WO2000019994A1 - Estrogen receptor ligands - Google Patents

Estrogen receptor ligands Download PDF

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Publication number
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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French (fr)
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WO2000019994A9 (en
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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
Publication of WO2000019994A1 publication Critical patent/WO2000019994A1/en
Publication of WO2000019994A9 publication Critical patent/WO2000019994A9/en

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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

Abstract

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 that is a carbocyclic or heterocyclic-5-member ring that has two double bonds or a 6-member aromatic ring. A plurality of selected substituents are bonded to the ring substantially independently of other substituents. The modular structure of these compounds allows for synthesis of a very large numer of substituent structural variations, substituent combinations and substituent positioning on the core. The structural variants of the ER ligands of this invention exhibit a spectrum of selective affinities for ERα and ERβ and a spectrum of agonist/antagonist properties.

Description

ESTROGEN RECEPTOR LIGANDS This invention was made at least in part through United States government funding through the National Institutes of Health (PHS 5R37 DK15556) and the U.S. Army (DAMD17- 97-1-7076). The United States government has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATIONS This application takes priority under 35 U.S.C § 119(e) from U.S. provisional application serial number 60/102,881 filed October 2, 1999 which is incorporated in its entirety by reference herein.
BACKGROUND OF THE INVENTION 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].
The molecular target of estrogens is the estrogen receptor (ER), of which there are now known to be two subtypes, ER-α and ER-β, that have different patterns of tissue expression and somewhat different ligand binding specificities [Mosselman, S. et al. (1996), "ERβ: 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 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.
10:1167-1177]. These co-regulators have interaction functions that tether ER to the RNA polymerase II preinitiation complex, as well as enzymatic activities to modify chromatin structure [Glass, C.K. et al. (1997), "Nuclear receptor coactivators," Curr. Opin. Cell. Biol. 9:222-232]. Each cell type and each gene presents to an ER(subtype)-ligand complex a unique combination of these effector components - various estrogen response elements and co- regulators - that appear to underlie, in part, the cell and gene selectivity of various estrogens [Katzenellenbogen, J.A. et al. (1996), "Tripartite steroid hormone receptor pharmacology: interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones," Mol. Endocrinol. 10:119-131]. 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.
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]. For patients with estrogen-responsive tumors, 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]. .. Among 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; Solrnssenr UN. (1945), "Synthetic estrogens and the relation between their structure and their activity," Chem. Rev. 37:481-598], Minor changes in the structure and stereochemistry of these ligands can have profound effects on both their affinity for ER, as well as their biocharacter (i.e., agonist vs antagonist balance). Major efforts have been directed toward optimizing ER ligand structure to obtain desired profiles of tissue selectivity, and even so, the ideal profile for various uses has not yet been achieved [Grese, T.A. et al. (1997), "Molecular deteπninants of tissue selectivity in estrogen receptor modulators," Proc. Νatl. Acad. Sci. USA 94:14105-14110; Grese, T.A. et al. (1998), "Synthesis and pharmacology of conformationally restricted raloxifene analogues: highly potent selective estrogen receptor modulators," J. Med. Chem. 41:1271-1283].
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, Ν. (1995) New Eng. J. Med. 332.:1638-1639 and Katzenellenbogen, B.S. (1991) J. Natl. Cancer Inst.83 :1434-1435]. In contrast, the stimulatory effects of tamoxifen in bone cells can be beneficial for the prevention of osteoporosis in postmenopausal women [Katzenellenbogen, B.S. (1996) Biol.Reprod. 54:287-293]. Pure antiestrogens, such as ICI 164,384 also show promise for hormonal therapy for estrogen-positive breast cancer, but exhibit detrimental effects on other estrogen positive tissues (bone, central nervous system and the cardiovascular system). A selective endocrine profile, as yet not achieved, which effects the
Figure imgf000006_0001
Tamoxifen Estradiol ICI 164,348
desired inhibitory response in targeted tumor cells, while avoiding detrimental inhibitory or stimulatory effects in other tissues, is preferred in a drug for use in hormonal therapy for estrogen-positive breast cancer.
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]. More recently, analogous techniques employing solid-phase organic synthesis have been applied to the development of non-peptide libraries [Bunin, B.A. and Ellman, J.A. (1992), "A general and expedient method for the solid-phase synthesis of 1 ,4-benzodiazepine derivatives," J. Am. Chem. Soc. 114:10997- 10998; Bunin, B.A. et al. (1994), "The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library," Proc. Natl. Acad. Sci. USA 91:4708-4712; Hobbs- Dewitt, S. et al. (1 93), "'Diversomers': an approach to nonpeptide, nonoligomeric chemical diversity," Proc. Natl. Acad. Sci. USA 90:6909-6913; Beebe, X. et al. (1992), "Polymer- supported synthesis of 2,5-disubsti ted tetxahydrofurans," J. Am. Chem. Soc. 114:10061-10062; Chen, C. et al. (1994), "Analogous organic synthesis of small-compound libraries: validation of combinatorial chemistry in small-molecule synthesis," J. Am. Chem. Soc. 116:2661-2662; and
Zuckerman, R.N. et al. (1994), "Discovery of nanomolar ligands for 7-transmembrane G-protein coupled receptors from a diverse (N-substituted)glycine peptoid library," J. Med. Chem. 37:2678-2685]. Combinatorial techniques have been applied to the synthesis of pyrazoles from 1,3-diketones on solid support [Mamnzik, A.L. and Felder, E.R. (1996) Tetrahedron Lett. 37:1003] and to the synthesis of various heterocycles from α,β-unsaturated ketones [Marzinzik,
A.L. and Felder, E.R.(1998) J. Org. Chem. 63:723-727]. Solid-state synthesis of tetra- and penta-substituted pyrroles has been reported [Mjalli A.M.M. et al. (1996) Tetrahedron Lett. 37:2943-2946].
For the most part, 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. For example, 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]. However, 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.
SUMMARY OF THE INVENTION 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:
Figure imgf000008_0001
where x is 0 or 1 and where the core ring A is a carbocyclic or heterocyclic ring having two double bonds, if it is a 5-membered ring (x = 0) or that is aromatic, if it is a 6-membered ring (x = 1). 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.
In one aspect, 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:
(R6) ;≠)
R2 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.
Dependent upon the core structure, the position of the heteroatom(s) relative to a particular R group and/or the placement of double bonds, the core can accommodate up to 3 to 6 substituents. Substituent Rl is required in the compounds of this invention. All 5-membered ring cores accommodate a minimum of 3 substituents (R1 and two of R2 , R3, R4 and R'). It is preferred that both R1 and R3 or Rl and R4 be present and that they are both non-hydrogen substituents. It is more preferred that R1, R\ and R3 or R1, R2, and R4 be present and that each is a non-hydrogen substituent. Certain cores may accommodate one (e.g., pyrroles) or two (e.g., cyclopentadienes) additional substituents, R5 and R6 (which can be hydrogens), on the ring atoms. Bonds to these substituents are indicated by dashed lines and each possible R^ and R6 is placed within parentheses to indicate that they are not present in all core structures and that the position of R6 can be varied in certain cores. Typically there will only be one R6 substituent in a given 5- membered ring core, which, however, may be at any ring position. In cyclopentadienones, CR4R6 or CR5R6 can represent C=O.
In a second aspect, 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:
Figure imgf000010_0001
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.
Dependent upon the core structure, the position of the heteroatom(s) relative to a particular R group and/or the placement of double bonds, the aromatic core can accommodate 4 to 6 substituents. Substituent R1 is required in the compounds of this invention. Six-membered ring cores accommodate a minimum of 4 substituents (R1 and three of R2 , R3, R4 , R3 and R5'). Substituent RD' is selected from the same groups as R5 and may be the same or different from R\ RJ or R1 and R4 be present and that they are both non-hydrogen substituents. It is more preferred that R1, R2, and R3 or R1, R2, and R4 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 R1 are indicated by dashed lines to show that they may be absent dependent upon heteroatom placement.
Substituent R1 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., CF3 or CF2CF3. The R1 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 R1 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 R1 group is p-hydroxyphenyl. In general, the R1 phenyl group can carry any substituent that can be metabolically converted into a p-OH group, e.g., OCH3, O-COCH3, etc.
Substituents R\ R3 and R4, 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 Rl ), 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 CF3 or CF2CF3. 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 R2, R3, R4 are lower alkyl or alkenyl groups, and phenyl and o-, m-, or p-substituted phenyl groups. More preferred R2 are ethyl and propyl (straight-chain or branched) groups. More preferred R4 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 R3 are phenyls and substituted phenyls, including o-, m-, or p-hydroxyphenyl, o-, m-, or p-alkoxyphenyl, o-, m-, or p-halophenyl.
Substituents R5 or R5' when present, e.g., in pyrroles and cyclopentadienes and other 5- member ring systems (Table 1) or aromatic systems (Table 2), can be selected from any of the groups defined for R\ R3 and R4 and may be hydrogens. R5 and R5' may be the same as or different than any of R1, R2, R\ or R4. Substituents R5 or R5' may be the same or different than each other.
R6 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 R6 groups can include halogen and hydroxy group substitution. One or more of any -CH2 - groups in Rδ can be replaced with -CO- groups. R6 is preferably a lower alkyl or hydrogen.
In specific embodiments, the R'-R4 (and R5, R5- or R6 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(CH2)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.,
Figure imgf000013_0001
where RA (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-.
In general 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(CH2)n-CH(OH)-CH(OH)-RB 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, RB is H or -(CH2)m-CH3ι where m is an integer from 0 to about 6. Diols can also be alicyclic; Amides:
-(X)x(CH2)nCO-NRR' where 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, and 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; and
Sulfoxides:
-(X)x(CH2)nSOR' where X is O or S, x is 0 or 1, n is an integer from 1 to about 12, including those with n = 1 to about 5 and those with n= 6 to about 10, and 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 CF3 and CF2CF3;
Sulfones:
-(X)x(CH2)πSO2R' where X is O or S, x is 0 or 1, n is an integer from 1 to about 12, including those with n = 1 to about 5 and those with n= 6 to about 10, and 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 CF3 and CF2CF3.
See Scheme 17 for exemplary basic and polar substituents.
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 R2-R6 or Ry substituent or can be a substituent on any of R'-R6 or R3'.
In five-membered ring compounds any two substituents on a given ring carbon can be linked to form a spiro-ring. For example, substituents R3 and R6 or R4 and R6 on the same ring atom can together form a carbon chain -(CH2 )„- where n is 3 to about 6 to form a spiro ring system with the parent cycle A. Carbons in the R5 R6 chain or R4/R6 chain may also be substituted, e.g., with halogens or lower alkyl, alkenyl or alkynyl groups, and one or two of the CH2 groups of the chain may be replaced with an -CO-, -0- , -S- or an -NH-.
Substituents on adjacent ring atoms, e.g., the pairs Rl and R2 , R2 and R3 , R3 and R4, R1 and R3, R 5' or R6, 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 R2 can be linked to a phenyl substituent at R' or R3 .
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.
In specific embodiments, 5- and 6-member ring ER ligands of this invention can contian two substituted or non-substituted phenyl rings in addition to R'. In other specific embodiments, R1 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. In other specific embodiments, R1 is a p-substituted phenyl group, where the substituent is OH or OR where R is a lower alkyl group, R2 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.
In more preferred embodiments, the ER ligands of this invention have core structures as listed in Tables 1 and 2 where the substituents R'-R6 and R5' are as defined above. Structural variants in addition to those listed in Table 1 and 2 may be obtained by interchanging the positions of R2, R3, R4, and R5. ER ligands are those compounds which exhibit measurable binding affinity for the estrogen receptor in assays as described herein.
Exemplary compounds of this invention are provided in Scheme 18. .. 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. In those cases in which the ER ligand selectively interacts with an ER subtype or variant, 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.
Pharmaceutical 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., ERα or ERβ), 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., ERα or ERβ), 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.
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB illustrate transcriptional activation by ERα and ERβ, respectively, in response to the pyrazole compound 38 b. Human endometrial cancer (HEC-1) cells were transfected with expression vectors for ERα (Fig. 1A) or ERβ (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 β-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 ERα and ERβ response with 10 nM E2. See: J. Sun et al. (1999) Endocrinology 140 (2):800-804.
Figure 2 illustrates transcriptional activation by ERα and ERβ in response to two pyrazoles XXX (solid lines) and XXXI (dashed lines). HEC-1 cells were transfected with expression vectors for ERα (diamonds) and ERβ (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 β-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 ERα and ERβ response with 1 nM E2. .. Figures 3 A and 3B are transcriptional activation profiles for ERα (Fig. 3A) and ERβ (Fig. 3B) in response to pyrazole XXXX. HEC-1 cells were transfected with expression vectors for ERα or ERβ 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 β-galactosidase activity from an internal control plasmid. Values are the mean ± SD for three or more separate experiments.
DETAILED DESCRIPTION OF THE INNENΗON 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):
Figure imgf000019_0001
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.
In part because they allow some flexibility in orientation of substituent groups and in part because their synthesis is amenable to solid-phase methods, five-member and six-member carbocyclic and heterocyclic rings were selected for preparation of ER ligands based on the illustrated pharmacophore.
Two substructural motifs noted in known ER ligands are 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.
Figure imgf000020_0001
Motif A. Homobibenzyl Motif B. Syn-Bibenzyl
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. The representative cores listed are distinct from one another in the position of the R1 substituent on the selected rings with respect to heteroatoms (and or double bonds) and other substituents therein, in the cyclopentadienes with respect to the tetrahedral carbon, or in the cyclopentadienones with respect to the C=O group on the ring. Dependent upon the selection of a particular R'-R6 or R substituent distinct structures may be obtained by interchange of substituents.
In specific embodiments, ER ligands of this invention can have the structures:
Figure imgf000021_0001
where 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;
Figure imgf000021_0002
where x is 0 or 1 and 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 R3 and can be a polar or basic substituent and other variables are defined as in the Summary above;
Figure imgf000021_0003
where, x is 0 or 1 , 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;
Figure imgf000022_0001
where x is 0 or 1, 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;
Figure imgf000022_0002
where x is 0 or 1 , X is N, NH, NR\ S or 0 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;
Figure imgf000022_0003
where x is 0 or 1, X is N, NH, NR5, 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;
Figure imgf000023_0001
where one of 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;
Figure imgf000023_0002
where R -R are defined as in the Summary above;
Figure imgf000023_0003
where R2 and R4 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;
Figure imgf000023_0004
where R2 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
Figure imgf000024_0001
where 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 R2 are straight-chain or branched lower alkyl groups having up to 6 carbons atoms.
Specific compounds having the illustrated structures are listed in Scheme 18.
Pyrazoles of particular interest having significant ER binding affinity (RBAs) include those having the structure:
Figure imgf000024_0002
where R2 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-CH3, 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 (RB As) include those having the structure:
Figure imgf000025_0001
where R2 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-CH3, 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:
Figure imgf000025_0002
where R2 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-CH3, 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.
In specific embodiments, ER ligands of this invention can have the structures:
Figure imgf000026_0001
where the 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;
Figure imgf000026_0002
where 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 R3 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;
Figure imgf000027_0001
where 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;
Figure imgf000027_0002
Figure imgf000028_0001
or
Figure imgf000028_0002
where 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;
or
Figure imgf000029_0001
Figure imgf000029_0002
where 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;
Figure imgf000030_0001
where 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 R2 is defined as in the Summary above, but is preferably a lower alkyl groups having up to about 6 carbon atoms; and
Any of:
Figure imgf000030_0002
or
Figure imgf000031_0001
where 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 .
Five- and six-membered ring core compounds of this invention substituted with a basic or polar side chain are of particularly interest as potential SEEMS (selective estrogen receptor modifiers) which can display agonist or antagonist activity (or a mixture of these activities) that can vary from tissue to tissue. 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:
Figure imgf000031_0002
a basic or polar group can be substituted at R, R2, 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 R2 are lower alkyl groups having up to about 6 carbon atoms.
Synthesis ofER Ligands
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 rα-formaldehyde affords the 4,5- disubstituted imidazole 2 [Bredereck, H., Gompper, R. & Hayer, D. (1959). Imidazole aus α- Diketonen Chem. Ber. 92, 338-343], which upon deprotection with BBr in CH2C12 affords the imidazole 3 in good yield. A similar reaction using 4-methoxybenzaldehyde affords the 2,4,5- trisubstituted imidazole 4 [Lombardino, J.G. & Weisman, E.H. (1974), "Preparation and antiinflammatory activity of some nonacidic trisubstituted imidazoles,". J. Med. Chem. 17, 1182- 1 188; Schubert, V.H., Giesemann, G., Steffen, P. & Bleichert, J. (1962), "p-Aryl- und p- Alkoxyphenyl-imidazole," J. Prat. Chem. 18, 192-202; Hayes, J.F., Mitchell, M.B. & Wicks, C. (1994), "A novel synthesis of 2,4,5-triarylimidazoles," Heterocycles 38, 575-585]. To prepare tetrasubstituted systems, the sodium salt of imidazole 4 is alkylated with ethyl, propyl, and butyl iodide, and then deprotected to afford free phenols 6a-d.
Unsymmetrical, imidazoles (IM1 core) are synthesized as outlined in Schemes IB and 2. Scheme IB illustrates the synthetic approach to N-ethyl imidazole 12. Reaction of 4-methoxy- deoxybenzoin (7) [Gardner, P.D. (1956), "Organic peracid oxidation of some enol esters involving rearrangement, "J. Am. Chem. Soc. 78, 3421-3424] with bromine and a trace of A1C13 in Et2O gives α-bromoketone 8 [Jenkins, S.S. (1934) "The grignard reaction in the synthesis of ketones. IV. A new method of preparing isomeric unsymmetrical benzoins,". J. Amer. Chem. Soc. 56, 682-684] which, upon reaction with sodium azide in acetone, affords the corresponding azide 9. The azido-ketone 9 is treated with one equivalent of Et3N and imine 10 in THF. Removal of solvent and excess Et3N followed by treatment of the crude intermediate 2,5- dihydro-2-hydroxyimidazole with TFA in CH Cl2, according to the procedure of Patonay
[Patonay, T. & Hoffman, R.V. (1995), "Base-Promoted Reactions of α-Azido Ketones with Aldehydes and Ketones: A Novel Entry to α-Azido-β-hydroxy Ketones and 2,5-Dihydrp-5- hydroxyoxazoles, "Journal of Organic Chemistry 60, 2368-2377], results in the formation of N- ethyl imidazole 11. Deprotection with BF3-SMe2 in CH2C12 produces imidazole 12 in good yield.
The 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-α-bromobutyrophenone (13) wifh/?-anisidine in acetone gives the α-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 BF3-SMe7 in CH2C12 produces the free phenol 17.
The methods illustrated in Schemes 1A, IB and 2 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 imidazole ring core structures. Details of syntheses of representative imidazole are provided in the Examples.
Thiazoles - The synthesis of representative thiazoles is shown in Scheme 3. Thioamide 19 (when R' = Me), derived from 4-alkoxybenzonitrile (18 when R = Me) [Taylor, E.C. & Zoltewicz, J.A. (1960), "A new synthesis of aliphatic and aromatic thioamides from nitriles,"J Am. Chem. Soc. 82, 2656-2657] is condensed with 4'-alkoxy-α-bromoacetophenone (20 when R = Me) or 4'-alkoxy-α-bromobutyrophenone (13 when R' = Me) in refluxing DMF to give the 2,4- disubstituted thiazole, e.g., 21a, [Dolling, K., Zaschke, H. & Schubert, H. (1979), "Kristallin- flussige Thiazole, "J. Prat. Chem. 321, 643-654] or 2,4,5-trisubstituted thiazole, e.g., 21b, respectively. Deprotection with BBr3 affords moderate yields of the free phenols, e.g., 22a and 22b. Details of a representative syntheses are provided in the Examples.
The methods illustrated in Scheme 3 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 thiazole ring core structures.
Oxazoles - Representative oxazoles can be synthesized, as outlined in Schemes 4A and B. Reaction of the lithium anion of dithiane 23 with 7-methoxybenzyl bromide gives the alkylated product (24 when R = Me) which upon hydrolysis affords 4'-alkoxy-deoxybenzoin (25 when R = Me ) [Katrizky, A.R., Boulton, A.J. & Short, D.J. (1960), "Interaction at a distance in conjugated systems. Part III. Effect of aryl and heteroaryl groups on the infrared intensities of C=C and C+C stretching bands," J Chem. Soc. , 1519-1523] in excellent yield. Conversion to the bromide (26 when R = Me) [Cowper, R.M. & Stevens, T.S. (1940), "Mechanism of the reaction between arylamines and benzoins, "J. Chem. Soc. , 347-349] and azide (27 when R = Me ) is accomplished as described for analogous compounds 8 and 9 above. The azido-ketone
27 is then treated with one equivalent of Et3N and 7-anisaldehyde, and then with TFA to afford oxazole (28 when R = Me ) [Strzybny, P.P.E., van ES, T. & Backeberg, O.G. (1969), "Reaction of α-acyloxyketones with ammonium acetate, "J. South African Chem. Inst. 22, 158-164]. Oxazole 30 results from the condensation of bromo-ketone 26 with -methoxybenzamide in refluxing toluene (Scheme 4B) analogous to the thiazole synthesis discussed above.
Deprotection of 28 and 30 with BF3-SMe2 gives oxazoles 29 and 31, respectively. Details of a representative synthesis are provided in the Examples.
The methods illustrated in 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]. The method of 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. Condensation of the diketone with hydrazine hydrochloride or N- substituted hydrazine hydrochlorides in refluxing DMF/THF (1 : 1) affords the 3,5-disubstituted pyrazole 34a or 1 ,3,5-trisubstituted pyrazoles 34b-d; yields can be higher with aryl-substituted hydrazines than with hydrazine itself [van Steenis, J. (1946), "The nitration of dianisoylmethane and p-methoxydesoxybenzoin, " Chem. Ber. 29-46; Hergenrother, P.M. (1991), "New Developments in Thermally Stable Polymers, " Rec. Travl Chim. Pays-Bas. 110, 481-491; Ando, W., Sato, R., Yamashita, M., Akasaka, T. & Miyazaki, H. (1983), "Quenching of singlet oxygen by l,3,5-triaryl-2-pyraazolines, "J Org. Chem. 48, 542-546]. Deprotection of 34a-d with BBr3 affords the free phenols 35a-d.
The introduction of a 4-alkyl substituent was accomplished through the alkylation of diketone 33 with TBAF and an alkyl iodide (e.g., ethyl iodide) to afford 36. [Tewari, S.C. & Rastogi, S.N. (1979), "Studies in antifertility agents: Part XXH: 1 ,2-diethyl- 1 ,3-bis-(p- hydroxyphenyl)-l-propene, " Ind. J. Chem. 18B, 62-64; Clark, J.H. & Miller, J.M. (1977),
"Hydrogen bonding in organic synthesis. Part 6. C-Alkylation of β-dicarbonyl compounds using tetralkylammonium fluorides, " J. Chem. Soc, Perkin l , 1743-1745] . Conversion of diketone 36 to the corresponding pyrazoles is accomplished as with the unsubstituted case, to afford pyrazoles 38a-d. Details of representative syntheses are provided in the Examples.
Alternatively, 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 R1 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.
The methods illustrated in Schemes 5A-D 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 pyrazole ring core structures.
Isoxazoles - An illustrative preparation of an isoxazole is shown in Scheme 6 [Perkins, M., Beam, C.F., Dyer, M.C.D. & Hauser, CR. (1988), "3-(4-Chlorophenyl)-5-(4- methoxyphenyl)isoxazole, " Org. Syn. Coll. Vol VI, 278-281]. Double deprotonation of the ketoxime (39 when R2 is H) derived from 4-methoxyacetophenone with n-BuLi, followed by addition of methyl 4-methoxybenzoate affords the 3,5-disubstituted isoxazole e.g., 40 [Ichinose,
N., Mizuno, K., Tami, T. & Otsuji, Y. (1988), "A novel NO insertion into cyclopropane ring by use of NOBF4. Formation of 2-isoxazolines, " Chem. Lett. , 233-236]. Deprotection with BBr3 afforded the free phenol 41 [Murthy, A.K., Rao, K.S.R.K.M. & Rao, N.V.S. (1968) "Isoxazolylphenols and their absorption spectra " Aus. J. Chem. 21, 2315-2317]. Details of a representative synthesis are provided in the Examples.
The methods illustrated in Scheme 6 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 isoxazole ring core structures.
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 P2S5 /chloranil results in isothiazole 43.
The methods illustrated in Schemes 7A 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 isothiazole ring core structures.
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 α, β-unsaturated ketones are formed by treatment with ethanolic KOH. The α, β-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. See: Khanna, I.K. et al. (1997) J. Med. Chem. 40 :1619-1633 and Stetter, H. (1976) Angewandte Chemie Int'l Ed. Eng. 15: 639-647. Several exemplary diketones 53-56 are listed in Scheme 8. These diketones can be converted into furans (Scheme 8), thiophenes (Scheme 9) or pyrroles (Schemes 10A-D).
Furans- Acid catalyzed cychzation of the diketones, illustrated in Scheme 8 for diketones
53-56 give furans 57-59 in 85-93% yields (Wu, A. et al. (1997) Synthetic Comm. 27:2087-2091). Also illustrated is addition of a halogen substituent (Y) to the furan ring. The synthesis of additional exemplary furans 210-214 is illustrated in Scheme 8A where a synthesis of 1 ,4- diketone precursors is illustrated at the top of the scheme. An alternate approach to 1,4-diones is illustrated in Scheme 8B which uses enolate chemistry employing α-bromoketones as electrophiles. Desoxyanisions were treated with one equivalent of potassium bis(trimethylsilyl) amide followed by addition of α-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.
Thiophenes-Txeatment of diketones as illustrated in Scheme 9 for diketones 54 and 56 with Lawesson's Reagent gives thiophenes 60 and 61 in about 85% yields [Kiebooms, R.H.L. et al. (1997) J. Org. Chem. 62:1473-1480]. Also illustrated is addition of a halogen substituent (Y) to the furan ring.
Pyrroles- Acid catalyzed cychzation of diketones in the presence of a selected primary amine as illustrated in Schemes 10A-B and 10D for diketones (such as 53 and 55) results in the formation of N-substituted pyrroles PR3 (e.g., 62 where R' = R3 = C6H4-OCH3, R2 = C2H5 and R4 = C6H5 ) and PR2 [Khanna, I.K. et al. (1997) J. Med. Chem. 40:1619-1633]. Reaction of the a 1 ,4-diketone, e.g., 54, with ammonium acetate in acetic acid results in a pyrrole PR2 where R2 is
H (such as 64 where R1 = R3 = C6H4-OCH3, and R4 = C6H5) in Scheme IOC. Deprotonation of the N-H pyrrole with sodium hydride followed by alkyl iodide addition (illustrated for ethyl iodide) gives the N-R2 -substituted analog . Scheme 10C also illustrates a method for introducing a halogen onto the pyrrole ring.
Scheme 10D illustrates a synthesis of a pyrrole of core structure PR1 (with N-R1). It is apparent from an overview of Schemes 10A-D that a variety of different pyrroles with different relative positions of substituents R'-R5 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.
The methods illustrated in Schemes 8, 8A, 9, and 10A-D can be employed or readily adapted by one of ordinary skill in the art using well-known techniques and methods for synthesis and with appropriate choice of starting materials for the preparation of the fucans, thiophenes and pyrroles of this invention.
Cyclopentadienes and Cyclopentadienones: 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., R4 and R6 , 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. Also, 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.
Scheme 1 ID illustrates another alternative synthesis of cyclopentadienes of this invention as applied to cyclopentadiene ligands 230-235 with R2 = C2H5. Cyclopentadienyl ligands with R2 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 R2 is n-propyl can also be made by this method. In this method, a thiazolium salt catalysed addition of an aldehyde to an α, β- unsaturated ketone under Stetter conditions gives the corresponding 1,4-diketone. Intramolecular aldol condensation of the 1,4-diketone using methanolic potassium hydroxide gives cyclopentenones. Cyclopentadienes are derived from Gringard and dehydration reaction on the cyclopentenones. However, 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. Dehydration of the tertiary alcohol and removal of the trimethylsilyl group were achieved under acidic work-up conditions following the Grignard reaction to give desired cyclopentadienes. The cyclopentadienes obtained were found to be very sensitive to air, heat and acidic impurities in solvents.
The methods illustrated in Schemes 11 A-D can be employed or readily adapted by one of ordinary skill in the art using well-known techniques and methods for synthesis and with appropriate choice of starting materials for the preparation of the cyclopentadienes and cyclopentadienones of this invention.
Base Substituents: 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.
The methods illustrated in Schemes 12A-D can be employed or readily adapted by one of ordinary skill in the art using well-known techniques and methods for synthesis and with appropriate choice of starting materials for the preparation of various amine-substituted ER ligands of this invention.
Six-membered ring heterocycles
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. As illustated in 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.
The method illustrated in Scheme 13 A can be employed or readily adapted by one of ordinary skill in the art using well-known techniques and methods for synthesis and with appropriate choice of starting materials for the preparation of the various substituted pyridazines ER ligands.
Pyrimidines- the pyrimidines are classified into two groups (see below) for convenience of description of their synthesis. The first group or Class-I compounds can be prepared as depicted in Scheme 13B. The following numbering applies to Scheme 13B. The reaction of ketone 1 with triflic anhydride in the presence of a nitrile 2 proceeds via a (trifluoromethanesulfonyl)carbenium ion to furnish the pyrimidines 3 in good yields. Deprotection of the phenolic methyl ether in 3 under mild acid conditions leads to the formation
Figure imgf000041_0001
Pyrimidines : Class-) Class-ll For the synthesis of Class-II compounds of pyrimidines the synthetic sequence illustrated in Scheme 13C is employed. The following numbering applies to Scheme 13C. However, the use of a ketone 5, flanked by two methylene groups resulted in the formation of two separable regio-isomeric pyrimidines, obtained by the ring closure at either of the methylene carbons. Deprotection of the phenolic methyl ethers following the same strategy as in Scheme 13A lead to the formation of the regio-isomeric pyrimidines 6 and 7, respectively. The reaction, when extended to synthesize the pyrimidine 8 (with three phenolic groups), led to the formation of mostly the regio-isomer 9 with only trace amounts of the desired isomer 8, which in addition was difficult to separate from the excess anisonitrile used in the reaction. Another synthetic procedure that can be applied to the synthesis the desired pyrimidine 8 is illustrated in Scheme
13D. This method can generally be applied to the synthesis of various pyrimidines of this invention.
The methods illustrated in Schemes 1 A-D can be employed or readily adapted by one of ordinary skill in the art using well-known techniques and methods for synthesis and with appropriate choice of starting materials for the preparation of a variety of Pyrimidine ER ligands of this invention.
Pyrazines-ths synthesis of the pyrazines follows a simple strategy as depicted in Scheme 13E. The following numbering refers to Scheme 13E. Condensation of diketone 10 with the substituted efhylenediamine 11 under acidic conditions furnished the pyrazines 13 in moderate yields. Deprotection of the phenolic methyl ethers of 13 under mild acid conditions furnished in good to excellent yields the pyrazines 13. By another route, based on the condensation of α-hydroxy ketones with ammonium acetate in ethanol, as depicted in Scheme 13F, a mixture of the pyrazines were synthesized, which on deprotection under similar conditions as above gave the pyrimidines 13, 14 (Scheme 13F) and an inseparable mixture of the pyrimidines 15 and 16(Scheme 13F).
Ouinoxalines-the synthesis of the quinoxalines follows a simple strategy as depicted in Scheme 14A. The following numbering refers to Scheme 14A. Condensation of the phenylenediamine 1 with α-diketone 2 under acidic conditions furnished in moderate yields a
1 : 1 mixture of the regio-isomeric quinoxalines 3. Removal of the phenolic methyl ether- .using boron triflouride dimethyl sulfide then furnished the deprotected quinoxalines 4 in good yields.
Ouinazolines:-t e synthesis of the quinazolines can be carried out using the strategy described in Scheme 14B.
Cinnolines-t s synthesis of cinnolines can be carried out using the strategy described in
Scheme 14C.
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. The synthesis proceed on the resin-tethered species and after formation of the desired substituted ring structure, it is released from the resin (i.e., solid support). Scheme 15A illustrates distinct syntheses for compounds where R3 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 R2 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.
Estrogen Receptor Binding
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. Lamb and/or rat uterine cytosol ER preparations were used as described in Katzenellenbogen, J.A. et al. (1977) "Estrogen photoaffinity labels. 1. Chemical and radiochemical synthesis of hexestrol diazoketone and azide derivatives; photochemical studies in solution." Biochemistry 16:1964-1970. In some cases, a mixture of regioisomers were prepared and the binding affinity of the mixture was assessed. In particular in the unsymmetrical pyrazole cases (Rl ≠ R3), isomers were formed. In the cases so far studied, the isomers have been found to be formed in comparable amounts, so that the ratios of isomers in the mixtures are likely to be between 2:1 and 1 :2. In some cases, the mixtures have been separated and the individual isomers have been tested for binding as pure compounds and significant differences in binding affinity have been found. In most cases, it has not yet been determined which regioisomeric structure corresponds to which separated regioisomer.
If the isomer ratios are within 1 :2 or 2:1, then any binding affinity measured for a mixture could never be less than one-third the affinity of the pure high affinity isomer. In the worst case, if one isomer were inactive, and the other active isomer were present as the 1 part in a 1 :2 mixture, then when the high binding isomer was pure, it 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.
Without wishing to be bound thereby, the following analysis of the ER binding affinities of individual compounds is provided:
Imidzoles, Oxazoles and Thiazoles - The receptor binding data for several imidazoles are shown in Table 3. Although the members of this series have rather low affinity, there is an increase in RBA with the addition of alkyl substituents at the 1 -position (6a-d); this trend reaches a maximum for propyl 6c, reversing for the butyl substituent 6d. Such trends are well known both in steroidal systems (11 β- and 16α-substituents) [Anstead, G.M., Carlson, K.E. & Katzenellenbogen, J.A. (1997). The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62, 268-303], as well as in other non-steroidal ligand series (such as 2-phenylindoles [von Angerer, E., Prekajac,
J. & Strohmeier, J. (1984). 2-Phenylindoles. Relationship between structure, estrogen receptor affinity, and mammary tumor inhibiting activity in the rat J Med. Chem. 27, 1439-1447], tetrahydrochrysenes [Hwang, K.J., ONeil, J.P. & Katzenellenbogen, J.A. (1992). 5,6,11,12- Tetrahydrochrysenes: Synthesis of rigid stilbene systems designed to be fluorescent ligands for the estrogen receptor. J. Org. Chem. 57, 1262-1271] etc.), and probably represent the filling of a preformed pocket of limited volume in the receptor by this substituent [Anstead, G.M., Carlson,
K.E. & Katzenellenbogen, J.A. (1997) supra]. The principal difference in binding, however, is between the tetra-substituted imidazoles (6b-d, 12, 17) and the di- or tri-substituted imidazole (3 and 6a), the tetrasubstituted ones having much higher affinity. There is little difference in binding between imidazoles 12 and 17, which have a different arrangement of nitrogen atoms in the heterocyclic core, but display their four substituents in an identical fashion. The overall low binding affinity of the imidazoles as a class might arise from the high inherent polarity of this heterocyclic system. The dipole moment for imidazole is very large, 5.56 D, and this may be unfavorable for binding to the estrogen receptor.
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
(22a vs. 22b). The oxazole 29 has undetectable affinity for ER. The isomer 31, however, does have measurable though low binding. In contrast to imidazoles, 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). However, in the imidazole series, 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.
Durani et al. (1989) "Structure-activity relationship of antiestrogens: a study using triarylbutenone, benzofuran, and triarylfuran analogues as models for triarylethylenes and triarylpropenones" J. Med. Chem. 32:1700-1707 reported receptor affinity and biological activity data for several structural classes including triarylfurans.
Furans- RBA data and differential binding affinities for ERα and ERβ for several furans are given in Table 5 Furan 204, for example, exhibits relatively high RBA. Several furans exhibit significant binding strength preference for ERα compared to ERβ. Furan 203, for example, binds to ERα about 70-fold more strongly than it does to ERβ.
Pyrazoles and Isoxazoles - The RBA data for the 1 ,2-azoles are presented in Tables 6A- B. Immediately apparent is the relatively high binding affinity of pyrazoles 38b and 38d. An interesting comparison can be made among compounds 35a, 35b, 38a, and 38b. The disubstituted progenitor 35a has very low affinity; addition of a third substituent, 1 -phenyl in 35b or 4-ethyl in 38b, causes only a 2-fold or 3-fold increase in binding affinity, respectively. By contrast, addition of the fourth substituent (to give 38b) causes either an 800- or 500-fold increase in binding affinity, respectively. Clearly, this is not additive behavior - two groups that each alone raise binding affinity 2- and 3-fold, together raise binding not 6-fold but 1500-fold. This suggests that high binding affinity is achieved when there is a detailed and proper match between the peripheral substituents and several subsites on the receptor. In the azole system, it appears that enhanced binding is associated with a tetrasubstituted ring. Consistent with this is the lower affinity of the isoxazole 41, whose affinity is similar to the most closely related trisubstituted pyrazole 38a.
There are other interesting trends in the pyrazole series: Replacement of the N-phenyl substituent (38b) with an N-benzyl group (38c) causes a significant 100-fold reduction in binding. Both of these compounds are tetrasubstituted pyrazoles. The decrease in binding affinity in 38b vs. 38c again suggests the need for a detailed match between ligand substituents and receptor subsites: the extra "kink" in the benzyl substituent in 38c may be repositioning the peripheral substituents in a less favorable geometry. The addition of a hydroxyl group at the para position of the N-phenyl substituent (compound 38d vs. 38b) has only a minor effect on binding, indicating that polarity is well tolerated in this region of the receptor.
Differential ligand binding affinities for ERα and ERβ can be measured using purified preparations of human ERα and ERβ as described in Example IB. Using this assay, Pyrazole compound 38b was found to bind to ERα three-fold more strongly than to ERβ. 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 (Table 1) are given in Table 7. Overall, these pyrazole isomers have lower ER affinity (RBA) compared to pyrazoles of core structure PAl by an average of about 2-fold. However, 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.
Pyrazoles with basic side groups
Table 8 presents ER binding affinity data for pyrazole of the indicated formula where one of R, R2, R"or R'"is a cyclic amine group, i.e., a piperidinylethoxy group:
Figure imgf000049_0001
and otherwise R2 is ethyl, and R is OH, R"and R'"are H or OH groups, as shown in Table 8.
The presence of this basic side group has been associated with mixed agonist/antagonist activity. As indicated in Table 8, the pyrazole 301 in which the basic group is substituted at R'" (with R2 = ethyl, R and R" both = OH) gives a very high affinity ER ligand.
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%.
Pyridazines- All pyridazines (see. Table 13 A) that have been assessed for binding to ER have exhibited no measurable binding affinity. 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. In addition, pyrimidines of structure PM4 where R2 = methyl,^1 and R5 = pOH-phenyl and R3 = -CH2-C6H4 or R3 = -CH2-C6H4-pOH exhibited relatively low RBA of 0.032% and 0.013% respectively. The pyrimidine in which R3 = -CH2- C6H4 exhibited no clear preference for binding to an ER subunit (ERα = 1.26% and ERβ = 0.696%). The pyrimidine in which R3 = -CH2-C6H4-pOH exhibited a preference for binding to ERα (ERα = 0.417% and ERβ = 0.076%).
A pyrimidine that can be characterized as having structure PM3 where R1 and RJ are pOH-phenyl groups, R4 is a phenyl group and R5 is ethyl exhibited a reasonable RBA of 1.00% with a clear preference for binding to ERα with ERα = 9.5% and ERβ = 3.24%.
Pyrazines- A mixture of two pyrazines regioisomers of structure PZ1 was assessed for relative ER binding affinity. The isomers where those where R2 was ethyl, R! was pOH-phenyl and either R4 or R5 was p-OH-phenyl. RBA of 2.63% was measured for this mixture and a apparent preference for binding to ERα was observed (ERα = 7.94%> and ERβ = 2.24%). A pyrazine of the same structure with R2 = ethyl, and all of R1, R4 and R3 = p-OH-phenyl was found to have a significantly lower RBA of 0.263%. This pyrazine exhibited a similar apparent preference for for binding to ERα with ERα = 7.41 % and ERβ = 2.51 %.
Quinoxalines- two mixtures containing quinoxaline regioisomers of structure QX2 (Table 2) were assessed for ER relative binding affinity (RBA) and binding affinity to the individual ER subunits. The pair of regioisomers, where R2 = ethyl, R1 = p-OH-phenyl with either R' = OH or R" = OH, exhibited a relatively low RBA of 0.20% with an apparent preference for binding to
ERβ (ERα = 0.537%) and ERβ = 0.933%). The pair of regioisomers, where R2 = n-propyl, R1 = p-OH-phenyl with either R' = OH or R" = OH, exhibited a much lower RBA of 0.014% and again exhibited a clear preference for binding to ERβ (ERα = 0.03%) and ERβ = 0.224%).
Agonist/Antagonist Character ofER Ligands Compounds are tested as ER agonists/antagonists in transcriptional activation assays in cells expressing ERα or ERβ. Cells are transfected with an expression plasmid for ERα or ERβ 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. 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. The 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 ERα or ERβ and in different cells types. Using the (ERE)3-pS2-CAT reporter, 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 ERα and ERβ. 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.
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 ERα and ERβ. 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 ERα and ERβ, 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
ERα potency selective agonist compared to estradiol. The pyrazole exhibited a 120-fold higher potency in activating transcription via ERα than via ERβ. In contrast, estradiol exhibits significantly lower activation selectivity between ERα and ERβ. Similar ERα potency-selective character was observed for this pyrazole in other cell types and with other estrogen-responsive promoters. As noted above pyrazole compound 38b was found to bind to ERα three-fold more strongly than to ERβ. Thus, 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 ERα compared that exhibited by the pyrazole with ERβ. These results suggest that factors beyond ligand-receptor interaction, such as receptor-coactivator interactions are likely important determinants of transcriptional potency.
Figure 2 is a graph of transcriptional activation by ERα (diamonds) and ERβ.(squares) in response to pyrazole 334 and pyrazole 336. Both of the pyrazoles assayed are potent in activating transcription under the assay conditions through ERα, but are weak or very weak transcriptional activators through ERβ. Both of these pyrazoles are ERα-potency selective agonists. Pyrazole 336 exhibits no activation through ERβ, even at the highest concentrations used. This pyrazole can be classified as an ERα-specific agonist. For both pyrazoles tested, the difference in ERα and ERβ binding affinities parallels the observed potency selectivity or specificity.
The transcriptional activity profiles of pyrazole 301, which carries a basic piperidinylethoxy group, were examined for ERα and ERβ, respectively in HEC-1 cells as described in the Examples. Figures 3 A and 3B are graphs of the transcriptional profiles (CAT activity) of pyrazole 301 for ERα and ERβ, respectively. Pyrazole 301 displayed no agonist activity on ERβ (Fig. 3B). However, on ERα this compound was a partial agonist, reaching an efficacy level nearly half that of estradiol at 1 nM (Fig. 3 A). Interestingly, as the concentration of compound 301 increases, the ERα agonist activity returns to near basal levels. At high concentrations, pyrazole 301 acts as an antagonist through both ERα and ERβ., its potency as an antagonist through ERα being about 10-fold higher than through ERβ, which is consistent with its higher affinity for the ERα subtype (see Table 8). Pyrazole 301 is unusual, however, in that it exhibits a biphasic agonist-antagonist dose response through ERα. Many compounds exhibit partial agonist activity on ERα, and they are often more complete antagonists on ERβ than on ERα. 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.
The 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.
Pharmaceutical Compositions and Methods
This invention is also directed to pharmaceutically acceptable esters and salts of the ER ligands of various formulas and structures 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.
Pharmaceutically acceptable 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. For example, 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.
Pharmaceutical 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
(agonists, antagonists and or mixed agonist-antagonist as appropriate). 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. In particular, 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. Alternatively, 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.
The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives.
In general, pharmaceutical compositions of this invention can contain from 0.001-99%)
(by weight) of one or more of the ER ligands disclosed herein. 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. For administration by injection or infusion, 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. This means that 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.
The magnitude of 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. In making the compositions of the present invention, 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.
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. Some examples of 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.
The 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. The term "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.
As a pH adjusting reagent for preparing the pharmaceutical composition, 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-sodium hydroxide, and aspartic acid-sodium hydroxide. Among them, 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., ERα or ERβ), 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., ERα or ERβ), 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. For example, ERβ 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 ERβ-mediated responses should block this response without blocking desired responses to estrogens that are mediated by ERα, 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 β 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. Endocrinology 138:4613-4621; Dotzlaw H, Leygue E, Watson PH, Murphy LC 1997 Expression of estrogen receptor-β in human breast tumors. J. Clin. Endocrinol. Metab. 82:2371-2377; Kuiper GGJM, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson J 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor β. Endocrinology 139:4252-
4263; Kuiper GGJM, Gustafsson J-A 1997 The novel estrogen receptor-beta subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS Letters 410:87-90; Katzenellenbogen BS, Korach KS 1997 Editorial: A new actor in the estrogen receptor drama-Enter ER-β. Endocrinology 138:861-862; Montano MM, Jaiswal AK, Katzenellenbogen BS 1998 Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-α and estrogen receptor-β. J Biol Chem 273:25443- 25449; Register TC, Shively CA, Lewis CE 1998 Expression of estrogen receptor alpha and beta transcripts in female monkey hippocampus and hypothalamus. Brain Res 788:320-322; Register TC, Adams MR 1998 Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor beta. J Steroid Biochem Molec Biol 64:187-191).
The estrogen subtypes, ERα and ERβ, are the products of two different genes. However, variant forms of both ER subtypes are known. ERβ 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 subtypeβ (ERβ) studied with ERβ and ERα 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-α and estrogen receptor-β. J Biol Chem 273:25443-25449). In addition, gene transcripts with deleted exons and altemate exon splicing, which may be translated into proteins, are known. These variant ER forms can have different transcription regulating activities, and can respond differently to different ER ligands (Chaidarun S, Alexander J 1998 A tumor-specific truncated estrogen receptor splice variant enhances estrogen-stimulated gene expression. Mol Endocrinol 12:1355-1366; Leygue ER, Watson PH, Murphy LC 1996 Estrogen receptor variants in normal human mammary tissue. J Natl Cancer Inst 88:284-290; Miksicek PJ, Lei Y, Wang Y 1993 Exon skipping gives rise to alternatively spliced forms of the estrogen receptor in breast tumor cells. Breast Cancer Res Treat 26: 163-174; Pfeffer U, Fecarotta E, Vidali G 1995 Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissue and in MCF-7 cells. Cancer Res 55:2158-2165; Zhang QX, Borg A, Fuqua SAW 1993 An exon 5 deletion variant of the estrogen receptor frequently co-expressed with wild-type estrogen receptor in human breast cancers. Cancer Res 53:5882-5884).
Various mutant forms of ERs have been characterized, and some of these show different responses to ER ligands (Wrenn CK, Katzenellenbogen BS 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 Chem 268:24089-24098; Montano MM, Ekena KE, Krueger K, Keller AL, Katenellenbogen BS 1996 Human estrogen receptor ligand activity inversion mutants: Receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens. Mol Endocrinol 10:230-242). 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,
Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491-1494).
Some of the actions of 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,
Kachkache M, Balsan S 1993 Cell signaling and estrogens in female rat osteoblasts: A possible involvement of unconventional nonnuclear receptors. J Bone Miner Res 8:1365-1376; Marino M, Pallottini V, Trentalance A 1998 Estrogens cause rapid activation of IP3-PKC-a signal transduction pathway in HEPG2 cells. Biochem Biophys Res Commun 245:254-258; Mermelstein PG, Becker JB, Surmeier DJ 1996 Estradiol reduces calcium currents in rats neostriatal neurons via a membrane receptor. J Neuroscience 16:595-604; Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 9:404-410; Pappas TC, Gametchu B, Yannariello-Brown J, Collins TJ, Watson CS 1994 Membrane estrogen receptors in GH3/B6 cells are associated with rapid estrogen-induced release of prolactin. Endocrine 2:813-822; Wehling M 1997 Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59:365-393; Zheng J, Ramirez VD 1997. Demonstration of membrane estrogen binding proteins in rat brain by ligand blotting using a 17b-estradiol-[125I] bovine serum albumin conjugate. J Steroid Biochem Molec Biol 62:327- 336). Although it is not known, as yet, which ER subtypes are important in regulating nongenomic responses, subtype-selective ER ligands may enable regulation of these responses in a tissue and cell selective manner.
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.
The following examples are provided to further illustrate the invention and are in no way intended to limit the scope of the invention.
EXAMPLES
Example 1 :
Measurement of relative ER Binding Affinities
A. ER ligand binding assays can be performed as previously reported [Katzenellenbogen,
J.A. et al. (1977) "Estrogen photoaffinity labels. 1. Chemical and radiochemical synthesis of hexestrol diazoketone and azide derivatives; photochemical studies in solution." Biochemistry 16:1964-1970] using lamb and/or rat uterine cytosol diluted to approximately 1.5 nM of receptor, which was incubated with buffer of several concentrations of uniabeled competitor together with 10 nM [3H]estradiol for 18-24 hours. Free ligand was removed by adsorption to dextran-coated charcoal. Uniabeled competitors were prepared in 1:1 DMF:TEA to ensure solubility.
B.
Differential ligand binding affinities for ERα and ERβ can be determined by competitive radiometric binding assays using 10 nM [3H]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 ERα (amino acids 304-554) and ERβ (203-452) ligand binding domains expressed in E. coli or using full length ERα and ERβ expressed in Baculovirus (commerically available).
Example 2:
Cell assays for ER Ligand Activity
Compounds are tested as ER agonists/antagonists in transcriptional activation assays in cells expressing ERα or ERβ. Cells are transfected with an expression plasmid for ERα or ERβ 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. 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. The 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 ERα or ERβ and in different cells types.
Using the (ERE)3-pS2-CAT reporter, 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 ERα and ERβ. 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.
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 ERα and ERβ. 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 ERα 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 ERα than via ERβ. In contrast, estradiol exhibits significantly lower activation selectivity between ERα and ERβ. Similar ERα 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 ERα three-fold more strongly than to ERβ. Thus, 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 ERα compared that exhibited by the pyrazole with ERβ. These results suggest that factors beyond ligand-receptor interaction, such as receptor-coactivator interactions are likely important determinants of transcriptional potency.
CHEMICALS, MATERIALS, AND PLASMID CONSTRUCTIONS
Cell culture media were purchased from GIBCO (Grand Island, NY). Calf serum was from Hyclone Laboratories (Logan, UT) and fetal calf serum was from Atlanta Biologicals (Atlanta, GA). ,4C-Chloramphenicol (50-60 Ci/mmol) and [Η]E, were from DuPont, NEN Research Products (Boston, MA). The expression vector for human ERα (pCMV5-hER) was constructed previously as described (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. Chem. 268:24089- 24098). The expression vector pCMN5-ERβ was constructed by inserting the full-length cDΝA encoding human ERβ (530) residues, pΝGVl-ERβ (Mosselmen et al. (1996) supra) and including the additional 53 Ν-terminal amino acids as found in Genebank accession number AF
051427), into the BamHI site of pCMV5. 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. Νatl. 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. Chem. 268:24089-24098], C3-Ti-LUC, which contains - 1030 to +58 of the human complement C3 promoter fused to the firefly luciferase reporter gene (Νorris, 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, ΝJ) or pCMNβ (Clontech, Palo Alto, CA) which contains the β-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.
Cell culture and transient transfections Human endometrial cancer (HEC-1) cells, Chinese hamster ovary (CHO) cells and HeLa cells are maintained in culture and transfected as described (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. 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. 9:814-825; Mclnemey, E.M. and Katzenellenbogen, B.S. (1996), "Different regions in activation function-1 of the human estrogen receptor required for antiestrogen- and estradiol-dependent transcription activation," J. Biol. Chem. 271:24172-24178). Transfection of HEC-1 cells in 60-mm dishes utilizes 0.4 ml of a calcium phosphate precipitate containing 2.5 μg of the reporter gene plasmid, 100 ng of ER expression vector, and carrier DNA to a total of 5 μg DNA. CAT or luciferase activity, normalized for the internal control β-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),
"Different regions in activation function-1 of the human estrogen receptor required for antiestrogen- and estradiol-dependent transcription activation," J. Biol. Chem. 271:24172- 24178).
Example 3 :' Chemical Syntheses
General Methods All reactions using water- or air-sensitive reagents were conducted under an Ar atmosphere with dry solvents. Solvents were distilled under N2 as follows: CH2C12 from CaH2, THF from sodium benzophenone ketyl, DMF from MgSO4, and Hexanes from CaSO4. Triethyl amine was distilled over CaH2. All other reagents were purchased from commercial suppliers and used without further purification. Reactions were all monitored by TLC, performed on 0.25 mm silica gel glass plates containing F-254 indicator. Visualization on TLC was achieved by UV light (254 nm), iodine vapors, or phosphomolybdic acid indicator. Flash chromatography was performed using Woelm 32-63 μm silica gel packing unless otherwise noted.
'H NMR and 13C NMR spectra were recorded on a Varian U400, Varian U500 or Varian INOVA 750. Electron ionization (El) spectra were obtained using a Finnigan-MATCH5 spectrometer at 70 eV. Fast atom bombardment (FAB) were recorded on a VG ZAB-SE spectrometer. High pressure liquid chromatography (HPLC) was performed on a SpectraPhysics
PI 00 solvent delivery system with ultraviolet detection at 254 nm. Elemental analysis was performed by the Microanalytical Service Laboratory at the University of Illinois.
Compound numbers listed refer to those in the Schemes.
General Demethylation Procedure using BBr3. To a stirring solution of the methyl- protected heterocycle (1 eq.) in CH2C12 at -78 °C was added a solution of BBr3 (4-5 eq.) as a IN solution in CH2C12. The reaction were allowed to warm to room temperature and stirred for 18 h. After quenching with H2O, the layers were separated and the aqueous layer extracted with EtOAc (3 x 5 mL). The combined organic layers were dried over
Figure imgf000067_0001
filtered and concentrated to afford the crude phenols. Flash chromatography afforded the demethylated products.
General Demethylation Procedure using BF3 Me? To a stirring solution of the methyl protected heterocycle (1 eq.) in CH2C12 (8 mL) at room temperature was added BF3-SMe2 complex (75 eq.). After stirring for 24 h, solvent and excess reagent were evaporated under nitrogen stream in hood. Residue was taken up in EtOAc and washed with H2O and sat. NaCl. Organic extract was dried over NajSO^ filtered and solvent removed under reduced pressure. The resulting residue was purified though a silica plug, eluting with EtOAc. Solvent evaporation afforded the deprotected products.
Imidazoles 4,5-Di(4-methoxyphenyl)-lH-imidazole (2). To 4,4'-dimethoxybenzil (1) (2.0g, 7.4 mmol) and -formaldehyde (1.0 g, 11.1 mmol) was added formamide (50 mL). The bright yellow suspension was heated to reflux (220 °C) for 2 h. The reaction mixture was then cooled to room temperature then to 0°C. The crystals that formed were filtered and recrystallized from EtOAc to afford 2 (2.4 g, 86%). mp 183-184°C (lit [16] mp 183-184°C);
4,5-Di(4-hydroxyphenyl)-lH-imidazole (3). Imidazole 2 (100 mg, 0.35 mmol) afforded 3
(52 mg, 59%o) by the general BBr3 demethylation procedure.
2,4,5-Tή(4-methoxyphenyl)-lH-imida∑ole (4). A suspension of 4,4'-dimethoxybenzil (1) (4.0 g, 15 mmol) and -anisaldehyde (20 mL, 164 mmol) and formamide (100 mL) was heated to reflux (220 °C) for 2 h, during which time the reaction mixture became homogeneous. The reaction was then cooled to 0°C and the precipitated product 4, was filtered. The light yellow powder was recrystallized from MeOH/H2O to afford 3.80 g of 4 [Hayes, J.F., Mitchell, M.B. & Wicks, C (1994), "A novel synthesis of 2,4,5-triarylimidazoles, " Heterocycles 38, 575-585] (66%). mp 89-91 °C (lit [Hayes, J.F., Mitchell, M.B. & Wicks, C (1994), "A novel synthesis of 2,4,5-triarylimidazoles, " Heterocycles 38, 575-585] mp 88-94°C).
General N-Alkylation Procedure for Imidazoles. A solution of imidazole 4 (200 mg, 0.52 mmol) in THF (10 mL) and DMF (1.5 mL) was cooled to 5°C NaH (31 mg, 0.78 mmol) was added as 60% dispersion in mineral oil. The reaction mixture was warmed to room temperature for 1 h and respective alkyl halide (0.04 mL, 0.62 mmol) was added. The resulting suspension was heated to reflux for 12 h, then cooled to room temperature. The light precipitate was filtered and the.filtrate was concentrated under vacuum to a yellow solid which was flashed on silica (30%) EtOAc/Hexanes) to afford alkylated products 5b-d
1 -Ethyl-2,4,5-tri(4-methoxyphenyl)-imidazole (5b), 1 -Propyl-2,4,5-tri(4-methoxyphenyl)- imidazole (5c), l-Butyl-2,4,5-tri(4-methoxyphenyl)-imidazole (5d) in 80-90%) yields.
2,4,5-Tri(4-hydroxyphenyl)-lH-imidazole (6a). According to the general BBr3 demethylation procedure above, imidazole 4 (3.0 g, 7.8 mmol) afforded 6a as a green-orange solid that darkened upon exposure to air (1.8 g, 68%>). mp 203-205 °C.
l-Ethyl-2,4,5-tri(4-hydroxyphenyl)-imidazole (6b). According to the general BBr3 demethylation procedure above, imidazole 5b (185 mg, 0.46 mmol) afforded 5b (107 mg, 62%). mp 150-153 °C;
l-Propyl-2,4,5-tri(4-hydroxyphenyl)-imidazole (6c). According to the general BBr3 demethylation procedure above, imidazole 5c (170 mg, 0.40 mmol) afforded 6c (86 mg, 55%). mp 172-175 °C
l-Butyl-2,4,5-tri(4-hydroxyphenyl)-imidazole (6d). According to the general BBr3 demethylation procedure above, imidazole 5d (190 mg, 0.43 mmol) afforded 6d (78 mg, 46 %). mp 153-155 °C (dec).
l-Ethyl-2,5-(4-methoxyphenyl)-4-phenyl imidazole (11). Azido-ketone 9 (50.0 mg, 0.187 mmol) and imine 10 (92.0 mg, 0.564 mmol) were dissolved in THF (15 mL). Et3N (29.0 μL, 0.208 mmol) was added via syringe and reaction stirred at room temperature for 48 h. The reaction mixture was then poured into H2O and extracted with CH2C12, organic fractions were pooled, dried over Na^SO,, filtered and solvent removed under reduced pressure. The intermediate, 2,5-dihydro-2-hydroxyimidazole, used in next step without further purification or characterization, was taken up CH,C12 (10 mL). Solution was cooled to 0 °C and TFA (14.4 μL, 0.187 mmol) was added via syringe. Reaction stirred at 0 °C for 36 h. The mixture was diluted with CH2C12 (10 mL) and washed with H20, sat. NaHCO3, and sat. NaCl successively. Organic fraction was dried over Na,SO4, filtered and solvent removed under reduced pressure. Purification by flash column chromatography (1:2 EtOAc: Hexanes) and recrystallization from CH2C1 Hexanes afforded imidazole 11 as a white solid (24.6 mg, 34% yield from azide 9).
l-Ethyl-2,5-(4-hydroxyphenyl)-4-phenyl imidazole (12). Imidazole 11 (12.0 mg, 0.031 mmol) was demethylated according to the general BF3-SMe2 procedure to afford imidazole 12 as an off-white powder (10.6 mg, 95%>).
5-Ethyl-l,4-(4-methoxyphenyl)-2-phenyl imidazole (16). Keto-amide 15 (110.0 mg, 0.273 mmol) and ammonium acetate (105.0 mg, 1.362 mmol) were heated to reflux in acetic acid (10 L) for 48 h. Acetic acid was removed under reduced pressure, resulting residue was taken up in EtOAc, washed with sat. NaHCO3, H20, and sat. NaCl. Organic extracts were dried over Na2SO4, filtered and solvent removed. Product was purified by flash column chromatography (1:4 EtOAc -.Hexanes) and recrystallization from CH2C1 Hexanes to give imidazole 16 as a white solid (25.7 mg, 25%).
5-Ethyl-l,4-(4-hydroxyphenyl)-2-phenyl imidazole (17). Imidazole 16 (25.0 mg, 0.065 mmol) was demethylated as outlined in general BF3-SMe2 procedure above to give deprotected imidazole 17 as an off-white powder (20.2 mg, 87%>).
Thiazoles
2,4-Di(4-methoxyphenyl)-thiazole (21a). A suspension of thioamide 19 (1.3 g, 7.9 mmol) and α-bromo-4'-methoxy-acetophenone (20) (1.8 g, 7.9 mmol) in DMF (10 mL) was heated to reflux for lh, until it became homogeneous. The heat was removed and the reaction was stirred for 15 h at room temperature. The reaction mixture was poured into H2O (50 mL) and the solid precipitate was filtered to afford crude 21a. Recrystallization from CH3NO2 afforded pure 21a as light yellow crystals (1.8 g, 81%). 5-Ethyl-2,4-di(4-methoxyphenyl)-thiazole (21b). A suspension of thioamide 19 (975 mg, 5.8 mmol) and α-bromo-4'-methoxy-butyrophenone (13) (1.5 g, 5.8 mmol) in DMF (10 mL) was heated to reflux for 4 h, until it became homogeneous. The heat was removed and the reaction was poured into H2O (50 mL). The water was extracted with EtOAc (3 x 10 mL) and the combined organic layers were washed with sat. LiCl (10 mL), then brine (10 mL). After drying over MgSO4, the reaction mixture was filtered, and concentrated to a yellow powder. Flash chromatography (10%) EtOAc/Hexanes) afforded 21b as a light yellow powder (1.1 g, 51%).
2,4-Di(4-hydroxyphenyl)-thiazole (22a). Thiazole 21a (1.0 g, 3.6 mmol) was demethylated using BBr3 as outlined in the general procedure above to afford 22a (430 mg, 45%). mp 218-221 °C;
2,4-Di(4-hydroxyphenyl)-5-ethyl-thiazole (22b). Thiazole 21b (1.0 g, 2.7 mmol) was demethylated according to the general BBr3 procedure to afford 22b (460 mg, 58%). mp 246- 247 °C;
Oxazoles 2,4-(4-Methoxyphenyl)-5-phenyl oxazole (28). Azido-ketone 27 (0.18 g, 0.673 mmol) andp-anisaldehyde (0.25 mL, 2.05 mmol) were dissolved in THF (15 mL). Et3N (94.0 μL, 0.674 mmol) was added via syringe and reaction stirred at room temperature for 48 h. The reaction mixture was then poured into H2O and extracted with CH2C12, organic fraction was dried over Na SO4, filtered and solvent removed under reduced pressure. Resulting intermediate 2,5- dihydro-5-hydroxyoxazole, used in next step without further purification or characterization, was taken up CH2C12 (10 mL). Solution was cooled to 0 °C and TFA (54.0 μL, 0.701 mmol) was added via syringe. Reaction stirred at 0 °C for 36 h. The mixture was diluted with CH2C12 (10 mL) and washed with H2O, sat. NaHCO3, and sat. NaCl successively. Organic extracts were combined, dried over NajSO^ filtered and solvent removed under reduced pressure . Purification by flash column chromatography (1 :2 EtOAc -.Hexanes) and recrystallization from
CH2C12/Hexanes afforded oxazole 28 as a white solid (72.4 mg, 30%> yield from azide 27). mp 125-128 °C (lit. [Strzybny, P.P.E., van ES, T. & Backeberg, O.G. (1969), "Reaction of α- acyloxyketones with ammonium acetate, "J. South African Chem. Inst. 22, 158-164] mp 126-127 °C);
2, 4-(4-Hydroxyphenyl)-5 -phenyl oxazole (29). Oxazole 28 (22.0 mg, 0.062 mmol) was demethylated according to the general BF3-SMe2 procedure above to give deprotected oxazole 29 as an off-white powder (18.8 mg, 93%o).
2,5-(4-Methoxyphenyl)-4-phenyl oxazole (30). A solution of bromo-ketone 26 (87.0 mg, 0.285 mmol) and p-methoxybenzamide (43.0 mg, 0.285 mmol) in toluene was heated to reflux for 36 h. Toluene was removed under reduced pressure and resulting residue purified by flash column chromatography (1 :4 EtOAc: Hexanes). Recrystallization of desired product from
CH2C12/Hexanes afforded oxazole 30 as a colorless solid (52.9 mg, 52%). mp 147-149°C;
2,5-(4-Hydroxyphenyl)-4-phenyl oxazole (31). Oxazole 30 (22.0 mg, 0.062 mmol) was demethylated according to the general BF3-SMe2 procedure above to give deprotected oxazole 31 as an off-white powder (18.1 mg, 89%>).
General Procedure for Pyrazole Synthesis. A suspension of diketone (1 eq.) and appropriate hydrazine hydrochloride (3-5 eq.) in a 3:1 mixture DMF-.THF was heated to reflux for 16-24 h with reaction progress being monitored by TLC for disappearance of starting material. The reaction mixtures was cooled to room temperature and poured into iced sat. LiCl solution (10 mL) and EtOAc (10 mL). The layers were separated and the organic layer was washed with brine (10 mL), dried over MgS04, filtered and concentrated. Purification by flash column chromatography (EtOAc/Hexanes systems) afforded the pyrazoles.
3,5-Di(4-methoxyphenyl)-lH-pyrazok (34a). Diketone 33 (91 mg, 0.32 mmol) and hydrazine (0.1 mL, 3.2 mmol) were reacted as outlined in general pyrazole procedure to afford 34a [van Steenis, J. (1946), "The nitration of dianisoylmethane and p-methoxydesoxybenzoin, " Chem. Ber. , 29-46] as an off-white solid (32.6 mg, 38%). mp 172-175°C (lit [van Steenis, J. (1946)>."The nitration of dianisoylmethane and p-methoxydesoxybenzoin, " Chem. Ber. , 29-46] mp 174°C).
l-Phenyl-3,5-di(4-methoxyphenyl)-pyrazoh (34b). Diketone 33 (100 mg, 0.35 mmol) and phenyl hydrazine hydrochloride (500 mg, 3.5 mmol) were reacted as outlined in general pyrazole procedure above to afford 34b [Ando, W., Sato, R., Yamashita, M., Akasaka, T. &
Miyazaki, H. (1983), "Quenching of singlet oxygen by l,3,5-triaryl-2-pyraazolines, "J. Org. Chem. 48, 542-546] (30 mg, 25%). mp 159-161 °C (lit [Ando, W., Sato, R., Yamashita, M., Akasaka, T. & Miyazaki, H. (1983), "Quenching of singlet oxygen by l,3,5-triaryl-2- pyraazolines, "J. Org. Chem. 48, 542-546] mp 163°C).
l-Benzyl-3,5-di(4-methoxyphenyl)-pyrazole (34c). Diketone 33 (300 mg, 1.07 mmol) and benzylhydrazine dihydrochloride (335 mg, 2.13 mmol) were reacted as outlined in the general pyrazole procedure above to afford 34c (179 mg, 45%>).
l,3,5-Tri(4-methoxyphenyl)-pyrazole (34d). Diketone 33 (100 mg, 0.35 mmol) and 4- methoxyphenyl hydrazine hydrochloride (92.2 mg, 0.53 mmol) were reacted as outlined in the general pyrazole procedure above to afford 34d as a white solid (112 mg, 85%o).
3,5-Di(4-hydroxyphenyl)-lH-pyrazok (35a). Pyrazole 34a (20 mg, 0.07 mmol) was demethylated with BBr3 according to the general procedure to afford 35a [Hergenrother, P.M. (1991), "New Developments in Thermally Stable Polymers, " Rec. Travl. Chim. Pays-Bas. 110, 481-491] as an off-white solid (11 mg, 63%).
l-Phenyl-3,5-di(4-hydroxyphenyl)-pyrazole (35b). Pyrazole 34b (20 mg, 0.06 mmol) was demethylated with BBr3 according to the general procedure to afford 34b [Hergenrother, P.M. (1991), "New Developments in Thermally Stable Polymers, " Rec. Travl. Chim. Pays-Bas. 110, 481-491] as an off-white solid ( 11.5 mg, 58%). WO 00/1 ι9v9w944 PCT/US99/22747
J -Benzyl-3 ,5-di(4-hydroxyphenyl)-pyrazole (35c). Pyrazole 34c (178 mg, 0.48 mmol) was demethylated with BBr3 according to the general procedure to afford 35c as a yellow film (100 mg, 66%).
l,3,5-Tri(4-hydroxyphenyl)-pyrazole (35d). Pyrazole 34d (112 mg, 0.29 mmol) was demethylated with BBr3 according to the general procedure to afford 35d (44.8 mg, 45%) as an off-white solid.
4-Ethyl-3,5-di(4-methoxyphenyl)-lH-pyrazole (37a). Diketone 36 (100 mg, 0.32 mmol) and hydrazine (0.12 mL, 3.2 mmol) were reacted as outlined in the general pyrazole procedure above to afford 37a as a while solid (69 mg, 70%).
l-Phenyl-4-ethyl-3,5-di(4-methoxyphenyl)-pyrazole (37b). Diketone 36 (100 mg, 0.35 mmol) and phenyl hydrazine hydrochloride (140 mg, 0.96 mmol) were reacted as outlined in the general pyrazole procedure above to afford 37b as an orange solid (109 mg, 87%).
l-Benzyl-4-ethyl-3,5-di(4-methoxyphenyl)-pyrazole (37c). Diketone 36 (200 mg, 0.64 mmol) and benzylhydrazine dihydrochloride (188.2 mg, 0.97 mmol) were reacted as outlined in the general pyrazole procedure above to afford 37c as colorless film (80 mg, 31%).
4-Ethyl-l ,3 ,5-tri(4-methoxyphenyl)-pyrazole (37d). Diketone 36 (50 mg, 0.16 mmol) and 4-methoxyphenyl hydrazine hydrochloride (140 mg, 0.96 mmol) were reacted as outlined in the general pyrazole procedure above to afford 37d as an orange solid (11 mg, 23%>).
4-Ethyl-3,5-di(4-hydroxyphenyl)-lH-pyrazole (38a). Pyrazole 37a (69 mg, 0.22 mmol) was demethylated according to the general BBr3 procedure to afford 38a as a white solid (35 mg, .l-Phenyl-4-ethyl-3,5-di(4-hydroxyphenyl)-pyrazole (38b). Pyrazole 37b (100 mg, 0.26 mmol) was demethylated according to the general BBr3 procedure to afford 38b as a white solid (50 mg, 54%).
l-Benzyl-4-ethyl-3,5-di(4-hydroxyphenyl)-pyrazole (38c). Pyrazole 37c (80 mg, 0.20 mmol) was demethylated according to the general BBr3 procedure to afford 38c contaminated with a brominated side product. The two compounds were separated using RPHPLC (30:70, H,O:MeOH, Partisil ODS2 C-18 prep column) (50 mg, 54%).
4-Ethyl- 1 ,3 ,5-tri(4-hydroxyphenyl)-pyrazole (38d). Pyrazole 37d (10 mg, 0.03 mmol) was demethylated according to the general BBr3 procedure to afford 38d (9.9 mg, 100%).
3,5-Di(4-methoxyphenyl)isoxazole (40). To a solution of oxi e 39 (1.0 g, 6 mmol) in
THF (20 mL) at 0°C was added nBuLi (9.11 mL, 13.3 mmol) as a solution in Hexanes. The clear solution was stirred for 30 min at 0°C then methyl 4-methoxybenzoate (498 mg, 3 mmol) was added as a solution in THF (5 mL) over 5 min. The reaction mixture was stirred at 0°C for 30 min, then warmed to room temperature. 5 N HCl (10 mL) was added and the biphasic reaction mixture was brought to reflux overnight (12 h). Upon cooling to 0°C, isoxazole 40
[Ichinose, N., Mizuno, K., Ta i, T. & Otsuji, Y. (1988), "A novel NO insertion into cyclopropane ring by use of NOBF4. Formation of 2-isoxazolines, " Chem. Lett. , 233-236] precipitated and was collected via filtration (450 mg, 27%). mp 174-177°C (lit [Ichinose, N., Mizuno, K., Tami, T. & Otsuji, Y. (1988), "A novel NO insertion into cyclopropane ring by use of NOBF4. Formation of 2-isoxazolines, " Chem. Lett. , 233-236] mp 176-177°C);
3,5-Di(4-hydroxyphenyl)isoxazole (41). Isoxazole 40 (300 g, 1.1 mmol) was demethylated according to the general BBr3 procedure to afford 41 [Murthy, A.K., Rao, K.S.R.K.M. & Rao, N.V.S. (1968) "Isoxazolylphenols and their absorption spectra, " Aus. J. Chem. 21, 2315-2317] as a white solid (152 mg, 56 %). mp 267-269°C (lit [Murthy, A.K., Rao, K.S.R.K.M. & Rao, N.V.S. (1968) "Isoxazolylphenols and their absorption spectra, " Aus. J. Chem. 21, 2315-2317] mp 255°C);
General procedure for the synt esis of pyridazines:
A stirred solution of 1,4-dione (55.0 mg, 0.12 mmol) in hydrazine hydrate (5 mL) with a minimal amount of ethanol (to dissolve dione), was heated to reflux overnight. The reaction was allowed to cool to room temperature and then diluted with ethyl acetate (15 mL). The solution was transferred to a separatory and the aqueous layer washed with ethyl acetate. The organic extracts were pooled and washed with water, sat. sodium chloride, dried over sodium sulfate and filtered. Solvent was removed under reduced pressure to yield crude 4,5-dihydropyridazine. Flash column chromatography (1 :4 EtOAc:hexanes) afforded pure 4,5-dihydropyridazine, which was taken up into methylene chloride and left exposed to air overnight. Any remaining methylene chloride was removed under reduced pressure to afford crude pyridazine. Purification by flash column chromatography (1:1 EtOAc -.hexanes) followed by recrystallization (EtOAc :Hex) gave pure pyridazine.
Methoxy-protected pyridazine were deprotected according to the BF3 SMe2 general demethylation procedure as described above to afford pyridazine analogs.
General procedure for the synthesis of pyrimidines:
To a well-stirred solution of the ketone (1 mmol) and the nitrile (2.2 mmol) in an. dichloroethane (5 ml) was slowly added triflic anhydride (1.1 mmol). The reaction mixture was stirred at room temperature for 24hr, satd. bicarbonate solution was added and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were washed with brine and dried (an. Na^SO . The solvent was removed in vacuo and the crude product was purified by flash column chromatography over silica gel using 30% ethyl acetate-hexane as eluent to furnish the pyrimidines.
General procedure for the synthesis of pyrazines: Method A (Scheme 13E): A magnetically stirred solution of the α-diketone (lmmol) and the diamine (1 mmol) in acetic acid (1.5 ml) was refluxed for 3.5-4.0 hr. The reaction mixture was cooled, poured into ice and extracted with ethyl acetate (3x5ml). The combined organic phases were washed with brine and dried (an. Na,SO4). Evaporation of the solvent and purification of the residue over a silica gel column using 20% ethyl acetate-hexane as eluent furnished the pyrazines.
Method B (Scheme 13F) To a stirred mixture of the α-hydroxy ketones ( lmmol each) in ethanol ( 6 ml) was added ammonium acetate (3 mmol). The reaction mixture was refluxed for 4hr, cooled and poured into ice, the precipitated solid filtered off and was washed with cold water. The residue was purified over a silica gel column using 20% ethyl acetate-hexane as eluent to furnish the mixture of pyrazines.
General protocol for deprotection of phenolic methyl ethers:
To a stirred solution of the protected pyrimidine (1 mmol) or pyrazine (1 mmol) in dichloromethane was added boron triflouride-dimethylsulfide complex (10 mmol phenolic gp.) and the reaction stirred at room temperature for 24-36 hr. 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. Na2SO4). Evaporation of the solvent and purification of the residue over a silica gel column using 50%> ethyl acetate- hexane or ethyl acetate as eluent furnished the free phenolic pyrimidine or pyrazine.
General experimental procedure for synthesis of quinoxalines:
Step 1: A well stirred mixture of the o-phenylenediamine dihydrochloride (1 mmol) and the α-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. NaιSO4) 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.
Na^SO,,). Evaporation of the solvent and purification of the residue over a silica gel column using ethyl acetate as eluent furnished the deprotected quinoxalines.
Those of ordinary skill in the art will appreciate that starting materials, reagents, reaction conditions, methods, techniques, purification and isolation methods other those specifically detailed herein can be employed or readily adapted in view of well-know principles to make and use the compounds of this invention. All art-known equivalents of starting materials, reagents, reaction conditions, methods, techniques, purification and isolation methods described herein are intended to be encompassed by this invention.
All references cited herein are incorporated in their entirety herein to the extent that they are not inconsistent with the disclosure herein
TABLE 1: EXEMPLARY STRUCTURES OF FIVE-MEMBERED RTNG CORES
IMIDAZOLES
Figure imgf000079_0001
IM1 IM2
Figure imgf000079_0002
IM3 IM4
OXAZOLES/THIAZOLES
Figure imgf000079_0003
X = O, OA1 X = O, OA2 X = O, OA3 X = S, TA1 X = S, TA2 X = S, TA3 Table 1 (continued)
PYRAZOLES
Figure imgf000080_0001
PA1 PA2 PA3
ISOXAZOLE/ISOTHIAZOLE
Figure imgf000080_0003
Figure imgf000080_0002
X = 0, 101 X = 0, I02 X = 0, I03 X = S, IS1 X = S, IS2 X = S, IS3
CYCLOPENTADIENES
Figure imgf000080_0004
Table 1 (continued)
CYCLOPENTADIENEONES
Figure imgf000081_0001
FURANS/THIOPHENES
Figure imgf000081_0002
X = 0, F1 X = O, F2 X = O, F3 X = S, T1 X = S, T2 X = S, T3
Figure imgf000081_0003
PR1 PR2 PR3 TABLE 2: EXEMPLARY STRUCTURES OF SLX-MEMBERED RING CORES
BENZENES
Figure imgf000082_0001
PYRIDINES
Figure imgf000082_0002
Figure imgf000082_0003
PY3 PY4 PY5 Table 2 (Continued)
PYRIMIDINES
Figure imgf000083_0001
Figure imgf000083_0002
PM1 PM2 PM3
Figure imgf000083_0003
PYRAZINES
R^
Figure imgf000083_0004
PZ1 PZ2 Table 2(Continued)
PYRIDAZINES
Figure imgf000084_0001
PZD3 PZD4
Table 2 (Continued)
Quinoxalines
Figure imgf000085_0001
QX1 QX2
Quinazoiines
Figure imgf000085_0002
Table 2(Continued)
Cinnolines
Figure imgf000086_0001
CN1 CN2
Phthalazines
Figure imgf000086_0002
Table 3 : ESTROGEN RECEPTOR BINDING DATA FOR IMIDAZOLES 3, 6A-D,
12 AND 17
Figure imgf000087_0001
Compound R4 R3 R2 R1 RBA
3 4'-HO-C6H4 4'-HO-C6H4 H H O.001
6a 4'-HO-C6H4 4'-HO-C6H4 H 4'-HO-C6H4 0.007
6b 4'-HO-C6H4 4'-HO-C6H4 C2H5 4'-HO-C5H4 0.38
6c 4'-HO-C6H4 4'-HO-C6H4 C3H7 4'-HO-C6H4 0.62
6d 4'-HO-C6H4 4'-HO-C6H4 C4H9 4'-HO-C6H4 0.17
12 C6H5 4'-HO-C6H4 C2H5 4'-HO-C6H4 0.25
17 4'-HO-C6H4 C2H5 4'-HO-C6H4 C6H5 0.37
Table 4: ESTROGEN RECEPTOR BINDING DATA FOR THIAZOLES 22AB AND
OXAZOLES 29 AND 31
Figure imgf000088_0001
Compound X R3 R4 RBA
22a s 4'-HO-C6H4 H 0.018
22b s 4'-HO-C6H4 C2H5 0.041
29 O 4'-HO-C6H4 C6H5 <0.001
31 O H5 4'-HO-C6H4 0.027
Table 5: ER BINDING AFFINITIES FOR EXEMPLARY FURANS
Figure imgf000089_0001
R2 R' R" R'" BA(%) ER ( ERβ(%) %)
201 C2H5 OH H OH 5.01 67.6 6.31
202 n-C3H7 OH H OH 3.89
203 C2H5 OH OH OH 5.89 214 3.02
204 n-C3H7 OH OH OH 9.33 85.1 2.4
H OH H OH 0.04
H OH OH H 0.13
200 C2H5 OH OH H 0.71 15.1 2.51
Table 6A: ESTROGEN RECEPTOR BINDING AFFINITY DATA FOR PYRAZOLES AND ISOXAZOLE
Figure imgf000090_0001
Compound X R4 R2 RBA
35a N H H 0.009
35b N C6H5. H 0.028
35c N C6H5CH2. H <0.007
35d N pHOC^ H 0.059
38a N H C2H5 0.015
38b N C6H5. C.H5 14.0
38c N C6H5CH2. C2H5 0.150
38d N pHOCΛ C2H5 19.0
41 O ~ C2H5 0.006
Table 6B : ESTROGEN RECEPTOR BINDING AFFINITIES REPRESENTATIVE FOR PYRAZOLES
Figure imgf000091_0001
R2 R R4 %RBA
H OH H 0.009
H OH C6H5 0.028
H OH C6H5CH2 O.007
H OH p-HOC6H4 0.059
C2H5 OH H 0.015
C2H5 OH C6H5 14
C2H5 OH C6H5CH2 0.47
C2H5 OH p-HOC6H4 20
CH3 OH C6H5 1.6
C2H5 OH CH2CH2OH 1.2
C3H7 OH C6H5 25
Table 7: ER BINDING AFFINITIES FOR EXEMPLARY PYRAZOLE ISOMERS
Figure imgf000092_0001
R' R" R'" R2 RBA(%)
210 H H OH C2H5 0.008 ± 0.005
211 OH H H C2H5 0.43 ± 0.07
212 OH H OH C2H5 5.6
213 OH H OH n-C3H7 15
214 OH OH OH C2H5 13
Table 8: ER BINDING AFFINITIES FOR PYRAZOLES WITH BASIC SIDE
GROUPS.
Figure imgf000093_0001
R R" R'" R2 RBA% RBA, ERα RBA, ERβ cytosol
OH PI OH C2H5 2.0
OH H OH PI 0.013
PI OH OH C2H5 0.40
301 OH OH PI C2H5 32 ± 6.4 5.1 ± 1.6 0.18 ± 0.17
Table 9: RELATIVE ER BINDING AFFINITY DATA FOR CYCLOPENTADIENES
Figure imgf000094_0001
R' R2 R3 RBA RBA ERα ERβ
H C2H5 C2H5 0.028 0.372 0.355
OH C2H5 C2H5 1.047
H C2H5 C6H5 0.05 1.26 0.50
OH C2H5 C6H5 1.20 8.32 7.08
235 OH C2H5 p-OH-C6H5 8.91 5.25 1.66
OH n-C3H7 C6H5 0.06 0.33 0.708
OH n-C3H7 p-OH-C6H5 1.12 8.51 1.66
Scheme \f\
Figure imgf000095_0001
/ Hi where R1 = RJ = R4 p-OH-C6H4- or p-MeO-C&II<
Scheme IB
Figure imgf000096_0001
A i where Rl = R! - p-OH-C4H4 or ?- eO-C5H, R: = Ξ:
Scheme 2
Figure imgf000096_0002
1SR = Me
6F, ■ SMe, 15
17R = H (87%)
where R1 = R3 = □-OH-C H1 or p-MβO-C5H.
Mλ RJ - C4H,-
R2 = Et Scheme 3
a
Figure imgf000097_0001
2la RΛ = K, R' = Me (31%)
BBr-j 21b R" = Ec, R' = Me (35%) CH^CU 7 a R.1 = K. R' = H (*5%)
22b R1 = Et, R' = H (53%) r/t/ where R' = R3 = p-OH-CjH, or p-MeO-C^ 1 = H or Et
Figure imgf000097_0002
Figure imgf000098_0001
Figure imgf000099_0001
= Me, X = II
R = i-Pi, X = H "" = l:t, X = 11 8b R = Bu, X= H '01 = X = H R = Bu, X = OH 102 = IEl, X = Oil 38d R = ABu. X = H '?3 R = i-Bu. X = OH 104 = l'r. X = θll
Sche e 5B Synthesis of C(4) alkyl pyrazole analogs, [this scheme is here and visible in "page layout", but not in "normal' view? It also prints fine.]
Scheme 5C
R
For synthesis of: N-N particularly: R1 = C6H4-OR, R
// \ = CH3, H
/-~^\ R4' A R2 R3 = X, C6H4-OR' R' = CH3 , H
( PA2J R3 R4 = C6H5, CgH4- OR", R" = CH3, H
Figure imgf000100_0001
1. BuLi (2 equiv) / THF
2. O O
3. HCl
Figure imgf000100_0002
ArB(OH)2, Pd(Ph3P)4
Na2C03, H20 / DME
80 °C, 72 h
Figure imgf000100_0003
Figure imgf000101_0001
Scheme 6
Figure imgf000102_0001
39
R -.H aac: r— 40 H = Me (30%) 7 i 41fl = H(5ό%) J =H
Scheme 7
Figure imgf000102_0002
42 43
P2S5 / chioranil
Figure imgf000102_0003
44
scneme 8
Figure imgf000103_0001
R4CHO thazolium cat.
Et3N, EtOH pTsOH Toluene
Figure imgf000103_0002
Figure imgf000103_0003
, R2 = H, R3 = p-CH30-C6H4, R4 = C2H5 30-C6H4, R^ = H, rV 3 = - , p-CH30-C6H4 30-C6H4 30-CsH4, R2 H, R ?3° = = p-CH30-C6H4,
HO-C6H4 = C6H5
Figure imgf000103_0004
Scheme 8A
Figure imgf000104_0001
Figure imgf000104_0002
R=ethyl or propyl
1 KHMDS(1 1)
Figure imgf000104_0004
Figure imgf000104_0005
Figure imgf000104_0003
R'=OCH3, R2= H R3= ethyl R'=H, R2=OCH3, R3= ethyl R'= H, R2= OCH3, R3= propyl R'=R2=OCH3, Rα= ethyl R'=R2=OCH3, R3= propyl
Figure imgf000104_0006
R'= OH, R2=H,R3= ethyl 210 R"=OCH„R'"=H,R2= ethyl R'=H, R2= OH, R3= ethyl 211 R"=H, R'"= OCH,, R2= ethyl R'=H, R2= OH, R3= propyl 212 R"= H, R'"= OCH„ R2= propyl R'= R2= OH, R3= ethyl 213 R"= R"= OCHj, R2= ethyl R'= R2= OH, R3= propyl 214 R"=R'"=OCH„R2= propyl Scheme 8B alternative 1 ,4-dione synthesis, particularly: R1 = C6H5, C6H4-0R, R = CH3, H
R2 = CgH5, C6H4-0R', R' = CH3, H R4 = C6H5, C6H4-OR", R" = CH3, H
Figure imgf000105_0001
alternative
Figure imgf000105_0002
R = CgHs, CgH4-OR', R' = CH3, H R = CgHs, CgH-OR", R" = CH3, H
Scheme 9
Figure imgf000106_0001
60 R1 = R4 = p-CH30-C6H4, R2 = H, R3 = C6H 5
61 R1 R3 = p-CH30-C6H4, R2 - H, R4 = C 'fi6Hπ5
Figure imgf000106_0002
T2 Synthesized by choice of starting diketone i.e.
Figure imgf000106_0003
Schemes 10A-D
Figure imgf000107_0001
Figure imgf000107_0002
Figure imgf000107_0003
1. NaH
2. R2I
PR2
PR2
Figure imgf000107_0004
or NCS (III)
R5 = Y if I, then Y = Br if II, then Y = l if lll, then Y = Cl
Figure imgf000107_0005
Sche
Figure imgf000108_0001
H+/Δ
Figure imgf000108_0002
96A 94
R' 'MgBr @
Figure imgf000108_0003
95A
1. NaH
© 2. R6X
3. BBr3 (to deprotect)
Figure imgf000108_0004
97
95B
® SeO?
Figure imgf000108_0005
96B Scheme 11 B
regioisomers
Figure imgf000109_0001
98
1. LiAIH4
2. H+
Figure imgf000109_0002
97
Scheme 1 1 C General Route for the Synthesis of Cyclooeπtadienes
Figure imgf000110_0001
Scheme 1 1D General Route for the Synthesis of Cyclopentadienones
Figure imgf000110_0002
R = Et or n-Pr " o iΛΛ^ IZA
Figure imgf000111_0001
Figure imgf000112_0001
f-BιιC> .0
Br PP .T I DIΛD j ,OH o. Br 1) BuLi -N NH
2) IBυ02C-N=N-CO?fBu
O' ^ o O^OJBu 3) HCl
7 (05%) 8 (82%)
Figure imgf000112_0002
Scheme 1 B • Synthesis of N(l ) basic side chain containing pyia/.olc 2.
Figure imgf000113_0001
Schβme 12C - Synthesis of C(4) basic side chain containing pyrazole 3. 20
Scheme 13A Pyridazine synthesis particularly. R = C6H5, C6H4-OR, R = CH3, H
R2 = C6H5, C6H4-OR', R' = CH3, H
R4 = C6H5, C6H4-OR", R" = CH3, H
Figure imgf000114_0001
particularly: R1
Figure imgf000114_0002
R3 = C6H5, C6H4-OR', R' = CH3, H R = CgHs, CgH -OR , R = CH3, H
Scheme 13B Synthesis of Pyπmidiπes Class- 1
Figure imgf000115_0001
2 3 R = Et 72-74% R = Et 95-97% R = π-Pr 97-99% R = n-Pr 84-95% X = H or OMe X = H or OH
Scheme 13 C Synthesis of Pynmidmes Class-ll
Figure imgf000115_0002
90%
Figure imgf000116_0001
Scheme 13E. Synthesis of Pyrazines
Figure imgf000117_0001
13
Scheme 13F Synthesis of Pyrazines
Figure imgf000117_0002
R = Et or π-Pr 13
Figure imgf000117_0003
14 15 16
Figure imgf000118_0001
BF3.SMe2
Figure imgf000118_0002
Scheme 14t3: Synthesis of Quinazoiines
Figure imgf000118_0003
POCL3.
Figure imgf000118_0004
R = Et or π-Pr Scheme 14C Synthetic route to cinnolines
Figure imgf000119_0001
R = Et or π-Pr
Scheme 14D: Synthetic route to phthalazines
R = Et or π-Pr Scheme 15A: Combinatorial Synthesis: Pyrazoles
Figure imgf000120_0001
NH2NHR4 NH2NHR4 108 108
Figure imgf000120_0002
Scheme 15B: Combinatoπal Synthesis: Pyrazoles(ll)
Figure imgf000121_0001
103 105
NH2NHR4 108
Figure imgf000121_0002
© R^B(OH)2122 Pd(Ph3P)4
R^ = aryl ^ Na2C03 eg C6H5, H20/DME
remove from support deprotect
Figure imgf000121_0003
Figure imgf000121_0004
Scheme 16: Combinatorial Synthesis: Oxazoles, Thiazoles, imidazoles
Figure imgf000122_0001
Figure imgf000122_0002
Figure imgf000122_0003
Scheme 16: Combinatorial Synthesis: Oxazoles, Thiazoles, Imidazoles (cont)
Figure imgf000123_0001
Figure imgf000123_0003
Figure imgf000123_0002
Scheme 17: Exemplary Basic and Polar Substituents
Figure imgf000124_0001
(CH2)3-CF2CF3
Figure imgf000124_0002
^ O-(CH2)10- ^
Figure imgf000124_0003
|-(CH2)β-N n = ' 1-12
,0-(CH2)5 S02-(CH2)3-CF2CF3 tt m = = 1-6
-(CH2 2)^,6 ■o
Figure imgf000124_0004
0-(CH2)7^ N-
/
Figure imgf000124_0005
Scheme 18 : Exemplary ER Ligands
Figure imgf000125_0001
RBA = 0.016%
Figure imgf000125_0002
RBA = 0.50%
Figure imgf000125_0003
RBA = 0.68 % (1 :1 mixture of isomers) Scheme 18 (Continued)
Figure imgf000126_0001
RBA = 20%
Figure imgf000126_0002
( Csy i
Figure imgf000127_0001
Figure imgf000127_0002

Claims

We claim:
An estrogen receptor ligand having the structure:
Figure imgf000128_0001
wherein x is 0 or 1 and when x = 0 the core ring A is a 5 -membered ring structure that is doubly unsaturated or when x is 1 the core ring is a 6-membered ring structure which is aromatic wherein the ring can be a carbocyclic ring or a heterocyclic ring having one or two non-carbon heteroatom and wherein:
Rl can be selected from the group consisting of phenyls and substituted phenyls wherein the non-hydrogen phenyl group substituents can include, without limitation, basic or polar groups, halogens, hydroxy groups, lower alkyl, alkenyl, alkynyl, and alkoxyl groups, lower ethers, ketones, or thioethers, and substituted lower alkyl, alkenyl or alkynyl groups, where the substituents can be halogens or hydroxy groups;
R\ R3 and R\ can be the same or different, and can be selected from the group consisting of hydrogen, a basic or polar group, a phenyl or substituted phenyl group, lower alkyl, alkenyl or alkynyl where the lower alkyl, alkenyl or alkynyl groups may be substituted, with a basic or polar group, a phenyl, hydroxyls or halogens, lower ethers, ketones or thioethers, and halogens;
R5 or R5' , when present, can be selected from any of the groups defined for R2, RJ and R4 or may be hydrogens, R and may be the same as or different than any of R , R-, R , oor
R4 and R5 or R5' may be the same or different than each other; and
R6, when present, can be hydrogen, a basic or polar group, a lower alkyl, alkenyl, alkynyl, or alkoxy groups, which may be substituted with lower alkyl, alkenyl or alkynyl groups, lower ether or thioethers or halogens and one or more of any -CH2 - groups in R6 can be replaced with -CO- groups.
2. The estrogen receptor ligand of claim 1 which has the structure:
Figure imgf000129_0001
wherein A* is a 5 -membered ring structure that is doublv unsaturated.
The estrogen receptor ligand of claim 1 which has the structure:
Figure imgf000129_0002
wherein A" is a 6-membered ring structure which is aromatic.
4. The estrogen receptor ligand of claim 1 that is a pyrazole.
5. The estrogen receptor ligand of claim 1 that is a cyclopentadiene.
6. The estrogen receptor ligand of claim 1 that is a furan.
7. The estrogen receptor ligand of claim 1 that is a pyrimidine.
8. The estrogen receptor ligand of claim 1 wherein R1 is a p-OH-phenyl group.
9. The estrogen receptor ligand of claim 8 wherein R3 is a p-OH-phenyl group.
10. The estrogen receptor ligand of claim 8 wherein R: is a lower alkyl group.
11. The estrogen receptor ligand of claim 8 wherein R3 is a phenyl group substituted with a basic or polar group.
12. The estrogen receptor ligand of claim 1 wherein R2 is an ethyl or an i-propyl group.
13. A pharmaceutical composition comprising an estrogen receptor of claim 1 in an amount sufficient to exhibit an effect on a hormone-dependent disorder.
14. A method for treating a hormone-dependent disorder which comprises the step of administering to a patient suffering from that disorder the pharmaceutical composition of claim 13.
15. The method of claim 14 wherein the hormone-dependent disorder is hormone-responsive breast cancer.
16. A method for treating estrogen responsive disorders and physiological conditions which comprises the step of administering to a patient suffering from the disorder a pharmaceutical composition of claim 14.
1 . A method for selective regulation of a cellular activity under the control of estrogen receptor which comprises administering a composition comprising an amount of an estrogen ligand of claim 1 sufficient to effect such cellular activity.
18. The estrogen receptor ligand of claim 1 which exhibits RBA of about 1% or more.
19. The estrogen receptor ligand of claim 1 which exhibits RBA of about 10% or more.
20. The estrogen receptor ligand of claim 1 which exhibits selective affinity for one of ERα or ERβ.
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WO2003055860A1 (en) * 2001-12-21 2003-07-10 Vernalis (Cambridge) Limited 3,4-diarylpyrazoles and their use in the therapy of cancer
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US8710038B2 (en) 2004-09-17 2014-04-29 Exelixis, Inc. Pyrazole kinase modulators and methods of use
US8217073B2 (en) 2005-09-27 2012-07-10 Myrexis, Inc. Pyrrole derivatives as therapeutic compounds
US7470715B2 (en) 2005-12-22 2008-12-30 Pfizer Inc. Estrogen modulators
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