Influence of new hydroxylated triphenylethylene (TPE) derivatives on estradiol binding to uterine cytosol

Twelve homologous triphenyl acrylonitrile derivatives with a p-OH or p-CH3 group on one or more of the phenyl rings were synthesized in order to assess the relative influence of each position on binding to the estrogen receptor (ER) and on inhibition of prostaglandin synthetase (PGS). Their relative binding affinities (RBAs) for [3H]estradiol (E2)-labeled ER were compared at 0 and 25 degrees C in mouse and rat uterus cytosol with those of tamoxifen derivatives, cyclofenil and diethylstilbestrol. RBAs in both species were closely correlated (r = 0.92) although the RBAs were about twice as high in the mouse as in the rat. The unsubstituted skeleton had an RBA of much less than 0.1 (estradiol = 100). An OH-group in R1 or R2 (Fig. 1) engendered very low affinity whereas an OH-group in R gave rise to a compound with an RBA equivalent to that of E2, emphasizing the importance of this position in the interaction with ER. Compounds with an additional OH-group in R1 or R2 were significantly better competitors than E2. No further increase in RBA was noted with the trihydroxy derivative. The effect of the introduction of a hydrophobic CH3-group decreased affinity as expected in R, but also in position R1 unless a second OH-group was present in R2. None of the 12 test-compounds competed significantly for binding to the "anti-estrogen binding site" in rat kidney supernatant. Although polar groups were not necessary for inhibition of PGS, inhibition was enhanced by the presence of a hydroxy group in R or R1 (but not R2). Even greater inhibition was obtained by the further introduction of a CH3-group in R1 or R respectively. The conformations of these derivatives are compared to those of known estrogen ligands and anti-inflammatory agents in order to obtain further information on these protein recognition sites.     

Synthesis and receptor binding of polynuclear organometallic estradiol derivatives

Twelve novel organometallic derivatives of estradiol were synthesized with the aim of utilizing organometallic cold bioprobes as radioisotopic labels substitutes for steroid hormone receptor assays. For this purpose, we envisaged the attachment of several stable cobalt, molybdenum, osmium carbonyl clusters (tetra- and pentanuclear species) at estradiol 17 alpha-, 16 alpha-, 2- or 4-positions. The binding affinity of these new complexes for uterine estradiol receptor has been measured by the competitive binding method. The results show that the 17 alpha-position can tolerate substitution by bulky organometallic groups (especially in the case of cobalt and molybdenum carbonyl clusters). Estradiol derivatives which are functionalized at C-4 and C-16 alpha bind estradiol receptor with reasonable affinity and the RBA values are the same for the complexed and uncomplexed hormones. The 2- position is more sensitive to organometallic substitution and the complexation at the 2- alkyne results in a dramatic decrease of the RBA values. These results show that the attachment of polynuclear moieties in estradiol 17 alpha-, 4- and 16 alpha-, positions gives rise to compounds which are of potential utility in a new non-radioisotopic receptor assay since the metal-carbonyl markers are readily detected by high-sensitivity Fourier-transform infra-red spectroscopy.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

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.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

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.     

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.     

Biological activities of 4-fluoro estrogen analogues

As part of a study on the biological activity of gamma-emitting estradiol analogues, a series of fluorinated and/or brominated analogues of estradiol were synthesized. 4-Fluoro-1,3,5(10-estratrien-3,17 beta-diol (4-fluoro-estradiol) and 16 alpha-bromo-1,3,5(10)-estratrien-3,17 beta-diol (16 alpha-bromo-estradiol) had relative binding affinities (RBA) for the rabbit uterine cytosol receptor which were comparable to those of estradiol. However, while 4-fluoro-estradiol stimulated the increase in uterine weight of immature mice to the same amount as estradiol, 16 alpha-bromo-estradiol was less effective in this assay indicating that its metabolism is probably more rapid than that of estradiol. 16 beta-Bromo-1,3,5(10)-estratrien-3,17 beta-diol had a low RBA and was ineffective in stimulating uterine weight at the doses used. 4-Fluoro-16 alpha-bromo-estradiol had a lower RBA than either the 4-fluoro- or 16 alpha-bromo-estradiol and all the 4-fluoro-seleno-estrogens tested possessed low RBA's.     

18F-labeled difluoroestradiols: preparation and preclinical evaluation as estrogen receptor-binding radiopharmaceuticals

A-ring fluorination of estradiol (ES) at position 2 or 4 decreases the rate of metabolism by blocking the formation of catechol estrogens, one of the major metabolic pathways of ES. We postulate that adding a 2- or 4-fluoro substituent to 16alpha-[18F]fluoroestradiol (FES), a positron emission tomography (PET) radiopharmaceutical used for estrogen receptor (ER) imaging, should prolong its blood circulation time, and thus, improve its localization in ER-rich target tissues. On such account, we prepared a series of FES derivatives substituted with a fluorine atom at C2 or C4, with or without an 11beta-OMe group, and we tested their binding affinities for the ER and different serum proteins including rat alphafetoprotein (AFP) and human sex hormone-binding globulin (SHBG). Labeling at the 16alpha-position was accomplished via nucleophilic substitution with [18F]F(-) on the reactive 16beta,17beta-cyclic sulfate intermediates. Decay corrected yields varied between 30 and 50% for a total synthesis time of 120 min, providing final products with specific activities >3000 Ci/mmol. The 18F-labeled analogs were evaluated for their biodistribution in immature female rats. Substitutions with the 4-F have little effect on binding affinities. Addition of the 2-F diminishes ER and AFP-binding affinities while augmenting the affinity for the SHBG. Addition of the 11beta-OMe decreases all binding affinities, particularly to AFP and SHBG. In contrast, biodistribution of the corresponding [16alpha-18F]fluoro analogs in immature female rats revealed that the presence of the 11beta-OMe group improves ER-mediated uterus uptake, with the 4,16alpha-[16alpha-18F]difluoro-11beta-methoxyestradiol showing the highest uptake values (15% ID at 1-h post-injection). These data suggest that the addition of both a 4-F and 11beta-OMe group onto FES may provide an improved radiopharmaceutical for PET imaging of ER densities in breast cancer patients.     

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.     

Preparation and biological evaluation of 18F-labeled benzamide analogs as potential dopamine D2 receptor ligands

Three ¹⁸F-labeled benzamide derivatives were prepared and evaluated as potential ligands to study the dopamine D₂ receptor phenomenon. The compounds are analogs of iodobenzamide, eticlopride and raclopride and are labeled with an N-2-[¹⁸F]fluoroethyl functionality on the pyrrolidine ring. The compounds were tested in vitro for binding affinity and found to exhibit somewhat lower affinity than the non-fluorinated analog. In vivo distribution studies revealed that all compounds were more highly bound to plasma proteins than was raclopride. In addition, compartmentation of radioactivity demonstrated nonspecific binding to be the predominate retention in the brain as reflected by the low caudate to cerebellum ratios for these compounds. These three ¹⁸F-labeled benzamide derivatives are inferior to raclopride and iodobenzamide for studies of the D₂ receptor system using positron emission tomography.     

Synthesis, characterization, and estrogen receptor binding affinity of flavone-, indole-, and furan-estradiol conjugates

Different flavone-, indole-, and furan-17beta-estradiol conjugates, linked via alkyl spacer chains extending from the 17alpha-position of the estradiol moiety, were synthesized by Pd-catalyzed cross-coupling reactions. Structures were assigned based on spectroscopic data. In vitro competitive binding assays for the estrogen receptor (alpha-ER), using [(3)H]estradiol (RBA=100) as a competitor, revealed that a two-carbon alkyl linker combined with a flavone conjugate provided the highest binding affinity (RBA approximately 9), warranting further studies on their potential use as selective estrogen-receptor modulators (SERMs) for hormone-replacement therapies.     

Triphenylethylene antiestrogen binding sites (TABS) specificity

The relative binding affinities (RBA) of various compounds for the triphenylethylene antiestrogen binding sites (TABS) were examined. The ability of tamoxifen to inhibit the binding of [3H]tamoxifen to salt extracted (0.4 M KCl) TABS from rat liver nuclei was used as a standard by which other compounds were compared (tamoxifen RBA, 100; Kd approximately 1 nM). Nafoxidine was the most effective triphenylethylene compound used (RBA 333; Kd approximately 0.3 nM) whereas the RBA of zuclomiphene and enclomiphene was not different from tamoxifen. MER-29 was the weakest inhibitor of the triphenylethylene derivatives (RBA 10; Kd approximately 10 nM). Trifluoperazine, chlorpromazine and the anti-calmodulin drugs W-13 and W-12 had RBA's of 25, 1, 1 and 0.1 respectively. The binding affinities of cholesterol and 7-ketocholesterol were significant (Kd approximately 22 nM) while the steroid hormones, estradiol, testosterone, progesterone and corticosterone displayed not observable affinity. Various compounds obtained from Merrill Dow Pharmaceuticals and the Eli Lilly Company which contained alklaminoethoxy side chains linked to aromatic ring structures had RBA's ranging from 1-0.3. We conclude, as other investigators have also concluded, that the similar binding affinities of various triphenylethylene antiestrogens for TABS and their divergent activities as antiestrogens makes it unlikely that TABS are directly involved in estrogen antagonism. The moderate but significant affinity of TABS for trifluoperazine and other drugs thought to be involved in calmodulin regulation indicates that TABS may be a linked in some way to calmodulin function. The binding of cholesterol and 7-ketocholesterol is also significant and may indicate that TABS are involved in cholesterol metabolism.     

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Identification of a lead pharmacophore for the development of potent nuclear receptor modulators as anticancer and X syndrome disease therapeutic agents

A series of tetrahydroisoquinoline-N-phenylamide derivatives were designed, synthesized, and tested for their relative binding affinity and antagonistic activity against androgen receptor (AR). Compound 1b (relative binding affinity, RBA = 6.4) and 1h (RBA = 12.6) showed higher binding affinity than flutamide (RBA = 1), a potent AR antagonist. These two compounds also exerted optimal antagonistic activity against AR in reporter assays. The derivatives were also tested for their activities against another nuclear receptor, farnesoid x receptor (FXR), with most compounds acting as weak antagonists, however, compound 1h behaved as a FXR agonist with activity slightly less than that of chenodeoxycholic acid (CDCA), a natural FXR agonist.     

17 alpha-allyl estradiol analogues as candidates for development of high-affinity fluorescein-estradiol conjugates

In order to develop stable, high-affinity fluorescein-estradiol conjugates, the fluorescein moiety must be leashed to the estradiol molecule at a point which interferes least with estradiol's binding to the receptor. Because of the high affinity of 17 alpha-substituted estradiol (e.g. ethynyl estradiol), we investigated a series of 17 alpha-substituted estradiol compounds to determine the optimal properties of a leash at this position. Twelve estradiol derivatives bearing a three-carbon 17 alpha side chain with or without a terminal functional group and with varying degrees of unsaturation were synthesized. Initial comparison of the receptor binding affinities of some of these derivatives suggested that three factors might reduce affinity: internal hydrogen bonding of the 17 beta-hydroxyl proton with an oxygen atom of the 17 alpha side chain; hydrophilicity of the ligand; or steric interference of the side chain with receptor binding. Further comparisons were designed to evaluate the relative contribution of these factors. The results suggest that the relative affinities of these 17 alpha-substituted estradiol derivatives are influenced primarily by the steric interference of the side chains and also by their hydrophilicity. Internal hydrogen bonding involving the 17 beta-hydroxyl proton does not seem to have a profound effect.     

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.