`
`71
`
`Selective Estrogen Receptor Modulators (SERMs)
`
`T. A. Grese* and J. A. Dodge
`
`Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA
`
`recognized. The widespread use of “ anlicstrogens ”
`
`and estronc, have
`Abstract: Naturally occurring estrogens, such as 17$-estradiol
`traditionally been thought to play a central role in the development and maintenance ol
`the female reproductive system and secondary sexual characteristics. In recent years, thet
`beneficial effects on the skeleton,
`the cardiovascular system, and the central nervous
`system, as well as the cancer risks associated with long term exposure have also been
`such as tamoxifen for the prevention
`and treatment of breast cancer has revealed that such compounds, while functioning as estrogen antagonists in
`mammary tissue, actually mimic the effects of estrogen in other tissues. The search for more selective agents
`has led to the development of raloxifene, a Selective Estrogen Receptor Modulator, which functions as an
`estrogen antagonist in the breast and uterus and as an estrogen agonist
`in the skeleton and cardiovascular
`system. Recent progress in the development of SERMs is the subject of this review, with an emphasis on
`structure activity relationships and on their effects in non-traditional target tissues.
`
`Introduction
`
`The central role played by endogenous estrogens, such as
`17B-estradiol, 1, and estrone, 2,
`in the development and
`maintenance of the female sex organs, mammary glands, and
`other sexual characteristics has long been recognized [1,2].
`Recently,
`their involvement
`in the growth and function of a
`number of other tissues, such as the skeleton, the cardiovascular
`system, and the central nervous system,
`in botl males and
`females has been recognized [3,4].
`
`The primary site of estrogen biosynthesis in the adult female
`is the ovary. After the menopause,
`the ovarian production of
`estrogens declines dramatically producing a wide range of
`primary and secondary physiological effects [5,6]. The decline
`in levels of circulating estrogens has also been linked to a
`number of pathological conditions
`including osteoporosis
`[7,8], coronary artery disease [9,10], depression [11,12]. and
`Alzheimer's disease [12,13]. Estrogen replacement
`therapy
`(ERT) has proveneffective in reducing the frequency and severity
`of these pathologies, but
`the increased risk of endometrial
`cancer observed with ERT has necessitated the development of
`therapeutic regimens in which the uterine effects of estrogen are
`opposed by progestin treatment (hormone replacement
`therapy
`or HRT)
`[14,15]. Side-effects of progestin treatment, such as
`resumption of menses, central nervous system disturbances, and
`the possibility of attenuated cardiovascular benefits, have
`unfortunately resulted in decreased patient compliance [16.17].
`
`Furthermore, recent studies which confirm the increased risk of
`breast and endometrial cancer associated with long term ERT or
`HRT have Jed to the search for treatment alternatives [18,19].
`
`importance of estrogen in the development and
`The
`maintenance of the female reproductive system has !ed to the
`pharmaceutical development of a variety of steroidal and non-
`steroidal compounds which interact with the estrogen receptor
`(ER) as contraceptives and for the treatment of breast cancer,
`uterine dysfunction, and other reproductive disorders. Several
`reviews of ER-modulators, with a particular emphasis on their
`utility in the treatment of breast cancer, have been recently
`published
`[20,21]. Early synthetic
`estrogens
`such
`as
`diethylstilbesirol (DES), 3, and hexestrol, 4, were once widely
`utilized as estrogen substitutes, but due to concerns similar to
`those encountered with the natural hormones and other side
`
`their utility has diminished. The discovery that
`effects
`compounds such as MER-25, 3, antagonize the action of
`estrogen in breast
`tissue led to intensive pharmaceutical
`research, culminating in the development of tamoxifen, 6,
`which has found great utility in the treatment of breast cancer
`[22]. Early concerns
`that
`the
`long-term use of
`these
`“antiestrogens" would lead to increased risks of osteoporosis
`and cardiovascular disease have been dispelled by
`the
`paradoxical
`finding that some compounds (i.e.
`tamoxifen and
`raloxifene, 7) actually mimic the effects of estrogen in skeletal
`and cardiovascular tissues, although others (i.e.
`JCJ] 182780,
`8b) do not
`[23]. Findings such as
`these have led to a
`
`Table 1.
`
`Classification of Estrogen Receptor Modulators
`
`Classification
`
`Uterine Stimulation
`
`Bone/Cardiovascular
`
`Example
`
`2nd Generation SERMs Pure Antiestrogens
`
`Estrogen Agonists
`
`Partial Agonists (1st Generation SERMs)
`
`agonist
`
`agonist
`
`agonist
`
`antagonist/neutral
`
`| 7B-estradiol, 1
`
`tamoxifen. 6
`
`raloxifene, 7
`
`IC] 182780. 8b
`
`1381-6128/98 $15,004.00
`
`© 1998 Benthain Science Publishers B.V.
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`72 Current Pharmaceutical Design, 1998, Vol. 4, No. 1
`
`Grese and Dodge
`
`Me OH
`
`
`
`
`
`HO
`
`17B-estradiol, 1
`
`estrone, 2
`
`DES, 3
`
`oN Nt
`
`
`
`
`
`MER-25,5
`
`hexestrol, 4
`
`tamoxifen, 6
`
`
`
`raloxifene, 7
`
`Fig. (1). Representative estrogen receptor modulators.
`
`reclassification of estrogen receptor ligands (Table 1) [24] on
`the basis of
`their effects in various traditional and non-
`traditional target tissues. An explosion of research to understand
`the molecular basis for this specificity [25] and a race to develop
`these “designer estrogens” or Selective Estrogen Receptor
`Modulators (SERMs) as pharmaceutical products has also taken
`place [26]. The prototypical 2nd generation SERM, raloxifene,
`7,
`is currently undergoing clinical evaluation for the prevention
`and
`treatment of postmenopausal
`osteoporosis
`[27].
`Nevertheless,
`it should be noted these distinctions may be
`somewhat arbitrary, since there is likely to be a continuum of
`activities from full agonist
`to full antagonist and the relative
`activity of an individual compound may be different for each
`tissue or animal species examined.
`
`In this review, we will discuss the known pharmacology of
`various structural classes of estrogen receptor modulators,
`particularly with respect
`to their effects in non-traditional
`tissues. We will describe the structure-activity relationships of
`these compounds, where such data is available, concentrating
`upon how elements of structure contribute to their tissue-specific
`actions. Finally, we will provide a brief overview of the current
`theories which have been developed to account
`for
`tissue-
`specificity of ER-modulators.
`
`OH
`
`HO
`
`“(CHp)xR
`
`ICI 164384, 8a X = 10, R = CON(Me)n-Bu
`ICI 182780, 8h X=9, R =SO(CH,),CF,CF,
`
`Steroidal ER Modulators and the Estrogen
`Pharmacophore
`
`Natural and synthetic steroidal estrogens have shown great
`utility and significant therapeutic benefits in the replacement of
`endogenous hormones in postmenopausal women [7-10,14-18].
`Although most studies have focused on the efficacy of ERT or
`HRT in the prevention of osteoporosis, cardiovascular disease,
`and disorders of the urogenital
`tract [28], recent reports have
`also described benefits in the central nervous system, including
`improvements
`in cognitive
`function,
`and palliation of
`Alzheimer's disease and postmenopausal depression [11-
`13,29,30].
`
`ERT and/or HRT have been demonstrated to provide a variety
`of cardiovascular benefits, resulting in a 40-50% reduction in the
`relative risk of coronary disease and atherosclerosis [31,32].
`The effects of estrogens on cardiovascular risk factors include
`raising serum levels of high-density lipoprotein (HDL)
`cholesterol and apolipoprotein A-I, and lowering levels of low-
`density lipoprotein (LDL)
`cholesterol,
`lipoprotein (a),
`endothelin-1, and apolipoprotein B [33,34]. Estrogen has also
`been demonstrated to have direct and indirect effects on blood
`
`vessel walls
`
`including increased nitric oxide synthesis,
`
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`
`
`Estrogen Receptor Modulators
`
`Current Pharmaceutical Design, 1998, Vol. 4, No.1 73
`
`inhibition of vascular smooth muscle cell proliferation, and
`increased vasodilation [35]. Currently it
`is
`felt
`that
`the
`combination of these effects on serum lipids and on vascular
`tone are responsible for the overall cardioprotective effects of
`estrogen therapy.
`
`function
`In the prevention of osteoporosis, estrogens
`primarily as antiresorptive agents, leading to decreased turnover
`of both cortical and cancellous bone [36,37]. As with other
`antiresorptive agents,
`this benefit
`is partially offset by a
`subsequent decrease in bone formation, however the overall
`result of ERT or HRT is a substantial increase in bone mineral
`density and a decrease in fracture incidence [38,39]. Although
`ERs have been detected in both osteoblasts and osteoclasts, it is
`currently unclear if the effects of estrogens on bone metabolism
`are direct or indirect [40].
`
`Steroidal Estrogens
`
`Early efforts to identify selective estrogens focused on
`changes in the parent steroid to elicit tissue specific biological
`responses. For example,
`the estrogen metabolites estriol, 9, and
`17o@-estradiol, 10, were found to be time-dependent mixed
`agonist-antagonists of estrogen, which
`stimulate
`early
`uterotrophic responses but have little effect on true uterine
`
`‘hypertrophy and hyperplasia unless administered chronically at
`high doses [41,42]. Estriol causes significantly less uterine
`hyperplasia than 17B-estradiol and inhibits the development of
`breast cancer in rodents [43]. In addition, ]7@-estradiol has been
`shown to exert
`a neuroprotective
`effect
`in
`a human
`neuroblastoma cell
`line
`(SK-N-SH)
`[44]. The estrogen
`metabolite, 2-methoxyestradiol, 11, has been implicated in the
`angiogenesis of vascular tissue and a number of analogs have
`been reported which potently inhibit
`tubulin polymerization
`[45,46]. The estrogen analog 17a-ethynylestradiol (EE), 12,
`has been extensively studied for its bone, uterine, and lipid
`effects due,
`in large part,
`to an enhanced oral activity profile
`relative to 17$-estradiol.
`
`Improvements in tissue selectivity have been observed with
`a family of D-ring halogenated estrones (such as 13) which have
`demonstrated potent
`lipid lowering yet diminished uterine
`hypertrophy relative to estrone [47]. Other attempts to attenuate
`the estrogenic activity of steroids via opening of the steroid
`nucleus, such as 9,1 1-seco steroids, 14, have met with limited
`success [48,49].
`Recently, the components of Premarin® (the most prescribed
`form of ERT) have been evaluated for their lipid lowering effects,
`These conjugated equine estrogens contain sulfate esters of two
`distinct estrogen structural classes; (1) ring B saturated steroids
`
`Me 9H
`
`
`
`Me GH
`
`14
`
`HO
`17@-estradiol, 10
` HO
`
` HO
`
`
`
`estriol, 9
`
`equilin, 15
`
`|7a-dihydroequilinen, 18
`Fig. (2). Steroidal estrogen receptor modulators.
`
`tibolone, 19
`
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`74 Current Pharmaceutical Design, 1998, Vol. 4, No. 1
`
`Grese and Dodge
`
`including traditional sex steroid hormones such as estrone, 17B-
`estradiol, and 17@-estradiol, and (2) ring B unsaturated estrogens
`such as equilin (Eq), 25, equilenin (Eqn), 16, 17f-
`dihydroequilenin (178-DHEqn), 17, 17B-dihydroequilin (17B-
`DHEgq), 17a-dihydroequilenin (17a-DHEgn), 18, and 17a-
`dihydroequilin (17a-DHEq).
`[In 1991, Bhavnani and co-workers
`examined these individual steroids,
`in their unconjugated form,
`to determine their relative binding affinities for the estrogen
`receptor and their 7a vivo effects on uterine hypertrophy in the
`immature rat
`[50].
`In thts
`study,
`the majority of equine
`components mimicked estrogen in their ability to increase
`uterine weight relative to vehicle treated animals. The notable
`exception to this uterotrophic response was !7a-DHEqn which
`did not cause a significant effect at the dose examined (2 mg/kg).
`More recently,
`the sulfate ester conjugate of 17a-DHEgn has
`been shown to
`lower
`serum cholesterol
`and increase
`
`hippocampal dendritic spine density in rats, and improve arterial
`vasomotor function in macaques [51].
`
`Work from our own laboratories on the relative effects of
`conjugated equine estrogens on bone versus uterus has shown
`that 17a-DHEanis a partial estrogen agonist [52]. In this study,
`uterotrophic effects were observed after 4 days of oral dosing for
`Eq, 15, Eqn, 16, 17B-DHEqn, 17, and I17a-DHEgqn, 18.
`Increases in uterine wet weight relative to ovariectomized (OVX)
`controls ranged from 263% for Eq to 100% for
`|7a-DHEqn.
`Serum cholesterol levels were lowered with similar potencies for
`all equine estrogens [52]. Bone mineral density measurements
`indicated that 17@-DHEgn effectively prevented osteopenia in a
`dose-dependent
`fashion after 5-weeks of oral administration
`(59.9% of ovariectomy-induced bone loss was prevented at
`|
`mg/kg, 119 % at 10 mg/kg).
`In addition, an average uterine
`weight gain of 100.4% relative to OVX controls was observed at
`the 1 mg/kg dose [52], These data demonstrate that 17a-DHEgn
`is a full estrogen agonist on bone, but a partial agonist on the
`uterus in the OVX rat and further highlight
`the structural
`significance of both the stereochemistry at the 17-position and
`unsaturation in the B-ring,
`
`is a unique steroid that possesses
`Tibolone (OD-14), 19,
`estrogenic, progestenic and androgenic properties. At doses of
`less
`than 2.5 mg/day, OD-14 appears
`to reduce skeletal
`remodeling without producing concomitant
`endometrial
`stimulation [53]. However, because of its estrogenic activity,
`endometrial hypertrophy over
`the
`long term remains
`a
`possibility.
`
`Pure Antiestrogens
`
`While estrogen agonists, partial agonists, and SERMs can
`mimic the pharmacology of
`the natural hormone, pure
`antiestrogens (e. g., 8a,b) represent a class of therapeutic
`agents which are devoid of estrogen agonism regardless of the
`target
`tissue. Initially introduced by Wakeling in 1988,
`these
`compounds demonstrate an absence of estrogenic activity on the
`rat uterus, vagina, and hypothalamic-pituitary axis as well as
`effectively antagonizing the stimulatory effects of estrogen
`[54]. In non-reproductive tract tissue, pure antiestrogens behave
`like estrogen antagonists as well, For example,
`(CI 164,384,
`8a, and IC] 182,790, 8b, exhibited no capacity for lowering
`serum cholesterol or sparing bone loss in the OVX rat model
`[55]. Recent data suggests that
`ICI 182,780 has significantly
`complex effects on rat skeletal tissue [56]. For example,
`loss of
`
`cancellous bone is observed in intact rats after administration of
`
`the compound whereas no bone loss is observed in OVX rats,
`
`Estradiol Pharmacophore
`
`Recently, Katzenellenbogen, et. al. have combined literature
`ER binding affinity data for a large numberof steroidal estrogen
`analogs with molecular modeling and receptor sequence analysis
`to develop a detailed picture of the estradiol pharmacophore
`{S7]. Their study recognizes the important contributions of the
`two hydroxy groups ofestradiol to receptor binding, with the 3-
`hydroxy acting primarily as
`a hydrogen bond donor and
`contributing approximately 1.9 kcal/mol
`to the binding free
`energy, while the 17B-hydroxy functions primarily as
`a
`hydrogen bond acceptor and contributes approximately 0.6
`keal/mo!
`[S8]. The preferred distance between the hydroxy
`functionalities appears to be somewhat flexible, possibly due to
`the inclusion of water molecules in the binding cavity [57].
`Large, preformed, hydrophobic pockets apparently exist within
`the ligand binding domain which are able to accommodate large
`substituents at the L1B- and 7a-positions [59]. Smaller pockets
`appear to exist at the 16B- and 17B-positions, while the 16c-
`position and the aromatic A-ring are relatively intolerant of
`substitution [57]. These properties of the ER ligand binding
`cavity, which were primarily determined empirically, appear to
`be supported by the recently reported X-ray crystal structure of
`the ER ligand binding domain complexed with estradiol [60].
`
`Triphenylethylenes
`
`The most thoroughly investigated class of non-steroidal ER
`modulators
`are
`the
`triphenylethylenes
`(JTPE’s),
`such as
`tamoxifen, 6. and clomiphene, 20, A commonstructural motif
`which is
`incorporated in many classes of molecules with
`estrogen antagonist activity involves the attachment of
`a
`sidechain containing a hydrogen bond acceptor to an ER binding
`core unit. This theme is illustrated for the triphenylethylenes via
`the progression from DES to MER-25 and tamoxifen. Originally
`investigated for contraceptive activity,
`the strong estrogen
`antagonist activity observed with many of these compounds in
`mammary tissue, has led to their development for treatment of
`breast cancer [20d]. The success of tamoxifen in this arena, has
`led to the investigation and development of a wide variety of
`analogs. To date, SAR work in this series has been confined
`primarily to the investigation of antagonist effects in mammary
`and uterine tissue [20]. Recently, reports of estrogen agonist
`effects of some of
`these compounds in the skeletal and
`cardiovascular system have begun to appear [26].
`In general,
`although they partially antagonize the effects of estrogen on the
`uterus, the members of this structural class tend to induce some
`level of uterine stimulation in the absence of endogenous
`estrogen, therefore they have been classified as partial agonists
`or first generation SERMs [61].
`
`to be utilized clinically was
`the first TPE’s
`One of
`clomiphene, 20. Although it was originally developed as a
`contraceptive, clomiphene has been mainly utilized for
`the
`induction of ovulation in anovulatory women [62]. Its effects on
`uterine tissue are complex, and are at least partly complicated by
`its availability as
`a mixture of double-bond isomers
`(zuclomiphene and enclomiphene), but
`it appears to cause
`significant stimulation of uterine epithelia in the rat [63,64].
`
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`Estrogen Receptor Modulators
`
`Current Pharmaceutical Design, 1998, Vol. 4, No.1 75
`
`in
`Clomiphene has been reported to reduce serum cholesterol
`rats, similar to estrogen, however this may not be an ER-
`mediated effect
`[63]. Clomiphene has also been reported to
`inhibit bone resorption im vitro [65] and to protect against bone
`loss in both OVX rats [66] and in postmenopausal women [67].
`Interestingly,
`the individual
`isomers of clomiphene have been
`reported to have similar effects on bone metabolism, while the
`uterine effects are primarily induced by zuclomiphene [68].
`Recently a clomiphene analog, MDL-103,323, 21, with
`antiproliferative activity in breast cancer assays has been
`reported to protect against bone loss in OVX rats with minimal
`uterine stimulation [69].
`
`Consistent with the importance of the hydroxyl moieties of
`estradiol
`for receptor binding, tamoxifen, 6, binds only weakly
`to the ER, however evidence suggests
`that
`the primary
`biologically active species may be its 4-hydroxy metabolite
`[70,71]. Estrogen antagonist effects in mammary tussue have
`been demonstrated in a variety of cell
`lines and animal models
`[20d]. Tissue specific estrogen agonist effects have been
`demonstrated in the OVX rat model of estrogen deficiency, where
`
`tamoxifen reduced serum cholesterol by 50% at doses of 0.1-10
`mg/kg and protected against bone loss with an EDsg of 0.1
`mg/kg [61,72,73].
`dn vitro effects on bone resorption and
`osteoclast viability have also been demonstrated [64,74].
`In a
`primate model,
`tamoxifen was shown to significantly inhibit
`the progression of coronary artery atherosclerosis [75].
`
`Due to the widespread use of lamoxifen in the treatment of
`breast cancer, a large body of clinical evidence with respect to
`its effects in other tissues has also accumulated [22].
`In the
`cardiovascular
`system,
`tamoxifen
`has been shown to
`significantly reduce risk factors of disease including LDL
`cholesterol,
`lipoprotein (a), and fibrinogen in postmenopausal
`women with little or no effect on triglycerides or HDL
`cholesterol
`[76,77]. A corresponding decrease in mortality due
`to cardiovascular disease has also been reported [78]. Clinical
`effects on the skeleton have included the preservation of bone
`mineral density at the lumbar spine, femoral neck, and forearm
`in postmenopausal women |77,79] as well as an estrogen-like
`reduction in serum markers of bone turnover
`[79b-d,80].
`Interestingly,
`in premenopausal women decreases in bonc
`
`a NMes
`
`NEt,
`Oo” és
`
`Py NEty
`
`O
`
`tamoxifen, 6
`
`clomifene, 20
`
`MDL-103.323, 21
`
`NMe,
`07 2
`
`gtseas
`
`NMe,
`
`gfer
`
`ff
`
`!
`
`idoxifene, 23
`
`a
`
`toremifene, 24
`
`oe NMe,'T
`
`—
`
`OH
`droloxifene, 22
`
`(HO},P(Q)0
`
`Fig. (3). Triphenylethylene estrogen receptor modulators.
`
`TAT=59. 25
`
`TMI, 26
`
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`76 Current Pharmaceutical Design, 1998, Vol. 4, No. |
`
`Grese and Dodge
`
`mineral] density have been observed with tamoxifen treatment
`[79a,e].
`
`Notwithstanding the positive effects described above for
`tamoxifen, there continues to be considerable concern about the
`increased risk of endometrial cancer which has been associated
`
`with tamoxifen use [81,82]. Estimations of the magnitude of
`this risk vary, however the average value of about a five-fold
`increasé is similar to that observed with ERT [83]. Significant
`stimulation of uterine endometrial tissue is also observed in the
`OVX rat model
`[61,84] and DNA adduct formation has been
`observed in both rats and humans [85,86]. Tamoxifen has also
`been shown'to induce liver cancer in rats [87].
`
`The growing concern over the potential cancer causing or
`cancer promoting effects of tamoxifen has recently led to the
`development of a number of tamoxifen analogs. The hypothesis
`that metabolic hydroxylation of tamoxifen at
`the 4-position is
`in some way responsible for
`these effects has
`led to the
`investigation of agents in which this metabolic pathway is
`blocked [20d]. Examples of this strategy include droloxifene,
`22, and idoxifene, 23, which have been reported to show
`decreased
`levels
`of DNA adduct
`formation
`and
`
`In toremifene, 24, chlorination
`hepatocarcinogenicity [88,89].
`of the aliphatic substituent of tamoxifen also appears to reduce
`DNA-adduet formation [90]. Alternative strategies for modifying
`the metabolic fate and/or tissue distribution observed with
`
`tamoxifen are represented by TAT-59, 25, and tamoxifen
`methiodide or TMI, 26. To date these compounds have been
`most extensively evaluated for the treatment of breast cancer
`{90,91}, however recently reports on their effects in non-
`traditional target tissues have begun to appear.
`
`the 3-position of the
`In droloxifene, 22, hydroxylation al
`TPE core leads to an altered metabolic profile and decreased
`estrogen agonist activity relative to tamoxifen [92,93]. In OVX
`rats, droloxifene has been reported to reduce serum cholesterol
`40-46% and to protect against
`loss of bone mineral density,
`similar to tamoxifen but with reduced uterine stimulation [94]. In
`a head-to-head comparison with tamoxifen, droloxifene was
`found to be at least 10-fold less potent in terms of its effects on
`serum cholesterol and bone density, even thoughit has a tenfold
`higher binding affinity to the ER [95]. Estrogenic cffects in the
`skeleton have also been observed by histomorphometry at both
`cancellous and cortical bone sites [96]. Recently, droloxifene
`has been observed to induce apoptosis of both MCP-7 cells and
`osteoclasts in culture, while estrogen has similar effects on
`osteoclasts but
`is mitogenic to MCF-7 cells [97]. This tissue-
`specific difference has led to the hypothesis that a common
`mechanism may account
`for both the estrogen agonist and
`antagonist activities of droloxifene. Although droloxifene has
`been evaluated clinically for efficacy in breast cancer treatment
`[91a], its effects on other estrogen target tissues in humans have
`not yet been reported,
`
`ldoxifene, 23, was designed to reduce both metaboltc
`oxidation and N-demethylation, via iodination of the 4’-
`position and replacement of
`the dimethylamino group of
`tamoxifen with a pyrrolidine ring, respectively [98]. As with
`droloxifene,
`idoxifene has been evaluated clinically for breast
`cancer treatment [91b], and a preliminary report describing its
`effects on serum cholestero! and bone density in the OVX rat has
`also appeared [99].
`It has also been reported to be less
`uterotréphic than tamoxifen [100].
`
`the
`recently been approved for
`Toremifene, 24, has
`treatment of breast cancer and has demonstrated clinical effects
`on serum cholesterol and bone mineral density which are similar
`to those of tamoxifen in postmenopausal breast cancer patients
`[101,102]. Although it has been reported to be less uterotrophic
`in the rat
`[103],
`its estrogenic effects on the uterus
`in
`postmenopausal women have been reported to be comparable to
`those of tamoxifen [104],
`
`Tamoxifen methiodide, 26, was designed to inhibit crossing
`of the blood-brain barrier,
`in order to avoid possible estrogen
`antagonist effects
`in the central nervous
`system [91d].
`Interestingly,
`this compound has recently been reported to
`selectively stimulate creatinine kinase activity in bone cells but
`not uterine cell
`lines, while tamoxifen and estrogen stimulate
`this activity in both [105]. Similar effects have been described
`in vivo, although correlation of these effects with bone density
`and uterine stimulation have not yet been reported [106].
`
`Two new TPE’s which contain carboxylic acid functionality
`in place of the amine side chain moiety have also been reported.
`GW5638, 27, has been deseribed as a bone-selective estrogen
`agonist, and has demonstrated decreased uterine stimulation,
`relative to tamoxifen,
`in OVX rats [107,108].
`Interestingly,
`amide analogs of 27 showed an increased tendency toward
`uterine stimulation both i vivo and imvitro [107]. In OVX rats,
`27 was observed to maintain bone mineral density at both the
`lumbar spine and the proximal tibia with an efficacy similar to
`that of !7B-estradiol or
`tamoxifen at doses of 1-10 mag/kg
`[107,108]. It has also been shown to reduce serum cholesterol in
`OVX rats with a maximal efficacy of 20-30% [108,109]. The
`magnitude of this effect, although similar to that observed with
`17B-estradiol, appears to be somewhat muted in comparison to
`the more bioavailable 17q@-ethynyl estradiol and other TPE’s,
`implying perhaps that multiple mechanisms may be involved in
`the regulation of serum ltpid concentrations by these compounds
`{110}. Hydroxytamoxifen acid, 28, a tamoxifen metabolite, has
`also been reported to have bone-selective effects in the OVX rat
`Fld],
`
`CO,H
`
`oe
`
`07 ~co,H
`
`a
`
`_
`
`ea Ss
`
`SO
`
`HO
`
`GW5638, 27
`
`28
`
`Fig. (4). Acidic triphenylethylenes.
`
`Several groups have recently reported the application of
`parallel synthesis techniques for the preparation of TPE libraries
`[112],
`It
`ts expected that
`the ready availability of more
`structurally diverse members of this class,
`together with the
`development of molecular biological assays predictive of sn
`vive tissue selectivity, will
`lead to greater understanding of the
`SAR of these compounds in multiple tissues.
`
`AstraZeneca Exhibit 2023 p. 6
`
`
`
`Estrogen Receptor Modulators
`
`Benzothiophenes
`
`In order to avoid the problems associated with double bond
`isomerization of the TPE’s, a variety of cyclic frameworks have
`been investigated for their ER modulating properties. Out of
`these
`structure-activity
`studies,
`raloxifene,
`7,
`a
`benzothiophene-containing compound with a unique profile of
`biological activily emerged [26b,c].
`.
`.
`Raloxifene has been shown to bind the estrogen receptor
`with high affinity and to function as
`a polent estrogen
`antagonist
`in mammary tumor cells and in rat models of
`mammary cancer [113,114].
`In contrast,
`in the cardiovascular
`
`Current Pharmaceutical Design, 1998, Vol. 4, No. 1 77
`
`system, raloxifene functions primarily as an estrogen agonist.
`In cell culture, raloxifene has demonstrated estrogen-like effects
`on vascular smooth muscle cells and on the inhibition of LDL
`oxidation [115,116]. In the OVX rat model, raloxifene has been
`shown to reduce serum cholesterol by 50-75% after 1-5 weeks of
`daily dosing [117,118]. Most
`importantly,
`in postmenopausal
`women treated daily withraloxifene, significant reductions in
`total serum cholesterol and LDL cholesterol have been observed
`after both eight weeks
`and two
`years
`‘
`,
`8
`exs a
`pera eee
`Similarly,
`the effects of raloxifene on the skeleton seem to
`parallel
`those observed with estrogen,
`Jn vitre studies have
`shown similar effects of
`raloxifene and 178-estradiol on
`
`Table 2.
`
`ER Binding and Inhibition of MCF-7 Celt Proliferation by 2-Aryl Raloxifene Analogs [127a]
`
` MCF-7Inhib. [Cgg (nM)°
`
`
`
`
`
`
`
`
`
`
`:
`
`[
`
`;
`
`,
`
`,
`
`
`estradiol
`
`
`
`4-OHtam?
`0.36
`0.5
`
`4-OH
`6-OH
`raloxifene, 7
`0.34
`02
`
`none
`none
`i
`Ta
`<0,002
`300
`4-OMe
`6-OMe-
`Tb
`<0.002
`300
`4-OMe
`6-OH ~
`Te
`0.073
`1000
`4-OH
` 6.OMe
`74
`0.008
`250
`4-OH
`“none
`Te
`0.003
`35
`none
`6-OH
`7
`0.062
`25
`4-Cl
`~~ 6-OH |
`7p.
`"0.046
`L
`4-OH
`6-Cl
`7h
`0.006
`1000
`4-Me
`6-OH
`Ti
`0.07
`50
`40H
`p 6Me C—O
`7
`nad
`300
`
`
`4F
`i.
`“6-OH
`7k
`0.19 >
`4-OH
`7 t ~~ 7-0H a
`1 :
`- 0.02
`-
`“300 oo
`™m OH 4-OH
`0.002
`190
`
`
`In
`5-OH
`4-OH
`O10
`100°
`“To
`6-OH
`2-OH
`0.057 ag
`'
`
`
`Dp
`6-OH
`3-OH
`0.16
`12
`4
`5-F,6-OH
`4-OH
`ae 0.098
`3
`
`
`Tr
`5-Me,6-OH
`-4-OH
`007
`npf
`
`
`
`
`_
`Is
`5,7-di(Me),6-OH
`4-OH
`0.005
`500
`1
`6-OH
`"2. Me,4-OH
`0.41 — :
`
`
`Tu
`6-OH
`3-Me,4-OH
`0.13
`i : 1
`a
`Wy
`6-OH —
`3-Cl,4-OH
`0.12
`OO
`
`
`— 63
`|
`~—
`TW
`6-OH
`3-F,4-OH
`0.20
`
`
`
` 6-OH 7x
`3,5-di(Me),4-OH
`“RBA = relative binding affinity by competition with 3-17 -estradiol.
`Paverage of at least 2 determinations, Values are + 10%. “Dose required 10 give 50% inhibition
`of a maximally effective ero | ty dose of 17B-estradiol. Average of at least
`3 determinations. Values are + 10%.
`“NA = not active at
`the doses tested.
`°4-
`Hydroxytamoxifen, the primary biologically active metabolite of tamoxifen. ND = not determined
`
`:
`
`a
`
`|
`
`!
`
`
`
`
`
`
`
`
`
`
`
`:
`
`.
`
`.
`
`AstraZeneca Exhibit 2023 p. 7
`
`
`
`78 Current Pharmaceutical Design, 1998, Vol. 4, No. 1
`
`Gresé und Dodge
`
`osteoclastogenesis and On creatinine kinase activity in human
`osteoblast cells
`[119,120].
`In rats,
`several
`studies have
`demonstrated a protective effect against ovariectomy-induced
`osteopenia at doses as
`low as 0.1 mg/kg [117,121,122].
`Positive effects on bone mineral density at both cortical and
`cancellous bone sites have been reported, as have positive
`effects on bone strength [121,123].
`Interestingly, although
`raloxifene suppresses bone resorption in the rat with efficacy
`which is approximately equal
`to that of estrogen, bone
`formation appears to be suppressed to a Jesser degree, resulting
`in a nel gain in bone mass with raloxifene [121,123b]. These
`positive results in animal studies, have now been confirmed with
`clinical studies in postmenopausal women [27]. Significant
`positive effects on histomorphometric parameters, bone
`markers, and on bone mineral density at both the lumbar spine
`and hip were observed after two years of raloxifene treatment
`(27,124].
`
`In terms of its pharmacology, raloxifene is distinguished
`from the TPE’s primarily on the basis ofits effects on the uterus,
`where a qualitative difference has been observed [61,125].
`In a
`direct comparison with tamoxifen, droloxifene, and idoxifene,
`raloxifene was a significantly more effective antagonist of
`estrogen action in the immature female rat uterus [61a]. In this
`assay the TPE’s functioned as partial agonists,
`inhibiting the
`effects of estrogen on uterine weight gain only to the level of
`their own intrinsic agonist activity, while raloxifene functioned
`essentially as a complete antagonist. Similarly,
`in OVX rats, the
`TPE’s have been found to induce a larger maximal stimulation of
`uterine weight and to induce uterine eosinophilia while
`raloxifene did not
`[61]. Although raloxifene has also been
`reported to stimulate a modest
`increase in uterine wet weight,
`this increase is not dose related and is not coincident with
`
`increases in other measures of uterine hypertrophy, and has
`therefore been attributed to water
`retention [117],
`In
`postmenopausal women, raloxifene has been reported to show
`no stimulatory effects on the uterus, even after 2 years of
`treatment [27,126],
`
`SAR studies in the raloxifene series have centered on
`
`the amine-
`modifications of the 2-arylbenzothiophene [127],
`containing side chain [113,128], and the carbonyl hinge [129],
`The 2-arylbenzothiophene unit appears to be the primary sile of
`ER binding, mimicking the interactions of 17B-estradiol with
`the receptor. On the basis of binding and an vitro activation data
`(Table 2) the 6-hydroxy of raloxifene is believed to imitate the
`3-hydroxy of estradiol, while
`the 4’-hydroxy roughly
`approximates the 17B-hydroxy [127a]. The recently published
`crystal structures of raloxifene and 17B-estradio! bound to the ER
`confirm this interpretation [60]. Consistent with the estradiol
`pharmacophore (vide supra), the 6-hydroxy