Estrogen receptor binding affinity and uterotrophic activity of triphenylhaloethylenes

Radiohalogenated estrogens have considerable potential for estrogen receptor-directed imaging and therapy for cancers which contain such receptors. In an effort to evaluate the potential of the triphenyl ethylene structure for such purposes we have synthesized 3 series of 2-halosubstituted triphenylethylenes containing oxygen functions in the 4 position of both aromatic rings attached to carbon 1 of the ethylene and tested their uterotrophic activity and competition for rat uterine low salt extractable, "cytosol" estrogen receptor. Most active, both as competitors for estradiol binding to estrogen receptors and by their ability to stimulate uterine growth are the 1,1-bis-4-hydroxyphenyl derivatives although the 1,1-bis-4-acetoxyphenyl derivatives also show good receptor affinity and demonstrate uterotrophic activities. However, since uterine cytosol contains enzymes which hydrolyze the acetates to the free phenols even during the incubation in the cold used for the competitive binding studies, a significant portion of the competition shown by the diacetates is probably due to their hydrolysis products, the free phenols. The 1,1-bis-4-methoxyphenyl derivatives are weak competitive binders and demonstrate uterotrophic activity only when administered at the higher, 20 micrograms, doses. Comparing the relative activities of various halogens at the 2 position, in each series the bromo and chloro derivatives generally were of similar activity and significantly more active than the corresponding iodo derivative. The non-halogen substituted derivatives were very good competitors for estrogen receptor binding but less active with regard to uterine growth stimulation, providing evidence that in vivo the vinyl halides would appear to be relatively stable to simple dehalogenation. Since they show reasonably good apparent affinities for the estrogen receptor and apparent in vivo stability, reflected by estrogenic activity, these halogen substituted triphenylethylene derivatives appear to be promising substrates for investigations of estrogen receptor directed imaging and therapy.     

Estrogen A-ring structure and antioxidative effect on lipoproteins

The oxidative modification of lipoprotein particles is an important step in atherogenesis. Estrogens are known to be powerful antioxidants independently of their binding to the estrogen receptors and the hormonal functions. We explored the structural determinants for the antioxidant activity of a large number of estrogen derivatives (n=43) in an aqueous lipoprotein solution in vitro by monitoring formation of conjugated dienes. Our results indicate that estrogen derivatives with an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups provide strongest antioxidant protection of low density lipoprotein (LDL) and high density lipoprotein (HDL). The electron donating methoxy groups may enhance the antioxidant effect by weakening the phenolic OH bond and providing stability to the formed phenoxyl radical. With some exceptions, compounds completely lacking unsubstituted hydroxyl groups in the A-ring exhibited no antioxidant effect, e.g. the most hydrophilic "tetrol" compound with three unsubstituted A-ring hydroxyl groups had no antioxidant effect. Moreover, additional hydroxyl groups in the B-, C- or D-ring seemed to weaken the antioxidant effect. Accordingly, both the presence of unsubstituted hydroxyl groups and adjacent substituents, as well as the lipophilicity of the derivatives determine the antioxidant activity of estrogen derivatives in aqueous lipoprotein solutions.     

Amide derivatives of 9,11-seco-estra-1,3,5(10)-trien-11-oic acid as modified orally active estrogen agonists with moderate antagonistic activity

Synthesis of amide derivatives of 9,11-seco-estra-1,3,5(10)-trien-11-oic acid containing alkyl and aromatic amine residues has been carried out with an aim to prepare orally active estrogen antagonists. Modification of the estradiol molecule in the form of C-seco-amide derivatives has led to their high oral absorption. Compounds 7 an n-propyl amide, 8 an n-butyl amide, and 16 a p-anisidyl amide of C-seco-estrane showed significant estrogen antagonistic activity (>20%), while, the majority of compounds possessed high estrogen agonistic activity on oral administration at 10mg/kg dose in rats.     

Diaryl naphthyl methanes a novel class of anti-implantation agents

Diaryl naphthyl methanes and the corresponding 1, 2, 3, 4- and 5, 6, 7, 8-tetrahydro naphthyl methane derivatives have been synthesized as novel estrogen receptor binding ligands. The secondary and tertiary amino alkoxy derivatives of diaryl naphthyl and tetrahydro naphthyl methane interact with the estrogen receptor to elicit promising estrogenic, antiestrogenic and implantation inhibition activities in rats. The most active compounds in this series are 7, 9 and 20, cent percent active in preventing implantation in rats at 2.5 mgkg(-1) dose.     

Inhibition of mammary carcinoma growth by retinoidal benzoic acid derivatives

The growth of many breast carcinoma cell lines is inhibited by vitamin A, and derivatives as well as synthetic retinoids. New retinoidal derivatives have recently been synthesized. These retinoidal benzoic acid derivatives displayed enhanced potency in their ability to reverse hamster tracheal keratinization and inhibit ornithine decarboxylase induction in mouse epidermis. We therefore screened a series of analogues of these compounds for their ability to inhibit human breast carcinoma cell proliferation utilizing three estrogen receptor-positive and two estrogen receptor-negative cell lines. The compound (E)-4-2-(5,6,7,8)tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)prop enyl benzoic acid (Ro 1374-10) was approximately 2-3 orders of magnitude more potent than all-trans-retinoic acid in inhibiting breast carcinoma cell proliferation while the compound SRI-6409-40, which differs from Ro 1374-10 only by the position of a methyl group, was 50-fold more potent than Ro 1374-10. All of the compounds tested displayed were inactive against the estrogen receptor-negative breast carcinoma lines.     

Synthesis and receptor-binding examinations of the normal and 13-epi-D-homoestrones and their 3-methyl ethers

An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the possibility for facile entry into the 13alpha-estrone series. Combination of this synthetic methodology with an isolation step carried out by means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy D-homoestrone derivatives in both the normal and the 13alpha-estrone series were then synthesized and tested in vitro in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference compound 3,17beta-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values for 13alpha-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13alpha-D-homoestrone derivatives are estrogen receptor-selective molecules.     

Design, synthesis, and evaluation of cyclofenil derivatives for potential SPECT imaging agents

To develop technetium- and rhenium-labeled nonsteroidal estrogen imaging agents for estrogen receptor (ER) positive breast tumors, two groups of rhenium and technetium cyclofenil derivatives were synthesized and characterized. The binding affinities of the rhenium complexes for ERs were determined. The tricarbonyl rhenium complex showed the highest binding affinity for ERs (81.2 for ERβ, 16.5 for ERα). Tricarbonyl technetium cyclofenil complexes were obtained in high radiochemical purity and radiochemical yields. The results of studies of their octanol/water partition and in vitro stability are presented. These results demonstrate that these radiolabeled cyclofenil derivatives may be considered as potential breast cancer imaging agents.     

Synthesis and cytotoxic activity of estrogen alpha-methylene-delta-lactones

Several estrogen derivatives containing the alpha-methylene-delta-lactone group as part of the D-ring were prepared as anti-tumor agents. The compounds were highly toxic towards HeLa S3 cells grown in culture.     

Structure-based drug design: synthesis, crystal structure, biological evaluation and docking studies of mono- and bis-benzo[b]oxepines as non-steroidal estrogens

Mono- and bis-benzo[b]oxepine derivatives have been rationally synthesized to meet the molecular requirement for interaction with estrogen receptor. Bis-benzo[b]oxepines (7 and 9) and mono-benzo[b]oxepine (10) acquire geometry with phenolic groups disposed in a fashion to stimulate estrogen receptor. Structure-based investigation, in vivo activity and docking studies have been described and correlated to demonstrate a practical approach for suitable ligand design.     

Estrogen receptor binding tolerance of 16 alpha-substituted estradiol derivatives

In order to examine the tolerance of the estrogen receptor for 16 alpha-substituents in estradiol, we have synthesized various 16 alpha-substituted estrogens and determined their binding affinity for receptor by a competitive radiometric binding assay. The substituents ranged from small, single-atom substituents (halogens), two-atom substituents (halomethyl groups), to larger alkyl groups and ultimately alkyl groups bearing various functionality, including fluorescent (nitrobenzoxadiazole, NBD) and photoreactive (nitroazidophenyl, NAP) groups. The estrogen receptor seems to have a moderate tolerance for bulky substituents: All of the halogen and halomethyl substituents bind with an affinity at least 50% that of estradiol; in the three atom alkyl series, the affinity declined markedly from propargyl (44%) and allyl (38%) to propyl (5%), suggestive of detailed steric constraints or a preference for unsaturation. The larger, more highly functionalized derivatives ranged in affinity from 0.1-7%, with the highest affinity binders being benzyl (5%) and 4-phenoxy-2(E)-butenyl (7%); most of the lowest affinity ones were the bulky fluorescent and photoreactive derivatives. Thus, the estrogen receptor has good tolerance for estradiol derivatives substituted at the 16 alpha-position with nonpolar groups of moderate bulk; however, with groups of larger bulk, affinity is much lower and becomes highly dependent upon the polarity and detailed structure of the substituents.     

Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis.     

Subcellular distribution of native estrogen receptor alpha and beta isoforms in rabbit uterus and ovary

The association of estrogen receptors with non-nuclear/cytoplasmic compartments in target tissues has been documented. However, limited information is available on the distribution of estrogen receptor isoforms, specially with regard to the newly described beta isotype. The subcellular localization of estrogen receptor alpha and beta isoforms was investigated in rabbit uterus and ovary. Native alpha and beta subtypes were immunodetected using specific antibodies after subjecting the tissue to fractionation by differential centrifugation. The ovary expressed alpha and beta estrogen receptors in predominant association to cytosolic components. However, in the uterus, a substantial proportion of the total estrogen binding capacity and coexpression of the two isoforms was detected in mitochondria and microsomes. The mitochondrial-enriched subfraction represented an important source of 17beta-estradiol binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of estrogen receptor alpha but mainly with estrogen receptor beta proteins. Using macromolecular 17beta-estradiol derivatives in Ligand Blot studies, we could confirm that both alpha and beta isoforms were expressed as the major estrogen binding proteins in the uterus, while estrogen receptor alpha was clearly the dominant isoform in the ovary. Other low molecular weight estrogen receptor alpha-like proteins were found to represent an independent subpopulation of uterine binding sites, expressed to a lesser extent. This differential cellular partitioning of estrogen receptor alpha and beta forms may contribute to the known diversity of 17beta-estradiol effects in target organs. Both estrogen receptor alpha and beta expression levels and cellular localization patterns among tissues, add complexity to the whole estrogen signaling system, in which membrane and mitochondrial events could also be implicated.     

3-Alkyl-2-phenylbenzo[b]thiophenes: nonsteroidal estrogen antagonists with mammary tumor inhibiting activity.

A number of 2-(4-hydroxyphenyl)benzo[b]thiophenes with a hydroxy group in position 5 or 6 and a short alkyl group at C-3 were synthesized and studied for their estrogen receptor affinities. Relative binding affinities (RBA) for the calf uterine estrogen receptor ranged from 3 to 60 (17β-estradiol = 100). The highest RBA values were found with ethyl derivatives [3 (5-OH): 60; 7 (6-OH): 28]. In accord with their receptor affinity, all benzothiophenes exhibited endocrine activity in the immature mouse uterine weight test. At doses of 0.25–7.0 mg/kg body weight, they showed partial estrogen antagonism and usually weak estrogenic effects. All compounds entered tests with hormone-sensitive human MCF-7 breast cancer cells. At concentrations of 1μM and higher, most of the derivatives displayed significant inhibition of cell growth. These results prompted us to test them in vivo for cytostatic activity using hormone-dependent MXT mouse mammary tumors. The 5-hydroxy derivatives 3 and 4 strongly inhibited the growth of these tumors. After 4 weeks of treatment with 3 × 4.2 mg/kg of compound 3, the average tumor weight was reduced by 83% vs control (tamoxifen at equimolar dose: 74%). The 6-hydroxy derivative 7 required higher doses (25 mg/kg) to give rise to the same effect. At the end of therapy, no increase of uterine weight due to an estrogenic effect was observed. We assume therefore that the antineoplastic activity of these compounds in this tumor model is due mainly to their estrogen antagonism.     

m-Carborane bisphenol structure as a pharmacophore for selective estrogen receptor modulators

A series of m-carborane derivatives was prepared based upon the structures of antiestrogenic drugs and their activities were evaluated by estrogen receptor alpha (ERalpha) binding assay and transactivation assay using human breast cancer cell line, MCF-7 cells. The m-carborane bisphenol 5 exhibited about a thousand times more potent ER agonistic activity than the o-carborane bisphenol 11. The m-carborane bisphenol structure appears to be a favorable hydrophobic pharmacophore for the development of novel selective estrogen receptor modulators (SERMs).     

Synthesis and biological evaluation of 2- and 4-hydroxymestranol

The synthesis of 2-hydroxy and 4-hydroxymestranol by oxidation of mestranol with m-chloroperbenzoic acid is described. The oral estrogenicity and contragestational activity of these and related catechol estrogen derivatives in the rat is also presented.     

Synthesis of some analogs of estradiol

As part of a search for estradiol derivatives designed for conjugation to carboxyl or amine functions of anti-cancer agents or suitable derivatives thereof, estradiol analogs with side chains at the C-16 or -17 position were prepared for biological assay. These analogs include several which have a substituted nitrogenous function at C-17. The avidity of some of these analogs for binding to estrogen receptor was found to be of a low order.     

Hydroxylated 2,3-diarylindenes: synthesis, estrogen receptor binding affinity, and binding orientation considerations

In order to develop high affinity, fluorescent ligands for the estrogen receptor based on 2-arylindenes, it is important to understand how this non-steroidal estrogen is oriented within the binding site and to know how hydroxyl substituents affect binding. To investigate these issues a series of dihydroxyl-substituted 2,3-diphenylindenes were prepared by the cyclization of appropriately substituted alpha-benzyldesoxybenzoins, and their binding affinities for the estrogen receptor measured by a competitive radiometric binding assay. Introduction of a p-hydroxyl group in the 2-phenyl ring of two 2,3-diphenyl-6-hydroxyindene systems causes a 3-fold increase in binding affinity, whereas, p-hydroxylation in the 3-phenyl ring of these systems causes a 2-fold reduction in binding affinity. The parallel change in binding affinity in these two systems suggests a consistent binding orientation of the 2,3-diarylindene systems, which, on the basis of earlier studies, has the indene system corresponding to the A/B-ring system of estradiol. This orientation model and the enhanced affinity of the p-hydroxy 2-ring derivatives are suggestive of a new hydrogen bonding site below the D-ring binding site. Changes in receptor binding affinity upon hydroxylation in triphenylacrylonitrile ligands for the estrogen receptor, reported by others, do not show such parallelism, suggesting that different derivatives may not be bound in congruent orientations. A m-hydroxyl substituent in ring-3 of the 2,3-diarylindene has very little effect on receptor binding. In designing fluorescent 2,3-diarylindene ligands for the estrogen receptor, 3-ring hydroxylation may be useful in reducing non-specific binding and in modifying electron donation to the fluorophore with only modest or no reduction in binding affinity. p-Hydroxylation of the 2-ring, although increasing receptor binding, is not consistent with the electron accepting nature required of this ring.     

Fatty acid esterification of lipoprotein-associated estrone in human plasma and follicular fluid

Estrogen fatty acid esters constitute a unique family of extremely hydrophobic hormonal derivatives which are exclusively transported in lipoprotein particles in plasma. In estradiol, the fatty acyl residues are conjugated at the 17beta-hydroxyl of the steroid D-ring, leaving the phenolic 3-hydroxyl group unsubstituted and, therefore, preserving antioxidative efficacy. The 17beta-fatty acid derivative of estradiol is proposedly an even more efficient antioxidant protecting LDL and HDL than the parent steroid. Previous studies have established that the enzyme lecithin:cholesterol acyltransferase which catalyzes the fatty acid esterification of 3beta-hydroxyl group of cholesterol, also catalyzes the formation of estrogen 17beta-esters. Estrone, the principal estrogen in the postmenopausal female, has a keto group at carbon-17 and has been thought unable to form fatty acid esters. However, we detected hydrophobic derivatives of estrone following incubations with human plasma and ovarian follicular fluid. These derivatives accumulated in HDL and LDL during incubation showing chemical characteristics similar to estrone-3-fatty acid esters. Liquid chromatographic-mass spectrometric analyses established the presence of unhydrolyzed estrone esters consisting of different fatty acid species, the major one being estrone-3-linoleate, in human HDL particles following incubation of estrone with plasma. These extremely hydrophobic estrone conjugates could, in theory, represent a storage form of this estrogen.     

Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates

Sulfamoylated derivatives of the endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2 (7)), such as 2-methoxy-3-O-sulfamoyl estrone (2-MeOEMATE (1)), display greatly enhanced activity against the proliferation of human cancer cells and inhibit steroid sulphatase (STS), another current oncology target. We explore here the effects of steroidal D-ring modification on the activity of such 2-substituted estrogen-3-O-sulfamates in respect of inhibition of tumour cell proliferation and steroid sulphatase. The novel 17-deoxy analogues of 2-MeOEMATE and the related 2-ethyl and 2-methylsulfanyl compounds showed greatly reduced inhibition of MCF-7 proliferation. Introduction of a 17alpha-benzyl substituent to such 2-substituted estrogen sulfamates also proved deleterious to anti-proliferative activity but could, in one case, enhance STS inhibition with respect to the parent substituted estrone sulfamate. In contrast, selected 17-oxime derivatives of 2-MeOEMATE displayed an enhanced anti-proliferative activity. These results illustrate that enhanced in vitro anti-cancer activity can be achieved in the 2-substituted estrogen sulfamate series and highlight, in particular, the importance of potential hydrogen bonding effects around the steroidal D-ring in the activity of these molecules. The SAR parameters established herein will assist the future design of anti-proliferative and anti-endocrine agents as potential therapeutics for both hormone dependent and independent cancers.