Estradiol Analogs Research Articles

Differential effects of estradiol and its analogs on cyclin D1 and CDK4 expression in estrogen receptor positive MCF-7 and estrogen receptor-transfected MCF-10AEwt5 cells.

Estradiol stimulates the growth of a majority of human breast tumors containing the estrogen receptors (ERs), proteins that mediates estrogen function. Opposing effects of estradiol have been found in cells expressing endogenous ER and those containing a transfected ER. To understand the role of estradiol structure in diverse estrogenic responses related to cell cycle regulation, we evaluated the effects of estradiol and its analogs on cell cycle progression, and the expression of cyclin D1 and the cyclin dependent kinase 4 (CDK4) in ER-positive MCF-7 and ER-transfected MCF-10AEwt5 cells. Four analogs of estradiol, with a re-positioned or a deleted hydroxyl group, were used. Our results show that estradiol and all of the analogs facilitated cell cycle progression of MCF-7 cells. In contrast, only estradiol inhibited the cell cycle progression of MCF-10AEwt5 cells significantly. Western blot analysis revealed that cyclin D1 protein increased to the maximal level by 6 h after the initiation of cell cycle from G1 phase of MCF-7 cells. The least effective analog in inducing cyclin D1 was 3-hydroxyestratriene. However, this analog was most effective at inducing CDK4, contributing to its efficacy in facilitating MCF-7 cell cycle. In contrast to MCF-7 cells, the level of cyclin D1 protein was not influenced significantly by estradiol or its analogs in MCF-10AEwt5 cells. Sucrose gradient sedimentation analysis of ER from MCF-7 cells showed that the major peak of [3H]-estradiol bound to ER could be displaced by a 10-fold excess of unlabelled estradiol or any of the analogs. In contrast, several analogs were less effective than unlabelled estradiol in competitive displacement of [3H]-estradiol bound to ER from MCF-10AEwt5 cells. These data indicate that the induction of cyclin D1 is an important part of the growth stimulatory effects of estrogens in MCF-7 cells, but it may not be involved in growth inhibition of MCF-10AEwt5 cells. Our results also show that estrogenic compounds interact with ER from MCF-10AEwt5 cells with altered ligand binding affinity, possibly due to the absence or dysfunction of certain transcription factors or ER-associated proteins that co-regulate ER function.

Three-dimensional quantitative structure-activity relationship study of nonsteroidal estrogen receptor ligands using the comparative molecular field analysis/cross-validated r2-guided region selection approach.

A newly developed comparative molecular field analysis (CoMFA) technique, the cross-validated r2-guided region selection (CoMFA/q2-GRS) method, has been used to build a quantitative structure-activity relationship (3D-QSAR) for nonsteroidal estrogen receptor (ER) ligands. Ligands included in this study belong to a series of diethylstilbestrol (DES) and indenestrol analogues whose affinities for the mouse ER (mER) have been determined in our laboratory. The final model utilized 30 compounds and yielded a q2GRS (cross-validated r2, guided region selection) of 0.796, as compared to a q2 of 0.720 for conventional CoMFA, with a standard error of prediction of 0.594 at 3 principal components. This model was used to visualize steric and electrostatic features of the ligands that correspond with ER binding affinity. Results obtained from the CoMFA steric and electrostatic plots of this model have also been compared to information from the ER binding affinities of substituted estradiol analogues. This is in an effort to determine structural features of compounds in the CoMFA analysis that may correspond to those of the estradiol analogues and to further clarify the mode of binding of nonsteroidal ER ligands.

Synthesis of analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth.

A new series of estradiol analogs was synthesized in an attempt to improve on the anticancer activity of 2-methoxyestradiol, a naturally occurring mammalian tubulin polymerization inhibitor. The compounds were evaluated as inhibitors of tubulin polymerization and the binding of [3H]colchicine to tubulin, as well as for in vitro cytotoxicity in human cancer cell cultures. Overall, the most potent of the new compounds were 2-(2',2',2'-trifluoroethoxy)-6-oximinoestradiol, 2-ethoxy-6-oximinoestradiol, and 2-ethoxy-6-methoximinoestradiol. These agents lacked significant affinity for the estrogen receptor. The cytotoxicities of the compounds correlated in general with their abilities to inhibit tubulin polymerization, thus supporting inhibition of tubulin polymerization as the primary mechanism causing inhibition of cell growth.

Synthesis of 14,17-propano analogues of estradiol

Stereocontrolled syntheses of the 14α,17α-propano and 14β,17β-propano analogues of estradiol are described, together with those of numerous derivatives in which additional functionality is incorporated into the bridged system. Intramolecular aldol condensation of 17β-acetoxy-3-methoxy-20-oxo-19-nor-17α -pregna-1,3,5(10)-triene-14-carbaldehyde 1 furnishes 3-methoxy-171-oxo-14,17α-prop-172 -enoestra-1,3,5(10)-trien-17β-yl acetate 2, which is transformed into 14,17α-propanoestra-1,3,5(10)-triene-3,17β-diol 17. In the first of two synthetic approaches to the 14β,17β-propano series, cycloaddition of methyl propiolate to 3-methoxyestra-1,3,5(10),14,16-pentaen-17-yl acetate 25 gives a 14,17-bridged intermediate 26, in which the latent propyne equivalency of the dienophile is elaborated through selective functional group transformations to give 17-acetoxy-3-methoxy-20-oxo-19-nor-14β -pregna-1,3,5(10)-triene-14-carbaldehyde 50 and the derived product 52 of intramolecular aldol condensation. The second approach entails regioselective functionalisation of 14-allyl-3-methoxy-14β-estra-1,3,5(10)-trien-17-one 54 at C-2′ or C-3′ to give intermediates for intramolecular closure between the chain terminus and C-17, leading to 14,17β-propano-14β-estra-1,3,5(10)-triene-3,17α-diol 64. The results of competitive binding assays of the hormone analogues 17 and 64 toward the estradiol receptor are reported, and compared with those of bridge-functionalised derivatives.

Synthesis, uterotrophic, and antiuterotrophic activities of some estradiol derivatives containing thiadiazole, thiazoline, and thiazolidinone moieties.

The effect of structural modification on the biological activity of hormones has been studied on five novel series of estradiol analogs bearing a variety of substituents at the 2-position of the steroidal nucleus. The synthesized compounds include 2-[2-(5-substituted amino-1,3,4-thiadiazol-2-yl)vinyl]estradiol 17 beta-acetate 5-9, 2-aroylmethylestradiols 10-12, 2-[2-aryl-2-(substituted thiocarbamoylhydrazono)ethyl]estradiols 13-18 and their cyclic thiazoline 19-24, and thiazolidinone derivatives 25-30. Among the products, the p-hydroxybenzolmethylestradiol 12 exhibited the highest antiestrogenic activity of 63%. It also elicited 34% of the uterotrophic activity of estradiol.

A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.

Enantioselective synthesis of ferrocene analogs of hexestrol and estradiol; recognition towards estradiol receptors

We describe herein the synthesis of the first non-steroidal ferrocene analogs of estradiol, in which the five-membered ring (D) is replaced by a cyclopentadienyl ligand. The control of the three chiral elements for3-((2-hydroxymethyl) ferrocenyl)-4-(4-hydroxyphenyl)hexane ( 4a,4b) and 3-((2-methyl)ferrocenyl)-4-(4-hydroxyphenyl)- hexane: ( 5a,5b) was achieved by asymmetric cyclopalladation of N,N-dimethylaminomethylferrocene. The relative configurations were unambiguously determined from X-ray structure analyses on the racemic materials at each key step of the synthesis. All the ferrocene derivatives synthesized are recognized by the estradiol receptors. The best results are obtained for molecules possessing the same ethyl group disposition as that found in meso-hexestrol.

Medical contraindications and issues for consideration in the use of once-a-month injectable contraceptives

A number of once-a-month injectable contraceptives have been studied over the last 30 years and at least three different formulations are currently in use worldwide. Each of these formulations contains an ester of the natural estrogen, estradiol, and a synthetic progestin. This is in contrast to the combined oral contraceptives which contain synthetic analogs of both estradiol and progesterone. This paper considers medical contraindications or precautions related to the use of once-a-month injectable formulations and suggests some considerations for their programmatic use, particularly in the developing world. Because once-a-month injectables contain both an estrogen and a progestin, the precautions related to their use are similar to those of combined oral contraceptives; however, since the estrogen component is a natural estrogen, administration is patenterai and the circulating levels reach peaks that are in the range of those of the normal preovulatory phase of the menstrual cycle, some of these precautions may be very conservative. Absolute contraindications to the use of monthly injectables are current or suspected pregnancy, and estrogen-responsive tumors of the breast or genital tract. Other factors have been termed precautions for use rather than absolute or relative contraindications. Primary precautions are conditions under which a woman should probably not use monthly injectables, but if she does, she should be monitored closely. These include current or individual history of cardiovascular disease, current liver disease or liver tumors, and over age 40 if a smoker. Secondary precautions to use are those which should be considered very carefully and balanced against the risks of not using the particular method. These include suspicion of breast or genital tract cancer until cancer is ruled out, unexplained abnormal vaginal bleeding, use of drugs which might affect metabolism and decrease effective levels of circulating steroids, breastfeeding, and having combinations of several risk factors for cardiovascular disease. These precautions are categorized as proposed, based on the fact that most women in the developing world face a risk from pregnancy that is probably far greater than the risks from any of the present formulations of steroidal contraceptives. A key consideration during programmatic use of monthly injectables is attention to compliance with follow-up schedules to minimize the risk of pregnancy. Because little information on monthly injectables is available that is directly related to these precautions, additional research studies will be required in order to reconsider whether some of the listed precautions are valid or others should be considered.

Synthesis, binding affinities and uterotrophic activity of some 2-substituted estradiol and ring-A-fused pyrone derivatives

A series of estradiol analogs has been synthesized and examined as potential estrogens. Nuclear modifications included a variety of substituents at the 2 position of estradiol, which was previously thought to be inhibitory for activity, and inclusion of the 3-phenolic hydroxyl group in a γ-pyrone and 3′-formylchromone rings fused to ring A of estradiol. The estrogen relative binding affinities and in vivo assays for uterotrophic activity in rats showed that all the tested compounds were capable of displacing [ 3H]E 2 from the estrogen receptor sites by different degrees. The highest inhibition of [ 3H]E 2 binding (78%) to the estrogen receptor was displayed by 2-acetylestradiol which was also a potent uterotrophic agent. Omission of the free 3-hydroxyl functionality by inclusion in a γ-pyrone ring produced a chromone derivative that was capable of inhibiting [ 3H]E 2 binding by 60% and displayed a uterotrophic response of 97%. Further nuclear modification by introduction of thiosemicarbazone moieties decreased the uterotrophic activity, the highest activity being elicited by the p-bromophenyl thiosemicarbazone derivative. The diketone 2-benzoylacetylestradiol 17β-acetate, 2-(3′-benzylideneacetyl)estradiol and 2-[3′-(3-anisylidene)acetyl]estradiol exhibited high inhibition of binding affinity while eliciting ≈ 50% the uterotrophic activity of estradiol.

Acid-mediated rearrangements of 14,17-ethenoestra-1,3,5(10)-trien-17-ols: synthesis of 14,16-ethano-19-norsteroids

3-Methoxy-171-methyl-l 4,17α-ethenoestra-1,3,5(10)-trien-17β-ol 1 undergoes a 16(17 → 171)abeo-rearrangement in the presence of boron trihalides, to give the corresponding 16α-methyl-14β,16β-ethano 17-ketone. Similar rearrangements are described for epimeric 14,17-bridged estradiol analogues, leading to 16β-methyl-14α,16α-ethano 17-ketones. By contrast, acid-mediated reactions of 3-methoxy-14,17α-ethenoestra-1,3,5(10)-trien-17β-ol 11 display reagent dependence, leading to products of competing 16(17 →r;171)abeo-and 15(14 → 172)abeo-rearrangements. Conversion of certain rearrangement products into 14,16-ethano analogues of estradiol and 19-nortestosterone are described, and X-ray crystallographic structure determinations are reported for three ring-D-bridged compounds, and a novel spiro compound arising from rearrangement of the 14α,17α-etheno 17β-alcohol 11.

17α -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 17α. substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17α. side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17α 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-17β-oxirane 3-benzyl ether and subsequent debenzylation ( 4 to 18), by cross-coupling of alkynes ( 21 to 24), by alkylation of 17αethynylestradiol 3,17 -bis (tetrahydropyranyl ether) and subsequent acidic hydrolysis ( 25 to 28), or by reacting estrone either with appropriate aryllalkynyllithium 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 17α. 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 .

Structure of 3-hydroxy-1-methyl-1,3,5(10)-estratriene-11,17-dione

C19H22O3, Mr = 298.39, orthorhombic, P2(1)2(1)2(1), a = 11.332 (6), b = 14.596 (7), c = 9.567 (4) A, V = 1582.6 (5) A3, Z = 4, D chi = 1.252 Mg m-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.0778 mm-1, F(000) = 640, T = 273 K, R = 0.067 for 1762 unique observed reflections where F greater than 2 sigma (F). The structure was studied to observe the effect of 1-methyl and 11-keto substitution. The 3-hydroxyl is hydrogen bonded to O11 at 2.76 A. The B ring is in a 7 alpha, 8 beta half-chair conformation. The molecule twists about the C(9)--C(10) bond to relieve the steric interaction between the 1-methyl and 11-keto groups. The C1--C10--C9--C11 torsion angle is 55 vs 33.5 degrees, the average of 38 estradiol analogue structures.

Synthesis of 7α- and 7β-carboxymethyl-9(11)-ene derivatives of estrone and estradiol

As part of a search for derivatives designed to conjugate to amino groups, either of a protein carrier for antibody production or of an immobilized side-chain on a polymer for affinity chromatography, functionalized estrone and estradiol analogues were prepared. These modified steroids were obtained via the introduction of a carboxymethyl side-chain at the C-7α and C-7β position on an adrenosterone derivative and were then aromatized on the A ring. These new compounds are unsaturated at the C-9 (11) position, which could be useful for a second modification.

Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site.

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved delivery through biological membranes. 32. Synthesis and biological activity of brain-targeted delivery systems for various estradiol derivatives.

Brain-targeted delivery systems based on the dihydropyridine in equilibrium pyridinium salt redox interconversion were synthesized for estradiol, estradiol 3-benzoate, and ethynylestradiol. Initial biological evaluation indicated that while all four compounds synthesized exerted central estrogenic activity as measured by serum LH suppression, only the delivery systems based on the 17-substituted estradiol and ethynylestradiol demonstrated prolonged action (greater than 12 days). The 17-(1-methyl-1,4-dihydronicotinic acid ester) of ethynylestradiol behaved in a similar manner to the previously described estradiol analogue in various assays. Tissue distribution studies in rats showed that administration of the ethynylestradiol derivative resulted in high sustained levels of the corresponding pyridinium salt in the central nervous system (CNS) while blood levels of the oxidized metabolite rapidly fell. The sustained brain levels were associated with a prolonged release of ethynylestradiol. By 24 h, posttreatment, no ethynylestradiol was found by HPLC in the blood while levels of over 20 ng/g of tissue were detected in the CNS. This enhanced central delivery gave a dose- and time-dependent LH suppression, which indicated a three- to fivefold increased potency compared with the corresponding estradiol derivative.

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.

Biological activities of 4-fluoro estrogen analogues

As part of a study on the biological activity of γ-emitting estradiol analogues, a series of fluorinated and/or brominated analogues of estradiol were synthesized. 4-Fluoro-1,3,5(10-estratrien-3,17β-diol (4-fluoro-estradiol) and 16α-bromo-1,3,5(10)-estratrien-3,17β-diol (16α-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, 16α-bromo-estradiol was less effective in this assay indicating that its metabolism is probably more rapid than that of estradiol. 16β-Bromo-1,3,5(10)-estratrien-3,17β-diol had a low RBA and was ineffective in stimulating uterine weight at the doses used. 4-Fluoro-16α-bromo-estradiol had a lower RBA than either the 4-fluoro- or 16α-bromo-estradiol and all the 4-fluoro-seleno-estrogens tested possessed low RBA's.

Biological activity of [ 127I] and [ 125I] estradiol analogs in vitro and in vivo

In order to develop a means by which the receptor status of breast cancers could be studied in vivo. 127I and 125I analogs of estrogens were synthesized and their abilities to bind to uterine receptors, to translocate these receptors to the nucleus, and to bind in vivo to mammary tumors in rats were studied. 6-[ 127I]-Iodoestra-1,3,5(10),6-tetraene-3,17β-diol ( 2a), 16β[ 127I]-iodoestra-1,3,5(10)-triene-3,17β-diol ( 1) and 16α[ 127I]-iodoestra-1,3,5(10)-triene-3,17β-diol ( 3a) were capable of binding to the 8S cytosol receptor, translocating the cytosol receptor to the nucleus, in vitro, and increasing uterine weight in vivo. 16α-[ 125I]-Iodoestradiol ( 3b) was found to bind to high affinity 8S cytosol receptors but 6-[ 125I]-iodoestratetraene ( 2b) appeared to bind only non-specifically to macromolecules sedimenting in the 4S region. When these compounds ( 2b) and ( 3b) labeled with 125I, were administered to rats with DMBA-induced mammary tumors, the radioactivity as determined by imaging techniques was noted primarily in the liver and intestines. However, some rat tumors appeared to concentrate compound ( 2b). In at least one such tumor, the iodinated estrogen ( 2b) was bound non-specifically to 4S proteins.

Behavioral effects of ethynyl estrogens in the female rat

The synthetic analogues of estradiol, ethynyl estradiol and mestranol, are used in oral contraceptives. Their effects on food intake and sexual behavior were evaluated in female rats, and compared with those of estradiol. It was found (Experiment 1) that both ethynyl estradiol and mestranol reduced food intake reliably, and more than estradiol. Water intake and body weight followed similar trends. Ethynyl estradiol, but not mestranol, plus progesterone stimulated proceptive and receptive behavior in ovariectomized female rats. Daily administration of ethynyl estrogens without progesterone showed similar trends (Experiment 3). It is hoped that these studies provide guidelines for further work on the effects of synthetic estrogens on sub-primate and primate behavior.

A series of optical, structural and isomeric analogs of estradiol: A comparative study of the biological activity and affinity to cytosol receptor of rabbit uterus

Competitive binding between optical, structural and spatial isomers of estradiol with estradiol receptors of cytosol from rabbit uterus has been studied. The concept is put forward that the 3–17 distance and the conformation of substituents in the molecule of estrogen, is of major importance for the specific binding with the rabbit cytosol receptor to take place and for the biological activity to be displayed. It is established that, for the binding with the receptor to be effective, the steric agreement is indispensable between the steroid molecule and the binding site of the receptor. l-estradiol-17β which has the same 3–17 distance as natural estradiol, but an opposite configuration of the asymmetric centres, was found to have a low affinity for the receptor. The affinity of d, l-estradiol-17β (racemic mixture of d- and l-estradiol-17β) is only 50%. The ability of the optical isomers to bind with the receptor correlates with their biological activity. Alteration in the configuration of asymmetric centre in the molecule of estradiol decreases the binding and the biological activity. The binding and biological activity also decrease if the D-ring becomes six-membered. It is shown that removal of any of the hydroxyl groups decreases the affinity for the cytoreceptor and the estrogenic activity. The binding disappears if both hydroxyl groups are removed, indicating that 3–17 hydroxyl groups are indispensable for the specific binding with the receptor and manifestation of the biological activity. A seemingly illogical fact that d, l-8-iso-D-homoestradiol-17αβ has a higher estrogenic activity and a higher affinity to the receptor than d, l-D-homoestradiol-17αβ of natural configuration, may be explained in terms of 3–17 hydroxyl groups distances.