blotting. The results of the present study demonstrated that psoralidin may reduce IL­1ß­induced chondrocyte apoptosis. Psoralidin pretreatment also reversed the inhibitory effects of IL­1ß on B­cell lymphoma 2 (Bcl­2) expression, and decreased the IL­1ß­induced expression of Bcl­2­associated X protein, matrix metalloproteinase (MMP)­1 and MMP­13. Furthermore, psoralidin decreased IL­1ß­induced caspase­3 and ­9 activity, NO release, ROS production and nuclear factor (NF)­κB nuclear translocation. In addition, the NF­κB inhibitor pyrriolidine­dithiocarbamate exerted similar effects to psoralidin, thus suggesting that IL­1ß induced proapoptotic effects in rat chondrocytes via an NF­κB­dependent pathway. Since psoralidin could protect chondrocytes from IL­1ß­induced apoptosis and MMP expression, the present results suggested that psoralidin may be considered a drug candidate for the treatment of osteoarthritis.

Design, synthesis and biological evaluation of novel indole-xanthendione hybrids as selective estrogen receptor modulators.

Ground breaking clinical therapeutic advances in the treatment of breast cancer (BC) is the introduction of selective estrogen receptor modulators (SERMs). We have expeditiously designed and synthesized indole-xanthendione hybrids by coalescing the indole nucleus with xanthendione. All the compounds were first screened for anti-proliferative activity, cytotoxicity and ER-α binding affinity by utilizing ER-α dominant T47D BC cell lines, PBMCs and ER-α competitor assay kit. From this study, two representative compounds 6e and 6f showing most promising activity were advanced for gene expression studies for targeting ER-α. Cell imaging experiment undoubtedly indicate that both the compounds were able to cross cellular bio membrane and accumulate thus instigating cytotoxicity. RT-PCR and Western blotting experiments further strengthened that both compounds altered the expression of mRNA and receptor protein of ER-α, thereby forestalling downstream transactivation and signalling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that indole moiety of the compounds 6e and 6f helps in the anchoring of the xanthendione moiety in the hydrophobic region of the cavity thus enabling the compound to bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these finding collectively imply that compound 6e and 6f represents a novel potent ER-α antagonist and in the development of SERMs for the management of BC.

Structurally Novel Antiestrogens Elicit Differential Responses from Constitutively Active Mutant Estrogen Receptors in Breast Cancer Cells and Tumors.

Many estrogen receptor α (ERα)-positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy-resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602-13. ©2017 AACR.

Design, synthesis and biological evaluation of novel 2-methoxyestradiol analogs as dual selective estrogen receptor modulators (SERMs) and antiangiogenic agents.

2-methoxyestradiol is a novel agent showing both anti-angiogenic and vascular disrupting properties. In this study, a series of 11α-substituted 2-methoxyestradiol analogs have been designed and synthesized targeting dual ERα and microtubulin. Biological evaluation was performed on their anti-proliferative activities against 5 different cell lines. The results indicated that most compounds exhibited good activities, in which compound 24c and 30c showed the best activity with low micromolar IC50 (2.73 µM -7.75 µM) in all cell lines. The investigation of ER affinity showed that the majority of the compounds displayed good activity at the concentration of 50 µM. In further mechanism study, it was observed that 24c and 30c could induce G2/M cell cycle arrest as well as significant anti-estrogenic activity. In CAM assay, compound 24c and 30c presented significantly anti-angiogenesis activity comparable with 2-methoxyestradiol. Overall, based on biological activities data, 24c and 30c can be identified as a potential lead molecule which might be of therapeutic importance for cancer treatment.

Risk assessment of the endocrine-disrupting effects of nine chiral pesticides.

The increased release of chiral pesticides into the environment has generated interest in the role of enantioselectivity in the environmental fate and ecotoxicological effects of these compounds. However, the information on the endocrine disrupting effects (EDEs) of chiral pesticides is still limited and discrepancies are also usually observed among different assays. In this study, we investigated the enantioselectivity of EDEs via estrogen and thyroid hormone receptors for nine chiral pesticides using in vitro and in silico approaches. The results of the luciferase reporter gene assays showed 7 chiral pesticides possessed enantioselective estrogenic activities and 2 chiral pesticides exerted thyroid hormone antagonistic effects. Proliferation assays in MCF-7 and GH3 cells were also used to verify the results of the dual-luciferase reporter gene assays. At last, the molecular docking results indicated that the enantioselective EDEs of chiral pesticides were partially due to enantiospecific binding affinities with receptors. Our data not only show enantioselective EDEs of nine chiral pesticides, but also would be helpful to better understanding the molecular biological mechanisms of enantioselectivity in EDEs of chiral pesticides.

A Semi-automated Approach to Create Purposeful Mechanistic Datasets from Heterogeneous Data: Data Mining Towards the in silico Predictions for Oestrogen Receptor Modulation and Teratogenicity.

The need to find an alternative to costly animal studies for developmental and reproductive toxicity testing has shifted the focus considerably to the assessment of in vitro developmental toxicology models and the exploitation of pharmacological data for relevant molecular initiating events. We hereby demonstrate how automation can be applied successfully to handle heterogeneous oestrogen receptor data from ChEMBL. Applying expert-derived thresholds to specific bioactivities allowed an activity call to be attributed to each data entry. Human intervention further improved this mechanistic dataset which was mined to develop structure-activity relationship alerts and an expert model covering 45 chemical classes for the prediction of oestrogen receptor modulation. The evaluation of the model using FDA EDKB and Tox21 data was quite encouraging. This model can also provide a teratogenicity prediction along with the additional information it provides relevant to the query compound, all of which will require careful assessment of potential risk by experts.

2D-NMR investigation and in vitro evaluation of antioxidant, antigenotoxic and estrogenic/antiestrogenic activities of strawberry grape.

Strawberry grape is considered beneficial due to its extensive phytochemical properties. To expand the knowledge about the chemical constituents and the biological activities of the whole plant, 2D-NMR investigation has been carried out on pulp, peel, seeds, stalks and leaves. Catechin and epicatechin were identified as the main constituents of the seed extract, quercetin and ferulic acid were detected in the leaves and malvidin and cyanidin glucopyranoside in the peels. The leaf, stalk and seed extracts were found to be very rich in phytochemicals and were tested for their ability to reduce the mutagenicity and genotoxicity of standard agents via Salmonella mutagenicity assay and SOS chromotest, respectively. Moreover, the estrogen/antiestrogen-like activity was evaluated on the MCF-7 estrogen-responsive cells. Seed and stalk extracts had an elevated antimutagenic/antigenotoxic activity. Stalk extracts highly reduced the proliferative effect of natural estrogen, 17ß-estradiol.

Non-allowed Pharmacologically Active Substances in Physical and Sexual Performance Enhancing Products.

BACKGROUND: Recently, a large amount of physical and sexual performance enhancing products have started to be freely sold mainly on internet web sites as dietary supplements. However, there a high suspicion that pharmacologically active substance, prohibited in these products, can be present to provide the expected effect. METHODS: A simple and rapid systematic toxicological analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry has been applied after a liquidliquid extraction at acidic, neutral and alkaline pH with chloroform-isopropanol (9:1 v/v). The assays were validated in the range from 10 mg to 250 mg/g products showing a good linearity for the calibration curves (r2 ≥0.99). Mean extraction recoveries of analytes from different products were always higher than 90% and intra-assay and inter-assay precision and accuracy were always better than 15%. RESULTS: The developed method was applied to the analysis of products with a high percentage of sales in websites and smart and sexy shops. In twelve of eighty supplements, anabolic steroids, antiestrogenic drugs, psychoactive substances and sildenafil and analogs were identified and quantified. CONCLUSION: Eventual health hazards caused by the hidden presence of pharmacologically active substances in physical and sexual performance enhancing products are reported.

Development of the ß-lactam type molecular scaffold for selective estrogen receptor α modulator action: synthesis and cytotoxic effects in MCF-7 breast cancer cells.

The estrogen receptors (ERα and ERß) which are ligand inducible nuclear receptors are recognized as pharmaceutical targets for diseases such as osteoporosis and breast cancer. There is an increasing interest in the discovery of subtype Selective Estrogen Receptor Modulators (SERMs). A series of novel ß-lactam compounds with estrogen receptor modulator properties have been synthesized. The antiproliferative effects of these compounds on human MCF-7 breast tumor cells are reported, together with binding affinity for the ERα and ERß receptors. The most potent compound 15g demonstrated antiproliferative effects on MCF-7 breast tumor cells (IC50 = 186 nM) and ERα binding (IC50 = 4.3 nM) with 75-fold ERα/ß receptor binding selectivity. The effect of positioning of the characteristic amine containing substituted aryl ring (on C-4 or N-1 of the ß-lactam scaffold) on the antiproliferative activity and ER-binding properties of the ß-lactam compounds is rationalized in a molecular modeling study.

Evaluation of the removal of antiestrogens and antiandrogens via ozone and granular activated carbon using bioassay and fluorescent spectroscopy.

Antiestrogens and antiandrogens are relatively rarely studied endocrine disrupting chemicals which can be found in un/treated wastewaters. Antiestrogens and antiandrogens in the wastewater treatment effluents could contribute to sexual disruption of organisms. In this study, to assess the removal of non-specific antiestrogens and antiandrogens by advanced treatment processes, ozonation and adsorption to granular activated carbon (GAC), the biological activities and excitation emission matrix fluorescence spectroscopy of wastewater were evaluated. As the applied ozone dose increased to 12 mg/L, the antiestrogenic activity dramatically decreased to 3.2 µg 4-hydroxytamoxifen equivalent (4HEQ)/L, with a removal efficiency of 84.8%, while the antiandrogenic activity was 23.1 µg flutamide equivalent (FEQ)/L, with a removal efficiency of 75.5%. The removal of antiestrogenic/antiandrogenic activity has high correlation with the removal of fulvic acid-like materials and humic acid-like organics, suggesting that they can be used as surrogates for antiestrogenic/antiandrogenic activity during ozonation. The adsorption kinetics of antiestrogenic activity and antiandrogenic activity were well described by pseudo-second-order kinetics models. The estimated equilibrium concentration of antiestrogenic activity is 7.9 µg 4HEQ/L with an effective removal efficiency of 70.5%, while the equilibrium concentration of antiandrogenic activity is 33.7 µg FEQ/L with a removal efficiency of 67.0%. Biological activity evaluation of wastewater effluents is an attractive way to assess the removal of endocrine disrupting chemicals by different treatment processes. Fluorescence spectroscopy can be used as a surrogate measure of bioassays during ozonation.