Synthesis and biological activities of nucleoside-estradiol conjugates

Nucleosides were coupled to estradiol via a 17alpha-ethynyl spacer group using Pd(II) as a catalyst. The conjugates were evaluated in vitro for estrogen receptor (ER) binding affinity and cytotoxicity against cell lines with and without ER. The highest receptor binding affinities (RBA approximately 3) were observed with conjugates coupled via a relative long spacer group, while none of the conjugates exhibited cytotoxicity against either cell lines.     

7alpha- and 17alpha-substituted estrogens containing tridentate tricarbonyl rhenium/technetium complexes: synthesis of estrogen receptor imaging agents and evaluation using microPET with technetium-94m

To develop technetium and rhenium-labeled imaging agents for estrogen receptor (ER) positive breast tumors, we have prepared tridentate metal tricarbonyl chelates substituted at the 7alpha- and 17alpha-positions of estradiol. Some of the Re(CO)(3) conjugates have high binding for the ER in vitro. The in vivo biodistribution of the highest affinity of these novel metal tricarbonyl conjugates, prepared as the (94m)Tc labeled analogue, was evaluated by tissue dissection and microPET imaging. Although target tissue-selective uptake was not apparent, it is notable that microPET imaging identified the stomach as a major site of activity deposition, a site that might have been missed by standard tissue distribution studies.     

Synthesis and structure–activity relationships of novel hybrid ferrocenyl compounds based on a bicyclic core skeleton for breast cancer therapy

Breast cancer is the most frequent cancer in women worldwide, and incidence is increasing year by year. Although current selective estrogen receptor modulators (SERMs) have clear advantages in the treatment of hormone-responsive breast cancer, they are ineffective for ER(−). In this study, we describe the design and synthesis of a series of dual-acting estrogen receptor (ER) and histone deacetylase (HDAC) inhibitors with incorporation of the ferrocenyl moiety, leading to novel hybrid ferrocenyl complexes (FcOBHS–HDACis) for breast cancer therapy. It is worth to note that these ferrocenyl conjugates could not only potently inhibit HDACs and the proliferation of ERα positive (ER(+)) breast cancer cells (MCF-7), but also show significant antiproliferative effect on ER(−) breast cancer cells (MDA-MB-231). Thus, the FcOBHS–HDACi conjugates represent a novel approach to the development of efficiently dual-acting agents for treatment of breast cancer.     

Synthesis and biological evaluation of prodigiosene conjugates of porphyrin,estrone and 4-hydroxytamoxifen

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene–tamoxifen conjugates.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

SAR study of tyrosine–chlorambucil hybrid regioisomers; synthesis and biological evaluation against breast cancer cell lines

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta- and para-tyrosine-chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine-chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER(+) and ER(-)) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure L-ortho, L-meta- and L-para-tyrosine phenolic regioisomers. The synthesis of all tyrosine-chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERα)] are discussed in relation to the biological activity.     

Synthesis and biological activities of phthalocyanine-estradiol conjugates

Phthalocyanine-based photosensitizers, coupled via a 17alpha-ethynyl group to estradiol using Pd(II) as a catalyst, were synthesized and evaluated for their estrogen receptor binding affinity and in vitro photocytotoxicity. The highest receptor binding affinities (RBA=8-13) were observed with lipophilic conjugates coupled via a relative long spacer group while the sulfonated analogues showed little binding affinities (RBA <2). The highest photocytotoxicity was observed with the sulfonated conjugates, the nature of the spacer group did not have a pronounced effect.     

Tethered indoles as functionalizable ligands for the estrogen receptor

To create ligands for the estrogen receptor that contain pendant groups for tethering to a poly(amido)amine (PAMAM) dendrimer, we have explored a class of N-substituted 2-phenyl indoles. Attachment of tethers of different length and chemical nature to this non-steroidal indole scaffold gave high affinity ligand-tether conjugates that can be easily functionalized. To further explore the utility of this system, an indole-conjugated dendrimer was prepared and evaluated as an estrogen receptor ligand.     

BODIPY-17α-ethynylestradiol conjugates: Synthesis,fluorescence properties and receptor binding affinities

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.     

Design, synthesis, cytocidal activity and estrogen receptor α affinity of doxorubicin conjugates at 16α-position of estrogen for site-specific treatment of estrogen receptor positive breast cancer

Doxorubicin (DOX) is an important medicine for the treatment of breast cancer, which is the most frequently diagnosed and the most lethal cancer in women worldwide. However, the clinical use of DOX is impeded by serious toxic effects such as cardiomyopathy and congestive heart failure. Covalently linking DOX to estrogen to selectively deliver the drug to estrogen receptor-positive (ER(+)) cancer tissues is one of the strategies under investigation for improving the efficacy and decreasing the cardiac toxicity of DOX. However, conjugation of drug performed until now was at 3- or 17-position of estrogen, which is not ideal since the hydroxyl groups at this position are important for receptor binding affinity. In this study, we designed, prepared and evaluated in vitro the first estrogen-doxorubicin conjugates at 16α-position of estradiol termed E-DOXs (8a-d). DOX was conjugated using a 3-9 carbon atoms alkylamide linking arm. E-DOXs were prepared from estrone using a seven-step procedure to afford the desired conjugates in low to moderate yields. The antiproliferative activities of the E-DOX 8a conjugate through a 3-carbon spacer chain on ER(+) MCF7 and HT-29 are in the micromolar range while inactive on M21 and the ER(-) MDA-MB-231 cells (>50μM). Compound 8a exhibits a selectivity ratio (ER(+)/ER(-) cell lines) of >3.5. Compounds 8b-8d bearing alkylamide linking arms ranging from 5 to 9 carbon atoms were inactive at the concentrations tested (>50μM). Interestingly, compounds 8a-8c exhibited affinity for the estrogen receptor α (ERα) in the nanomolar range (72-100nM) whereas compound 8d exhibited no affinity at concentrations up to 215nM. These results indicate that a short alkylamide spacer is required to maintain both antiproliferative activity toward ER(+) MCF7 and affinity for the ERα of the E-DOX conjugates. Compound 8a is potentially a promising conjugate to target ER(+) breast cancer and might be useful also for the design of more potent E-DOX conjugates.     

3D QSAR of novel estrogen–RGD peptide conjugates: Getting insight into structural dependence of anti-osteoporosis activity and side effect of estrogen in ERT

To explore the structural dependence of the oral potency and side effect of estrogen–RGD peptide conjugates, here six novel conjugates were prepared via introducing RGD-tetrapeptides into both 3- and 17-positions of estradiol, and introducing RGD-octapeptides into 3-position of estrone. In an ovariectomized mouse model they exhibited higher anti-osteoporosis activity and lower side effect than estrogen. For 3D QSAR analysis the anti-osteoporosis activities of nine known conjugates estrogen–RGD tetrapeptide conjugates were also provided. Using Cerius² module their 3D QSAR analysis was performed, four equations with high r² values were established, and the structural dependence of the oral potency and side effect of them was elucidated.     

Isocratic separation of estrogen conjugates on DEAE-sephadex.

Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model $\text{y}\text{=}\text{a}\text{+}\text{b}\text{1}\text{x}$ where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.     

The naphthol selective estrogen receptor modulator (SERM), LY2066948, is oxidized to an o-quinone analogous to the naphthol equine estrogen, equilenin

o-Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o-quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o-quinone (t(1/2)=3.9 ± 0.1h) which like 4-hydroxyequilenin-o-quinone (t(1/2)=2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o-quinones, which were characterized by LC-MS/MS. Incubations of LY2066948 or equilenin with rat liver microsomes also gave detectable o-quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N-dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o-quinone GSH conjugates were detected in equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, no o-quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o-quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.     

Identification of some plasma metabolites of 6, 7-3-H-estrone glucosiduronate in male dogs.

Following the constant infusion of 6, 7-3-H-estrone glucosiduronate in male dogs for a period of 120 minutes, the radioactive metabolites present in the plasma were separated by solvent partition, DEAE-Sephapadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and steroids and their derivatives. Most of the radioactivity in the plasma was identified as estrone glucosiduronate. The major metabolite present was estradiol-17-beta-3-glucosiduronate. Small amounts of estradiol-17-alpha-3-glucosiduronate and free estrone were also identified. Three other minor conjugates were separated, but positive identification could not be made.     

Establishment and characterization of HER2-positive cell line derived from pleural effusion of human breast scirrhous carcinoma

We have established a cell line derived from a pleural effusion of breast scirrhous carcinoma. This cell line presented immunohistochemically negative for estrogen receptor and slightly positive for progesterone receptor, and positive for c-erb B/HER2/neu. However the original tumor was found to be positive for estrogen receptor and progesterone receptor and slightly positive for c-erbB/HER2/neu expression. Enzyme and electrochemiluminescence immunoassays of tumor markers in the conditioned medium by the cell line revealed they secrete CA1 5-3, NCC-ST-439 and HER2 protein. We believe the cell line will contribute to the therapeutic study of malignant breast cancer.     

Identification of novel metabolites of the xenoestrogen 4-tert-octylphenol in primary rat hepatocytes

A number of environmental pollutants, including 4-tert-alkylphenols, can mimic the actions of endogenous steroids and have the potential to disrupt the endocrine function in humans and animals. The biotransformation of a 4-tert-alkylphenol in isolated rat hepatocytes was studied in order to determine the possible fate and activity of these xenoestrogens in higher vertebrates. Hepatocytes were incubated with 30 microM 4-(1',1',3', 3'-tetramethylbutyl)[U-(14)C]phenol (4-tert-octylphenol; t-OP) for up to 60 min. Radiolabelled metabolites were detected by radio-HPLC and the structures determined by gas chromatography-mass spectrometry (GC-MS) analysis of the conjugated or aglycone products. After a 15 min incubation, over 97% of t-OP was metabolised to a complex mixture of metabolites. The initial metabolites formed were identified as products of hydroxylation of the aromatic ring to form catechols and methylated catechols, as well as glucuronide conjugates of the catechol metabolites or parent phenol. These products were further metabolised by hydroxylation of the alkyl chain followed by glucuronide conjugation of the alkoxy group. The conjugated metabolites of t-OP are unlikely to retain estrogen receptor activity, however t-OP is metabolised by some pathways that are similar to that of estrogen catabolism, namely by ortho-hydroxylation to form catechols, methylation by catechol O-methyltransferases and ring conjugation by uridine diphosphoglucuronosyl transferases. Further investigations are needed to determine whether 4-tert-alkylphenols can alter circulating sex steroid profiles by acting as substrates of enzymes determining estrogen metabolism and excretion.