Novel steroidal pure antiestrogens

A series of steroidal estrogen antagonists with no intrinsic estrogenicity in rat uterotrophic-antiuterotrophic tests has been discovered. The compounds are derivatives of estradiol containing amidoalkyl side chains at the 7 alpha-position. The most potent compounds are N-n-butyl-N-methyl-11-(3,17 beta-dihydroxyestra- 1,3,5(10)-trien-7 alpha-yl) undecanamide and N-2,2,3,3,4,4,4-heptafluorobutyl-N-methyl-11-(3,17 beta-dihydroxyestra- 1,3,5(10)-trien-7 alpha-yl) undecanamide. Structure activity relationships are discussed.     

Estrogens, antiestrogens and cell proliferation

The classical estrogen receptor model does not sufficiently account for the tumor-promoting activity of estrogens or for the antiproliferative effect of anti-estrogens in estrogen-dependent tumors. Particular difficulties not readily accommodated within the model are that hormonal autonomy can supervene without loss of the estrogen receptor and that antiestrogen effects are highly context-dependent, without apparent differences in the estrogen receptor itself or in metabolic transformation of antiestrogens. Recent studies suggest that estrogens may promote cell proliferation, in part, through the mediation of growth factors and that antiestrogens may exert some of their effects by mechanisms unrelated to the estrogen receptor.     

Newly discovered orally active pure antiestrogens

In order to develop orally active pure antiestrogens, we incorporated the carboxy-containing side chains into the 7alpha-position of the steroid scaffold and found that 17-keto derivative CH4893237 (12b) functioned as a pure antiestrogen with its oral activity much superior to clinically used pure antiestrogen, ICI182,780. Results from the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing in mice attributed to both improved absorption from the intestinal wall and metabolic stability in liver.     

Relative mitogenic activities of various estrogens and antiestrogens

The abilities of a variety of estrogens and antiestrogens to stimulate DNA synthesis in the prepuberal rat uterus were compared. One microgram of each compound was administered in vivo via a single intraperitoneal injection. DNA synthesis was assayed in vitro in isolated nuclei 24 h later. The relative mitogenicities of the steroidal estrogens were: 16 alpha-E2 less than 17 alpha-E2 = E3 = 16-EpiE3 less than 16 beta-E2 = 17 beta-E2. The potencies of several nonsteroidal estrogens were also tested. Indenestrol A was as potent at 17 beta-E2, whereas indanestrol and dimethylstilbestrol had weaker activities. The antiestrogens, nafoxidine and 4-hydroxytamoxifen, were both potent stimulators of DNA synthesis. The abilities of an estrogen to stimulate increases in uterine wet weight, DNA polymerase alpha activities, and DNA synthesis in uterine nuclei 24 h after injection were closely correlated. Because the magnitude of the stimulation of DNA synthesis was greatest, its measurement is the most sensitive of these assays of uterotrophic activity.     

Strategies for breast cancer therapy with antiestrogens

Current clinical research is focused upon the application of adjuvant therapy for the treatment of breast cancer. Combination chemotherapy is the most successful adjuvant therapy for premenopausal patients whereas the antiestrogen tamoxifen (1 or 2 yr) is successful in postmenopausal disease. We have developed a unifying strategy for the treatment of breast cancer. The thesis is based upon the application of continuous adjuvant therapy with tamoxifen in a low estrogen environment. Chemotherapy causes a chemical castration in premenopausal patients. In contrast, tamoxifen causes an increase in steroidogenesis. A combination of both approaches will work against each other until ovarian failure occurs. Patients should be checked for castration to provide a low estrogen environment in which tamoxifen, a competitive antagonist of estrogen action, can effectively work. Laboratory evidence using carcinogen-induced rat mammary tumor models demonstrates the efficacy of long-term therapy. Studies with the human breast cell line MCF-7 grown in athymic mice show that tamoxifen is a tumoristatic agent so that once the therapy is stopped, tumors can be regrown by estrogen administration. Patients should receive continuous tamoxifen therapy to prevent the growth-stimulating effects of adrenal steroids, environmental and phyto-estrogens.     

Interaction of non-steroidal antiestrogens with dopamine receptor binding

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.     

Species-specific pharmacology of antiestrogens: role of metabolism

The nonsteroidal antiestrogen tamoxifen exhibits a paradoxical species-specific pharmacology. The drug is a full estrogen in the mouse, a partial estrogen/antiestrogen in humans and the rat, and an antiestrogen in the chick oviduct. Inasmuch as tamoxifen has antiestrogenic effects in vitro, differential metabolism of tamoxifen to estrogens might occur in the species in which it has an estrogenic pharmacology. Tamoxifen or its metabolite 4-hydroxytamoxifen could lose the alkylaminoethane side chain to form the estrogenic compound metabolite E or bisphenol. Sensitive metabolic studies with [3H]tamoxifen in chicks, rats, and mice identified 4-hydroxytamoxifen as the major metabolite, but no potentially estrogenic metabolites were observed. Athymic mice with transplanted human breast tumors can be used to study the ability of tamoxifen to stimulate target tissue or tumor growth. Estradiol caused the growth of transplanted MCF-7 breast cancer cells into solid tumors and a uterotrophic response. However, tamoxifen does not support tumor growth when administered alone, although it stimulates uterine growth. Since a similar profile of metabolites is sequestered in human and mouse tissues, these studies strongly support the concept that the drug can selectively stimulate or inhibit events in the target tissues of different species without metabolic intervention.     

Modes of inhibition of protein kinase C by triphenylacrylonitrile antiestrogens

Protein kinase C (PKC) I (gamma), II (beta) and III (alpha) subspecies' activities are inhibited by three triphenylacrylonitrile (TPE) antiestrogens at micromolar concentrations. TPE 1 (having a p-hydroxy and a p-diethylaminoethoxy group on the 3-, and 3'- phenyl rings respectively) and TPE 2 (having a p-diethylaminoethoxy group on both the 3-, and 3'- phenyl rings) are competitive with the mechanism of activation by phosphatidylserine (PS). TPE 3 (having p-hydroxy groups on each of the three phenyl rings) is non-competitive with PS and inhibits the Ca2+- and PS-independent phosphorylation of protamine sulfate by PKC subspecies. This evidence suggests that PKC activity can be inhibited by different routes depending on the TPE structure: diethylaminoethoxy side chain-substituted TPEs (TPE 1 and 2) interact with PS as well as with the regulatory domain, whereas the trihydroxylated derivative (TPE 3) inhibits the enzyme by interacting with the catalytically active site.     

Differential binding of antiestrogens by rat uterine and chick oviduct cytosol

Analysis of the interactions of two synthetic estrogen antagonists, tamoxifen and CI 628, with rat uterine and chick oviduct cytosol revealed significant differences in the antiestrogen binding properties of these tissues. In the rat uterus CI 628, tamoxifen and estradiol were bound to a similar number of saturable binding sites and estradiol could completely inhibit the binding of tritiated antiestrogens to these sites. In contrast, high affinity, saturable antiestrogen binding sites in chick oviduct were present at three times the concentration of estradiol binding sites and estradiol could only partially inhibit the binding of tritiated antiestrogens to these sites. It is concluded that antiestrogens bind to the estrogen receptor in both tissues and that chick oviduct has an additional saturable antiestrogen binding site distinct from the classical estrogen receptor site.     

The evolution of nonsteroidal antiestrogens to become selective estrogen receptor modulators

The discovery of the first nonsteroidal antiestrogen ethamoxytriphetol (MER25) in 1958, opened the door to a wide range of clinical applications. However, the finding that ethamoxytriphetol was a “morning after” pill in laboratory animals, energized the pharmaceutical industry to discover more potent derivatives. In the wake of the enormous impact of the introduction of the oral contraceptive worldwide, contraceptive research was a central focus in the early 1960’s. Numerous compounds were discovered e.g., clomiphene, nafoxidine, and tamoxifen, but the fact that clinical studies showed no contraceptive actions, but, in fact, induced ovulation, dampened enthusiasm for clinical development. Only clomiphene moved forward to pioneer an application to induce ovulation in subfertile women. The fact that all the compounds were antiestrogenic made an application in patients to treat estrogen responsive breast cancer, an obvious choice. However, toxicities and poor projected commercial returns severely retarded clinical development for two decades. In the 1970’s a paradigm shift in the laboratory to advocate long term adjuvant tamoxifen treatment for early (non-metastatic) breast cancer changed medical care and dramatically increased survivorship. Tamoxifen pioneered that paradigm shift but it became the medicine of choice in a second paradigm shift for preventing breast cancer during the 1980’s and 1990’s. This was not surprising as it was the only medicine available and there was laboratory and clinical evidence for the eventual success of this application. Tamoxifen is the first medicine to be approved by the Food and Drug Administration (FDA) to reduce the risk of breast cancer in women at high risk. But it was the re-evaluation of the toxicology of tamoxifen in the 1980’s and the finding that there was both carcinogenic potential and a significant, but small, risk of endometrial cancer in postmenopausal women that led to a third paradigm shift to identify applications for selective estrogen receptor (ER) modulation. This idea was to establish a new group of medicines now called selective ER modulators (SERMs). Today there are 5 SERMs FDA approved (one other in Europe) for applications ranging from the reduction of breast cancer risk and osteoporosis to the reduction of menopausal hot flashes and improvements in dyspareunia and vaginal lubrication. This article charts the origins of the current path for progress in women’s health with SERMs.     

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.     

Estrogen receptors,antiestrogens, and non-small cell lung cancer

This review considers data on expression of different types of estrogen receptors (ERα and ERβ) in in vitro cultured cells of non-small cell lung cancer and also in human and animal lung tumors. Estrogens are shown to play an important role in genesis and development of non-small cell lung cancer because the estrogen-stimulated cell proliferation as well as antiestrogen-caused inhibition of proliferation occurred only in the cells expressing different types of estrogen receptors. In general, the situation is similar to that observed in breast cancer, but in the cells of non-small cell lung cancer not ERα are expressed in more than half of cases but ERβ. Just estrogen receptors β play the crucial role in inducing cell proliferation in response to estrogens, and ERβ is a prognostic marker of a favorable course of non-small cell lung cancer. Data on the interactions between ER and EGFR signaling pathways, as well as on the additive antitumor effect of antiestrogens (tamoxifen and fulvestrant) combined with tyrosine kinase inhibitors (gefitinib, erlotinib, and vandetanib) are considered. The review also includes data on the influence of estrogens on genesis and development of lung cancer in humans and animals and the frequency of ERα and ERβ expression in non-small cell lung cancer in tissues from patients of the two sexes. Problems of quantitative determination of α and β estrogen receptors in the tumor cells are also discussed.     

The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

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.     

Design of novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.     

The intratumoral aromatase model: studies with aromatase inhibitors and antiestrogens

Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which androstenedione was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm³, the animals were grouped for continued supplementation with androstenedione only (control) or for treatment with letrozole 10 μg per day (long-term), tamoxifen 100 μg per day (long-term), letrozole alternating to tamoxifen (4-week rotation), tamoxifen alternating to letrozole (4-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3–4 weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (34 weeks). Furthermore, alternating letrozole and tamoxifen treatment every 4 weeks was less effective than letrozole alone. Tumors doubled in 17–18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 μg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 μg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented androstenedione but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.     

Exploring the molecular basis of action of ring D aromatic steroidal antiestrogens

Salpichrolides are natural plant steroids that contain an unusual six‐membered aromatic ring D. We recently reported that some of these compounds, and certain analogs with a simplified side chain, exhibited antagonist effects toward the human estrogen receptor (ER), a nuclear receptor whose endogenous ligand has an aromatic A ring (estradiol). Drugs acting through the inhibition or modulation of ERs are frequently used as a hormonal therapy for ER(+) breast cancer. Previous results suggested that the aromatic D ring was a key structural motif for the observed activity; thus, this modified steroid nucleus may provide a new scaffold for the design of novel antiestrogens. Using molecular dynamics (MD) simulation we have modeled the binding mode of the natural salpichrolide A and a synthetic analog with an aromatic D ring within the ERα. These results taken together with the calculated energetic contributions associated to the different ligand‐binding modes are consistent with a preferred inverted orientation of the steroids in the ligand‐binding pocket with the aromatic ring D occupying a position similar to that observed for the A ring of estradiol. Major changes in both dynamical behavior and global positioning of H11 caused by the loss of the ligand–His524 interaction might explain, at least in part, the molecular basis of the antagonism exhibited by these compounds. Using steered MD we also found a putative unbinding pathway for the steroidal ligands through a cavity formed by residues in H3, H7, and H11, which requires only minor changes in the overall receptor conformation. Proteins 2015; 83:1297–1306. © 2015 Wiley Periodicals, Inc.