Estrogen and androgen receptor binding affinity of 10 beta-chloro-estrenen derivatives

10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.     

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.     

Estrogen receptor ligands. Part 4: The SAR of the syn-dihydrobenzoxathiin SERAMs

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.     

Synthesis and biological activity of 17 beta-substituted estradiol

The dialkylaminoethoxy side chain in triphenylethylene antiestrogens is required for their antiestrogenic activity. Without this side chain the compounds lose their antiestrogenic activity and become essentially estrogenic. Estradiol substituted at the 17 beta-position with dialkylaminoethoxy, dialkylaminoethylamino, or dialkylaminoethylthiol were synthesized and tested for their ability to displace estradiol for its receptor. All of the derivatives tested exhibited low binding affinities to the estrogen receptor, with RBA values ranging between 0 to 1.2 (estradiol = 100). The mouse and rat uterine weight test revealed only low estrogenic activity for this class of compounds. None of the estradiol derivatives synthesized showed antiestrogenic activity.     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.     

Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 (1986) 301) indicated preferential binding of a hydroxylated metabolite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of TTR could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [125I]T4. Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxybiphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) greater than trichlorophenols greater than dichlorophenols greater than monochlorophenols. Structures with chlorine in both ortho positions to the hydroxyl group were more efficient competitors. The relative affinity of binding of pentachlorophenol (PCP) to TTR was about twice that of T4. Scatchard analysis showed that PCP mainly decreased the affinity constant K11 while the binding capacity R1 was not altered, indicating a competitive type of inhibition. PCP was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of PCP (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding site of TTR.     

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.     

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.     

Antiestrogenic activity of vitamin A in in vivo uterotrophic assay

All-trans-retinoic acid (ATRA), the primary active metabolite of vitamin A, was examined for its antiestrogenic activity in rats using an in vivo uterotrophic assay. All rats were ovariectomized 2 weeks prior to receiving 5 mg/kg/day ATRA or 0.3 micro g/kg/day ethynyl estradiol (EE) subcutaneously once a day for 3 consecutive days. Rats were sacrificed 1, 3, 6, 12 or 24 h after the last treatment. EE increased uterine weight and the coinjection of ATRA with EE significantly suppressed this effect 3 and 24 h after treatment. mRNA expression was examined during this 24-h period and the mRNA expression levels of estrogen receptor alpha (ER alpha), retinoic acid receptor beta (RAR beta), retinoid X receptor gamma (RXR gamma) and cellular retinol-binding protein I (CRBP I) were found to have significantly increased in the ATRA+EE group compared with those in the EE group. This is the first report on the antiestrogenic activity of ATRA determined using an in vivo adult rat uterotrophic assay. The up-regulation of RAR or RXR mRNA expression level was probably responsible for the antiestrogenic activity of ATRA.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

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.     

Direct and reversible inhibition of estradiol-stimulated prolactin synthesis by antiestrogens in vitro

The antiestrogens tamoxifen and monohydroxytamoxifen inhibited the estradiol-stimulated increase in prolactin synthesis by dispersed cells in culture derived from immature rat pituitary glands. Monohydroxytamoxifen had a relative binding affinity for the estrogen receptor similar to that of estradiol, whereas tamoxifen's relative binding affinity was approximately 3%. This was consistent with the observation that monohydroxytamoxifen was 30 times more potent than tamoxifen as an antiestrogen in vitro. To avoid the possibility that tamoxifen was fractionally metabolized to monohydroxytamoxifen by the pituitary cells, the p-methyl, p-chloro, and p-fluoro derivatives of tamoxifen that are unlikely to be converted to monohydroxytamoxifen were tested for activity. The substitution did not have a detrimental effect on their ability to inhibit the binding of [3H]estradiol to either rat uterine or pituitary gland estrogen receptors. Similarly, the derivatives of tamoxifen inhibited estradiol-stimulated prolactin synthesis at concentrations that were consistent with their relative binding affinities. Although it is clearly an advantage for tamoxifen to be metabolized to the more potent antiestrogen monohydroxytamoxifen, we have shown that this is not a prerequisite for the antiestrogenic actions of tamoxifen. With the direct actions of antiestrogens established, the pituitary cell system was validated for further structure-activity relationship studies. Overall, the inhibition of estradiol-stimulated prolactin synthesis by antiestrogens is competitive and reversible with estradiol, an effect that can be explained by interactions with the estrogen receptor system.     

Rapid synthesis of 4-benzylidene and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans as potential selective estrogen receptor modulators (SERMs) using McMurry coupling reaction

7-Methoxy-4-(4-methoxybenzylidene)-2-substituted phenyl-benzopyrans I and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans II carrying different alkylamino residues, designed as estrogen receptor (ER) binding ligands, were successfully synthesized through the McMurry coupling reaction of substituted benzaldehyde/substituted benzophenones and 2-hydroxyphenyl-7-methoxy-chroman-4-one in presence of lithium aluminum hydride and titanium (IV) chloride (LAH-TiCl(4)). Self-coupling of carbonyl reactants led to the formation of several side products. The prototypes were evaluated for their relative binding affinity (RBA), as well as their estrogenic and antiestrogenic activities. High order of estrogenic activity (>50% gain) observed with compounds 3, 7a, 7b, 7c, 8, and 10a and also their partial estrogen antagonistic activity (> or =15%) at the uterine level points toward successful designing of the compounds. Compounds 4, 7a, 7b, 7c, and 10a also possessed significant anticancer activity against human adenocarcinoma cell line (MCF-7 cell line) that may be related to their estrogen-dependent action.     

Nonsteroidal estrogens bearing acyl azide functions: potential electrophilic and photoaffinity labeling agents for the estrogen receptor.

In an effort to develop novel affinity labeling agents for the estrogen receptor, we have synthesized two nonsteroidal ligands, a 1-aroyl-2-aryl tetralin system (1) and a 2-aryl-3-aroylbenzo[b]thiophene system (2). These agents, patterned after the Lilly antiestrogens trioxifene and LY 117018, respectively, embody acyl azide functions as part of a benzoyl chromophore. The acyl azide group has weak acylating activity, suitable for electrophilic affinity labeling, but this function is also photoreactive and, in its particular embodiment within these ligands, it could provide an efficient photochemical route to the highly reactive singlet acyl nitrene. The tetralin system (1) was prepared in nine steps from 6-methoxy-1-tetralone, and the benzothiophene system (2) was prepared in four steps from a known substituted benzo[b]thiophene precursor. In competitive binding assays, both compounds show reasonable binding affinity for the rat and lamb uterine estrogen receptor: estradiol = 100%, 1 = 3%, and 2 = 12%. When assayed by indirect receptor consumption assays, both compounds appear to have substantial capacity for irreversible binding (electrophilic reaction) with the receptor. This reactivity, which suggests that acylation of the receptor has occurred, is photoreversible. The nature of this ligand-receptor interaction is being investigated further.     

Uterine estrogen receptor binding of catecholestrogens and of estetrol (1,3,5(10)-estratriene-3,15alpha,16alpha,17beta-tetrol).

The binding affinities for the catecholestrogen metabolites of estradiol and of their methyl ethers for the rat uterine cytosol estrogen receptors were examined. Similarly the binding of the fetal estradiol metabolite, 15alpha-hydroxyestriol (estertrol) was also measured. All of the catecholestrogens showed binding affinities far in excess of their uterotrophic potency. This is different from estriol, the product of the alternative metabolic pathways and suggests that the direction of estradiol metabolism may have an important role in the modulation of estrogenic activity of the female sex hormone.     

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.     

Synthesis and estrogen receptor binding affinities of novel pyrrolo[2,1,5-cd]indolizine derivatives

A series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor. Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their structural resemblance to estrogen.     

Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats

Estradiol sulfamate (J995), estradiol-17beta with a substituted sulfamate group in position 3, has much higher systemic estrogenic activity after oral administration than 17beta-estradiol (E2) due to reduced hepatic metabolism of the drug. The lower dose necessary for achievement of adequate systemic estrogenic effects results in a substantial reduction of otherwise commonly observed hepatic side-effects. This makes J995 a strong candidate as an estrogen suitable for oral administration. The present study was performed to examine and compare the effects of J995 and E2 on the uterus after oral or subcutaneous administration to ovariectomized or ovariectomized+hypophysectomized female rats, in particular on the levels of the estrogen receptor (ER) (alpha+beta), ERalpha mRNA and insulin-like growth factor-I (IGF-I) mRNA. The ER levels were determined using a ligand binding assay and the mRNA levels using solution hybridization. The doses of J995 or E2 were chosen to achieve comparable uterotrophic activity. The rats were treated with hormones for 7 days and the treatment was initiated 14 days after surgery. We conclude that there are no major differences in the uterine response to treatment with J995 or E2 with respect to the effects on ER and ERalpha mRNA levels. The IGF-I mRNA level though, is more affected by J995 than by E2 after 7 days of treatment, indicating a prolonged effect of J995.     

Experimental study on the estrogen-like effect of mercuric chloride

Although mercuric chloride has toxicity on reproductive system, it is uncertain if such toxicity is induced by estrogen-like effect. To study whether mercuric chloride has the estrogen-like effect and its relevant mechanism, proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, peroxidase activity assay and estrogen receptor competitive binding assay were conducted to screen the estrogen-like effect of mercuric chloride. The MCF-7 cells proliferated in the stimulation of mercuric chloride and got to the peak at 10⁻⁷ mol/l concentration. And this proliferation could be completely blocked by estrogenic antagonist ICI182.780. In addition, mercuric chloride could increase the weight of uterus of ovariectomized SD rats and the peroxidase activity of uterus complying with dose-effect relationship. However, mercuric chloride could not affect the binding of estradiol (E₂) to estrogen receptor (ER). So mercuric chloride exhibits the estrogen-like effect through binding and activating ER rather than bind to ER by competing with E₂.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.