A series of unconjugated ferrocenyl phenols: prospects as anticancer agents

We recently reported that a ferrocenyl diphenol butene derivative showed a very strong cytotoxic effect on both hormone-dependent and -independent breast cancer cell lines. In order to obtain more information about the structure-activity relationship in the cytotoxicity of small ferrocene compounds, we have prepared a series of simple unconjugated ferrocenyl diphenol complexes (ortho,para; meta,para; para,para). These compounds retain a reasonable to good affinity for both estrogen receptor types, with higher values for the beta form, and superior binding for the para,para diphenol complex (RBA=28%). In vitro these complexes exhibit significant cytotoxic effects on hormone-independent prostate (PC3) and breast cancer cell lines (MDA-MB231), with IC50 values between 2.5 and 4.1 microM. This effect is more marked with PC3, the ortho,para diphenol complex proving the most effective. On the hormone-dependent MCF7 breast cancer cell line, the observed effect seems to be the result of two components, one cytotoxic (antiproliferative), the other estrogenic (proliferative). Electrochemical studies show that the cytotoxic effect of the complexes correlates with the ease of oxidation of the ferrocene group. All these complexes are much less cytotoxic than the ferrocenyl diphenol butene derivative.     

Synthesis,receptor binding,molecular modeling,and proliferative assays of a series of 17alpha-arylestradiols

A series of new derivatives of estradiol substituted at position 17alpha by various aryls has been synthesized. This was made possible by efficient activation methods for the addition of aryllithiums to the carbonyl group at position 17 of estrone by using tetramethylethylenediamine (TMEDA) or BF3 x OEt2. Their relative binding affinity (RBA) for the alpha and the beta forms of the estrogen receptor (ER) have been measured. All except one of the compounds synthesized had an RBA value of around 10 % which indicates a level of tolerance towards the bulky substituent at position 17. The lipophilicity values measured for these compounds are higher than that found for estradiol (E2). A study of their proliferative/antiproliferative effects was carried out on hormone-dependent (MCF7) and hormone-independent (MDA-MB231) breast cancer cell lines. It is interesting to note that all the compounds are estrogenic. The possibility of easily attaching an iodine at the end of a phenyl spacer opens up a route to new radiopharmaceuticals for use in radioimaging.     

Tamoxifen derivatives for delivery of the antitumoral (DACH)Pt group: selective synthesis by McMurry coupling,and biochemical behaviour

The goal of our study was to potentiate the effects of the ((R,R)-trans-1,2-diaminocyclohexane)-platinum(II) fragment [(DACH)Pt], known for its cytotoxic properties, either with tamoxifen (Tam), the most widely used antiestrogen in the treatment of hormone-dependent breast cancers, or with its active metabolite hydroxytamoxifen (hydroxy-Tam). We coupled Tam or hydroxy-Tam derivatives bearing a malonato group at the para position of the beta aromatic ring with the (DACH)Pt fragment. The malonato-Tam and malonato-hydroxy-Tam compounds were prepared through McMurry coupling of the appropriate ketones. The presence of the malonate group resulted in a pronounced stereospecificity in the reaction, since malonato-Tam was obtained only as the Z isomer, while malonato-hydroxy-Tam was obtained as an 80/20 E/Z mixture. Attribution of the isomeric structures was achieved by 2D NMR spectroscopy. The platinum complexes (DACH)Pt-malonato-Tam and (DACH)Pt-malonato-hydroxy-Tam were then prepared by coupling the barium salts derived from the malonato-Tam and malonato-hydroxy-Tam with the nitrate derived from (DACH)PtCl(2). Study of the biochemical properties of these two platinum complexes showed that, while the hydroxy-Tam complex is satisfactorily recognized by the estrogen receptor (relative binding affinity, RBA=6.4 %), the Tam complex is less well recognized (RBA=0.5 %). The effects of these complexes on two hormone-dependent breast cancer cell lines (MCF7 and MVLN) were studied in vitro. Both complexes showed an antiproliferative effect on MCF7 cells, and an antiestrogenic effect on MVLN cells. The observed effects appear to be essentially antihormonal, since incorporation of the (DACH)Pt fragment into the tamoxifen skeleton did not cause an increase in the cytotoxicity of the complexes.     

Steroid conjugates of dichloro(6-aminomethylnicotinate)platinum(II): effects on DNA, sex hormone binding globulin, the estrogen receptor, and various breast cancer cell lines

Esters of 6-aminomethylnicotinic acid with various steroidal alcohols were treated with K(2)PtCl(4) to give the N,N-chelated dichloroplatinum(II) complex conjugates 4. Their interaction with plasmid DNA was monitored by electrophoretic mobility measurements. Their affinities towards sex hormone binding globulin (SHBG) and towards the nuclear estrogen receptor ER(alpha) were assessed by competitive displacement radioassays. The inhibitory effect of 4 on breast tumour cells MCF-7 ER(+)/ER(-) and MDA-MB-231 was investigated in vitro. Conjugates with 3-O-linked estrogens 4 a,b or 17-O-linked androgens 4 g bound strongly to SHBG. The conjugate complex 4 b, featuring a 3-O-linked estradiol, also bound strongly and agonistically to the estrogen receptor. It also elicited distinct growth retardation of MCF-7 (ER(+)) cells, presumably by a mechanism different from that of cisplatin.     

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.     

Focused libraries of 16-substituted estrone derivatives and modified e-ring steroids: inhibitors of 17beta-hydroxysteroid dehydrogenase type 1

17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), an oxidoreductase which has a preferential reductive activity using NADPH as cofactor, converts estrone to estradiol and is expressed in many steroidogenic tissues including breast and in malignant breast cells. As estradiol stimulates the growth and development of hormone-dependent breast cancer, inhibition of the final step of its synthesis is an attractive target for the treatment of this disease. The parallel synthesis of novel focused libraries of 16-substituted estrone derivatives and modified E-ring pyrazole steroids as new potent 17beta-HSD1 inhibitors is described. Substituted 3-O-sulfamoylated estrone derivatives were used as templates and were immobilised on 2-chlorotrityl chloride resin to give resin-bound scaffolds with a multi-detachable linker. Novel focused libraries of 16-substituted estrone derivatives and new modified E-ring steroids were assembled from these immobilised templates using solid-phase organic synthesis and solution-phase methodologies. Among the derivatives synthesised, the most potent 17beta-HSD1 inhibitors were 25 and 26 with IC50 values in T-47D human breast cancer cells of 27 and 165 nm, respectively. Parallel synthesis resulting in a library of C5'-linked amides from the pyrazole E-ring led to the identification of 62 with an IC50 value of 700 nM. These potent inhibitors of 17beta-HSD1 have a 2-ethyl substituent which will decrease their estrogenic potential. Several novel 17beta-HSD1 inhibitors emerged from these libraries and these provide direction for further template exploration in this area. A new efficient diastereoselective synthesis of 25 has also been developed to facilitate supply for in vivo evaluation, and an X-ray crystal structure of this inhibitor is presented.     

Jostling for Position: Optimizing Linker Location in the Design of Estrogen Receptor‐Targeting PROTACs

Estrogen receptor‐α (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone‐sensitive ER‐positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof‐of‐concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER‐targeting PROTACs are composed of an estradiol on one end and a hypoxia‐inducing factor 1α (HIF‐1α)‐derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7α position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex).     

Bicyclic substituted hydroxyphenylmethanone type inhibitors of 17 β-hydroxysteroid dehydrogenase Type 1 (17 β-HSD1): the role of the bicyclic moiety

An attractive target that has still to be explored for the treatment of estrogen‐dependent diseases, such as breast cancer and endometriosis, is the enzyme responsible for the last step in the biosynthesis of estradiol (E2): 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1). It catalyzes the reduction of the weakly active estrone (E1) into E2, which is the most potent estrogen in humans. Inhibition of 17β‐HSD1 lowers intracellular E2 concentrations and thus presents a therapeutic target for estrogen‐dependent pathologies. Recently, we reported a new class of highly active and selective 17β‐HSD1 inhibitors: bicyclic substituted hydroxyphenylmethanones. Here, further structural variations on the bicyclic moiety are described, especially focusing on the exchange of its hydroxy function. Twenty‐nine novel inhibitors were synthesized and evaluated for 17β‐HSD1 inhibition in a cell‐free and cellular assay, for selectivity toward 17βHSD2 and estrogen receptors (ER) alpha and beta, as well as for metabolic stability. The best compound exhibited IC₅₀ values of 12 nM (cell‐free assay) and 78 nM (cellular assay), high selectivity for 17β‐HSD1, and reasonable metabolic stability. A molecular docking study provided insight into the protein–ligand interactions of this compound with 17β‐HSD1.     

β-Diketonate versus β-Ketoiminate: The Importance of a Ferrocenyl Moiety in Improving the Anticancer Potency

Herein we present a library of fully characterized β-diketonate and β-ketoiminate compounds that are functionalized with a ferrocenyl moiety. Their cytotoxic potential has been determined by screening against human breast adenocarcinomas (MCF-7 and MDA-MB-231), human colorectal carcinoma p53 wild type (HCT116 p53^+/+) and normal human prostate (PNT2) cell lines. The ferrocenyl β-diketonate compounds are more than 18 times more cytotoxic than the ferrocenyl β-ketoiminate analogues. Against MCF-7, compounds functionalized at the meta position are up to nine times more cytotoxic than when functionalized at the para position. The ferrocenyl β-diketonate compounds have increased selectivity towards MCF-7 and MDA-MB-231, with several complexes having selectivity index (SI) values that are more than nine times (MCF-7) and more than six times (MDA-MB-231) that of carboplatin. The stability of these compounds in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) has been assessed by NMR spectroscopy and mass spectrometry studies, and the compounds show no oxidation of the iron center from Fe^II to Fe^III. Cytotoxicity screening was performed in both DMSO and DMF, with no significant differences observedin their potency.     

The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.     

Putative biomarkers and targets of estrogen receptor negative human breast cancer

Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER), progesterone receptor (PR), and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self-renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.     

Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status

Potential antiproliferative effects of tocotrienols, the major vitamin E component in palm oil, were investigated on the growth of both estrogen-responsive (ER+) MCF7 human breast cancer cells and estrogen-unresponsive (ER-) MDA-MD-231 human breast cancer cells, and effects were compared with those of α-tocopherol (αT). The tocotrienol-rich fraction (TRF) of palm oil inhibited growth of MCF7 cells in both the presence and absence of estradiol with a nonlinear dose-response but such that complete suppression of growth was achieved at 8 μg/mL. MDA-MB-231 cells were also inhibited by TRF but with a linear dose-response such that 20 μg/mL TRF was needed for complete growth suppression. Separation of the TRF into individual tocotrienols revealed that all fractions could inhibit growth of both ER+ and ER- cells and of ER+ cells in both the presence and absence of estradiol. However, the γ- and δ-fractions were the most inhibitory. Complete inhibition of MCF7 cell growth was achieved at 6 μg/mL of γ-tocotrienol/δ-tocotrienol (γT₃/δT₃) in the absence of estradiol and 10μm/mL of δT₃ in the presence of estradiol, whereas complete suppression of MDA-MB-231 cell growth was not achieved even at concentrations of 10μg/mL of δT₃. By contrast to these inhibitory effects of tocotrienols, αT had no inhibitory effect on MCF7 cell growth in either the presence or the absence of estradiol, nor on MDA-MB-231 cell growth. These results confirm studies using other sublines of human breast cancer cells and demonstrate that tocotrienols can exert direct inhibitory effects on the growth of breast cancer cells. In searching for the mechanism of inhibition, studies of the effects of TRF on estrogen-regulated pS2 gene expression in MCF7 cells showed that tocotrienols do not act via an estrogen receptor-mediated pathway and must therefore act differently from estrogen antagonists. Furthermore, tocotrienols did not increase levels of growth-inhibitory insulin-like growth factor binding proteins (IGFBP) in MCF7 cells, implying also a different mechanism from that proposed for retinoic acid inhibition of estrogen-responsive breast cancer cell growth. Inhibition of the growth of breast cancer cells by tocotrienols could have important clinical implications not only because tocotrienols are able to inhibit the growth of both ER+ and ER- phenotypes but also because ER+ cells could be growth-inhibited in the presence as well as in the absence of estradiol. Future clinical applications of TRF could come from potential growth suppression of ER+ breast cancer cells otherwise resistant to growth inhibition by antiestrogens and retinoic acid.     

Modelling and Phenotypic Screening of NAP-6 and 10-Cl-BBQ,AhR Ligands Displaying Selective Breast Cancer Cytotoxicity in Vitro

To exploit the interaction of the aryl hydrocarbon receptor (AhR) pathway in developing breast-cancer-specific cytotoxic compounds, we examined the breast cancer selectivity and the docking pose of the AhR ligands (Z)-2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6; 5 ) and 10-chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ; 6 ). While the breast cancer selectivity of 5 in vitro is known, we discuss the SAR around this lead and, by using phenotypic cell-line screening and the MTT assay, show for the first time that 6 also presents with breast cancer selectivity, notably in the triple-negative (TN) receptor breast cancer cell line MDA-MB-468, the ER+ breast cancer cell lines T47D, ZR-75-1 and the HER2+ breast cancer cell line SKBR3 (GI₅₀ values of 0.098, 0.97, 0.13 and 0.21 μM, respectively). Indeed, 6 is 55 times more potent in MDA-MB-468 cells than normal MCF10A breast cells (GI₅₀ of 0.098 vs 5.4 μM) and more than 130 times more potent than in cell lines derived from pancreas, brain and prostate (GI₅₀ of 0.098 vs 10–13 μM). Molecular docking poses of 5 and 6 together with analogue synthesis and phenotypic screening show the importance of the naphthalene moiety, and an ortho-disposed substituent on the N-phenyl moiety for biological activity.     

Non-steroidal aromatase inhibitors based on a biphenyl scaffold: synthesis, in vitro SAR, and molecular modelling

The synthesis and in vitro biological evaluation (JEG-3 cells) of a series of novel and potent aromatase inhibitors, prepared by microwave-enhanced Suzuki cross-coupling methodology, are reported. These compounds possess a biphenyl template incorporated with the haem-ligating triazolylmethyl moiety, either on its own or in combination with other substituent(s) at various positions on the phenyl rings. The most potent aromatase inhibitor reported herein has an IC(50) value of 0.12 nM, although seven of its congeners are also highly potent (IC(50)相似文献   

Small Structural Differences between Two Ferrocenyl Diphenols Determine Large Discrepancies of Reactivity and Biological Effects

The ferrocenyl diphenol complexes 1,1-bis(4′-hydroxyphenyl)-2-ferrocenyl-but-1-ene ( 1 ) and 1,2-bis(4′-hydroxyphenyl)-1-ferrocenyl-but-1-ene [(Z)- 2 ], which differ by the relative position of the two phenolic substituents, display dramatically different antiproliferative activities on cancer cells ( 1 is far more cytotoxic than 2 ). In this study, our goal was to discover the origin of this difference by comparing their reactivity and biological behaviour. In terms of common behaviour, we found that 1 and 2 are both efficient inhibitors of thioredoxin reductase (TrxR) in vitro after oxidation by a horseradish peroxidase/H₂O₂ system. However, as 1 is only a moderate inhibitor of TrxR in MDA-MB-231 cells, TrxR is probably not the major target responsible for the cytotoxicity of 1 . In terms of differences, we noted that 1 induced a significant redox imbalance characterised by lipid peroxidation and thiol oxidation, and a moderate decrease of the mitochondrial membrane potential in breast cancer cells, whereas 2 has almost no effect. These results underline the importance of the trans configuration in the ferrocenyl–double bond–phenol motif, which is present in 1 but is cis in (Z)- 2 .     

Bis‐arylidene Oxindoles as Anti‐Breast‐Cancer Agents Acting via the Estrogen Receptor

We report a new family of bis‐arylidene oxindole derivatives that show highly selective estrogen receptor (ER)‐mediated anticancer activity at low‐nanomolar concentrations in ER‐positive (ER+) breast cancer cells. In terms of cell growth inhibition, IC₅₀ values for these compounds in ER+ breast cancer cells are two to three orders of magnitude lower than in ER‐negative (ER?) breast cancer cells and non‐cancer cells. In comparison with known bis‐arylidene drugs, these compounds are at least three orders of magnitude more toxic than tamoxifen and 1.5–4‐fold more toxic than 4‐hydroxytamoxifen in ER+ MCF‐7 cancer cells. These oxindoles inhibit ER transactivation, and their anticancer activities are inhibited in ER‐depleted MCF‐7 cells. Some of these nonsteroidal molecules also exhibit essential properties of selective ER down‐regulation. From the development of two series of bis‐arylidene oxindole‐based compounds, we report a new series of anticancer agents for estrogen‐responsive breast cancer.     

Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)₂D₃ analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.