Proc. Natl. Acad. Sci. USA Vol. 94, pp. 14105–14110, December 1997 Pharmacology Molecular determinants of tissue selectivity in estrogen receptor modulators TIMOTHY A. GRESE*, JAMES P. SLUKA*, HENRY U. BRYANT,GEORGE J. CULLINAN,ANDREW L. GLASEBROOK, CHARLES D. JONES,KEN MATSUMOTO,ALAN D. PALKOWITZ,MASAHIKO SATO,JOHN D. TERMINE, MARK A. WINTER,NA N. YANG, AND JEFFREY A. DODGE* Endocrine Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285 Edited by Paul Talalay, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 3, 1997 (received for review July 3, 1997) ABSTRACT Interaction of the estrogen receptoryligand molecular conformation of individual ligands (11–14). The complex with a DNA estrogen response element is known to recent characterization of a new ER isoform, ERb, adds a regulate gene transcription. In turn, specific conformations of further layer of complexity, because ligands with different the receptor-ligand complex have been postulated to influence structural characteristics may exhibit different binding profiles unique subsets of estrogen-responsive genes resulting in differ- depending on which ER isoform is being evaluated (15, 16). ential modulation and, ultimately, tissue-selective outcomes. The To date, ER ligands have been grouped into four classes on the estrogen receptor ligands raloxifene and tamoxifen have dem- basis of in vivo pharmacology, and representatives of these classes onstrated such tissue-specific estrogen agonistyantagonist ef- indeed have been shown to exhibit unique profiles with respect to fects. Both agents antagonize the effects of estrogen on mammary transcriptional activation (9). Differential effects on mammary, tissue while mimicking the actions of estrogen on bone. However, uterine, cardiovascular, and skeletal tissues have been observed tamoxifen induces significant stimulation of uterine tissue for each of the four classes (17). At opposing ends of the whereas raloxifene does not. We postulate that structural dif- pharmacological spectrum are 17b-estradiol and ICI-164,384; the ferences between raloxifene and tamoxifen may influence the former is the natural ligand and a full agonist in target tissues conformations of their respective receptoryligand complexes, whereas the latter appears to be essentially a pure antagonist in thereby affecting which estrogen-responsive genes are modulated vivo (17, 18). In between these two extremes lie other ER ligands in various tissues. These structural differences are 4-fold: (A) the such as tamoxifen and raloxifene (Fig. 1), which exert tissue- presence of phenolic hydroxyls, (B) different substituents on the selective estrogen-like effects. Both act as estrogen antagonists in basic amine, (C) incorporation of the stilbene moiety into a cyclic mammary tissue, but mimic the agonist effects of estrogen on benzothiophene framework, and (D) the imposition of a carbonyl bone and in the cardiovascular system (19). In the uterus, ‘‘hinge’’ between the basic amine-containing side chain and the however, tamoxifen induces significant stimulation and appears olefin. A series of raloxifene analogs that separately exemplify to function as a partial estrogen agonist, whereas raloxifene each of these differences have been prepared and evaluated in a appears to function entirely as an antagonist. This uterine agonist series of in vitro and in vivo assays. This strategy has resulted in activity has been associated with an increased risk of endometrial the development of a pharmacophore model that attributes the cancer for patients treated with tamoxifen (20). Because of its differences in effects on the uterus between raloxifene and unique profile of tissue specificity, raloxifene has been charac- tamoxifen to a low-energy conformational preference imparting terized as a selective estrogen receptor modulator and is presently an orthogonal orientation of the basic side chain with respect to in clinical development for the prevention and treatment of the stilbene plane. This three-dimensional array is dictated by a postmenopausal osteoporosis (21, 22). Recently, a potential single carbon atom in the hinge region of raloxifene. These data mechanism for this tissue specificity involving selective interac- indicate that differences in tissue selective actions among ben- tion of the ER-raloxifene complex with a response element zothiophene and triarylethylene estrogen receptor modulators distinct from the classical estrogen response element has been can be ascribed to discrete ligand conformations. proposed (8). As part of our program to explore the pharmacol- ogy of raloxifene and related molecules, we have sought to Recent advances in understanding the mechanisms by which uncover the molecular features that account for its unique profile nuclear hormone receptors exert their effects on gene tran- of tissue specificity, particularly those features that differentiate scription have greatly enhanced the prospects for tissue- it from tamoxifen. Four major structural differences are apparent selective regulation of these processes (1–4). The recognition on inspection (Fig. 1): (A) the presence of the phenolic hydroxyls, that the estrogen receptor (ER) may interact with more than (B) the nature of the basic amine, (C) the incorporation of the one DNA response element, that these interactions may be stilbene into a cyclic benzothiophene framework, and (D) the mediated by various coactivators and corepressors, and that imposition of a carbonyl ‘‘hinge’’ between the basic amine- the nature of these interactions may be dependent on the containing side chain and the olefin. In this paper we directly specific ligand that is bound to the receptor allows for the compare the in vitro and in vivo pharmacology of a series of possibility of ligand-based transcriptional control (5–8). In- compounds that separately address each of these structural deed, it has been hypothesized that a series of ligand- features. We demonstrate that individual elements of raloxifene’s dependent conformations exist and that each of these confor- molecular structure are responsible for its enhanced tissue selec- mations may influence a unique subset of estrogen-responsive tivity relative to tamoxifen; that changes in these structural genes (9, 10). A logical extension of this hypothesis is the elements affect the profile of biological activities that previously dependence of ER conformation on the structural features and have been described for raloxifene; and that one of these struc- tural elements causes raloxifene to assume a distinct molecular The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in This paper was submitted directly (Track II) to the Proceedings office. accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: ER, estrogen receptor. © 1997 by The National Academy of Sciences 0027-8424y97y9414105-6$2.00y0 *To whom reprint requests should be addressed. e-mail: grese@ PNAS is available online at http:yywww.pnas.org. lilly.com. 14105 Downloaded by guest on September 28, 2021 14106 Pharmacology: Grese et al. Proc. Natl. Acad. Sci. USA 94 (1997) Table 2. Comparative effects on in vivo estrogen antagonism for various basic side chain modifications in the raloxifene nucleus In Vivo estrogen antagonism, maximal percent inhibition Compound R (Uterine wtybody wt) Tamoxifen 48* 1 91* 1g 81* FIG. 1. ER ligands tamoxifen and raloxifene. Differentiating struc- tural features are highlighted. 1h 3.8 conformation, which may, in turn, impact the conformation of the ER-raloxifene complex and in so doing be responsible for the unique tissue selectivity evidenced by this ER modulator. 1i 10 MATERIALS AND METHODS Chemistry. The compounds shown in Table 1 were prepared as previously described (23). Analogs bearing side-chain varia- tions, shown in Table 2, were prepared by Friedel–Crafts acyla- 1j 38* tion of 2-[4-methoxyphenyl]-6-methoxybenzothiophene with anisoyl chloride followed by selective O-demethylation and alky- lation of the resulting phenol with the appropriate electrophile 1k 36* under basic or Mitsunobu conditions (24, 25). Deprotection with y *Statistical significance relative to ethynyl estradiol-treated control ethanethiol aluminum chloride provided the target compounds. # Compound 2 (see Fig. 6) was prepared by a modification of the (P 0.05). method of Crenshaw involving the acid-mediated cyclization of 1 a compounds were fully characterized by H-NMR, mass spectra, an appropriately substituted -arylthiodeoxybenzoin (26). Com- andyor elemental analysis. Tamoxifen, 17a-ethynyl estradiol, and pound 3 (see Fig. 6) was prepared from a suitably protected 17b-estradiol were purchased from commercial sources. 11-thiacoumestrol by a method analogous to that described ER Binding. Affinity to the ER was determined in MCF-7 previously for coumestrol (27). Thus 3,9-dimethoxy-11- cell lysates by standard methods (29). Relative binding affin- thiacoumestan (28) was demethylated and converted to the b y ities were calculated as IC50 17 -estradiol IC50 compound. corresponding bis(tert-butyldimethylsilyl)ether. Low temperature MCF-7 Cell Proliferation. Effects on cellular proliferation reduction with diisobutylaluminum hydride followed by conden- were determined by standard methods (29) in MCF-7 breast sation of the resultant lactol with phenol provided the interme- adenocarcinoma cells. Cell cultures were treated in triplicate