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Chapter 15. Targeting the estrogen receptor with SERMs
SECTION
IV. IMMUNOLOGY,
ENDOCRINOLOGY
AND METABOLIC
DISEASES
Editor: William K. Hagmann Merck Research Laboratories, Rahway, NJ 07065
Chapter
15. Targeting
the Estrogen
Receptor
with SERMs
Chris P. Miller and Barry S. Komm Wyeth-Ayerst Research, Radnor, PA 19087 Introduction - Estrogens represent a diverse structural array of compounds that function via binding to two nuclear localized receptors - estrogen receptor r1 and 1%(ER) (1,2). Members of this group of ligands includes the classic steroid hormones 17uestradiol. estrone. and estriol; phytoestrogens such as coumestrol and gemstern; synthetic estrogens like diethylstilbestrol; and xenobiotics like bisphenols and several chlorinated hydrocarbon pesticides (3). A relatively diverse group of compounds, which have been classified as selective estrogen receptor modulators or SERMs, are also members of this estrogen family (4). When an estrogen binds to the ER a series of events occurs (e.g. receptor dimerization, receptor conformational changes, biochemical modifications, coactivator interaction, and DNA binding) culminating in What gene transcription which affects changes in cellular I tissue physiology. distinguishes the SERMs from the other estrogens is that they demonstrate both ER agonist and antagonist activity dependent upon the cell type and gene promoter targeted. The primary clinical application of estrogens is estrogen replacement therapy (ERT) in menopausal and postmenopausal women. Menopause is associated with a reduction in ovarian production of estrogens. Physiologically, this results in vasomotor instability (including an increase in hot flushes), vaginal dryness, incontinence, changes in blood lipid profile (e.g. increased LDL cholesterol), and a reduction in bone mass (osteoporosis). Replacement with estrogens alleviates all of these symptoms to some extent, however, unopposed estrogen replacement also results in unacceptable uterine stimulation and an increased risk of developing breast cancer. In order to alleviate the uterine stimulation, estrogens are combined with progestins. known as hormone replacement therapy (HRT). This completely eliminates the negative impact of estrogens on the uterus. However, the effect of estrogens on the breast appears to remain unaffected by this combination therapy. Alternative therapies for HRT are desired because of these associated negative side effects. When the first SERM, tamoxifen (1) was initially characterized, it was classified as an antiestrogen in the treatment of breast cancer. Subsequent to its development for the treatment of breast cancer, it was demonstrated that tamoxifen exhibited estrogen agonist effects on the skeleton and liver (5). Unfortunately, like ERT, it also stimulated the uterine endometrium resulting in an increased risk of endometrial cancer. In an effort to maximize the positive characteristics of tamoxifen and eliminate the negative effects, other SERMs have been developed. This second generation of compounds includes raloxifene (2), droloxifene (a), idoxifene (A), and levormeloxifene. Of this group, only raloxifene has been developed and marketed for Additionally, raloxifene modestly the treatment and prevention of osteoporosis. reduces LDL cholesterol, however it has been shown to exacerbate vasomotor instability. It does not stimulate the uterus and reduces the risk of developing breast cancer (6). Third generation SERMs, TSE-424 (3) and lasofoxifene (S), are in Phase III development and demonstrate an improved profile over raloxifene, however no SERM has been shown to function as an equivalent replacement for HRT. The current candidates lack uterine and breast stimulation, but also lack the vasomotor relief,
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vaginal lubrication, greater efficacy on the skeleton and improved lipid profiles associated with classical HRT. The benefit that newer SERMs or combination therapies will demonstrate to improve upon the current SERMs’ effectiveness will be known in the near future I /NV-O
c1
N-O
1
1; ‘I 1; ” IR--:
HO
HO
C
2 N-O
HO HO
5:
5
Structural basis for SERM selectivity - Most non-steroidal selective estrogens reported to date share a common pharmacophore consisting of 2 aryl groups separated by two atoms, often in a stilbene type arrangement. Additionally, SERMs typically bear a third aryl group possessing a 4-aminoethoxy substitution. It had been speculated that when selective estrogens bind to ER, their stilbene like cores mimic the action of 17!3estradiol where at least one of the two aryl groups of the core generally contains a phenol group and overlays with the A-ring phenol of an estratriene nucleus (e.g. 17pestradiol). The third aryl group which bears the 4-aminoethoxy side chain would then project from a position which corresponds to the 11 b-position of the estratriene nucleus (7). Indeed. recent X-ray co-crystallographic studies of 4-OH tamoxifen (a high affinity metabolite of tamoxifen (1)) or raloxifene with the ligand binding domain of human ERa demonstrate this to be the case (8,9). A similar binding orientation was observed for raloxifene in rat ERP (10). When either of the SERMs bound to the receptor, a large conformational change was induced wherein helix 12 was moved to a groove formed between helices 3 and 5. This is in contrast to the situation with the non-steroidal estrogen agonist diethylstilbestrol (DES) or 17P-estradiol which both lack an amine bearing side chain. When these estrogen agonists bind the receptor, helix 12 is folded over revealing a groove formed by residues from helices 3, 4, 5, and 12 (Figure 1) (8). This groove provides a binding site for the LXXLL motif of various nuclear receptor coregulators, which recognize this portion of the receptor (11). The net result for the SERM occupied receptor is that helix 12, displaced by the bulky SERM side chain, is in a position that blocks the interaction of the receptor with certain nuclear coactivator proteins. Interference of coactivator recruitment can interfere with cellular transcription and thus allow a SERM to behave as an antagonist on target genes within a given cell type. The particular balance of coregulatory proteins and target promoter(s) in a given cellular backdrop likely determines ultimate SERM selectivity (12).
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H12
Figure 1. Ribbon representation of X-Ray crystal structures of the ligand binding domains of ERa/DES with a co-crystallized coactivator fragment (left) and ERa/4-OH tamoxifen (right). The sidechain of 4-OH-tamoxifen has displaced helix 12 from the agonist bound conformation (8). It has been demonstrated that small structural modifications to SERM molecules often results in large differences in both in vitro and in vivo potency as well as tissue selectivity (13-15). Nevertheless, certain general determinants of tissue selectivity have been made. In particular, it has been observed that the nature and orientation of the side chain is critical to the estrogen’s profile. In a series of 2-phenyl benzothiophenes, it was demonstrated that small changes in the amine of the side chain resulted in large changes to the compound’s ability to stimulate uterine hypertrophy (15). Another key determinant of selectivity between structural classes is the relative orientation of the side chain. Templates, such as triphenylethylenes (e.g. tamoxifen) where the side chain is in the plane of the stilbene system tend to cause increased uterine hypertrophy compared to templates where the side chain is capable of orienting itself orthogonal to the stilbene plane. This orthogonal orientation may come about through attachment of the side chain to a hinge atom linker, or through placement of the side chain on a non-aromatic, cyclic system which allows for the side chain to occupy an axial position (15). Interestingly, in cases where the side chain is connected in a triphenylethylene, coplanar type arrangement, the nature of the terminal amine seems much less critical to compound selectivity, if necessary at all (16-18). MOLECULAR
CLASSES
OF SERMS
Triohenvlethvlenes (TPE’s) - Triphenylethylenes were among the first reported structural classes of SERM molecules and tamoxifen (1) and clomiphene (7, shown as E-isomer) are probably the best known examples. Tamoxifen has been widely used for decades as a treatment for breast cancer and its indication was recently expanded for the prevention of breast cancer in women at risk (19). While long-term use of tamoxifen was associated with a 45%-50% decrease in breast cancer incidences in longterm studies of women at increased risk for the disease, there was a concomitant increased incidence in uterine cancer, deep vein thrombosis and hot flushes (20). The hyperplastic effect of tamoxifen on the uterus is of some concern and considerable efforts have been expended modifying the structure in order to decrease its trophic action on uterine tissue. Toremifene (8) is currently marketed for the treatment of breast cancer but apparently also suffers from similar uterine liability (21). Droloxifene ;;; ;I; idoxifene (4) were both recently terminated from late stage clinical trials , .
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Additional triphenylethylenes under investigation include MDL-101,232 (9), a clomiphene analogue with extended sidechain and FC-1271a (IO) (a human rnetabolite of toremifene) (24-26). Both compounds displayed estrogen agonist activity on bone as well as in decreasing total cholesterol, however, both compounds also demonstrated significant uterine effects when dosed in ovariectomized animals (27,28). It appears that the triphenylethylene class of SERMs generally continues to suffer from increased uterine liability relative to many of the other SERM classes.
Benzothiophenes - The FDA approved raloxifene (2, Evista@) in December 1997 for the treatment and prevention of osteoporosis in postmenopausal women (29). In a double-blind randomized trial of 7705 postmenopausal, osteoporotic women (MORE study), raloxifene at 60 mg/day reduced the risk of vertebral fracture in women by 30-50% (depending on pre-existing fracture status of patient group) over a 36 month period but also increased the risk of deep vein thrombosis (30,31). In addition, a 90% reduction of invasive ER+ breast cancer incidences was reported in raloxifene treated groups (60 mg/day and 120 mg/day groups combined) and there was no increase in endometrial cancer among women in the raloxifene treated groups (32). In a separate study evaluating the effect of raloxifene (60 mglday) on hot flushes, it was demonstrated that raloxifene, like tamoxifen, increased the incidences of hot flushes in postmenopausal women (33). Currently raloxifene is being evaluated in a large 10 year study (STAR) to compare its profile to tamoxifen in effecting long-term reductions in the incidence of postmenopausal ER+ breast cancer among women with increased risk for the disease (34).
Several reports in the literature describe modifications to the benzothiophene core with the goal of improving potency and selectivity. Exchange of the carbonyl hinge at the three position of the benzothiophene with an N, 0, S atom, or methylene group resulted in compounds with increased antiestrogenic potency in an MCF-7 cell proliferation assay. Particularly noteworthy was the replacement of the carbonyl group
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with an oxygen atom resulting in 11 which displayed high antiestrogenic potency in a 4-day immature rat uterine assay (ED 50 = 0.006 mglkg S.C. against ethynyl estradiol (0.1 mg/kg p.0.) compared to an EDso = 0.05 mg/kg S.C. for raloxifene). However, the potency advantage dissipated somewhat when comparing oral doses of the two compounds in the same model (EDso = 0.25 mg/kg p.o. for 11 vs EDso = 0.55 mglkg p.o. for raloxifene) (35). Methylation of the 4’-phenol of 11 results in arzoxifene (IJ), a SERM with substantially enhanced oral activity relative to raloxifene. Evaluation of 12. in 6 month ovariectomized rats revealed that it was from 30 to 100 times more potent than raloxifene in preventing ovariectomy-induced effects on body weight, serum cholesterol, and bone density, while displaying marginal estrogen agonist activity on the uterus (36,37). The increased oral bioavailabilty is presumably due to decreased intestinal glucuronidation since the 4’-OH phenol is not available for conjugation (38). Additional recent modifications to the benzothiophene core of raloxifene include reduction of the double bond between the 2 and 3 position of the benzothiophene resulting in the racemic dihydroraloxifene analogue (l3) with 23 trans stereochemistry. This compound displayed receptor affinity and MCF-7 cell proliferation inhibition potency similar to raloxifene (39). Steroids - Since the principle endogenous ligand for the ER receptor is 178-estradiol, a steroid hormone containing the estratriene nucleus, it is not surprising that many attempts to modify the structure chemically in order to alter its pharmacological profile have been made. It has been shown that the placement of long aliphatic amide (l4) and sulfoxide (l5) containing side chains at the 7c( position resulted in compounds possessing what has been described as “pure” antiestrogen activity (40-42). It has also been demonstrated that the placement of similar aliphatic chains (or long aliphatic side chains with phenoxy linkers) into the 118 position of the estratriene nucleus (l6) can also result in compounds with such activity (43). While such compounds, most notably 15 (ICI-182780 (faslodex)), have advanced in the clinic for the treatment of ER-depezent breast cancer; their selective estrogen activity is more restricted compared to raloxifene or tamoxifen since that they do not display estrogen agonist like activity on the many parameters where ER agonist activity is desired, such as bone, cardiovascular or the CNS (44,45). Nevertheless, placement of side chains that more closely resemble those of raloxifene at the 118 position of the estratriene nucleus resulted in compounds such as 17 that prevented bone loss as well as lowered cholesterol in ovariectomized rats while demonstrating minimal uterine effects when compared to 17(3-estradiol (46,47). This result is consistent with the x-ray co-crystal of raloxifene and ERcx that places the raloxifene sidechain in a position of the receptor that is coincident with the 118 position of 178-estradiol (9).
14 R = (CH&&ON(Me)n-Bu 15 R = (CH2)sSO(CH2)&F2CF3
F= (CH2)$02(CH2)sC2F5
17
lndoles - The 2-phenyl substituted indole has been used as a non-steroidal estrogen scaffold (48-50). Conversion of these previously reported indole estrogen agonists to compounds with improved selectivity profiles was accomplished through the introduction of aliphatic side chains originating from the l-position and often terminating with amines, such as ZK-119010 (18) (5152). While initial reports described 18 as a compound that caused no uterine stimulation in mice, a later report
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demonstrated that the compound had some uterotrophic activity in immature female rats (5253). Longer chain compounds like 19 and 20 were subsequently identified with potent antiestrogenic activity in MCF-7 cell assays and did not display estrogen agonism in a mouse uterine wet weight assay (5455). More recently, 2-phenyl indoles with rigid side chains have been reported as molecules possessing highly desirable SERM-like profiles. TSE-424 (5) demonstrated potent antagonism of 178estradiol stimulated proliferation of the MCF-7 breast cancer cell line (I& = 0.19 nM) and displayed little or no uterine stimulation when dosed in either immature or ovariectomized rats. Additionally, in a 6-week ovariectomized model of rat osteopenia, 5 was able to protect against bone loss and lower total cholesterol with oral doses of 0.3 mglkg. Interestingly, 3 inhibited activity at hepatic lipase and ape(a) promoters in hepG2 cells similarly to 178-estradiol while raloxifene and tamoxifen were inactive. The SERM (5) has completed phase II clinical trials for the treatment and prevention of osteoporosis-in postmenopausal women and is presently advancing through phase III clinical trials (56,57). The piperidine-containing SERM ERA-923 (2J) competitively and potently inhibited binding of 176-estradiol on both ERa and ER8. In its agonist mode, 21 lowered cholesterol and protected against bone loss in an ovariectomized rat model while in MCF-7 cells, 21 inhibited estradiol-stimulated growth with an I&O = 0.7 nM. In nude mice implanted with MCF-7 breast tumor cells, 21 (3-10 mglkglday given orally) inhibited estradiol-stimulated growth. Additionally, 21 inhibited proliferation of a human endometrial line, EnCa-101, human ovarian BG-1 %ls, and an MCF-7 variant that is inherently resistant to tamoxifen. SERM (21) displays no uterotrophic effects when Two Phase I trials with 21 given to healthy, given alone to rats or mice. postmenopausal women have been completed and a Phase II evaluation in women with metastatic breast cancer is currently underway (5859).
‘O -OH k 18 R = (CH&-N(CH& fi R = (CH2)loCON(CH3)n-Bu 20 R = (CH2)gSO-(CH&,CF$Fs Napthalene Derivatives - Napthalene, dihydronapthalenes, and tetrahydronapthalene scaffolds have served as versatile cores for molecules demonstrating selective estrogen action. Molecules related to the dihydronapthalene trioxifene (22) were recently described where the carbonyl hinge was replaced by oxygen (23) or CH2 (24) (60-62). SERM (24) displayed little or no uterine stimulation in ovanectomized rats while protecting agarnst bone loss and reducing cholesterol with oral doses as low as 0.1 mglkg per day (61). Lasofoxifene, the tetrahydronapthalene (S), is currently in
22
23x=0 24 X=CH2
25
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phase III clinical trials as a salt of its (-) isomer (63). SERM (fi), closely related to the much earlier reported estrogen (25), protected against lumbar vertebral bone loss in ovariectomized rats with an EDSO of cl ug/kg/day p.o. (64-66). In human phase II clinical trials, S prevented bone loss in postmenopausal women with a dose of just 0.25 mg/day (67). The excellent oral potency of 5 is attributed to reduced intestinal glucuronidation of the phenol. Benzopvrans and Arvlcoumarins - Certain benzopyrans and arylcoumarins have been described as compounds having SERM activity. EM-800 (26) is the (S)-(+) bispivaloylated prodrug of its progenitor, EM-652 (27). In human breast cancer cell lines (ZR-75-1 and T-47D), 27, was from 27 to 60 times more potent as an antagonist than its (R) enantiomer (68). The ability of -27 to block both the 178-estradiol-dependent interaction of ERcr and ERj3 with the coactivator (SRC-1) and the in vitro interaction of SRC-1 with the ligand binding domains of both receptors suggested that the compound fully impedes both AF-1 and AF-2 ER function in these systems. This was regarded as Important to the molecule’s ultimate potential in treating breast cancer since the AF-1 domain of ER displays ligand-independent activity and mediates growth factors as well as the ras oncogene and MAPK pathways. 4-OH-tamoxifen, in contrast, only inhibits cellular transcription through the ligand dependent AF2 domain (69). In a comparison with raloxifene. S was tested for its ability to prevent bone loss and lower serum cholesterol in a 37-week rat ovariectomized model. It was concluded from the study that $@ was from 3-10 times more potent than raloxifene on preventing bone loss in the proxrmal tibia and femur and in reducing total cholesterol. Both compounds were similarly efficacious, but 26, unlike raloxifene, produced no discernable stimulation of the rat uterine epithelium (69). Arylcoumarins, exemplified by 28, have been recently described and reported to exhibit SERM activity. In a 3-day immature rat uterine assay, subcutaneous dosing of compound 28 at 1 mglkglday demonstrated no significant increase in uterine wet weight while?n the same assay at the same dose, raloxifene increased the wet uterine weights by 50% which was significantly different from control. Compound (28) also inhibited both estradiol induced proliferation of MCF-7 cells and IL-6 expression in human osteoblasts (70).
S R = COC(CH3)3 27 R=H
28
Constrained SERMs with tetracvclic cores - Recently, several SERMs have been described wherein the core consists of a tetracycle that simultaneously constrains the stilbene phenols into a coplanar arrangement as well as fixes the side chain into an axial conformation. The phytoestrogen coumestrol (29) acted as the precursor to the SERM (30) where the carbonyl of the lactone served as the attachment point for the basic side chains. The importance of the basic side chain for SERM selectivity is demonstrated by the fact that 30 is a potent antagonist of estrogen dependent stimulation of MCF-7 cells (I&O = 0.7 nM). Additionally, 30 did not stimulate uterine hypet-trophy in ovariectomized rats through a range of doses but is able to partially inhibit uterine hypertrophy caused by ethynyl estradiol in immature rats. Coumestrol, in contrast, failed to inhibit estrogen induced MCF-7 cell proliferation and caused uterine hypertrophy in an ovariectomized rat model (71,72). SAR in the series demonstrated that the preferred amine for optimal selectivity was a piperidine ring. Additionally, it was demonstrated that the napthalene fused compound (31) displayed
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excellent in vivo potency on several estrogen sensitive parameters including cholesterol levels, bone mineral density, and inhibition of estrogen stimulated uterine hypertrophy. Since 31 was not remarkably potent on certain in vitro measurements such as estrogen receptor affinity (ERa RBA = 0.22 vs 0.34 for raloxifene) or inhibition of estrogen stimulated MCF-7 cell proliferation (GO = 0.4 nM vs 0.4 nM for raloxifene) it is possible that the superior in vivo potency of 31 in preventing bone loss in the ovariectomized model of rat osteopenia (0.01 mglkglday vs 1.0 mg/kg/day for raloxifene) is evidence for its having increased bioavailability relative to other SERMs such as raloxifene (72). A molecule from a related series of indenobenzothiophenes (32) (which can be viewed as a conformationally locked version of raloxifene) proved itself a potent antagonist in an MCF-7 cell assay (ICSO = 0.1 nM) (73).
22
30x=0
31 X = CH=CH
32
Conclusion - The approval of raloxifene for the treatment and prevention of osteoporosis in postmenopausal women illustrates the value of SERMs for the treatment of at least one of the maladies associated with this patient population. Additional evidence from clinical studies indicate that raloxifene, like tamoxifen, lowers the risk of ER+ breast cancer, but unlike tamoxifen, raloxifene does not appear to increase the risk of endometrial cancer or uterine bleeding. Encouraged by raloxifene’s early success, much recent research dedicated to improving the pharmacological properties of the earlier generation SERMs has resulted in compounds with improved bioavailability and selectivity. To date, however, SERMs have not been shown to positively affect many other symptoms of the menopause and postmenopause period such as hot flushes, vaginal dryness, or urinary incontinence. Since these “quality of life” issues are what prompt many women to seek hormone replacement therapy in the first place, it is likely that the search for newer and better SERMs or SERM combinations will occupy the attentions of the research community for some time to come (74-76). References
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15s --
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P. Van de Velde, F. Nique, P. Planchon, G. Prevost, J. Bremaud, M.C. Hameau, V. Magnien, D. Philibert, and G. Teutsch, J.Steroid.Biochem.Molec.Biol., !&3, 449 (1996). J.A. Dodge, M.G. Stocksdale. L.J. Black, E.R. Rowley, A. Bekele, H.W. Cole, C.E. Brown, D.E. Magee, G.A. Dehoney, and H.U. Bryant, J.Bone.Miner.Res., 8 (suppl. l), S278 (1993). T.A. Grese and J.A. Dodge, Ann.Rep.Med.Chem., 1,181 (1996). Y. Bouali, J.G. Teutsch, F. Nique. P. Van de Velde, and D. Philibert. FR2757399 (1998). Y. Bouali, F. Nique. J.G. Teutsch, and P. Van de Velde, FR2757519 (1998). E. von Angerer, J. Prekajac. and J. Strohmeier, J.Med.Chem., 27, 1439 (1984). E. von Angerer, J. Prekajac, and M. Berger, Eur.J.CancerClin.Oncol., 21, 531 (1985). P. Hilgard. J. Stekar. W. Wuttke. M. Elliger, and W. Holtei, Prog.CancerRes.Ther., 35, 369 (1988). E. von Angerer. DE 3821148 (1998). E. von Angerer. N. Knebel, M. Kager, and B. Ganss, J.Med.Chem., 33, 2635 (1990). Y. Nishino. M.R. Schneider, H. Michna. and E. von Angerer, J.Endocrinol., 130, 409 (1991). Leichtl, E. Angerer. C. Biberger, R. Koop, and S. von E. Holler, J.Steroid.Biochem.Molec.Biol.. 49, 51 (1994). A. Biberger and E. von Angerer. J.Steroid.Biochem.Molec.Biol., 58, 31 (1996). C.P. Miller, M.D. Collini, B.S. Komm, Y.P. Kharode, and H.A. Harris, Abstracts of Papers: 219@ Am.Chem.Soc.Natl.Mtg., March 2000; American Chemical Society, San Francisco, CA; MEDI 329. B.S. Komm, C.P. Miller, and C.R. Lyttle, J.Bone.MineralRes, 15S:1093 (2000). L.M. Greenberger, B. Komm, C. Miller, T. Annable, R. Lyttle, and P. Frost, Symp.NewDrugsCanc.Ther. Amsterdam (2000). C.P. Miller, L.M. Greenberger, T. Annable, M.D. Collini, B.D. Tran, B.S. Komm, P. Frost, J.P. Yardley, C.R. Lyttle, and M. A. Abou-Gharbia, Abstracts of Papers: 2211h Am.Chem.Soc.Natl.Mtg., April 2001; American Chemical Society, San Diego, CA; MEDI 167. C.D. Jones, T. Suarez, E.H. Massey, L.J. Black, and F.C. Tinsley, J.Med.Chem., 22, 962 (1979). H.U. Bryant, G.J. Cullinan, J.A. Dodge, K.J. Fahey, CD. Jones, C.W. Lugar, and B.S. Muehl. US 5484795. A.D. Palkowitz, EP 729951 (1996). Bus.Ed.&HealthMed.Writers BIOWIRE2K. Lednicer, D.E. Emmert, SC. Lyster, and G.W. Duncan, J.Med.Chem., 12, 881 (1969). H.Z. Ke. V.M. Paralkar. W.A. Grasser, D.T. Crawford, H. Qi. H.A. Simmons, C.M. Pirie. K.L. Chidsey-Frink, T.A. Owen, S.L. Smock, H.K. Chen, W.S.S. Jee, K.O. Cameron, R.L. Rosati, T.A. Brown, P. Dasilva-Jardine, and D.D. Thompson, Endocrinology, 139, 2068 (1998). L.A. Sorbera. P.A. Leeson, and J. Castaner, DrugsFut., 3, 1066 (1998). K. Cameron, Abstracts of Papers: 219’” Am.Chem.Soc.Natl.Mtg., March 2000; American Chemical Society, San Francisco, CA; MEDI 326. S. Gauthier, C. Brigitte, J. Clutier, Y.L. Dory, A. Favre, D. Larouche, J. Mailhot, C. Ouellet, A. Schwerdtfeger, G. Leblanc, C. Martel, J. Simard, Y. Merand, A. Belanger, C. Labrie, and F. Labrie, J.Med.Chem., 40, 2117 (1997). F. Labrie, C. Labrie, A. Belanger, J. Simard, S. Gauthier, V. Lu-The, Y. Merand, V. Giguere, B. Candas, S. Luo, C. Martel, S.M. Singh. M. Fournier. A. Coquet, V. Richard, R. Charbonneau, G. Charpenet, A. Tremblay, G. Tremblay, L. Cusan. and R. Veilleux, J.SteroidBiochem.Mol.Biol., a. 51 (1999). B.M. Stein, D.W. Anderson, L.M. Gayo, M.S. Sutherland, M. Doubleday, G.I. Shevlin, A. Kois, S. Khammungkhune, R.K. Jalluri, S.S. Bhagwat, and J.A. McKie, W O 0039120 (2000). T.A. Grese, H.W. Cole, D.E. Magee, D.L. Phillips, P.K. Shetler, L.L. Short, A.L. Glasebrook, and H.U. Bryant, Biorg.Med.Chem.Lett., 6, 2683 (1996). T.A. Grese, L.D. Pennington. J.P. Sluka, M.D. Adrian, H.W. Cole, T.R. Fuson, D.E. Magee. D.L. Phillips, E.R. Rowley, P.K. Shetler, L.L. Short, M. Venugopalan, N.N. Yang, M. Sate. A.L. Glasebrook, and H.U. Bryant, J.Med.Chem., 4l, 1272, (1998). M.G. Bell, H.U. Bryant, A.L. Glasebrook, CD. Jones, B.S. Muehl, and M.A. Winter, Abstracts of Papers: 219’h Am.Chem.Soc.Natl.Mtg., March 2000; American Chemical Society, San Francisco, CA; MEDI 245. P. Kenemans, J.Epidem.Biostat., 3, 141 (1999). G. Greendale, Lancet. 353, 571 (1999). B. Feldman, Res.Nurs.Health, 8, 261 (1985).
IV. IMMUNOLOGY,
ENDOCRINOLOGY
AND METABOLIC
DISEASES
Editor: William K. Hagmann Merck Research Laboratories, Rahway, NJ 07065
Chapter
15. Targeting
the Estrogen
Receptor
with SERMs
Chris P. Miller and Barry S. Komm Wyeth-Ayerst Research, Radnor, PA 19087 Introduction - Estrogens represent a diverse structural array of compounds that function via binding to two nuclear localized receptors - estrogen receptor r1 and 1%(ER) (1,2). Members of this group of ligands includes the classic steroid hormones 17uestradiol. estrone. and estriol; phytoestrogens such as coumestrol and gemstern; synthetic estrogens like diethylstilbestrol; and xenobiotics like bisphenols and several chlorinated hydrocarbon pesticides (3). A relatively diverse group of compounds, which have been classified as selective estrogen receptor modulators or SERMs, are also members of this estrogen family (4). When an estrogen binds to the ER a series of events occurs (e.g. receptor dimerization, receptor conformational changes, biochemical modifications, coactivator interaction, and DNA binding) culminating in What gene transcription which affects changes in cellular I tissue physiology. distinguishes the SERMs from the other estrogens is that they demonstrate both ER agonist and antagonist activity dependent upon the cell type and gene promoter targeted. The primary clinical application of estrogens is estrogen replacement therapy (ERT) in menopausal and postmenopausal women. Menopause is associated with a reduction in ovarian production of estrogens. Physiologically, this results in vasomotor instability (including an increase in hot flushes), vaginal dryness, incontinence, changes in blood lipid profile (e.g. increased LDL cholesterol), and a reduction in bone mass (osteoporosis). Replacement with estrogens alleviates all of these symptoms to some extent, however, unopposed estrogen replacement also results in unacceptable uterine stimulation and an increased risk of developing breast cancer. In order to alleviate the uterine stimulation, estrogens are combined with progestins. known as hormone replacement therapy (HRT). This completely eliminates the negative impact of estrogens on the uterus. However, the effect of estrogens on the breast appears to remain unaffected by this combination therapy. Alternative therapies for HRT are desired because of these associated negative side effects. When the first SERM, tamoxifen (1) was initially characterized, it was classified as an antiestrogen in the treatment of breast cancer. Subsequent to its development for the treatment of breast cancer, it was demonstrated that tamoxifen exhibited estrogen agonist effects on the skeleton and liver (5). Unfortunately, like ERT, it also stimulated the uterine endometrium resulting in an increased risk of endometrial cancer. In an effort to maximize the positive characteristics of tamoxifen and eliminate the negative effects, other SERMs have been developed. This second generation of compounds includes raloxifene (2), droloxifene (a), idoxifene (A), and levormeloxifene. Of this group, only raloxifene has been developed and marketed for Additionally, raloxifene modestly the treatment and prevention of osteoporosis. reduces LDL cholesterol, however it has been shown to exacerbate vasomotor instability. It does not stimulate the uterus and reduces the risk of developing breast cancer (6). Third generation SERMs, TSE-424 (3) and lasofoxifene (S), are in Phase III development and demonstrate an improved profile over raloxifene, however no SERM has been shown to function as an equivalent replacement for HRT. The current candidates lack uterine and breast stimulation, but also lack the vasomotor relief,
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vaginal lubrication, greater efficacy on the skeleton and improved lipid profiles associated with classical HRT. The benefit that newer SERMs or combination therapies will demonstrate to improve upon the current SERMs’ effectiveness will be known in the near future I /NV-O
c1
N-O
1
1; ‘I 1; ” IR--:
HO
HO
C
2 N-O
HO HO
5:
5
Structural basis for SERM selectivity - Most non-steroidal selective estrogens reported to date share a common pharmacophore consisting of 2 aryl groups separated by two atoms, often in a stilbene type arrangement. Additionally, SERMs typically bear a third aryl group possessing a 4-aminoethoxy substitution. It had been speculated that when selective estrogens bind to ER, their stilbene like cores mimic the action of 17!3estradiol where at least one of the two aryl groups of the core generally contains a phenol group and overlays with the A-ring phenol of an estratriene nucleus (e.g. 17pestradiol). The third aryl group which bears the 4-aminoethoxy side chain would then project from a position which corresponds to the 11 b-position of the estratriene nucleus (7). Indeed. recent X-ray co-crystallographic studies of 4-OH tamoxifen (a high affinity metabolite of tamoxifen (1)) or raloxifene with the ligand binding domain of human ERa demonstrate this to be the case (8,9). A similar binding orientation was observed for raloxifene in rat ERP (10). When either of the SERMs bound to the receptor, a large conformational change was induced wherein helix 12 was moved to a groove formed between helices 3 and 5. This is in contrast to the situation with the non-steroidal estrogen agonist diethylstilbestrol (DES) or 17P-estradiol which both lack an amine bearing side chain. When these estrogen agonists bind the receptor, helix 12 is folded over revealing a groove formed by residues from helices 3, 4, 5, and 12 (Figure 1) (8). This groove provides a binding site for the LXXLL motif of various nuclear receptor coregulators, which recognize this portion of the receptor (11). The net result for the SERM occupied receptor is that helix 12, displaced by the bulky SERM side chain, is in a position that blocks the interaction of the receptor with certain nuclear coactivator proteins. Interference of coactivator recruitment can interfere with cellular transcription and thus allow a SERM to behave as an antagonist on target genes within a given cell type. The particular balance of coregulatory proteins and target promoter(s) in a given cellular backdrop likely determines ultimate SERM selectivity (12).
SERMs
Chap. 15
Miller.
Komm
151 -
H12
Figure 1. Ribbon representation of X-Ray crystal structures of the ligand binding domains of ERa/DES with a co-crystallized coactivator fragment (left) and ERa/4-OH tamoxifen (right). The sidechain of 4-OH-tamoxifen has displaced helix 12 from the agonist bound conformation (8). It has been demonstrated that small structural modifications to SERM molecules often results in large differences in both in vitro and in vivo potency as well as tissue selectivity (13-15). Nevertheless, certain general determinants of tissue selectivity have been made. In particular, it has been observed that the nature and orientation of the side chain is critical to the estrogen’s profile. In a series of 2-phenyl benzothiophenes, it was demonstrated that small changes in the amine of the side chain resulted in large changes to the compound’s ability to stimulate uterine hypertrophy (15). Another key determinant of selectivity between structural classes is the relative orientation of the side chain. Templates, such as triphenylethylenes (e.g. tamoxifen) where the side chain is in the plane of the stilbene system tend to cause increased uterine hypertrophy compared to templates where the side chain is capable of orienting itself orthogonal to the stilbene plane. This orthogonal orientation may come about through attachment of the side chain to a hinge atom linker, or through placement of the side chain on a non-aromatic, cyclic system which allows for the side chain to occupy an axial position (15). Interestingly, in cases where the side chain is connected in a triphenylethylene, coplanar type arrangement, the nature of the terminal amine seems much less critical to compound selectivity, if necessary at all (16-18). MOLECULAR
CLASSES
OF SERMS
Triohenvlethvlenes (TPE’s) - Triphenylethylenes were among the first reported structural classes of SERM molecules and tamoxifen (1) and clomiphene (7, shown as E-isomer) are probably the best known examples. Tamoxifen has been widely used for decades as a treatment for breast cancer and its indication was recently expanded for the prevention of breast cancer in women at risk (19). While long-term use of tamoxifen was associated with a 45%-50% decrease in breast cancer incidences in longterm studies of women at increased risk for the disease, there was a concomitant increased incidence in uterine cancer, deep vein thrombosis and hot flushes (20). The hyperplastic effect of tamoxifen on the uterus is of some concern and considerable efforts have been expended modifying the structure in order to decrease its trophic action on uterine tissue. Toremifene (8) is currently marketed for the treatment of breast cancer but apparently also suffers from similar uterine liability (21). Droloxifene ;;; ;I; idoxifene (4) were both recently terminated from late stage clinical trials , .
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Additional triphenylethylenes under investigation include MDL-101,232 (9), a clomiphene analogue with extended sidechain and FC-1271a (IO) (a human rnetabolite of toremifene) (24-26). Both compounds displayed estrogen agonist activity on bone as well as in decreasing total cholesterol, however, both compounds also demonstrated significant uterine effects when dosed in ovariectomized animals (27,28). It appears that the triphenylethylene class of SERMs generally continues to suffer from increased uterine liability relative to many of the other SERM classes.
Benzothiophenes - The FDA approved raloxifene (2, Evista@) in December 1997 for the treatment and prevention of osteoporosis in postmenopausal women (29). In a double-blind randomized trial of 7705 postmenopausal, osteoporotic women (MORE study), raloxifene at 60 mg/day reduced the risk of vertebral fracture in women by 30-50% (depending on pre-existing fracture status of patient group) over a 36 month period but also increased the risk of deep vein thrombosis (30,31). In addition, a 90% reduction of invasive ER+ breast cancer incidences was reported in raloxifene treated groups (60 mg/day and 120 mg/day groups combined) and there was no increase in endometrial cancer among women in the raloxifene treated groups (32). In a separate study evaluating the effect of raloxifene (60 mglday) on hot flushes, it was demonstrated that raloxifene, like tamoxifen, increased the incidences of hot flushes in postmenopausal women (33). Currently raloxifene is being evaluated in a large 10 year study (STAR) to compare its profile to tamoxifen in effecting long-term reductions in the incidence of postmenopausal ER+ breast cancer among women with increased risk for the disease (34).
Several reports in the literature describe modifications to the benzothiophene core with the goal of improving potency and selectivity. Exchange of the carbonyl hinge at the three position of the benzothiophene with an N, 0, S atom, or methylene group resulted in compounds with increased antiestrogenic potency in an MCF-7 cell proliferation assay. Particularly noteworthy was the replacement of the carbonyl group
Chap
15
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Miller,
K~IIIII!I
152 -
with an oxygen atom resulting in 11 which displayed high antiestrogenic potency in a 4-day immature rat uterine assay (ED 50 = 0.006 mglkg S.C. against ethynyl estradiol (0.1 mg/kg p.0.) compared to an EDso = 0.05 mg/kg S.C. for raloxifene). However, the potency advantage dissipated somewhat when comparing oral doses of the two compounds in the same model (EDso = 0.25 mg/kg p.o. for 11 vs EDso = 0.55 mglkg p.o. for raloxifene) (35). Methylation of the 4’-phenol of 11 results in arzoxifene (IJ), a SERM with substantially enhanced oral activity relative to raloxifene. Evaluation of 12. in 6 month ovariectomized rats revealed that it was from 30 to 100 times more potent than raloxifene in preventing ovariectomy-induced effects on body weight, serum cholesterol, and bone density, while displaying marginal estrogen agonist activity on the uterus (36,37). The increased oral bioavailabilty is presumably due to decreased intestinal glucuronidation since the 4’-OH phenol is not available for conjugation (38). Additional recent modifications to the benzothiophene core of raloxifene include reduction of the double bond between the 2 and 3 position of the benzothiophene resulting in the racemic dihydroraloxifene analogue (l3) with 23 trans stereochemistry. This compound displayed receptor affinity and MCF-7 cell proliferation inhibition potency similar to raloxifene (39). Steroids - Since the principle endogenous ligand for the ER receptor is 178-estradiol, a steroid hormone containing the estratriene nucleus, it is not surprising that many attempts to modify the structure chemically in order to alter its pharmacological profile have been made. It has been shown that the placement of long aliphatic amide (l4) and sulfoxide (l5) containing side chains at the 7c( position resulted in compounds possessing what has been described as “pure” antiestrogen activity (40-42). It has also been demonstrated that the placement of similar aliphatic chains (or long aliphatic side chains with phenoxy linkers) into the 118 position of the estratriene nucleus (l6) can also result in compounds with such activity (43). While such compounds, most notably 15 (ICI-182780 (faslodex)), have advanced in the clinic for the treatment of ER-depezent breast cancer; their selective estrogen activity is more restricted compared to raloxifene or tamoxifen since that they do not display estrogen agonist like activity on the many parameters where ER agonist activity is desired, such as bone, cardiovascular or the CNS (44,45). Nevertheless, placement of side chains that more closely resemble those of raloxifene at the 118 position of the estratriene nucleus resulted in compounds such as 17 that prevented bone loss as well as lowered cholesterol in ovariectomized rats while demonstrating minimal uterine effects when compared to 17(3-estradiol (46,47). This result is consistent with the x-ray co-crystal of raloxifene and ERcx that places the raloxifene sidechain in a position of the receptor that is coincident with the 118 position of 178-estradiol (9).
14 R = (CH&&ON(Me)n-Bu 15 R = (CH2)sSO(CH2)&F2CF3
F= (CH2)$02(CH2)sC2F5
17
lndoles - The 2-phenyl substituted indole has been used as a non-steroidal estrogen scaffold (48-50). Conversion of these previously reported indole estrogen agonists to compounds with improved selectivity profiles was accomplished through the introduction of aliphatic side chains originating from the l-position and often terminating with amines, such as ZK-119010 (18) (5152). While initial reports described 18 as a compound that caused no uterine stimulation in mice, a later report
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demonstrated that the compound had some uterotrophic activity in immature female rats (5253). Longer chain compounds like 19 and 20 were subsequently identified with potent antiestrogenic activity in MCF-7 cell assays and did not display estrogen agonism in a mouse uterine wet weight assay (5455). More recently, 2-phenyl indoles with rigid side chains have been reported as molecules possessing highly desirable SERM-like profiles. TSE-424 (5) demonstrated potent antagonism of 178estradiol stimulated proliferation of the MCF-7 breast cancer cell line (I& = 0.19 nM) and displayed little or no uterine stimulation when dosed in either immature or ovariectomized rats. Additionally, in a 6-week ovariectomized model of rat osteopenia, 5 was able to protect against bone loss and lower total cholesterol with oral doses of 0.3 mglkg. Interestingly, 3 inhibited activity at hepatic lipase and ape(a) promoters in hepG2 cells similarly to 178-estradiol while raloxifene and tamoxifen were inactive. The SERM (5) has completed phase II clinical trials for the treatment and prevention of osteoporosis-in postmenopausal women and is presently advancing through phase III clinical trials (56,57). The piperidine-containing SERM ERA-923 (2J) competitively and potently inhibited binding of 176-estradiol on both ERa and ER8. In its agonist mode, 21 lowered cholesterol and protected against bone loss in an ovariectomized rat model while in MCF-7 cells, 21 inhibited estradiol-stimulated growth with an I&O = 0.7 nM. In nude mice implanted with MCF-7 breast tumor cells, 21 (3-10 mglkglday given orally) inhibited estradiol-stimulated growth. Additionally, 21 inhibited proliferation of a human endometrial line, EnCa-101, human ovarian BG-1 %ls, and an MCF-7 variant that is inherently resistant to tamoxifen. SERM (21) displays no uterotrophic effects when Two Phase I trials with 21 given to healthy, given alone to rats or mice. postmenopausal women have been completed and a Phase II evaluation in women with metastatic breast cancer is currently underway (5859).
‘O -OH k 18 R = (CH&-N(CH& fi R = (CH2)loCON(CH3)n-Bu 20 R = (CH2)gSO-(CH&,CF$Fs Napthalene Derivatives - Napthalene, dihydronapthalenes, and tetrahydronapthalene scaffolds have served as versatile cores for molecules demonstrating selective estrogen action. Molecules related to the dihydronapthalene trioxifene (22) were recently described where the carbonyl hinge was replaced by oxygen (23) or CH2 (24) (60-62). SERM (24) displayed little or no uterine stimulation in ovanectomized rats while protecting agarnst bone loss and reducing cholesterol with oral doses as low as 0.1 mglkg per day (61). Lasofoxifene, the tetrahydronapthalene (S), is currently in
22
23x=0 24 X=CH2
25
Chap
SERMs
15
Miller.
Komm
155 -
phase III clinical trials as a salt of its (-) isomer (63). SERM (fi), closely related to the much earlier reported estrogen (25), protected against lumbar vertebral bone loss in ovariectomized rats with an EDSO of cl ug/kg/day p.o. (64-66). In human phase II clinical trials, S prevented bone loss in postmenopausal women with a dose of just 0.25 mg/day (67). The excellent oral potency of 5 is attributed to reduced intestinal glucuronidation of the phenol. Benzopvrans and Arvlcoumarins - Certain benzopyrans and arylcoumarins have been described as compounds having SERM activity. EM-800 (26) is the (S)-(+) bispivaloylated prodrug of its progenitor, EM-652 (27). In human breast cancer cell lines (ZR-75-1 and T-47D), 27, was from 27 to 60 times more potent as an antagonist than its (R) enantiomer (68). The ability of -27 to block both the 178-estradiol-dependent interaction of ERcr and ERj3 with the coactivator (SRC-1) and the in vitro interaction of SRC-1 with the ligand binding domains of both receptors suggested that the compound fully impedes both AF-1 and AF-2 ER function in these systems. This was regarded as Important to the molecule’s ultimate potential in treating breast cancer since the AF-1 domain of ER displays ligand-independent activity and mediates growth factors as well as the ras oncogene and MAPK pathways. 4-OH-tamoxifen, in contrast, only inhibits cellular transcription through the ligand dependent AF2 domain (69). In a comparison with raloxifene. S was tested for its ability to prevent bone loss and lower serum cholesterol in a 37-week rat ovariectomized model. It was concluded from the study that $@ was from 3-10 times more potent than raloxifene on preventing bone loss in the proxrmal tibia and femur and in reducing total cholesterol. Both compounds were similarly efficacious, but 26, unlike raloxifene, produced no discernable stimulation of the rat uterine epithelium (69). Arylcoumarins, exemplified by 28, have been recently described and reported to exhibit SERM activity. In a 3-day immature rat uterine assay, subcutaneous dosing of compound 28 at 1 mglkglday demonstrated no significant increase in uterine wet weight while?n the same assay at the same dose, raloxifene increased the wet uterine weights by 50% which was significantly different from control. Compound (28) also inhibited both estradiol induced proliferation of MCF-7 cells and IL-6 expression in human osteoblasts (70).
S R = COC(CH3)3 27 R=H
28
Constrained SERMs with tetracvclic cores - Recently, several SERMs have been described wherein the core consists of a tetracycle that simultaneously constrains the stilbene phenols into a coplanar arrangement as well as fixes the side chain into an axial conformation. The phytoestrogen coumestrol (29) acted as the precursor to the SERM (30) where the carbonyl of the lactone served as the attachment point for the basic side chains. The importance of the basic side chain for SERM selectivity is demonstrated by the fact that 30 is a potent antagonist of estrogen dependent stimulation of MCF-7 cells (I&O = 0.7 nM). Additionally, 30 did not stimulate uterine hypet-trophy in ovariectomized rats through a range of doses but is able to partially inhibit uterine hypertrophy caused by ethynyl estradiol in immature rats. Coumestrol, in contrast, failed to inhibit estrogen induced MCF-7 cell proliferation and caused uterine hypertrophy in an ovariectomized rat model (71,72). SAR in the series demonstrated that the preferred amine for optimal selectivity was a piperidine ring. Additionally, it was demonstrated that the napthalene fused compound (31) displayed
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excellent in vivo potency on several estrogen sensitive parameters including cholesterol levels, bone mineral density, and inhibition of estrogen stimulated uterine hypertrophy. Since 31 was not remarkably potent on certain in vitro measurements such as estrogen receptor affinity (ERa RBA = 0.22 vs 0.34 for raloxifene) or inhibition of estrogen stimulated MCF-7 cell proliferation (GO = 0.4 nM vs 0.4 nM for raloxifene) it is possible that the superior in vivo potency of 31 in preventing bone loss in the ovariectomized model of rat osteopenia (0.01 mglkglday vs 1.0 mg/kg/day for raloxifene) is evidence for its having increased bioavailability relative to other SERMs such as raloxifene (72). A molecule from a related series of indenobenzothiophenes (32) (which can be viewed as a conformationally locked version of raloxifene) proved itself a potent antagonist in an MCF-7 cell assay (ICSO = 0.1 nM) (73).
22
30x=0
31 X = CH=CH
32
Conclusion - The approval of raloxifene for the treatment and prevention of osteoporosis in postmenopausal women illustrates the value of SERMs for the treatment of at least one of the maladies associated with this patient population. Additional evidence from clinical studies indicate that raloxifene, like tamoxifen, lowers the risk of ER+ breast cancer, but unlike tamoxifen, raloxifene does not appear to increase the risk of endometrial cancer or uterine bleeding. Encouraged by raloxifene’s early success, much recent research dedicated to improving the pharmacological properties of the earlier generation SERMs has resulted in compounds with improved bioavailability and selectivity. To date, however, SERMs have not been shown to positively affect many other symptoms of the menopause and postmenopause period such as hot flushes, vaginal dryness, or urinary incontinence. Since these “quality of life” issues are what prompt many women to seek hormone replacement therapy in the first place, it is likely that the search for newer and better SERMs or SERM combinations will occupy the attentions of the research community for some time to come (74-76). References
1. 2. 3. 4. 5. 6 7.
8. 9.
C. Weinberger. V. Giguere, S. Hollenberg, M.G. Rosenfeld, and R. M. Evans, Cold Spring Harbor symp. Quant. Biol. 51, 759 (1986). G.J.M. Kuiper, E. Enmark, M. Pelto-Huikko, S. Nilsson, and J. Gustafsson, Proc. Natl. Acad. Sci. USA, 93, 5925 (1996). T.M. Wilson, Estrogen Antiestrogen Basic Clin. Aspects, 21 (1997). T.A. Griese and J.A. Dodge, Curr. Pharm. Design, 4. 71 (1998). W. Gradishar and V. Jordan, J. Clin. Oncol., 15, 840 (1997). H.U. Bryant and W.H. Dere, P.S.E.B.M., 217.45 (1998). A. Bouhoute and G. Leclercq, Biochem.Pharmacol., 47, 748 (1994). A.K. Shiau, D. Barstad. P.M. Loria, L. Cheng, P.J. Cushner. D.A. Agard. and G.L. Greene. Cell, 95, 927 (1998). A.M. Brzozowski, A.C.W. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, 0. Engstrom, L. Ohman, G.L. Greene, J.-A. Gustafsson,
and M. Carlquist, Nature, 389. 753 (1997).
Chap
10. 11. 12. 13.
14.
15.
16. 17.
18. 19. 20.
21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
32.
33. 34. 35.
36. 37. 38. 39. 40. 41. 42.
15
SERMs
M~llei-. Komm
15s --
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