WO2000019994A1 - Ligands se liant aux recepteurs d'oestrogenes - Google Patents

Ligands se liant aux recepteurs d'oestrogenes Download PDF

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WO2000019994A1
WO2000019994A1 PCT/US1999/022747 US9922747W WO0019994A1 WO 2000019994 A1 WO2000019994 A1 WO 2000019994A1 US 9922747 W US9922747 W US 9922747W WO 0019994 A1 WO0019994 A1 WO 0019994A1
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estrogen receptor
groups
ligands
ring
substituted
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PCT/US1999/022747
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WO2000019994A9 (fr
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John A. Katzenellenbogen
Benita S. Katzenellenbogen
Brian E. Fink
Shaun R. Stauffer
Deborah S. Mortensen
Viswajanani Jitendra Sattigeri
Ying Huang
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Board Of Trustees Of The University Of Illinois
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Priority to AU11996/00A priority Critical patent/AU1199600A/en
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Publication of WO2000019994A9 publication Critical patent/WO2000019994A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/08—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/32—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/24—Radicals substituted by oxygen atoms

Definitions

  • Estrogens are endocrine regulators of the female reproductive system that also have important effects in many non-reproductive tissues (bone, liver, cardiovascular system, CNS, etc.).
  • Many estrogen pharmaceuticals based on both natural and synthetic substances, have been developed as agents for regulating fertility, preventing and controlling hormone-responsive breast cancer, and menopausal hormone replacement. These substances display a spectrum of agonist to antagonist activity that can show remarkable tissue and cell selectivity [Grese, T.A. et al. (1997), "Molecular determinants of tissue selectivity in estrogen receptor modulators,” Proc. Natl. Acad. Sci. USA 94:14105-14110].
  • ER estrogen receptor
  • ER- ⁇ and ER- ⁇ The molecular target of estrogens is the estrogen receptor (ER), of which there are now known to be two subtypes, ER- ⁇ and ER- ⁇ , that have different patterns of tissue expression and somewhat different ligand binding specificities [Mosselman, S. et al. (1996), "ER ⁇ : Identification and characterization of a novel human estrogen receptor,” FEBS Lett 392:49-53; Kuiper, G.G. J.M. et al. (1996), “Cloning of a novel receptor expressed in rat prostate and ovary,” Proc. Natl. Acad. Sci. USA 93:5925-5930].
  • ER estrogen receptor
  • ER is a transcription factor that binds to specific estrogen response elements in the promoter region of estrogen-regulated genes and whose activity for transcription is modulated by the estrogen ligands [Katzenellenbogen, J.A. and Katzenellenbogen, B.S. (1996), "Nuclear hormone receptors: ligand-activated regulators of transcription and diverse cell responses," Chem. Biol. 3:529-536].
  • the capacity of ER-ligand complexes to activate gene transcription is mediated by a series of co-regulator proteins [Horwitz, K.B. et al. (1996), "Nuclear receptor coactivators and corepressors," Mol. Endocrinol.
  • Tissue specificity and differences in agonist/antagonist activity of ER ligands may also, at least in part, be attributed to differences in ligand activity with or affinity for different sub-types of the ER receptor.
  • estrogen-responsive One third of all breast carcinomas are hormone-responsive and nearly all of these are estrogen-positive [Henderson, I.C., Cannellos G.P. (1980) New Eng. J. Med. 320:17].
  • hormonal therapies are preferred over cytotoxic chemotherapy and radiotherapy regimens because of their lower toxicity and the possibility that further remissions can be achieved with sequential use of multiple endocrine regimens [Royce, C. (1993) Drugs of the Future 18:599-600]. ..
  • known ligands for ER the natural estrogens are the simplest of the steroidal hormones, distinguished by having a phenolic A-ring.
  • Synthetic estrogens especially those of non-steroidal nature, generally retain a phenolic function (at least for those of high potency), but otherwise span a remarkable range of structural motifs that encompass simple acyclic core structures of various lengths and sizes, as well as a variety of ring-size fused and non-fused carbocyclic and heterocyclic systems [Magarian, R.A. et al. (1994), "The medicinal chemistry of nonsteroidal antiestrogens: A review," Curr. Med. Chem. 1:61-104; Solrnssen r UN. (1945), "Synthetic estrogens and the relation between their structure and their activity,” Chem. Rev.
  • Tamoxifen the ER ligand most commonly employed in hormonal therapy for estrogen- positive breast cancer [Jordan, V.C. (1995) Breast Cancer Res. Treat. 36:267-285], is a mixed agonist antagonist for ER. This drug exhibits a number of side effects when used in breast cancer therapy.
  • the level of agonist-antagonist activity of tamoxifen is variable and tissue dependent [Katzenellenbogen, B.S. (1996) Biol.Reprod. 54:287-293 and Katzenellenbogen, J.A. et al. ( 1996) Mol. Endocrinol. 10 :119-131]. Tamoxifen may increase the incidence of liver and uterine cancer [Davidson, ⁇ . (1995) New Eng. J. Med.
  • Combinatorial chemistry is of significant current interest for the identification of drug candidates.
  • Combinatorial synthetic methods involve the parallel synthesis of a large collection of structurally related analogs to generate a library of compounds representing systematic structural variations that is then available for functional assessment.
  • Combinatorial libraries are most often screened for a selected biological activity or function. Assessment of the properties of the members of such libraries of structurally related compounds can provide valuable insight into the relationship between structure and the property or function assessed.
  • Combinatorial synthetic techniques have been applied extensively to the generation of large peptide libraries [Gallop, M.A. et al. (1994) "Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries.” J. Med. Chem. 37:1233-1251].
  • ER ligands currently under investigation are not well suited for synthesis by combinatorial approaches, because their preparation generally involves a series of carbon-carbon bond forming reactions that are not uniformly high yield, nor well adapted to solid phase synthetic methods.
  • combinatorial approaches using solid phase synthetic methods have been applied to the preparation of ER ligands having stilbene-like structures [Williard, R., et al. (1995) Curr. Biol. 2:45-51 and Brown, D.S. and Armstrong, R.W. (1996) J.Am. Chem. Soc. 118:6331-6332].
  • combinatorial approaches have had limited application to the preparation of ER ligands.
  • the present invention is based, at least in part, on the inventors' development of a simple modular pharmacophore for ER ligands consisting of a core structure linked to a plurality of independent substituents.
  • the identification of this modular generic structure for ER ligands led to the development of modular stepwise synthetic methods, adaptable to solid-phase chemistry, for the generation of a combinatorial library of potential ER ligands with systematic structural variation.
  • Structural variants are readily generated based on this pharmacophore by variation of the core structure and selection of the substituents to be linked to the core structure.
  • This invention provides non-steroidal estrogen receptor ligands having a modular structure that is amenable to solid phase synthesis and the application of combinatorial synthetic methods to prepare these estrogen receptor ligands.
  • ER ligands of this invention consist of a core scaffold to which a plurality of selected substituents can be bonded substantially independently of other substituents.
  • the modular structure of these compounds allows for synthesis of a very large number of substituent structural variations, substituent combinations and substituent positioning on the core.
  • the structural variants produced by combinatorial methods can be assessed for differences in ER binding affinity and differences in physiological function allowing selection, for example, of ER ligands with a desired spectrum of agonist antagonist properties.
  • the ability to rapidly identify and select ER ligands with differences in agonist antagonist properties allows the identification and selection of ER ligands optimized for a given clinical or pharmaceutical application.
  • the compounds of this invention consist of a core structure that carries up to 6 substituents which together provide for binding to and interaction with ER.
  • the core scaffold is a 5 -membered ring structure that is doubly unsaturated (two double bonds) or 6-membered ring structure which is aromatic (triply unsaturated).
  • the ring structure can be a carbocyclic ring or a heterocyclic ring have one or two non-carbon heteroatoms in the ring.
  • the core ring can be described by the general formula:
  • the positioning of double bonds and heteroatoms in the ring is not illustrated in the structure above, but various ring structures are illustrated in Tables 1 and 2. Substituents attached via dotted bonds are optional, dependent upon double bond and heteroatom placement. The substituents in parenthesis are potentially present in compounds that have 5-membered ring cores.
  • the core scaffold is a 5-membered doubly unsaturated ring structure which can be a carbocyclic ring, i.e. a cyclopentadiene, or a heterocyclic ring having one or two non- carbon elements, e.g., O, S or N, heteroatoms in the ring.
  • ER ligands of this structure have the general formula:
  • R 2 where the 5-membered ring A' can be a variety of carbocyclic and heterocyclic moieties with various positioning of two double bonds. Substituents attached with dotted lines are optional, dependent upon the position of double bonds and any heteroatoms in the ring. The possible double bonds of ring A' and the possible heteroatoms, which can be placed in various ring positions, are not shown in the structure above.
  • a variety of core ring A' structures illustrating placement of double bonds, heteroatoms and substituents are exemplified in Table 1.
  • Table 1 contains a number of exemplary core 5-membered ring structures illustrating positioning of R substituents on the 5-membered ring.
  • Core ring structures are exemplified by cyclopentadienes, cyclopentadienones, pyrazoles, imidazoles, oxazoles, thiazoles, isoxazoles, isothiazoles, furans, pyrroles, and thiophenes. Additional structures may be obtained by varying the relative placement of substituents and double bonds. Two adjacent R substituents on the 5- membered ring can together form a cyclic structure.
  • the core can accommodate up to 3 to 6 substituents.
  • Substituent R l is required in the compounds of this invention. All 5-membered ring cores accommodate a minimum of 3 substituents (R 1 and two of R 2 , R 3 , R 4 and R'). It is preferred that both R 1 and R 3 or R l and R 4 be present and that they are both non-hydrogen substituents. It is more preferred that R 1 , R ⁇ and R 3 or R 1 , R 2 , and R 4 be present and that each is a non-hydrogen substituent.
  • the core scaffold is a 6-membered aromatic ring structure which can be a carbocyclic ring, i.e. a benzene, or a heterocyclic ring, e.g., a pyrazine or a pyrimidine, having one or two non-carbon elements, e.g., 0, S or N, heteroatoms in the ring.
  • ER ligands of this structure have the general formula:
  • R * where the 6-membered aromatic A" ring can be a variety of carbocyclic and heterocyclic moieties. The possible locations of heteroatoms in the ring are not shown in the above structure. One or two of the substituents attached by dotted lines can be absent dependent upon the placement of heteroatoms.
  • a variety of core ring structures illustrating placement of heteroatoms and substituents in six-membered rings are exemplified in Table 2. Two adjacent R substituents on a 6-membered core ring can together form a cyclic structure, as illustrated by quinoxalines or quinazolines in Table 2.
  • the aromatic core can accommodate 4 to 6 substituents.
  • Substituent R 1 is required in the compounds of this invention.
  • Six-membered ring cores accommodate a minimum of 4 substituents (R 1 and three of R 2 , R 3 , R 4 , R 3 and R 5' ).
  • Substituent R D' is selected from the same groups as R 5 and may be the same or different from R ⁇ R J or R 1 and R 4 be present and that they are both non-hydrogen substituents.
  • R 1 , R 2 , and R 3 or R 1 , R 2 , and R 4 be present and that each is a non-hydrogen substituent.
  • Benzenes have six substituents.
  • Pyridines have five substituents.
  • Pyrimidines and pyrazines have four substituents. Bonds to substituents other than R 1 are indicated by dashed lines to show that they may be absent dependent upon heteroatom placement.
  • Substituent R 1 can be selected from the group consisting of phenyls and substituted phenyls wherein the non-hydrogen phenyl group substituents can include, without limitation, halogens (F, Cl, and Br, being preferred), hydroxy groups, lower alkyl, alkenyl, alkynyl, and alkoxyl groups (where the term "lower” indicates 1 to about 6 carbon atoms), lower ethers, ketones, or thioethers, and substituted lower alkyl, alkenyl or alkynyl groups (where the substituents can be halogens or hydroxy groups).
  • Substituted alkyl, alkenyl and alkynyl groups can include* perhalogenated groups, e.g., CF 3 or CF 2 CF 3 .
  • the R 1 phenyl ring can carry multiple substituents that can be the same or different. Phenyl ring substituents can be at any of the ortho- (o-), meta- (m-) or para- (p-) positions on the ring.
  • Preferred substituents of R 1 phenyl groups are halogens (particularly F, Cl and Br), methyl, ethyl, vinyl, methoxy, ethoxy and hydroxy groups.
  • Preferred substituted phenyl groups are para-substituted, particularly p-halogen- and p-hydroxy- substituted phenyls.
  • the most preferred R 1 group is p-hydroxyphenyl.
  • the R 1 phenyl group can carry any substituent that can be metabolically converted into a p-OH group, e.g., OCH 3 , O-COCH 3 , etc.
  • Substituents R ⁇ R 3 and R 4 can be the same or different, and can be selected from the group consisting of hydrogen, a phenyl or substituted phenyl group (where phenyl substitution is_ as described for R l ), lower alkyl, alkenyl or alkynyl (where the lower alkyl, alkenyl or alkynyl groups may be substituted, with a phenyl, hydroxyls or halogens), lower ethers, ketones or thioethers, and halogens (F, Cl, Br, or I). Where a halogen is directly attached to the core ring, Br and I are preferred halogens.
  • Substituted alkyl, alkenyl and alkynyl groups can include perhalogenated groups, e.g., perfluorinated groups, such as CF 3 or CF 2 CF 3 .
  • Substituted alkyl, alkenyl and alkynyl groups include those substituted with a phenyl ring or a substituted phenyl ring, e.g., benzyl, p-hydroxybenzyl, m-fluorobenzyl, etc.
  • Preferred R 2 , R 3 , R 4 are lower alkyl or alkenyl groups, and phenyl and o-, m-, or p-substituted phenyl groups.
  • R 2 are ethyl and propyl (straight-chain or branched) groups.
  • More preferred R 4 are phenyls, o-, — or p- hydroxyphenyl, o-, m-, or p-alkoxyphenyl, o-, m-, or p-halophenyl, and branched alkyl groups, e.g., a t-butyl group.
  • More preferred R 3 are phenyls and substituted phenyls, including o-, m-, or p-hydroxyphenyl, o-, m-, or p-alkoxyphenyl, o-, m-, or p-halophenyl.
  • R 5 and R 5' may be the same as or different than any of R 1 , R 2 , R ⁇ or R 4 .
  • Substituents R 5 or R 5' may be the same or different than each other.
  • R 6 when present, e.g. in cyclopentadienes, can be hydrogen, lower alkyl, alkenyl, alkynyl, or alkoxy groups, substituted lower alkyl, alkenyl or alkynyl groups, lower ether or thioethers or a halogen (particularly F, Cl and Br).
  • Substitution of alkyl, alkenyl and alkynyl R 6 groups can include halogen and hydroxy group substitution.
  • One or more of any -CH 2 - groups in R ⁇ can be replaced with -CO- groups.
  • R 6 is preferably a lower alkyl or hydrogen.
  • the R'-R 4 (and R 5 , R 5- or R 6 when present) can also carry or be basic or polar groups, e.g., an alkyl, phenyl or other substituent listed above that carries a basic or polar substituent or basic or polar groups directly linked to the core ring structure.
  • Basic and/or polar groups on ER ligands can provide the ligand with antagonist or mixed agonist/antagonist - properties.
  • Basic groups include without limitation: amines, and amine-substituted alkyl, alkenyl or alkoxy groups.
  • Amines can be alkyl, alicyclic or aromatic amines.
  • Basic groups specifically include: -(X) x (CH 2 ) n -NRR' where X is 0 or S, x is 0 or 1, n is an integer from 1 to about 10 and preferably 2 to 6, and R and R, can be the same or different and can be alkyl, aryl, or alicyclic. R and R' in these specified basic groups can together form an heterocyclic or a substituted heterocyclic ring, e.g.,
  • R A (which may represent multiple substituents) can be lower alkyl, alkenyl or alkynyl groups or substituted lower alkyl, alkenyl or alkynyl groups. Additionally, any alicyclic rings can contain one or more carbonyl groups -CO-.
  • any polar groups can be employed as substituents on any R groups or for direct linkage to the ring.
  • Preferred polar groups include halogens, perhalogenated alkyl, alkenyl or alkynyl groups, hydroxy groups, hydroxy-substituted alkyl, ethers or thioethers, diols, amides, sulfoxides, and sulfones, e.g.,:
  • Diols -(X) x (CH 2 ) n -CH(OH)-CH(OH)-R B where X is O or S, x is 0 or 1, n is an integer from 1 to about 6 and preferably 1 to about 4, R B is H or -(CH 2 ) m -CH 3 ⁇ where m is an integer from 0 to about 6.
  • Diols can also be alicyclic; Amides:
  • X is 0 or S
  • x is 0 or 1
  • n is an integer from 1 to about 12 and preferably 6 to about 10
  • R and R' can be the same or different and can be alkyl, aryl, alicyclic, substituted alkyl, substituted aryl or substituted alicyclic, or together form an heterocyclic or a substituted heterocyclic ring;
  • R' can be alkyl, aryl, alicyclic, substituted alkyl, substituted aryl or a substituted alicyclic ring where substituents include halogens, particularly F, Cl and Br and perhalogenated alkyl groups, such as CF 3 and CF 2 CF 3 ;
  • R' can be alkyl, aryl, alicyclic, substituted alkyl, substituted aryl or a substituted alicyclic ring where substituents include halogens, particularly F, Cl and Br and perhalogenated alkyl group, such as CF 3 and CF 2 CF 3 .
  • Each of the hydroxy-substituted alkyls and the above-listed amides, diols, sulfoxides and sulfones can be directly attached to the 5-membered core ring as an R 2 -R 6 or R y substituent or can be a substituent on any of R'-R 6 or R 3' .
  • any two substituents on a given ring carbon can be linked to form a spiro-ring.
  • substituents R 3 and R 6 or R 4 and R 6 on the same ring atom can together form a carbon chain -(CH 2 ) lake- where n is 3 to about 6 to form a spiro ring system with the parent cycle A.
  • Carbons in the R 5 R 6 chain or R 4 /R 6 chain may also be substituted, e.g., with halogens or lower alkyl, alkenyl or alkynyl groups, and one or two of the CH 2 groups of the chain may be replaced with an -CO-, -0- , -S- or an -NH-.
  • Substituents on adjacent ring atoms can be linked to form a saturated or unsaturated carbocyclic or heterocyclic ring structure fused to the parent cycle A, e.g., an alkyl substituent of R 2 can be linked to a phenyl substituent at R' or R 3 .
  • Substituents are generally selected independently of core ring size, as discussed above to achieve desired ER ligand characteristics, but are preferably also selected to provide stable compounds and facilitate ease of preparation.
  • 5- and 6-member ring ER ligands of this invention can contian two substituted or non-substituted phenyl rings in addition to R'.
  • R 1 is substituted at a ring atom directly adjacent to a ring atom substituted with a lower alkyl group, particularly an ethyl, n-propyl, i-propyl, i-butyl or n-butyl group.
  • R 1 is a p-substituted phenyl group, where the substituent is OH or OR where R is a lower alkyl group, R 2 is a lower alkyl group (up to C6) and the ligand contains in addition one or two substituted or non-substituted phenyl groups.
  • Preferred substituents on the additional phenyl rings are p-OH, m-OH, p-halogen, m-halogen, p-OR or m-OR where R is a lower alkyl group.
  • the ER ligands of this invention have core structures as listed in Tables 1 and 2 where the substituents R'-R 6 and R 5' are as defined above. Structural variants in addition to those listed in Table 1 and 2 may be obtained by interchanging the positions of R 2 , R 3 , R 4 , and R 5 .
  • ER ligands are those compounds which exhibit measurable binding affinity for the estrogen receptor in assays as described herein.
  • the non-steroidal ER ligands of this invention are useful in pharmaceutical compositions for the treatment of hormone-responsive disorders.
  • the non-steroidal ER ligands of this invention are particularly useful in pharmaceutical applications for treatment of estrogen- responsive disorders and conditions, as active ingredients of pharmaceutical compositions in combination with a pharmaceutically acceptable carrier or exipient.
  • the ER ligands may be combined with each other to achieve a desired pharmaceutical response or administered in combination with known estrogens or antiestrogens.
  • the ER ligand is present in the pharmaceutical compositions in an amount, or in combination with other ligands in a combined amount, sufficient to induce or inhibit estrogen response.
  • the amount of ligand (or combined amount of ligands) present in the pharmaceutical composition is in the range that induces or inhibits the desired selective response.
  • the invention also relates to methods of treating estrogen responsive disorders and physiological conditions employing pharmaceutical compositions comprising ER ligands of this invention alone or in combination.
  • This invention provides pharmaceutical compositions which comprise one or a mixture of ER ligands having structures disclosed herein in combination with a pharmaceutically acceptable carrier appropriate for the pharmaceutical application and compatible with the ER ligand.
  • ER ligands are present in these pharmaceutical compositions in an amount or in a combined amount sufficient to elicit a measurable positive effect on a symptom or condition associated with an estrogen-dependent disorder on administration to an individual suffering from the symptom or disorder.
  • compositions of this invention can also include other steroid or non- steroid ER ligands which may supplement or enhance the activity of the composition for a particular medical application.
  • Pharmaceutical compositions of this invention include those which are useful in the prevention and treatment of hormone-dependent cancers, including breast cancer, those useful for hormone-replacement therapy, those useful in the treatment of infertility, those useful for treatment of osteoporosis and those useful for providing cardiovascular, CNS ( suppress hot flashes, provide cognitive improvements, etc.) or related benefits.
  • Pharmaceutical compositions of this invention can be provided in a variety of dosage forms including without limitation pills for oral administration, solutions or emulsions for oral administration or for injection.
  • This invention also provides methods for the treatment of hormone-dependent disorders, including the treatment of hormone-responsive breast cancer, which comprise the step of administering to a patient having the disorder or symptoms thereof a pharmaceutical composition comprising one or a mixture of the ER ligands of this invention where the ER ligand or mixture - of ligands is present in the composition at a level or a combined level sufficient to effect a positive biological response.
  • ER ligands of this invention can exhibit agonist, antagonist or mixed agonist antagonist function in vitro and in vivo. These functions can be assessed for a given ER ligand or ligand mixture employing in vitro methods known in the art or as described in the Examples herein.
  • This invention also provides methods for generation of and testing of combinatorial libraries of potential ER ligands for ER binding affinity as well as for the assessment of agonist/antagonist character of a given ER ligand.
  • the ER ligands of this invention are useful in vitro and/or in vivo for selective activation or repression of expression, dependent upon the agonist or antagonist nature of the ligand, of a gene regulated by ER.
  • Gene activation or repression can be selective with respect to subtype of ER (e.g., ER ⁇ or ER ⁇ ), or variant of ER (e.g., splice variant forms, truncated or processed forms, covalently modified forms, etc.).
  • the ER ligands of this invention are also useful in vitro and/or in vivo for selective regulation of cellular activities under the control of ER.
  • Cellular activities may be regulated in a variety of ways by ER, subtypes of ER or variants of ER, e.g., up or down regulation of a given cellular process. Regulation is selective with respect to subtype of ER (e.g., ER ⁇ or ER ⁇ ), or variant of ER (e.g., splice variant forms, truncated or processed forms, covalently modified forms, etc.).
  • Cellular activities that may be regulated include both genomic (related to gene expression) or non-genomic activities (not directly related to gene expression, e.g., such as regulation of calcium flux, particularly in bone cells, hormone release, particularly prolactin release from pituitary cells, etc.).
  • the subtype-selective ER ligands of this invention can also be of general use in the investigation of ER and its functions. These ligands can be employed to better understand structure and conformation of ER (both subtypes) and to elucidate how ER subtypes interact with other molecules and to relate structure, conformation and interaction with other molecules to ER function.
  • FIGS. 1A and IB illustrate transcriptional activation by ER ⁇ and ER ⁇ , respectively, in response to the pyrazole compound 38 b.
  • Human endometrial cancer (HEC-1) cells were transfected with expression vectors for ER ⁇ (Fig. 1A) or ER ⁇ (Fig. IB) and an (ERE)3-pS2- CAT reporter gene and were treated with the indicated concentrations of estradiol (E2) or the pyrazole for 24 h.
  • Cat activity was normalized for ⁇ -galactosidase activity from an internal control plasmid. Values are the mean ⁇ SD for three or more separate experiments, and are expressed as a percent of the ER ⁇ and ER ⁇ response with 10 nM E2. See: J. Sun et al. (1999) Endocrinology 140 (2):800-804.
  • Figure 2 illustrates transcriptional activation by ER ⁇ and ER ⁇ in response to two pyrazoles XXX (solid lines) and XXXI (dashed lines).
  • HEC-1 cells were transfected with expression vectors for ER ⁇ (diamonds) and ER ⁇ (squares) and an (ERE) 3 -pS2-CAT reporter gene and were treated with indicated concentrations of ligand for 24 h.
  • CAT activity was normalized for ⁇ -galactosidase activity from an internal control plasmid. Values are the mean ⁇ SD for three or more separate experiments, and are expressed as a percent of the ER ⁇ and ER ⁇ response with 1 nM E2. ..
  • Figures 3 A and 3B are transcriptional activation profiles for ER ⁇ (Fig. 3A) and ER ⁇ (Fig. 3B) in response to pyrazole XXXX.
  • HEC-1 cells were transfected with expression vectors for ER ⁇ or ER ⁇ and an (ERE) 3 -pS2-CAT reporter gene and were treated with indicated concentrations of ligand for 24 h in the presence or absence of estradiol (1 nM).
  • CAT activity was normalized for ⁇ -galactosidase activity from an internal control plasmid. Values are the mean ⁇ SD for three or more separate experiments.
  • the pharmacophore model for ER ligands of this invention consists of a core structure onto which independent peripheral structural elements are attached.
  • a preferred pharmacophore is illustrated in which a phenolic unit (B) that is always preserved, a second aromatic group (C) that is usually present, and another substituent (D) or two (D'), one of which may be aromatic is attached to the core (A):
  • High ER binding affinity will be associated with certain geometric arrangements of the peripheral substituents (B-D'), so that they will be "in register” with their corresponding subsites in the ligand binding pocket in ER.
  • Peripheral group orientation can be accomplished by core elements that encompass some structural variety.
  • the core serves as a molecular scaffold whose function is to correctly orient the peripheral substituents with appropriate topology for high affinity ER binding. Further, the chemical nature of the core may effect the binding affinity and or influence the interaction of substituents with ER.
  • the homobibenzyl motif A exemplified in the known non-steroidal ligands benzestrol and raloxifene and the syn-bibenzyl motif B.
  • the A motif can be represented in various 3,5-diaryl-l,2-azoles (pyrazoles and isoxazoles) and various 2,4-diaryl-l,3-azoles (imidazoles, thiazoles, and oxazoles).
  • the B motif can be represented in various 4,5-diaryl-l,3-azoles, as well as various 3,4-diaryl-l,2-azoles and various 4,5-diaryl-l,2-azoles.
  • the structure of ER ligands of this invention expand from these basic motifs.
  • Tables 1 and 2 illustrate representative core five- and six-membered ring structures of the ER ligands of this invention.
  • the cores include five-member cyclic rings that are doubly unsaturated and which may contain one or two heteroatoms (particularly N, O or S) .
  • the cores also include six-member aromatic rings which may contain one or two heteroatoms (particularly N).
  • the selected cores can accommodate from three to six substituents which can be oriented by placement on ring elements.
  • Dependent upon the selection of a particular R'-R 6 or R substituent distinct structures may be obtained by interchange of substituents.
  • ER ligands of this invention can have the structures:
  • x is 0 or 1 and R is hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other substituents are defined as in the Summary above;
  • R is hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group, where R' is a phenyl ring substituent as defined above in the definition of R 3 and can be a polar or basic substituent and other variables are defined as in the Summary above;
  • R and R' can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • R and R' can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • x is 0 or 1
  • X is N, NH, NR ⁇ S or 0
  • R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • x is 0 or 1
  • X is N, NH, NR 5 , S or O and R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • X or Y is N and the other of X or Y is N, S or O
  • x is 0 or 1 and R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • R -R are defined as in the Summary above;
  • R 2 and R 4 are defined as in the Summary above and R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group;
  • R 2 is defined as in the Summary above, R and R' can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and R" can be a hydrogen, a halogen, a hydroxy, an alkoxy, or a basic or polar group; and
  • R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and R" can be a hydrogen, a halogen, a hydroxy, an alkoxy, or a basic or polar group.
  • Preferred R and R' are H, preferred R" is OH and preferred R 2 are straight-chain or branched lower alkyl groups having up to 6 carbons atoms.
  • RBAs ER binding affinity
  • R 2 is an ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl group
  • R" and R'" may be positioned at the meta ox para ring positions and can be selected independently of each other from the group p-H, p-OH, p-F, p-Br, p-CH 3 , m-OH, m-F, or m-Br.
  • Pyrazoles of this structure exhibiting generally higher ER affinity are those in which R'" and R" are both p-OH.
  • Furans of particular interest having significant ER binding aiffinity include those having the structure:
  • R 2 is an ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl group
  • R" and R'" may be positioned at the meta ox para ring positions and can be selected independently of each other from the group p-H, p-OH, p-F, p-Br, p-CH 3 , m-OH, m-F, or m-Br.
  • Furans of this structure exhibiting generally higher ER affinity are those in which R'" and R" are both p-OH.
  • Cyclopentadienes of particular interest include those having the structure:
  • R 2 is an ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl group
  • R" and R'" may be positioned at the meta ox para ring positions and can be selected independently of each other from the group p-H, p-OH, p-F, p-Br, p-CH 3 , m-OH, m-F, or m-Br.
  • Furans of this structure exhibiting generally higher ER affinity are those in which R'" and R" are both p-OH.
  • ER ligands of this invention can have the structures:
  • A is an aromatic ring with up to two heteroatoms in the ring, x is 0 or 1 and R is hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group, other substituents are defined as in the Summary above, and one or two of the indicated substituents may be absent due to heteroatom placement;
  • A" is an aromatic ring with up to two heteroatoms in the ring
  • R is hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group
  • R' is a phenyl ring substituent as defmed above in the definition of R 3 which may be at any ring position (preferably para or meta ring positions) and can be a polar or basic substituent, other substituents are defined as in the Summary above, and one or two of the indicated substituents may be absent due to heteroatom placement;
  • R and R can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group, other variables are defined as in the Summary above and one or two of the indicated substituents may be absent due to heteroatom placement;
  • R, R' and R" can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group and other variables are defined as in the Summary above;
  • R, R' and R" can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group, other variables are defined as in the Summary above;
  • R, R' and R" can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group where R 2 is defined as in the Summary above, but is preferably a lower alkyl groups having up to about 6 carbon atoms; and
  • R and R' can be the same or different and can be hydrogen, alkyl, alkenyl, alkynyl or substituted alkyl, alkenyl, alkynyl, or a basic or polar group, other substituents are as described above in the Summary .
  • SEEMS selective estrogen receptor modifiers
  • Basic or polar side chain can be substituted at several possible positions in the compounds of this invention. For example, in pyrazoles of the following structure:
  • a basic or polar group can be substituted at R, R 2 , R"or R'".
  • a preferred positioning of the basic or polar side chain is such that it would occupy a region of the ligand binding pocket normally occupied by such groups in known SERMS, as well as complete estrogens antagonists.
  • Basic side groups of particular interest for substitution into pyrazoles include those carrying alicyclic amine groups, e.g.,: -(X) x (CH,) n -NRR' where X is O or S, x is 0 or 1, n is an integer from 1 to about 10 and preferably 2 to 6, and R and R', can be the same or different and can be alkyl, aryl, or alicyclic. R and R in these specified basic groups can together form an heterocyclic or a substituted heterocyclic ring.
  • a preferred basic side group is a piperidinylethoxy group.
  • Preferred R 2 are lower alkyl groups having up to about 6 carbon atoms.
  • Imidazoles The synthesis of representative symmetrical members of the imidazole class (specifically core structure IM1 of Table 1) and their N-alkyl analogs is accomplished by a well known approach [Sarshar, S., Siev, D. & Mjalli, A.M.M. (1996). Imidazole libraries on solid support Tetrahedron Lett. 37, 835-838] as shown in Scheme 1A. Refluxing 4,4'- dimethoxybenzil (1) in formamide in the presence of r ⁇ -formaldehyde affords the 4,5- disubstituted imidazole 2 [Bredereck, H., Gompper, R. & Hayer, D. (1959).
  • N-aryl substituted imidazoles (IM2 core of Table 1), as exemplified by imidazole 17 can be synthesized as outlined in Scheme 2. Refluxing 4'-methoxy- ⁇ -bromobutyrophenone (13) wifh/?-anisidine in acetone gives the ⁇ -amino-ketone 14, which is converted into the benzamide 15 upon reaction with benzoyl chloride and base. Cychzation with ammonium acetate in refluxing acetic acid affords the 1,2,4,5 tetrasubstituted imidazole 16, which upon deprotection with BF 3 -SMe 7 in CH 2 C1 2 produces the free phenol 17.
  • Oxazole 30 results from the condensation of bromo-ketone 26 with -methoxybenzamide in refluxing toluene (Scheme 4B) analogous to the thiazole synthesis discussed above.
  • Schemes 4A and B can be employed or readily adapted using well-known methods and by appropriate choice of starting materials by one of ordinary skill in the art for the synthesis of ER ligands of this invention having oxazole ring core structures.
  • Pyrazoles - The synthesis of the pyrazoles is illustrated in Schemes 5A-C.
  • Scheme 5 A involves the condensation of a hydrazine with a 1,3-diketone [Marzinzik, A.L. & Felder, E.R. (1996), "Solid support synthesis of highly functionalized pyrazoles and isoxazoles; scaffolds for molecular diversity, " Tetrahedron Lett. 37, 1003-1006].
  • Beak Reitz, D.B., Beak, P., Famey, R.F. & Helmick, L.S. (1978), "Dipole-stabilized carbanions from thioesters. Evidence for stabilization by the carbonyl group, "J. Am. Chem. Soc. 100, 5428-5436] can be used to obtain 1,3-diketone 33 from the reaction of the methyl thioester 32 and lithium ⁇ tetramethylpiperidide.
  • ketone 90 can be reacted with 2 eq. of nitrobenzyl ester 91 and LiN(iPr) 2 to give the 1,3-diketone which is then taken to the pyrazole (e.g., 38) as further indicated in Scheme 5A (Path B).
  • Scheme 5B provides more detail of the syntheses of pyrazoles 200-204 via the method of Scheme 5A.
  • Scheme 5C presents a general method for synthesis of pyrazoles having core PA2 in which R 1 is attached to a ring. This scheme also illustrates a method for addition of I to the ring. Any halogen can be added by appropriate selection of reagent.
  • Scheme 5D provides more detail of the syntheses of pyrazoles 205-209 via the method of Scheme 5C.
  • isothiazoles Illustrative preparations of isothiazoles are shown in Schemes 7A and B. Reaction of the thioketone imine 42 with iodine results in cychzation to form isothiazole 43 (Scheme 7A). Alternatively, isoxazoles (as prepared in Scheme 6) can be reductively cleaved to form enaminoketone 44 which on treatment with P 2 S 5 /chloranil results in isothiazole 43.
  • Furans, Thiophens and Pyrroles Heterocycles having one heteroatom in the 5-membered ring core (e.g., furans, thiophenes and pyrroles) can generally be prepared by cychzation of appropriately substituted 1,4-diketones. Ring substitution is for the most part determined by selection of the 1 ,4-diketone.
  • the synthesis of 1,4-diketones is illustrated in Scheme 8. Starting with aldehydes and ketones that are commercially available or readily synthesized by well-known methods, substituted ⁇ , ⁇ -unsaturated ketones are formed by treatment with ethanolic KOH.
  • the ⁇ , ⁇ -unsaturated ketones are transformed using, for example, the S tetter reaction with appropriately substituted aldehydes in the presence of a thiazolium salt catalyst (e.g., 3-benzyl-5- (2-hydroxyethyl)-4-mefhyl-thiazolium chloride for aliphatic aldehydes or 3-ethyl-5-(2- hydroxyethyl)-4-methyl-thiazolium bromide for aromatic aldehydes) to form the desired diketones.
  • a thiazolium salt catalyst e.g., 3-benzyl-5- (2-hydroxyethyl)-4-mefhyl-thiazolium chloride for aliphatic aldehydes or 3-ethyl-5-(2- hydroxyethyl)-4-methyl-thiazolium bromide for aromatic aldehydes
  • Desoxyanisions were treated with one equivalent of potassium bis(trimethylsilyl) amide followed by addition of ⁇ -bromoketone to give the desired tetra-substituted diones in good yield.
  • This approach affords the 1 ,4-diones as mixtures of diastereomers, however, no separation of the stereoisomers is required, as these centers become non-stereogenic in the final products.
  • Scheme 10D illustrates a synthesis of a pyrrole of core structure PR1 (with N-R 1 ). It is apparent from an overview of Schemes 10A-D that a variety of different pyrroles with different relative positions of substituents R'-R 5 with respect to each other and with respect to the N in the ring can be obtained by appropriate substitution of starting 1,4-diketones. Furans, thiophene and pyrroles having methoxy substituents on substituted phenyl R groups (e.g. 58, 60 and 62) can be deprotected with boron tribromide to afford the demethylated products.
  • Furans, thiophene and pyrroles having methoxy substituents on substituted phenyl R groups e.g. 58, 60 and 62
  • boron tribromide to afford the demethylated products.
  • Scheme 11 A (including paths A, B, B' and C) illustrates representative syntheses of cyclopentadienes and cyclopentadienones of this invention.
  • Dieneone 93 produced for example by path A is cyclized to give the cyclic unsaturated ketone 94.
  • Additional non-hydrogen substituents e.g., R 4 and R 6 can be added to the five-membered ring as indicated in path C to ultimately give various cyclopentadienes (e.g., 95A- B).
  • the cyclic ketone 94 can be reduced via path B to a cyclopentadienone 96A.
  • a cyclopentadiene having two hydrogens 97 on the same ring carbon can be oxidized to give a cyclopentadiene 96B.
  • Scheme 1 IB illustrates a synthesis of cyclopentadienones of this invention and an alternative synthesis of cyclopentadienes.
  • a cyclic unsaturated ketone 98 is prepared by cobalt carbonyl catalyzed cychzation of a substituted alkyne and olefin. This reaction can result in the generation of regioisomers.
  • Ketone 98 is reduced to give cyclopentadiene 97.
  • Scheme 1 IC provides another general scheme for synthesis of cyclopentadienones.
  • Cyclopentadienyl ligands with R 2 that is a lower alkyl group, e.g., n-propyl can also be made by this method by selection of Grignard reagent.
  • Compounds 236 and 237 where R 2 is n-propyl can also be made by this method.
  • a thiazolium salt catalysed addition of an aldehyde to an ⁇ , ⁇ - unsaturated ketone under Stetter conditions gives the corresponding 1,4-diketone.
  • Cyclopentadienes are derived from Gringard and dehydration reaction on the cyclopentenones.
  • the cyclopentadienes were not stable to the conditions of deprotection to release free phenol. So the cyclopentenones were deprotected under mild conditiond (borontrifluoride-dimethylsulfide) to give the cyclopentenones.
  • the free phenols were then temporarily reprotected as their trimethylsilyl ehters using bis(trimethysilyl) cetamide which were then subjected to Gringnard reaction.
  • Schemes 12 A-D illustrate representative methods for introduction of basic amino substituents into five-membered ring ER ligands of this invention.
  • the schemes illustrate the synthesis of a base-substituted pyrazole. Intermediate 100 is reacted with a substituted 1,3 diketone to form the pyrazole.
  • Schemes 12B and 12C illustrate in more detail the synthesis of Scheme 12A for the introduction of a piperidinylalkoxy basic group.
  • Scheme 12B illustrates the introduction of the basic side chain at a ring nitrogen of a pyrazole.
  • Scheme 12C illustrates the introduction of the basic side chain at C(3) of the pyrazole ring.
  • Scheme 12 D illustrates introduction of a basic side chain on a phenyl substituent on the pyrazole ring.
  • Pyrida ⁇ ine- six-membered ring pyridazine analogues can also be prepared from- the 1,4- diones described above for synthesis of furans, thiophenes and pyrroles.
  • Scheme 13 A treatment of the diones with hydrazine hydrate followed by air oxidation affords the desired pyridazines.
  • Exemplary pyridazines synthesized by the illustrated methods are indicated in Scheme 13 A.
  • This method can generally be applied to the synthesis of various pyrimidines of this invention.
  • Cinnolines-t s synthesis of cinnolines can be carried out using the strategy described in
  • Phthalazines the synthesis of phthalazines can be carried out using the strategy described in Scheme 14D.
  • Combinatorial Methods Combinatorial chemistry can be employed to synthesize a variety of potential ER ligands having the 5 -member and 6-membered unsaturated ring core structures described herein. These solid phase methods allow the production of a combinatorial library of compounds, having varying substituents on the core structure, to test for ER binding and activity.
  • Schemes 15 A and B provides illustrative solid support syntheses of compounds having a heterocyclic ring structure, pyrazoles. These schemes exemplify the use of a resin P, e.g., the Merrifield resin, to tether a starting material.
  • Scheme 15A illustrates distinct syntheses for compounds where R 3 is aliphatic (path A) or aryl (path B). This scheme can be used to generate pyrazoles with three or four substituents.
  • Scheme 15B illustrates an altemate route to pyrazoles proceeding through a distinct intermediate 119 to a tethered pyrazole 109.
  • Path A in Scheme 15B illustrates halogen addition to the ring, e.g., 123.
  • the choice of paths in B depends on whether substituent R 2 is aliphatic or aryl.
  • Scheme 16 provides illustrative solid support syntheses of compounds having a. heterocyclic ring structure, oxazoles, thiazoles and imidazoles. Interestingly, a single intermediate 134 in Scheme 16 can be used to generate compounds of all three ring structures 145, 147, or 149.
  • ER ligands are those compounds which exhibit measurable binding affinity for the estrogen receptor. There are various ways to measure and quantify ER binding affinity. In this invention ER binding affinity is measured in competitive binding assays compared to estradiol. Binding affinity is expressed as a relative binding affinity (RBA) in percent compared to estradiol which is assigned an affinity of 100%. Substantial affinity for ER is indicated by an
  • RBA of about 0.1 % or more. Good affinity binding to ER is indicated by an RBA of about 1%- to about 10%. High affinity binding to ER is indicated by an RBA of about 10% or higher.
  • the binding affinities of substituted compounds of heterocyclic cores structures listed in Table 1 are shown in Tables 3-5 A-B, organized according to heterocyclic core structure.
  • the binding values were obtained from a competitive radiometric binding assay, using [ Hjestradiol as the tracer and dextran-coated charcoal to adsorb free tracer or hydroxyapatite to adsorb the ER-tracer complex; the values are expressed as relative binding affinities (RBA), in percent, with respect to estradiol assuming an affinity of 100%) for estradiol.
  • RBA relative binding affinities
  • Lamb and/or rat uterine cytosol ER preparations were used as described in Katzenellenbogen, J.A. et al. (1977) "Estrogen photoaffinity labels. 1.
  • any binding affinity measured for a mixture could never be less than one-third the affinity of the pure high affinity isomer.
  • the concentration in the binding assay would be 3-fold higher and its measured affinity also 3-fold higher than in the 1 :2 mixture. This means that in cases where mixtures have been examined for binding affinity, that one of the isomers present may have up to a 3-fold higher binding affinity than indicated by the measurement.
  • Table 4 shows the binding data for two thiazoles and oxazoles prepared. Although affinities are again very low, the more highly substituted thiazole again has the higher affinity
  • the oxazole 29 has undetectable affinity for ER.
  • the isomer 31 does have measurable though low binding.
  • thiazoles and oxazoles do not have very high dipole moments; so overall polarity is not likely to be the source of their low ER binding affinity, although heteroatom orientation appears to play a role (29 vs. 31).
  • the compounds with the highest affinities were all tetrasubstituted. Since it is only possible to trisubstitute a thiazole or oxazole, this core structure may be unable to present sufficient peripheral substituents to afford ligands with good ER binding affinities.
  • the low binding affinities of the imidazoles, thiazoles and oxazoles may be, at least in part, due to their overall structure which is expected to be rather planar. It has been reported that good ligands for the estrogen receptor need to have some degree of "thickness" in the central portion of the ligand [41]. When alkyl substituents are added to either the imidazoles or thiazoles, their RBA increases. This increased binding could be due to an increase in steric bulk around the central portion of the molecule, the result, in part, of a twisting of some of the aromatic substituents (see below) or to an increase in lipophilicity.
  • Furans- RBA data and differential binding affinities for ER ⁇ and ER ⁇ for several furans are given in Table 5
  • Furan 204 exhibits relatively high RBA.
  • Several furans exhibit significant binding strength preference for ER ⁇ compared to ER ⁇ .
  • Furan 203 for example, binds to ER ⁇ about 70-fold more strongly than it does to ER ⁇ .
  • Differential ligand binding affinities for ER ⁇ and ER ⁇ can be measured using purified preparations of human ER ⁇ and ER ⁇ as described in Example IB. Using this assay, Pyrazole compound 38b was found to bind to ER ⁇ three-fold more strongly than to ER ⁇ . This result indicates that certain ER ligands of this invention can exhibit differential ligand binding affinity to the different ER subtypes.
  • ER binding affinities of pyrazole isomers of core structure PA2 are given in Table 7.
  • RBA ER affinity
  • the structure-binding affinity pattern for both pyrazole isomers is quite similar. It is believed that these two core structure pyrazole isomers are binding in the same orientation in the ER binding pocket. Thus, it is possible to permute the position of heteroatoms in the azole ring without major effect on ER binding affinity provided that the peripheral substituents remain disposed with the same geometry and provided that one remains in the same azole series.
  • the pyrazole isomers are compounds with equivalent dipole moment and polarities.
  • Table 8 presents ER binding affinity data for pyrazole of the indicated formula where one of R, R 2 , R"or R'"is a cyclic amine group, i.e., a piperidinylethoxy group:
  • R 2 is ethyl, and R is OH, R"and R'"are H or OH groups, as shown in Table 8.
  • RBA of compounds of structures disclosed herein as potential ER ligands either prepared by solution methods or preferably prepared by combinatorial synthetic methods can be readily determined using testing methods disclosed herein. Differential binding affinity of compounds herein can also be readily determined using methods described herein.
  • Cyclopentadienes- The relative ER binding affinity data of cyclopentadienes 230- 237 are provided in Table 9.
  • the ER binding affinities of the cyclopentadiene ligands are generally lower than those of pyrazoles, but exhibit similar patterns of binding affinity as a function of substituents.
  • Cyclopentadiene 235 exhibits relative high ER binding affinity of 8.91%.
  • the pyridazines are much more polar than the other 5- and 6-member ring compounds. It is believed that the high polarity of the core is detrimental to ligand binding to ER.
  • Pyrimidines-R A values for several pyrimidines of structure PM4 are provided in Table 10.
  • the binding affinities of these pyrimidines for ER are generally lower than those of the 5- membered ring ligands. Again, however, the compounds exhibit similar patterns of binding affinity as a function of substituents.
  • Agonist/Antagonist Character of ER Ligands Compounds are tested as ER agonists/antagonists in transcriptional activation assays in cells expressing ER ⁇ or ER ⁇ . Cells are transfected with an expression plasmid for ER ⁇ or ER ⁇ together with an estrogen-responsive reporter gene construct e.g., (ERE) 3 -pS2-CAT, and treated with increasing concentrations of the test compound or with estradiol for comparison. Reporter gene expression is a measure of the capacity of ER complexed with various compounds to activate transcription, and it is followed as a function of concentration of the test compound. Potency and agonist character in activating transcription is measured relative to activation of the same system by estradiol.
  • an estrogen-responsive reporter gene construct e.g., (ERE) 3 -pS2-CAT
  • test compound The ability of the test compound to inhibit transcriptional activation by increasing concentrations of estradiol is also measured as a function of test compound concentration.
  • ability of a test compound to inhibit transcriptional activation by estradiol is a measure of antagonist character and antagonist potency of the test compound.
  • Transcriptional activation can be assessed with ER ⁇ or ER ⁇ and in different cells types.
  • CAT activity is measured as a function of the concentration of added test compound (typically ranging from 10 "12 - 10 "6 molar) in the presence or absence of the known stimulator (estradiol, typically ranging from 10 "12 - 10 "6 molar).
  • Agonist and/or antagonist character can be selective for ER ⁇ and ER ⁇ . Assays can be performed, for example, in human endometrial cancer (HEC-1) cells, Chinese hamster ovarian (CHO) cells, and HeLa cells. Agonist antagonist character can also be assessed with various promoters, e.g., the estrogen-responsive pS2 promoter, the simple TATA promoter, a non- consensus lactoferrin estrogen-responsive promoter, a heterologous thymidine kinase promoter and the complement C3 promoter which is an estrogen-responsive promoter that contains a non- consensus ERE.
  • HEC-1 human endometrial cancer
  • CHO Chinese hamster ovarian
  • Agonist antagonist character can also be assessed with various promoters, e.g., the estrogen-responsive pS2 promoter, the simple TATA promoter, a non- consensus lactoferrin estrogen-responsive promoter, a heterologous thymidine kinase promoter
  • the agonist/antagonist character of a given test compound relative to a selected ER ligand, e.g., estradiol, can be assessed using the transcriptional activation assays described.
  • a given compound may be a pure agonist activating expression and exhibiting no transcriptional inhibition, a pure antagonist suppressing stimulation of expression by known activators and not stimulating transcription themselves or a mixed agonist/antagonist showing both types of behavior.
  • Test compounds may exhibit selectivity in potency, where a given test compound stimulates transcription at lower concentration through one ER subtype than through the other ER subtype. Test compounds may exhibit selectivity in that they stimulate transcription or inhibit expression to a greater degree through one or the other of ER ⁇ and ER ⁇ . Test compounds can exhibit a different level of potency for activation compared to inhibition of stimulation of gene expression.
  • Figures 1A and B are graphs of transcriptional activation by ER ⁇ and ER ⁇ , respectively, i response to pyrazole compound 38b in HEC-1 cells using (ERE) 3 -pS2-CAT.
  • the figures plot CAT reporter activity as a function of the concentration of the ER ligand. Both figures also show the effect of estradiol (E2) on transcriptional activation by the ER subunits.
  • Pyrazole 38b is an
  • ER ⁇ potency selective agonist compared to estradiol.
  • the pyrazole exhibited a 120-fold higher potency in activating transcription via ER ⁇ than via ER ⁇ .
  • estradiol exhibits significantly lower activation selectivity between ER ⁇ and ER ⁇ .
  • Similar ER ⁇ potency-selective character was observed for this pyrazole in other cell types and with other estrogen-responsive promoters.
  • pyrazole compound 38b was found to bind to ER ⁇ three-fold more strongly than to ER ⁇ .
  • differences in relative binding of the ligand does not fully account for the significantly higher (120-fold) selectivity for activation exhibited by the pyrazole with ER ⁇ compared that exhibited by the pyrazole with ER ⁇ .
  • Figure 2 is a graph of transcriptional activation by ER ⁇ (diamonds) and ER ⁇ .(squares) in response to pyrazole 334 and pyrazole 336.
  • Both of the pyrazoles assayed are potent in activating transcription under the assay conditions through ER ⁇ , but are weak or very weak transcriptional activators through ER ⁇ . Both of these pyrazoles are ER ⁇ -potency selective agonists.
  • Pyrazole 336 exhibits no activation through ER ⁇ , even at the highest concentrations used. This pyrazole can be classified as an ER ⁇ -specific agonist. For both pyrazoles tested, the difference in ER ⁇ and ER ⁇ binding affinities parallels the observed potency selectivity or specificity.
  • FIGS. 3 A and 3B are graphs of the transcriptional profiles (CAT activity) of pyrazole 301 for ER ⁇ and ER ⁇ , respectively.
  • Pyrazole 301 displayed no agonist activity on ER ⁇ (Fig. 3B).
  • this compound was a partial agonist, reaching an efficacy level nearly half that of estradiol at 1 nM (Fig. 3 A).
  • the concentration of compound 301 increases, the ER ⁇ agonist activity returns to near basal levels.
  • pyrazole 301 acts as an antagonist through both ER ⁇ and ER ⁇ ., its potency as an antagonist through ER ⁇ being about 10-fold higher than through ER ⁇ , which is consistent with its higher affinity for the ER ⁇ subtype (see Table 8).
  • Pyrazole 301 is unusual, however, in that it exhibits a biphasic agonist-antagonist dose response through ER ⁇ . Many compounds exhibit partial agonist activity on ER ⁇ , and they are often more complete antagonists on ER ⁇ than on ER ⁇ . However, typically, as the concentration of ligand increases, a constant level of efficacy is reached in assays of agonist and antagonist activity. Pyrazole 301, in contrast, demonstrates agonist activity up to nearly 50%) that of estradiol, but its efficacy then decreases to only 10%o.
  • agonist character and antagonist character of compounds of structures disclosed herein as potential ER ligands either prepared by solution methods or preferably prepared by combinatorial synthetic methods can be readily determined using testing methods disclosed herein.
  • Acid addition salts are prepared by contacting compounds having appropriate basic groups therein with an acid whose anion is generally considered suitable for human or animal consumption.
  • Pharmacologically acceptable acid addition salts include, but are not limited, to the hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, and tartrate salts. All of these salts can be prepared by conventional means by reacting, for example, the selected acid with the selected basic compound.
  • Base addition salts are analogously prepared by contacting compounds having appropriate acidic groups therein with a base whose cation is generally considered to be suitable for human or animal consumption.
  • Pharmacologically acceptable base addition salts include but are not limited to ammonium, amine and amide salts.
  • esters of compounds of this invention are prepared by conventional methods, for example by reaction with selected acids.
  • Pharmaceutically acceptable esters include but are not limited to carboxylic acid esters RCOO-D (where D is a cationic form of a compound of this invention and where R is H, alkyl or aryl groups).
  • This invention is also directed to prodrugs and derivatives which on being metabolized will result in any of the ER ligands of this invention.
  • alkoxy or acetate groups can be metabolized to hydrogens.
  • Labile substituents may be protected employing conventional and pharmaceutically acceptable protecting groups removable on metabolism.
  • Pharmaceutically active compounds may be derivatized by conventional methods to provide for extended metabolic half-life, to enhance solubility in a given carrier, to provide for or facilitate slow- release or timed-release or enhance or affect other drug delivery properties.
  • compositions according to the present invention comprise one or more ER ligands of this invention in association with a pharmaceutically acceptable carrier or exipient adapted for use in human or veterinary medicine.
  • the carrier is generally selected, as is known in the art for the particular application and should be compatible with the active ingredients.
  • Such compositions may be prepared for use in conventional manner in admixture with one or more physiologically acceptable carriers or exipient.
  • the compositions may optionally further contain one or more other therapeutic agents which may, if desired, be known ER ligands
  • ER ligands are present in these pharmaceutical compositions in an amount or in a combined amount sufficient to elicit a measurable positive effect on a symptom or condition associated with an estrogen-dependent disorder on administration to an individual suffering from the symptom or disorder.
  • the ER ligands according to the invention may be formulated for oral, buccal, parenteral, topical or rectal administration.
  • the ER ligands according to the invention may be formulated for injection or for infusion and may be presented in unit dose form in ampules or in multidose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • Aqueous vehicles can be provided with pH control agents, electrolyte control or agents that enhance solubility of the active ingredients in the vehicle.
  • compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives.
  • compositions of this invention can contain from 0.001-99%)
  • ER ligands may be provided as pure regioisomers or as a mixture of regioisomers. Analogously, ER ligands may be provided as a mixture of enantiomeric forms or as a purified enantiomer.
  • the invention further provides a process for preparing a pharmaceutical composition which comprises bringing a ER ligand of the invention into association with a pharmaceutically acceptable exipient or carrier.
  • the carrier or exipient being selected as is known in the art for compatibility with the desired means of administration, for compatibility with the selected ER ligands and to minimize detrimental effects to the patient.
  • the daily dosage as employed for treatment of an adult human of approximately 70 kg body weight will range from 0.2 mg to 10 mg, preferably 0.5 to 5 mg, which can be administered in 1 to 4 doses, for example, depending on the route of adrninistration and the clinical condition of the patient.
  • These formulations also include formulations in dosage units.
  • the formulations are present in the form of a discrete pharmaceutical unit, for example, as tablets, dragees, capsules, caplets, pills, suppositories or ampules.
  • the active compound content of each unit is a fraction or a multiple of an individual dose.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses for 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains the amount of active compound which is given in one administration and which usually corresponds to a whole, one half, one third or one quarter of a daily dose.
  • a prophylactic or therapeutic dose of a particular compound will, of course, vary with the nature of the severity of the condition to be treated, the particular ER ligand compound and its route of administration. It will also vary according to the age, weight and response of the individual patient.
  • the compounds of the present invention are preferably formulated prior to administration.
  • the present pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients.
  • the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container.
  • the carrier When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, exipient or medium for the active ingredient.
  • the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10%) by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
  • Suitable carriers, exipient, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
  • compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.5 to about 150 mg, more usually about 0.1 to about 10 mg, of the active ingredient.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier.
  • any allowed for preparing medicines can be used, including but not limited to hydrochloric acid-sodium hydroxide, acetic acid-sodium acetate, glycine-sodium chloride-hydrochloric acid, potassium dihydrogenphosphate-disodium hydrogenphosphate, potassium hydrogenphthalate-sodium hydroxide, sodium secondary citrate-hydrochloric acid, sodium dihydrogen-phosphate-disodium hydrogenphosphate, sodium dihydrogenphosphate-dipotassium hydrogen-phosphate, potassium dihydrogenphosphate-dipotassium hydrogenphosphate, tartaric acid-sodium tartrate, lactic acid-sodium lactate, sodium barbital-sodium acetate-hydrochloric acid, succinic acid-boric acid, potassium primary citrate-sodium hydroxide, sodium primary citrate-borax, disodium hydrogenphosphate-citric acid, sodium acetate-hydrochloric acid, glutamic acid
  • hydrochloric acid-sodium hydroxide hydrochloric acid-sodium hydroxide, acetic acid-sodium acetate, glycine-sodium chloride-hydrochloric acid, tartaric acid-sodium tartrate, lactic acid-sodium lactate, sodium acetate-hydrochloric acid, glutamic acid-sodium hydroxide, and aspartic acid-sodium hydroxide.
  • This invention is further directed to therapeutic methods employing the ER ligands of this invention and pharmaceutical compositions containing them in the treatment of estrogen- dependent or estrogen-related disorders.
  • These methods comprise a step of administering to a patient having the disorder or symptoms thereof a pharmaceutical composition comprising one or a mixture of the ER ligands of this invention where the ER ligand or mixture of ligands is present in the composition at a level or a combined level sufficient to effect a positive biological response.
  • the present invention provides ER ligands that can be used in place of or in combination with currently known pharmaceuticals active in estrogen-dependent or estrogen- related disorders.
  • Certain ER ligands of this invention and certain ER ligands identified by the combinatorial synthetic methods and selective assays described herein can exhibit improved properties (enhanced activity and/or decreased undesired side-effects) for treatment of estrogen- dependent and estrogen-responsive disorders.
  • the ER ligands of this invention are useful in vitro and/or in vivo for selective activation or repression of expression, dependent upon the agonist or antagonist nature of the ligand or its potency, of a gene regulated by ER.
  • Gene activation or repression can be selective with respect to subtype of ER (e.g., ER ⁇ or ER ⁇ ), or variant of ER (e.g., splice variant forms, truncated or processed forms, covalently modified forms, etc.).
  • the ER ligands of this invention are also useful i vitro and/or in vivo for selective regulation of cellular activities under the control of ER.
  • Cellular activities may be regulated in a variety of ways by ER, subtypes of ER or variants of ER, e.g., up or down regulation of a given cellular process. Regulation is selective with respect to subtype of ER (e.g., ER ⁇ or ER ⁇ ), or variant of ER (e.g., splice variant forms, truncated or processed forms, covalently modified forms, etc.).
  • Cellular activities that may be regulated include both genomic (related to gene expression) or non-genomic activities (not directly related to gene expression, e.g., such as regulation of calcium flux, particularly in bone cells, hormone release, particularly prolactin release from pituitary cells, etc.).
  • the subtype-selective ER ligands of this invention can also be of general use in the investigation of ER and its functions. These ligands can be employed to better understand structure and conformation of ER (both subtypes) and to elucidate how ER subtypes interact with other molecules and to relate structure, conformation and interaction with other molecules to ER function.
  • Agents that can act selectively to stimulate or inhibit estrogen action through the individual ER subtypes can be useful in achieving selective regulation of specific responses and specific tissues.
  • ER ⁇ appears responsible for mediating the beneficial effects of estrogens in suppressing vascular cell overgrowth in response to blood vessel injury. Therefore, an ER ligand that antagonizes only ER ⁇ -mediated responses should block this response without blocking desired responses to estrogens that are mediated by ER ⁇ , such as maintenance of a favorable profile of blood lipids.
  • Preferred ER ligands of this invention which exhibit selective interaction with ER subtypes can be employed to selectively stimulate or inhibit estrogen action.
  • references that relate to tissue distribution of ER subtypes include: Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, Nilsson S 1998 Differential response of estrogen receptor a and estrogen receptor ⁇ to partial estrogen agonists/antagonists. Mol Pharmacol 54:105-112; Couse JF, Lindsey J, Grandien K, Gustafsson J-A, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-alpha and estrogen receptor-beta messenger ribonucleic acid in the wild type and ER-alpha knockout mouse.
  • the estrogen subtypes, ER ⁇ and ER ⁇ are the products of two different genes. However, variant forms of both ER subtypes are known. ER ⁇ variants having different N-terminal lengths that correspond to different transcriptional start sites are known ( McLnemey EM, Weiss KE, Sun J, Mosselman S, Katzenellenbogen BS 1998 Transcription activation by the human estrogen receptor subtype ⁇ (ER ⁇ ) studied with ER ⁇ and ER ⁇ receptor chimeras. Endocrinology 139:4513-4522; Montano MM, Jaiswal AK, Katzenellenbogen BS 1998 Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor- ⁇ and estrogen receptor- ⁇ .
  • ERs can be covalently modified by post-transcriptional events, such as phosphorylation, acetylation, and glycosylation. These modifications can also alter ER responsiveness to different ER ligands (Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor: Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 269:4458-4466; Kato SH, Endoh Y, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masucshige S, Gotoh Y, Nishida E,
  • estrogens appear to be non-genomic, and may involve action through ERs in the cell membrane. Examples of such responses are stimulation of calcium flux regulation in bone cells and prolactin release from pituitary cells (Lieberherr M, Grosse B,
  • Methods for selective regulation of cellular activities through ER employing the ER ligands of this invention can be used with variant, mutant and modified ERs as described herein and as known in the art.
  • the interaction of ER ligands of this invention with variant, mutant and modified ERs can be assessed as described herein for ER.
  • Differential ligand binding affinities for ER ⁇ and ER ⁇ can be determined by competitive radiometric binding assays using 10 nM [ 3 H]estradiol as tracer, and hydroxylapatite to adsorb bound receptor-ligand complex, as described previously [Carlson, K.E. et al. (1997) "Altered ligand binding properties and enhanced stability of a constitutively active estrogen receptor: evidence that an open-pocket conformation is required for ligand interaction," Biochemistry 36:14897-14905].
  • Differential assays are performed using purified preparations of human ER ⁇ (amino acids 304-554) and ER ⁇ (203-452) ligand binding domains expressed in E. coli or using full length ER ⁇ and ER ⁇ expressed in Baculovirus (commerically available).
  • Compounds are tested as ER agonists/antagonists in transcriptional activation assays in cells expressing ER ⁇ or ER ⁇ .
  • Cells are transfected with an expression plasmid for ER ⁇ or ER ⁇ together with an estrogen-responsive reporter gene construct e.g., (ERE) 3 -pS2-CAT, and treated with increasing concentrations of the test compound or with estradiol for comparison.
  • Reporter gene expression is a measure of the capacity of ER complexed with various compounds to activate transcription, and it is followed as a function of concentration of the test compound. Potency and agonist character in activating transcription is measured relative to activation of the same system by estradiol.
  • test compound The ability of the test compound to inhibit transcriptional activation by increasing concentrations of estradiol is also measured as a function of test compound concentration.
  • ability of a test compound to inhibit transcriptional activation by estradiol is a measure of antagonist character and antagonist potency of the test compound.
  • Transcriptional activation can be assessed with ER ⁇ or ER ⁇ and in different cells types.
  • CAT activity is measured as a function of the concentration of added test compound (typically ranging from 10 '12 - 10 "6 molar) in the presence or absence of the known stimulator (estradiol, typically ranging from 10 "12 - 10 "6 molar).
  • Agonist and/or antagonist character can be selective for ER ⁇ and ER ⁇ . Assays can be performed, for example, in human endometrial cancer (HEC-1) cells, Chinese hamster ovarian (CHO) cells, and HeLa cells.
  • Agonist/antagonist character can also be assessed with various promoters, e.g., the estrogen-responsive pS2 promoter, the simple TATA promoter, a non- consensus lactoferrin estrogen-responsive promoter, a heterologous thymidine kinase promoter and the complement C3 promoter which is an estrogen-responsive promoter that contains a non- consensus ERE.
  • various promoters e.g., the estrogen-responsive pS2 promoter, the simple TATA promoter, a non- consensus lactoferrin estrogen-responsive promoter, a heterologous thymidine kinase promoter and the complement C3 promoter which is an estrogen-responsive promoter that contains a non- consensus ERE.
  • the agonist antagonist character of a given test compound relative to a selected ER ligand, e.g., estradiol, can be assessed using the transcriptional activation assays described.
  • a given compound may be a pure agonist activating expression and exhibiting no transcriptional inhibition, a pure antagonist suppressing stimulation of expression by known activators and not stimulating transcription themselves or a mixed agonist/antagonist showing both types of behavior.
  • Test compounds may exhibit selectivity in potency, where a given test compound stimulates transcription at lower concentration through one ER subtype than through the other ER subtype. Test compounds may exhibit selectivity in that they stimulate transcription or inhibit expression to a greater degree through one or the other of ER ⁇ and ER ⁇ . Test compounds can exhibit a different level of potency for activation compared to inhibition of stimulation of gene expression.
  • Pyrazole compound 38b was found to be an ER ⁇ potency selective agonist compared to estradiol when assayed in HEC-1 cells using (ERE) 3 -pS2-CAT. It exhibited a 120-fold higher potency in activating transcription via ER ⁇ than via ER ⁇ . In contrast, estradiol exhibits significantly lower activation selectivity between ER ⁇ and ER ⁇ . Similar ER ⁇ potency-selective character was observed for this pyrazole in other cell types and with other estrogen-responsive promoters. Pyrazole compound 38b was found to bind to ER ⁇ three-fold more strongly than to ER ⁇ .
  • the expression vector pCMN5-ER ⁇ was constructed by inserting the full-length cD ⁇ A encoding human ER ⁇ (530) residues, p ⁇ GVl-ER ⁇ (Mosselmen et al. (1996) supra) and including the additional 53 ⁇ -terminal amino acids as found in Genebank accession number AF
  • the estrogen responsive reporter plasmids were (ERE) 3 -pS2-CAT, constructed as described previously (Kraus, W.L. et al. (1995), "Ligand- dependent, transcriptionally productive association of the amino-and carboxyl-terminal regions of a steroid hormone nuclear receptor," Proc. ⁇ atl. Acad. Sci. USA 92:12314-12318), (ERE) 2 - TATA-CAT [Wrenn, CD. and Katzenellenbogen, B.S. (1993), "Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast," J. Biol.
  • C3-Ti-LUC which contains - 1030 to +58 of the human complement C3 promoter fused to the firefly luciferase reporter gene ( ⁇ orris, J.D. et al. (1996), "Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity," Mol. Endocrinol. 10:1605-1616), and lactoferrin ERE-tk-CAT, which contains 2 copies of the non-consensus lactoferrin ERE fused to the thymidine kinase promoter and CAT reporter gene.
  • the plasmid pCHl 10 (Pharmacia, Piscataway, ⁇ J) or pCMN ⁇ (Clontech, Palo Alto, CA) which contains the ⁇ -galactosidase gene, was used as an internal control for transfection efficiency.
  • Expression vectors employed herein are comrnerically available or available through routine preparations using published information.
  • HEC-1 Human endometrial cancer
  • CHO Chinese hamster ovary
  • HeLa HeLa cells
  • J. Biol. Chem. 268:24089-24098 J. Biol. Chem. 268:24089-24098
  • Montano M.M. et al. (1995)
  • the carboxyl-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists Mol. Endocrinol.
  • CAT or luciferase activity normalized for the internal control ⁇ -galactosidase activity, is assayed as described (Montano, M.M. et al. (1995), "The carboxyl-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists," Mol. Endocrinol. 9:814-825; Mclnemey, E.M. and Katzenellenbogen, B.S. (1996),
  • Oxazole 30 (22.0 mg, 0.062 mmol) was demethylated according to the general BF 3 -SMe 2 procedure above to give deprotected oxazole 31 as an off-white powder (18.1 mg, 89%>).
  • Step 1 A well stirred mixture of the o-phenylenediamine dihydrochloride (1 mmol) and the ⁇ -diketone (1 mmol) in acetic acid were refuxed for 3.5-4.0 hr. The reaction mixture was cooled, poured into ice and extracted with ethyl acetate (3x10 ml). The combined extracts were washed with brine, dried (an. Na ⁇ SO 4 ) and concentrated. Purification of the residue over a silica gel column using 30% > ethyl acetate-hexane as eluent furnished an ⁇ 1 : 1 unseparable mixture of the quinoxalines.
  • Step 2 To a magnetically stirred solution of the isomeric mixture of protected quinoxalines (lmmol) in dichloromethane was added boron triflouride-dimethyl sulfide (10 mmol phenolic gp.) and the stirring continued for 2 days at room temperature. After quenching with water, the layers were separated and the aqueous layer extracted with ethyl acetate (3x10 ml). The combined extracts were washed with satd. bicarbonate solution, brine and dried (an.
  • Table 6A ESTROGEN RECEPTOR BINDING AFFINITY DATA FOR PYRAZOLES AND ISOXAZOLE
  • R R" R'" R 2 RBA% RBA, ER ⁇ RBA, ER ⁇ cytosol
  • R 2 H
  • R 3 p-CH 3 0-C 6 H 4
  • R 4 C 2 H 5 3 0-C 6 H 4
  • R ⁇ H
  • rV 3 -
  • R ethyl or propyl
  • R 2 OCH 3
  • R 2 OCH3
  • R 3 propyl
  • R ⁇ ethyl
  • R 3 propyl
  • T2 Synthesized by choice of starting diketone i.e.
  • R Et or ⁇ -Pr 13
  • R ⁇ aryl ⁇ Na 2 C0 3 eg C 6 H 5 , H 2 0/DME

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Abstract

L'invention concerne des ligands non stéroïdes se liant aux récepteurs d'oestrogènes qui possèdent une structure moléculaire qui peut faire l'objet d'une synthèse en phase solide ainsi que l'application de méthodes combinatoires synthétiques à des fins de préparation de ces ligands se liant aux récepteurs d'oestrogènes. Les ligands des RO de l'invention sont constitués d'un élément noyau structurant qui est un noyau carbocyclique ou hétérocyclique à 5 éléments possédant deux liaisons doubles ou un noyau aromatique à 6 éléments. Plusieurs substituants sélectionnés sont liés au noyau de façon sensiblement indépendante des autres constituants. La structure modulaire de ces composés permet de synthétiser un nombre très élevé de variations structurelles de substituants, de combinaisons de substituants et de positions de substituants sur le noyau. Les variantes structurelles des ligands des RO manifestent un éventail d'affinités sélectives avec ROα et ROβ et une gamme de propriétés d'agoniste/d'antagoniste.
PCT/US1999/022747 1998-10-02 1999-10-01 Ligands se liant aux recepteurs d'oestrogenes WO2000019994A1 (fr)

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US7235539B2 (en) 2002-01-23 2007-06-26 Wyeth 6-Hydroxyequilenins as estrogenic agents
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CN107474038B (zh) * 2017-07-17 2020-10-09 苏州楚凯药业有限公司 一类靶向雌激素受体荧光探针及其制备和使用方法
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US11970457B2 (en) 2018-10-10 2024-04-30 Vtv Therapeutics Llc Metabolites of [3-(4-(2-butyl-1-[4-(4-chloro-phenoxy)-phenyl]-1H-imidazol-4-yl)-phenoxy)-propyl]-diethyl-amine
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