Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Synthesis and biological activity of 11-Oxygenated and heterocyclic estrone analogs in pancreatic cancer monolayers and 3D spheroids.

Pancreatic Ductal Adenocarcinoma (PDAC), representing over 90 % of pancreatic cancer diagnoses, is an aggressive disease with survivability among the worst of all cancers due to its difficulty in detection and its high metastatic properties. Current therapies for PDAC show limited success at extending life expectancies, primarily due to cancer resistance and lack of patient-specific targeted therapies. This work highlights the design and evaluation of estrone-derived analogs with both heterocyclic side-chain functionality and 11-oxygenated functionality for use in pancreatic cancer. First-round heterocyclic analogs show preliminary promise in AsPC-1 and Panc-1 cell lines, with IC50 values as low as 10.16 ± 0.83 µM. Their success, coupled with design choices from other studies, led to the synthesis of novel 11-hydroxyl and 11-keto estrone analogs that show potent in-vitro toxicity against various pancreatic cancer models. The three most cytotoxic analogs, KA1, KA2, and KA9 demonstrated low micromolar activities in both MTT and CellTiter assays in three pancreatic cancer cell lines: AsPC-1, Panc-1, and BxPC-3, as well as in a co-culture of Panc-1 and pancreatic stellate cells. IC50 values for KA9 (4.17 ± 0.90, 5.28 ± 1.87, and 5.70 ± 0.65 µM respectively) shows consistency in all cell lines tested. KA9 is also able to cause an increase in caspases 3 and 7 activity, key markers for apoptosis, at non-cytotoxic concentrations. Additional work was performed by generating 3D pancreatic cancer spheroids to better modulate the pancreatic tumor microenvironment, and KA9 continued to show the best IC50 values (21.0 and 24.3 µM) in both cell types tested. KA9 was also able to prevent the growth of spheroids whereas the standard chemotherapy, Gemcitabine, could not, suggesting that it may be a potent analog for future development of treatments. Molecular dynamic simulations were also performed to confirm biological findings and uncovered that KA9's preferential binding location is in the active site pocket of key proteins involved in cytotoxicity.

Direct in vitro action of estrone on uterine and white adipose tissue in obesity.

The hypothesis whether estrone (E1) could exhibit a direct action at uterus and white adipose tissue (WAT), under obesity was tested. In uterine tissue of obese rats, E1 increased nitric oxide (NO) synthesis, and reduced reactive oxygen species (ROS) production. The anti-oxidative action of E1 was sustained under inflammatory stress or high glucose levels. ICI 182780 or G15 compounds were employed as ER or GPER antagonists respectively. The action of E1 on ROS release involved ER participation; instead GPER mediated the acute stimulation on NO production. The antioxidative effect depends on NO-ROS balance. NO synthase (NOS) blockage suppressed the reduction in ROS synthesis elicited by E1, effect mediated by cNOS and not by iNOS. On WAT explants, E1 reduced ROS and thiobarbituric acid reactive substances production, and diminished leptin release. In summary, the data provide evidence that, in uterus and WAT, E1 counteracts inflammatory and oxidative stress induced by obesity.

EGR3 and estrone are involved in the tamoxifen resistance and progression of breast cancer.

BACKGROUND: Tamoxifen (Tam) is an effective treatment for estrogen receptor (ER) positive breast cancer. However, a significant proportion of patients develop resistance under treatment, presenting a therapeutic challenge. The study aims to determine the role of early growth response protein (EGR) 3 in tamoxifen resistance (TamR) and elucidate its molecular mechanism. METHODS: TamR cell models were established and NGS was used to screening signaling alternation. Western blot and qRT-PCR were used to analysis the expression of ERα, EGR3, MCL1 and factors associated with apoptosis. CCK8, colony formation and apoptosis assay were used to analysis resistance to Tam. Immunofluorescence, chromatin immunoprecipitation, and dual luciferase assays were used to investigate mechanism of regulation. RESULTS: We observed that EGR3, a deeply rooted ERα response factor, showed increased upregulation in response to both estrone (E1) and Tam in TamR cells with elevated level of E1 and ERα expression, indicating a potential connection between EGR3 and TamR. Mechanically, manipulating EGR3 expression revealed that it imparted resistance to Tam through increased expression of the downstream molecule MCL1 (apoptosis suppressor gene) that it regulated. Mechanismly, EGR3 directly binds to the promoter of the anti-apoptotic factor MCL1 gene, facilitating its transcription. Furthermore, apoptosis assays revealed that E1 reduces Tam induced apoptosis by upregulating EGR3 expression. Importantly, clinical public database confirmed the high expression of EGR3 in breast cancer tissue and in Tam-treated patients. CONCLUSIONS: These findings shed light on the novel estrogen/EGR3/MCL1 axis and its role in inducing TamR in ER positive breast cancer. EGR3 emerges as a promising target to overcome TamR. The elucidation of this mechanism holds potential for the development of new therapeutic modalities to overcome endocrine therapy resistance in clinical settings.

Biochemical responses of freshwater microalgae Chlorella sorokiniana to combined exposure of Zn(â ¡) and estrone with simultaneous pollutants removal.

With the development of livestock industry, contaminants such as divalent zinc ions (Zn (Ⅱ)) and estrone are often simultaneously detected in livestock wastewater. Nevertheless, the combined toxicity of these two pollutants on microalgae is still unclear. Moreover, microalgae have the potential for biosorption and bioaccumulation of heavy metals and organic compounds. Thus, this study investigated the joint effects of Zn (Ⅱ) and estrone on microalgae Chlorella sorokiniana, in terms of growth, photosynthetic activity and biomolecules, as well as pollutants removal by algae. Interestingly, a low Zn (Ⅱ) concentration promoted C. sorokiniana growth and photosynthetic activity, while the high concentration experienced inhibition. As the increase of estrone concentration, chlorophyll a content increased continuously to resist the environmental stress. Concurrently, the secretion of extracellular polysaccharides and proteins by algae increased with exposure to Zn (Ⅱ) and estrone, reducing toxicity of pollutants to microalgae. Reactive oxygen species and superoxide dismutase activity increased as the increase of pollutant concentration after 96 h cultivation, but high pollutant concentrations resulted in damage of cells, as proved by increased MDA content. Additionally, C. sorokiniana displayed remarkable removal efficiency for Zn (Ⅱ) and estrone, reaching up to 86.14% and 84.96% respectively. The study provides insights into the biochemical responses of microalgae to pollutants and highlights the potential of microalgae in pollutants removal.

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 µM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs.

Cancers utilize sugar residues such as sialic acids (Sia) to improve their ability to survive. Sia presents a variety of functional group alterations, including O-acetylation on the C6 hydroxylated tail. Previously, sialylation has been reported to suppress EGFR activation and increase cancer cell sensitivity to Tyrosine Kinase Inhibitors (TKIs). In this study, we report on the effect of deacetylated Sia on the activity of three novel EGFR-targeting Cucurbitacin-inspired estrone analogs (CIEAs), MMA 294, MMA 321, and MMA 320, in lung and colon cancer cells. Acetylation was modulated by the removal of Sialate O-Acetyltransferase, also known as CAS1 Domain-containing protein (CASD1) gene via CRISPR-Cas9 gene editing. Using a variety of cell-based approaches including MTT cell viability assay, flow cytometry, immunofluorescence assay and in-cell ELISA we observed that deacetylated Sia-expressing knockout cells (1.24-6.49 µM) were highly sensitive to all CIEAs compared with the control cells (8.82-20.97 µM). Apoptosis and varied stage cell cycle arrest (G0/G1 and G2/M) were elucidated as mechanistic modes of action of the CIEAs. Further studies implicated overexpression of CIEAs' cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that Sia deacetylation renders lung and colon cancer cells susceptible to EGFR therapeutics and provide insights for future therapeutic interventions.

Antiproliferative and Antimetastatic Properties of 16-Azidomethyl Substituted 3-O-Benzyl Estrone Analogs.

Four diastereomers of 16-azidomethyl substituted 3-O-benzyl estradiol (1-4) and their two estrone analogs (16AABE and 16BABE) were tested for their antiproliferative properties against human gynecological cancer cell lines. The estrones were selected for additional experiments based on their outstanding cell growth-inhibiting activities. Both compounds increased hypodiploid populations of breast cancer cells, and 16AABE elicited cell cycle disturbance as evidenced by flow cytometry. The two analogs substantially increased the rate of tubulin polymerization in vitro. 16AABE and 16BABE inhibited breast cancer cells' migration and invasive ability, as evidenced by wound healing and Boyden chamber assays. Since both estrone analogs exerted remarkable estrogenic activities, as documented by a luciferase reporter gene assay, they can be considered as promising drug candidates for hormone-independent malignancies.

Bisphenol analogues inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis.

Bisphenols, estrogenic endocrine-disrupting chemicals, disrupt at least one of three endocrine pathways (estrogen, androgen, and thyroid). 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) is a steroidogenic enzyme that catalyzes the activation of estradiol from estrone in human placenta and rat ovary. However, whether bisphenols inhibit 17ß-HSD1 and the mode of action remains unclear. This study we screened 17 bisphenols for inhibiting human 17ß-HSD1 in placental microsomes and rat 17ß-HSD1 in ovarian microsomes and determined 3D-quantitative structure-activity relationship (3D-QSAR) and mode of action. We observed some bisphenols with substituents were found to significantly inhibit both human and rat 17ß-HSD1 with the most potent inhibition on human enzyme by bisphenol H (IC50 = 0.90 µM) when compared to bisphenol A (IC50 = 113.38 µM). Rat enzyme was less sensitive to the inhibition of bisphenols than human enzyme with bisphenol H (IC50 = 32.94 µM) for rat enzyme. We observed an inverse correlation between IC50 and hydrophobicity (expressed as Log P). Docking analysis showed that they bound steroid-binding site of 17ß-HSD1. The 3D-QSAR models demonstrated that hydrophobic region, hydrophobic aromatic, ring aromatic, and hydrogen bond acceptor are key factors for the inhibition of steroid synthesis activity of 17ß-HSD1.

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

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

Novel Indoline Spiropyrans Based on Human Hormones ß-Estradiol and Estrone: Synthesis, Structure, Chromogenic and Cytotoxic Properties.

The introduction of a switchable function into the structure of a bioactive compound can endow it with unique capabilities for regulating biological activity under the influence of various types of external stimuli, which makes such hybrid compounds promising objects for photopharmacology, targeted drug delivery and bio-imaging. This work is devoted to the synthesis and study of new spirocyclic derivatives of important human hormones-ß-estradiol and estrone-possessing a wide range of biological activities. The obtained hybrid compounds represent an indoline spiropyrans family, a widely known class of organic photochromic compounds. The structure of the compounds was confirmed by 1H and 13C NMR, IR, HRMS and single-crystal X-ray analysis. The intermolecular interactions in the crystals of spiropyran (3) were defined by Hirshfeld surfaces and 2D fingerprint plots, which were successfully acquired from CrystalExplorer (v21.5). All target hybrids demonstrated pronounced activity in the visible region of the spectrum. The mechanisms of thermal isomerization processes of spiropyrans and their protonated merocyanine forms were studied by DFT methods, which revealed the energetic advantage of the protonation process with the formation of a ß-cisoid CCCH conformer at the first stage and its further isomerization to more stable ß-transoid forms. The proposed mechanism of acidochromic transformation was confirmed by the additional NMR study data that allowed for the detecting of the intermediate CCCH isomer. The study of the short-term cytotoxicity of new spirocyclic derivatives of estrogens and their 2-formyl-precursors was performed on the HeLa cell model. The precursors and spiropyrans differed in toxicity, suggesting their variable applicability in novel anti-cancer technologies.

Description of Chemical Synthesis, Nuclear Magnetic Resonance Characterization and Biological Activity of Estrane-Based Inhibitors/Activators of Steroidogenesis.

Steroid hormones play a crucial role in several aspects of human life, and steroidogenesis is the process by which hormones are produced from cholesterol using several enzymes that work in concert to obtain the appropriate levels of each hormone at the right time. Unfortunately, many diseases, such as cancer, endometriosis, and osteoporosis as examples, are caused by an increase in the production of certain hormones. For these diseases, the use of an inhibitor to block the activity of an enzyme and, in doing so, the production of a key hormone is a proven therapeutic strategy whose development continues. This account-type article focuses on seven inhibitors (compounds 1-7) and an activator (compound 8) of six enzymes involved in steroidogenesis, namely steroid sulfatase, aldo-keto reductase 1C3, types 1, 2, 3, and 12 of the 17ß-hydroxysteroid dehydrogenases. For these steroid derivatives, three topics will be addressed: (1) Their chemical synthesis from the same starting material, estrone, (2) their structural characterization using nuclear magnetic resonance, and (3) their in vitro or in vivo biological activities. These bioactive molecules constitute potential therapeutic or mechanistic tools that could be used to better understand the role of certain hormones in steroidogenesis.

Estrone analogs as potential inhibitors targeting EGFR-MAPK pathway in non-small-cell lung cancer.

Lung cancer is the deadliest human cancer globally, with non-small-cell lung cancer (NSCLC) being the most frequent type. Epidermal growth factor receptor (EGFR), a central regulator of tumor progression is frequently overexpressed in NSCLC and is a key drug target along with its downstream pathways. Here, we describe the biological evaluation of previously synthesized estrone analogs as potent inhibitors of NCI-H226 cells. Two of the analogs, MMA307 and MM320, significantly inhibited the proliferation of NCI-H226 cells with IC50 doses of 2.88 ± 0.21 and 9.68 ± 0.24 µM, respectively, compared with the positive control and chemotherapy, sorafenib, IC50 of 20.62 ± 1.32 µM. Exposing NCI-H226 cells to IC50 concentration of MMA307 and MMA320 resulted in the downregulation of EGFR and phospho-EGFR expression levels, and suppression of activated MAPK-ERK1/2 signaling proteins; phospho-B-Raf, phospho-MEK1/2 , and phospho-ERK1/2 . Furthermore, the downregulation of cyclin D1 and concomitant upregulation of phospho-cyclin D1 and p21waf1/cip1 were observed after the compounds' addition to NCI-H226 cells resulting in G1 phase cell cycle arrest. MMA320 but not MMA307 downregulated the expression levels of Dyrk1B, a checkpoint kinase at the G1 -S phase transition of the cell cycle. Additionally, molecular dynamic simulations were performed and found that MMA307 and MMA320 have higher binding affinities than sorafenib in MEK, BRAF, cyclin D1 , and Dyrk1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B). To conclude, the present study is the first to report on the antiproliferative potential of novel estrone analogs and provide evidence that MMA307 and MMA320 are promising novel lead candidates for the development of antilung cancer drugs.

Synthesis, antiproliferative evaluation and in silico studies of a novel steroidal spiro morpholinone.

Estrogens play a pivotal role in the development of estrogen-dependent breast cancer and other hormone-dependent disorders. A common strategy to overcome the pathological effects of estrogens is the use of aromatase inhibitors (AIs), which bind to the enzyme and prevent the union with the natural substrate, decreasing the amount of estrogens produced. Several AIs have been developed, including inhibitors with a steroidal backbone and a nitrogen heterocycle in their structure. Encouraged by the notable results presented by current and clinical steroidal drugs, herein we present the synthesis of a steroidal spiro morpholinone derivative as a plausible aromatase inhibitor. The morpholinone derivative was synthesized over a six-step methodology starting from estrone. The title compound and its hydroxychloroacetamide derivative precursor were evaluated for their antiproliferative profile against estrogen-dependent and independent solid tumor cell lines: A549, HBL-100, HeLa, SW1573, T-47D and WiDr. Both compounds exhibited a potent antiproliferative activity in the micromolar range against the six cancer cell lines, with the hydroxychloroacetamide derivative precursor being a more potent inhibitor (GI50 = 0.25-2.4 µM) than the morpholinone derivative (GI50 = 2.0-11 µM). Furthermore, both compounds showed, in almost all cases, better GI50 values than the steroidal anticancer drugs abiraterone and galeterone. Docking simulations of the derivatives were performed in order to explain the experimental biological activity. The results showed interactions with the iron heme (derivative 3) and important residues of the steroidal binding-site (Met374) for the inhibition of human aromatase. A correlation was found between in vitro assays and the score obtained from the molecular docking study.

Chronic estrone exposure affects spermatogenesis and sperm quality in zebrafish (Danio rerio).

Estrone (E1) is a common environmental contaminant found in rivers and streams due to the farming of animals, such as swine and cattle. Our study evaluated the effects of chronic E1 exposure at environmentally relevant concentrations on spermatogenesis and the semen quality of zebrafish (Danio rerio). We exposed the fish to E1 at concentrations of 20, 200, and 2000 ng/L diluted in 0.001% ethanol (v/v) for 49 days. There were two control groups: one was exposed to water only and the other to ethanol at the same concentration used in the E1 groups. Following exposure, we analyzed the proportion of testicular cell types and other components (%), rate of cell proliferation and death, and sex steroid concentrations. Furthermore, we analyzed the expression of insulin-like growth factor 1 (IGF1), IGF2, IGF1 receptor (IGF1R), and inducible nitric oxide synthase and assessed the semen quality. E1 exposure increased spermatogonia, spermatids, Sertoli cells, Leydig cells, and the proportion of inflammatory infiltrate but decreased the spermatozoa amount. These changes were reflected by reductions in the gonadosomatic index and levels of 11-ketotestosterone in the testes. On the other hand, E1 exposure increased testicular estradiol, IGF1R expression, and nitric oxide production. After an evaluation using a computer-assisted sperm analysis (CASA) system, we observed reduced progressive motility, curvilinear velocity, and beat cross frequency of 20 and 2000 ng/L E1 groups. Our findings support that E1 causes deleterious effects on the testicular function and semen quality of D. rerio even at environmental concentrations. Thus, E1 concentrations should be monitored in surface waters for the purposes of fish conservation.

Chemical Synthesis and Biological Evaluation of 3-Substituted Estrone/Estradiol Derivatives as 17ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitors Acting via a Reverse Orientation of the Natural Substrate Estrone.

Estradiol (E2) plays an important role in the progression of diseases such as breast cancer and endometriosis. Inhibition of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), the enzyme that catalyzes the last step in the biosynthesis of the estrogenic hormone E2, therefore constitutes an interesting approach for the treatment of these two estrogen-dependent diseases. In order to obtain new inhibitors of 17ß-HSD1, the impact of a m-carbamoylphenyloxy group at position three of an estrane nucleus was evaluated by preparing three derivatives of estrone (E1) and E2 using a microwave-assisted synthesis of diaryl ethers. Their inhibitory activity was addressed on two cell lines (T-47D and Z-12) representative of breast cancer and endometriosis, respectively, but unlike T-47D cells, Z-12 cells were not found suitable for testing potential 17ß-HSD1 inhibitors. Thus, the addition of the m-carbamoylphenyl group at C3 of E1 (compound 5) did not increase the inhibition of E1 to E2 transformation by 17ß-HSD1 present in T-47D cells (IC50 = 0.31 and 0.21 µM for 5 and E1, respectively), and this negative effect was more obvious for E2 derivatives 6 and 10 (IC50 = 1.2 and 1.3 µM, respectively). Molecular docking allowed us to identify key interactions with 17ß-HSD1 and to highlight these new inhibitors' actions through an opposite orientation than natural enzyme substrate E1's classical one. Furthermore, molecular modeling experiments explain the better inhibitory activity of E1-ether derivative 5, as opposed to the E2-ether derivatives 6 and 10. Finally, when tested on T-47D and Z-12 cells, compounds 5, 6 and 10 did not stimulate the proliferation of these two estrogen-dependent cell lines. In fact, they reduced it.

Synthesis of estrone selenocyanate Compounds, anti-tumor activity evaluation and Structure-activity relationship analysis.

Introducing different functional groups into steroid can bring unexpected changes in biological activity of the steroid. Using estrone as a raw material, through the functional group conversion and modification of the 17-carbonyl, the structural fragments with selenocyano groups were instilled in the form of amide, ester, and oxime ester, respectively, and various 17-substituted estrone selenocyanate derivatives were synthesized. In addition, different 3-substituted estrone selenocyanate derivatives were synthesized by introducing different selenocyanoalkoxy fragments into the 3-position of estrone in the form of alkyl ether. Furthermore, the selenocyano-containing moieties were embedded into the 2-position of estrone by means of amide, affording diverse 2-selenocyanoamide-estrone derivatives. The antiproliferative activities of the target compounds were screened by selecting tumor cell lines related to the expression of human hormones. The results showed that the introduction of selenocyano group into estrone could endow estrone with significant biological activity of inhibiting the proliferation of tumor cells. Structure-activity relationship research showed that the cytotoxicity of 3-selenocyanoalkoxy-estrone was further increased with the extension of alkyl carbon-chain within 8 carbon chain lengths. In addition, the cytotoxicity of the products with selenocyano via the form of amide was stronger than that of ester or ether. Selenocyano moiety instilled at the 2-position of estrone in the form of amide was more cytotoxic than that of 17- or 3-position. Among them, compound 21a has better inhibitory activity on tested tumor cells than positive controls Abiraterone and 2-methoxyestradiol. Research showed that the compound 21c induced programmed apoptosis in Sk-Ov-3 cancer cells, and compound 17d inhibited significantly the growth of human cervical cancer zebrafish xenografts in vivo, offering useful insights into the synthesis of steroid antitumor drugs.

Substitutional Diversity-Oriented Synthesis and In Vitro Anticancer Activity of Framework-Integrated Estradiol-Benzisoxazole Chimeras.

Hybridization of steroids and other pharmacophores often modifies the bioactivity of the parent compounds, improving selectivity and side effect profile. In this study, estradiol and 3'-(un)substituted benzisoxazole moieties were combined into novel molecules by structural integration of their aromatic rings. Simple estrogen starting materials, such as estrone, estradiol and estradiol-3-methylether were used for the multistep transformations. Some of the heterocyclic derivatives were prepared from the estrane precursor by a formylation or Friedel-Crafts acylation-oximation-cyclization sequence, whereas others were obtained by a functional group interconversion strategy. The antiproliferative activities of the synthesized compounds were assessed on various human cervical, breast and prostate cancer cell lines (HeLa, MCF-7, PC3, DU-145) and non-cancerous MRC-5 fibroblast cells. Based on the primary cytotoxicity screens, the most effective cancer-selective compounds were selected, their IC50 values were determined and their apoptosis-inducing potential was evaluated by quantitative real-time PCR. Pharmacological studies revealed a strong structure-function relationship, where derivatives with a hydroxyl group on C-17 exhibited stronger anticancer activity compared to the 17-acetylated counterparts. The present study concludes that novel estradiol-benzisoxazole hybrids exert remarkable cancer cell-specific antiproliferative activity and trigger apoptosis in cancer cells.

Design and synthesis of hetero-steroids via ring-closing metathesis: Biological studies towards in vitro anticancer activity.

Here, we report a synthetic approach to hetero-steroids and also studied their biological activities as anticancer agents. A novel class of oxacycles containing estrone moiety were synthesized in this report. Allyl ether derived from estrone underwent Claisen rearrangement (CR) and again O-allylation and subsequent ring-closure gave A-ring-furan and oxepine fused derivatives in high yields. We used double bond isomerization and ring-closing metathesis (RCM) as key steps to assemble hetero steroids containing a mixture of regio isomers like benzofurans and benzoxepine moieties. The novel benzofuran and benzoxepine-based hybrid steroid derivatives were subjected to in vitro cytotoxicity analysis and were found to exert cancer cell-specific activity.

Augmenting Skeletal Muscle Estrogen Does not Prevent or Rescue Obesity-linked Metabolic Impairments in Female Mice.

AIMS: We developed a novel mouse model with increased skeletal muscle estrogen content via inducible, skeletal-muscle-specific aromatase overexpression (SkM-Arom↑). We proposed to examine the effect that increased skeletal muscle estrogen both in gonadally intact and ovariectomized (OVX) female mice has on preventing or rescuing high-fat diet (HFD)-induced obesity. METHODS: In the prevention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed a low-fat diet (LFD) or HFD for 13 weeks. SkM-Arom↑ was induced at the initiation of dietary treatment. In the intervention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed an HFD for 14 weeks before induction of SkM-Arom↑ for 6 weeks. Glucose tolerance, insulin action, adipose tissue inflammation, and body composition were assessed. Liquid chromatography-mass spectrometry was used to determine circulating and skeletal muscle steroid content. RESULTS: SkM-Arom↑ significantly increased skeletal muscle 17ß-estradiol (E2) and estrone (E1) in both experiments. Interestingly, this resulted in leakage of estrogens into circulation, producing a physiologically relevant E2 concentration. Consequently, bone mineral density (BMD) was enhanced and adipose tissue inflammation was reduced in the prevention experiment only. However, no benefits were seen with respect to changes in adiposity or metabolic outcomes. CONCLUSION: We show that increasing skeletal muscle estrogen content does not provide a metabolic benefit in gonadally intact and OVX female mice in the setting of obesity. However, a chronic physiological concentration of circulating E2 can improve BMD and reduce adipose tissue inflammation independently of a metabolic benefit or changes in adiposity.