`
`7|
`
`Selective Estrogen Receptor Modulators (SERMS)
`
`T. A. Grese* and J. A. Dodge
`
`Lilly Research Laboratories, Eli Lilly and Company, lrrdianapolis, Indiana 46285, USA
`
`
`
`l7[5—estradio| and estrone, have
`Abstract: Naturally occurring estrogens. such as
`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. their
`beneficial effects on the skeleton,
`the eardiovasctilar system, and the central nervous
`system, as well as the cancer risks associated with long term exposure have also been
`recognized. The widespread use of “ anticstrogcns " such as tamoxifen for the prevention
`and treatment of breast cancer has revealed that such compounds, while functioning as estrogen antagoiiists 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 Estrogeii 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 recognizctl
`[1,2].
`Recently,
`their involvement
`in the growth and ftinction of a
`number of other tissues, such as the skeleton, the cardiovascular
`system, and the central nervous system,
`in both males and
`females has been recognized [3,4].
`
`The primary site of estrogen biosyntliesis in the adult female
`is the ovary. After the menopause,
`the ovarian production of
`estrogens declines dramatically producing a wide range oi‘
`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 |l2.13]. Estrogen replacement
`therapy
`(ERT] has proven effective 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 progestiti treatment (hormone replacement
`therapy
`or HRT) [14,15]. Side—cfl'ects 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].
`
`Table 1.
`
`Classification of Estrogen Receptor Modulators
`
`Furtherniorc, recent studies which confirm the increased risk of
`breast and eridometrial cancer associated with long term ERT or
`HRT have led to the search for treatment alternatives [18,19].
`
`importance of estrogen in the development and
`The
`maintenance of the female reproductive system has led to the
`pharmaceutical development of a variety of steroidal and non-
`stcroidal compounds which interact with the estrogen receptor
`(ER) as coiitraceptives and for the treatment of breast cancer,
`uterine dysfunction, and other reproductive disorders. Several
`reviews of ER—modu|ators, with a particular emphasis on their
`utility in the treatment of breast cancer, have been recently
`published
`l20,21], Early synthetic
`cstrogens
`such
`as
`dietliylstilbcstrol (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
`\/lER—2S., 5. 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 tie.
`ICI 182780,
`8b) do not
`['23]. Findings
`such as
`these have led to a
`
`Classification
`
`Uterine Stimulation
`
`Bone/Cardiovascular
`
`Example
`
`Pure Antiestrogeris
`
`Estrogen Agonists
`
`Partial Agonists (1st Generation SERMS)
`
`Znd Gerieratioii SERMS
`
`agonist
`
`agonist
`
`agonisi
`
`l7]3—estradiol, 1
`
`tainoxiferi, 6
`
`raloxifcric, 7
`
`aiiiagonisr/neutral
`
`1C1 182780. 8b
`
`1381-6128/98 51515 fl()+.00
`
`© 1998 Bentham Science Publishers B.V.
`
`AstraZeneca Ex. 2023 p. 1
`Mylan Pharms. Inc. V. AstraZeneca AB IPR2016-01324
`
`
`
`Grese and Dodge
`
`OH
`
`0 \ 0
`
`HO
`
`DES, 3
`
`
`
`72 Current Phannnceutical Design, 1998, Vol. 4, No. I
`
`
`
`Me OH
`
`
`
`l7[i-estradiol, 1
`
`N Et
`
`0/\/
`
`1
`
`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.
`
`on
`
`HO
`
`"'(cH2)XR
`
`ici 154324, 8a x = to. R = CON(Me)n—Bu
`lCl l82780, 8h x = 9, R = SO(Cl-{2)3CF2CF3
`
`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—lO,l4-18].
`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 [1 l-
`13,2930].
`
`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~l, and lowering levels of low»
`density lipoprotein (LDL)
`cholesterol,
`lipoprotein (a),
`endothelin-l, 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 Phanmzceutical Design, I998, Vol. 4. No. I 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
`ln 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
`l70t-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 l7B—estradiol and inhibits the development of
`breast cancer in rodents [43]. In addition, l7ot-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 l7ot-ethynylestradiol
`(EE2),12,
`has been extensively studied for its bone, uterine, and lipid
`effects due,
`in large part,
`to an enhanced oral activity profile
`relative to 17B-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,ll-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
`
`
` McO
`
`14
`
` HO
`
`l7B—dihydroquiIinen, 17
`
`
`
`l7ot—estradiol, 10
`
`
`
`
`
`HO
`
`equilin, 15
`
`equilinen, 16
`
`
`
`I7ot-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 l7ot-estradiol, and (2) ring B unsaturated estrogens
`such as equilin (Eq), 15, equilenin (Eqn), 16,
`l7B-
`dihydroequilenin (l7B—DHEqn), 17,
`l7]3—dihydroequilin (l7B-
`DHEq),
`l7ot—dihydroequilenin (l7ot—DHEqn), 18, and l7ot—
`dihydroequilin (l70t—DHEq).
`In l99l, Bhavnani and co—workers
`examined these individual steroids,
`in their unconjugated form.
`to determine their relative binding affinities for the estrogen
`receptor and their in Villa effects on uterine hypertrophy in the
`immature rat
`[50].
`in this 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 l70t~DHEqn which
`did not cause a significant effect at the dose examined (2 mg/kg).
`More recently,
`the sulfate ester conjugate of l7ot—DHEqn 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
`l70t-DHEqn is a partial estrogen agonist [52]. In this study,
`uterotrophic effects were observed after 4 days of oral dosing for
`Eq, 15, Eqn. 16,
`l7B—DHEqn, 17, and l7ot—DHEqn, 18.
`Increases in uterine wet weight relative to ovariectomized (OVX)
`controls ranged from 263% for Eq to 100% for
`l7ot-DHEqn.
`Serum cholesterol levels were lowered with similar potencies for
`all equine estrogens [52]. Bone mineral density measurements
`indicated that
`l7ot~DHEqn effectively prevented osteopenia in a
`dose—dependent
`fashion after 5»weeks of oral administration
`(59.9% of ovariectomy~induced bone loss was prevented at
`l
`mg/kg,
`ll9 % at
`IO mg/kg).
`in addition, an average uterine
`weight gain of l0O.4% relative to OVX controls was observed at
`the 1 mg/kg dose [52]. These data demonstrate that
`l7ot~DHEqn
`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 l7-position and
`unsaturation in the B-ring.
`
`is a unique steroid that possesses
`Tibolone (OD—l4), 19,
`estrogenic, progestenic and androgenic properties. At doses of
`less
`than 2.5 mg/day, OD—l4 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,h) represent
`:1 class of therapeutic
`agents which are devoid of estrogen agonism regardless of the
`target
`tissue.
`lnitially 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,
`lCl 164,384.
`821, and lCl 182,790, Sb, exhibited no capacity for lowering
`serum cholesterol or sparing bone loss in the OVX rat model
`[55], Recent data suggests that
`lCl
`l82,780 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 number of steroidal estrogen
`analogs with molecular modeling and receptor sequence analysis
`to develop a detailed picture of the estradiol pharmacophore
`[57]. Their study recognizes the important contributions of the
`two hydroxy groups of estradiol to receptor binding, with the 3~
`hydroxy acting primarily as
`a hydrogen bond donor and
`contributing approximately 19 kcal/mol
`to the binding free
`energy, while the
`l7B~hydroxy functions primarily as
`a
`hydrogen bond acceptor and contributes approximately 0.6
`kcal/mol
`[58]. 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 llB- and 70t—p0Sill0nS [59]. Smaller pockets
`appear to exist at the I6[3- and l7fl—positions, while the 16(1-
`position and the aroiinatic 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
`(TPE’s),
`such as
`tamoxifen, 6. and clomiphene, 20. A common structural 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
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`Current PIzanm1ceuticaIDesign, I998. Vol. 4. N17. I 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 in 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—lf)3,323, 2], 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
`[70,7l]. Estrogen antagonist effects in mammary tissue 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 (11-10
`mg/kg and protected against bone loss with an ED50 of 0.1
`mg/kg [6l,72,73].
`In 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 tamoxifen in the treatment of
`breast cancer, a large body of clinical evidence with respect to
`its effects in other tissues has also accumulated [22].
`ln 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 estrogenalike
`reduction in serum markers of bone turnover
`[79b—d_.80].
`Interestingly,
`in premenopausal women decreases in bone
`
`O/\/ NMC2
`
`O/\/ NE‘:
`
`O/\//\/ NF-‘2
`
`\ O
`
`4
`
`\ 0
`
`Cl
`
`tainoxifen, 6
`
`clomifene, 20
`
`MD1,|()3,'32_‘<, 21
`
`
`
`idoxifene. 23
`
`
`
`NMe
`
`O/\/,
`
`2
`
`\ O
`
`H
`droloxifene, 22
`
`(HO)3P(_())O
`
`Fig. (3). Triphenylcthylene estrogen receptor modulators.
`
`TAT/59. 25
`
`TM]. 26
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`76 Current Phannaceutical Design, 1998, Vol. 4, No. I
`
`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
`increase 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
`hepatocarcinogenicity [88,89].
`In toremifene, 24, chlorination
`of the aliphatic substituent of tamoxifen also appears to reduce
`DNA-adduct 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
`[909]], however recently reports on their effects in non-
`traditional target tissues have begun to appear.
`
`the 3-position of the
`liydroxylation at
`ln droloxifeiie. 22,
`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]. ln
`:1 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 though it has a tenfold
`higher binding affinity to the ER [95]. Estrogenic effects 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 MCF-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
`[9la], its effects on other estrogen target tissues in humans have
`not yet been reported.
`
`ldoxifene, 23, was designed to reduce both metabolic
`oxidation and N-demethylation, via iodinatioti of the 4‘-
`position and replacement of
`the dimethylamino group of
`tamoxifen with a pyrrolidine ring, respectively [98]. As with
`(lroloxifene.
`idoxifene has been evaluated clinically for breast
`cancer treatment [‘)lb], and a preliminary report describing its
`effects on serum cholesterol and bone density in the OVX rat has
`also appeared [99].
`It has also been reported to be less
`uterotrophic than tamoxifen [I00].
`
`Grese and Dodge
`
`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
`[l0l,lO2]. 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 [lO4].
`
`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 |9ld].
`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 [l05]. Similar effects have been described
`in vivo, although correlation of these effects with bone density
`and uterine stimulation have not yet been reported [I06].
`
`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 described as a bone—selective estrogen
`agonist. and has demonstrated decreased uterine stimulation,
`relative to tamoxifen,
`in OVX rats [l07,lO8].
`Interestingly,
`amide analogs of 27 showed an increased tendency toward
`uterine stimulation both in vivo and in viii-o [107].
`ln 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 17B-estradiol or tamoxifen at doses of 1-10 mg/kg
`[lO7,lO8]. It has also been shown to reduce serum cholesterol in
`OVX rats with a maximal efficacy of 20-30% [l08,lO9]. The
`magnitude of this effect, although similar to that observed with
`17B-estradiol, appears to be somewhat muted in comparison to
`the more bioavailable I70:-ethynyl estradiol and other TPE‘s,
`implying perhaps that multiple mechanisms may be involved in
`the regulation of serum lipid concentrations by these compounds
`[1 10]. Hydroxytamoxifen acid. 28, a tamoxifen metabolite, has
`also been reported to have bone-selective effects in the OVX rat
`[lll].
`
`Com
`
`
`
`o/\ cola
`
`/
`
`\ \
`
`/
`\
`
`HO
`
`cwsms, 27
`
`23
`
`Fig. (4). Acidic triphcnylcthylencs.
`
`Several groups have recently reported the application of
`parallel synthesis techniques for the preparation of TPE libraries
`[112].
`it
`is expected that
`the ready availability of more
`structurally diverse members of this class,
`together with the
`development of molecular biological assays predictive of in
`viva tissue selectivity, will
`lead to greater understanding of the
`SAR of these compounds in multiple tissues.
`
`Astrazeneca Ex. 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 activity emerged [26b,c].
`
`Current Phannaceutical Design, 1998, Vol. 4, No. I 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 [1 l5,l 16]. in the OVX rat model, raloxifene has been
`shown to reduce serum cholesterol by 50-75% after 1-5 weeks of
`daily dosing [ll7,ll8]. Most importantly,
`in pofstmenopausal
`women treated daily with‘ raloxifene, significant reductions in
`total serum cholesterol and LDL cholesterol have been observed
`
`Raloxifene has been shown to bind the estrogen receptor
`with high affinity and to function as a potent estrogen
`antagonist
`in mammary tumor cells and in rat models of
`mammary cancer [ll3,ll4].
`In contrast,
`in the cardiovascular
`
`after both eight weeks and two years Oftreatmem [27]‘
`Similarly,
`the effects of raloxifene on the skeleton seem to
`parallel
`those observed with estrogen.
`In vitro studies have
`shown similar effects of
`raloxifene and l7[}—estradiol on
`
`Table 2.
`
`ER Binding and Inhibition of MCF-7 Cell Proliferation by 2-Aryl Raloxifene Analogs [1273]
`
` MCF-7 lnhib. [C50 (nM)‘
`
`estradiol
`
`4-oH—iam€
`
`
`
`
`6-OH
`
`T
`
`T
`
`
`0.2T TT
`T
`T
`
`
`0-36
`
`0.34
`
`T
`
`4—0H
`none
`
`rziloxifene, 7
`7a T
`
`4—OMe
`.
`T
`7bT TT
`4—OMe
`7c
`‘
`T
`T6T—oHTT
`7
`7d
`6-OMe
`4.ToHT
`7c
`T Tnom:
`4—0H
`T
`ll0n€
`4-Cl
`4-OH
`
`TT
`
`T
`
`T
`
`E
`
`T
`
`T
`
`0.073
`0.008
`T 0003
`
`T
`
`7i
`
`T
`
`T
`
`6T—TOH
`
`T
`
`4—Me
`
`4—UH
`
`T
`
`T
`
`T 0.07
`
`NAd
`
`1000
`250
`35TTTTT
`
`50
`
`300
`
`
`
`
`
`
`TT T6T.oii
`7k
`T 7
`T
`TT
`4L1:
`T
`0.19
`T
`T
`2.T3
`T
`
`
`
`
`
`
`T 71 7»OH 4—OH 0.02 T 300
`
` ,
`4.oii
`TT <0.002
`i90TT TTTTTT
`TT
`7
`100
`7n
`,
`5-00
`4—OH
`0
`0.10
`T
`70 T
`TT
`6-bit
`2.oii
`0.057
`
`7p
`6—OH
`3—OH
`0.16
`7q
`5-F,6~OH
`4»OH
`0.093
`4—OH
`
`TT
`
`T
`
`>
`
`T
`
`T
`
`T
`
`T
`
`to
`3.2
`
`
`3
`
` 5,7—d1T(Me),6-OH
`
`T
`
`4-OH
`2—MeT.4—ToH
`T3—Me,4»OH
`
`7:
`7Tu
`
`T
`
`T
`
`T
`
`T
`
`T
`
`6—OH
`6—OH
`
`t
`
`0.41
`0.l3
`
`r
`
`T
`
`T
`
`T
`
`T
`
`.
`
`TT
`
`500
`2
`i
`
`’
`
`
`
`
`
`2.3T
`0.12
`3.ci,4loii
`T6—OH
`_
`7v
`
`
`03 T
`T
`0.20
`3—F,4—OH
`6»OH
`T
`T 7w
`
`
`
`
` 0—oii T 3,5~di(Mc),z1—OH
`I’Avi-ragc of at least 2 determinations. Values are : |O‘7o.
`‘Dose iequired to give 50% inhibition
`“RBA : relative binding affinity by competition with 3H—l7B-€S|I'E1dlOl
`l7[i—estradiol. Average of zit_least
`3 determinations. Values are i lO% ‘INA : not active at
`the doses tested.
`“4—
`of a maximally effective (|0'H) dose of
`Hydroitytamoxifen. the primary biologically active metabolite of tamoxifen.
`-1 ND : not determined
`
`T
`
`T
`
`Astrazeneca Ex. 2023 p. 7
`
`
`
`78 Current Pharmaceutical Design, I 998, Vol. 4, No. I
`
`GI‘L’.\'€
`
`rind Dudpu
`
`osteoclastogenesis and on ereatinine kinase activity in human
`osteoblast cells
`[ll9,l20].
`In rats,
`several
`studies have
`demonstrated a protective effect against ovarieetomy—induced
`osteopenia at doses as
`low as 0.1 mg/kg [ll7,l2l,l22].
`Positive effects on bone mineral density at both cortical and
`cancellous botie sites have been reported. as have positive
`effects on bone strength [l2l,l23].
`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 lesser degree, resulting
`in a net gain in bone mass with raloxifene [l2l,l23b]. These
`positive results in animal studies. have now been confirmed with
`clinical studies in postmenopausal women [27]. Significant
`positive effects on histomorphomctric parameters, bone
`markers, and on bone mineral density at both the lumbar spine
`and hip were observed after two years of raloxifene treatment
`[27,l24].
`
`raloxifene is distinguished
`terms of its pharmacology,
`lti
`from the TPE‘s primarily on the basis of its effects on the uterus,
`where a qualitative difference has been observed [6l.l25].
`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 [ola]. 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 eosinopliilia 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 [l J7],
`ln
`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
`modifications of the 2—arylbenzothiophene [I27].
`the amine-
`containing side chain [l 13,128], and the carbonyl hinge [129]
`The 2«arylbenz.othiophene unit appears to be the primary site of
`ER binding, mimicking the interactions of 17B-estradiol with
`the receptor. On the basis of binding and in vilro activation data
`(Table 2) the 6-hydroxy of raloxifene is believed to imitate the
`3—hydroxy of estradiol, while
`the
`4’—hyclroxy
`roughly
`approximates the l7B—hydroxy [l27a]. The recently published
`crystal structures of raloxifene and l7B—estradiol bound to the ER
`confirm this interpretation [60]. Consistent with the estradi