BETA ISOFORM

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under Grant Nos. SIO RR0I90I2, GM-42641 , AI I0197S, and HLI 12639 awarded by the National Institutes of Health. The Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0002] The present application claims the benefit of priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application No. 61/963,031 , filed on November 21, 2013, the content of which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The field of the invention relates to compounds that function as ligands for estrogen receptors (ERs). In particular, the field of the invention relates to substituted (4'- hydroxyphenyDcycloalkane compounds that are specific agonists for the estrogen receptor beta (ERp) and the use of such compounds in pharmaceutical compositions for treating diseases and disorders associated with ERp activity.

[0004] Estrogens are important regulators of many physiological processes that include reproduction, cognition, cardiovascular health, and bone metabolism. {See, e.g., Deroo ei af., "Estrogen Receptors and Human Disease," J. Clin. Invest. 1 16:561- 570(2006). Based on their widespread role in a number of physiological processes, estrogens have been implicated in a number of diseases and disorders which include cell proliferative diseases and disorders (e.#. breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, and prostate cancer), neurodegenerative diseases and disorders, cardiovascular disease, and osteoporosis to name a few. (See id.). In many of these diseases and disorders, estrogen mediates its effects through the estrogen receptors (ERs). [0005] The ERs exist in 2 main forms, ERot and ERp, which have different tissue expression patterns. (See Mueller el ai (2001), "Estrogen receptors and endocrine diseases: lessons from estrogen receptor knockout mice," Curr. Opin. Pharmacol. I : 613- 61 ). ERa and ERP are encoded by separate genes, ESRI and ESR2, respectively, found at different chromosomal locations, and numerous mRNA splice variants exist for both ERa and ERp. (See, e.g., Hemyk el ai, "Estrogen receptor mutations in human disease," (2()()4) Endocr. Rev. 25:869-x9X). Because of their role in estrogen-related diseases, ERa and ERp have been targeted for development of specific ligands that modulate their activities. The ligand specificity of ERa and ERp differ, and a ligand that binds and functions as an agonist or antagonist for ERa may or may not bind and function as an agonist or antagonist for ERp.

[0006] One group of ligands for ERs that have been developed are the so-called "selective estrogen receptor modulators" or "SERMs" which include tamoxifen and raloxifene. Tamoxifen and raloxifene have been observed to exhibit tissue-specific estrogenic activity. For example, tamoxifen is an antagonist in the breast and has been a safe and effective adjuvant endocrine therapy for breast cancer for almost 20 years, but tamoxifen is an ER agonist in bone and uterus. (See. e.g., Deroo el ai, "Estrogen Receptors and Human Disease," J. Clin. Invest. 1 16:561-570 (2006)). Raloxifene exhibits greater agonist activity in bone and less agonist activity in the uterus. (See Fabian el ai, "Selective estrogen-receptor modulators for primary prevention of breast cancer," J. Clin. Oncol. 23:1644-1 55 (2005)). Whether a ligand is an ER agonist or antagonist in a particular tissue depends on several factors, including which form of the estrogen receptor predominates in the particular tissue, in other words ERa or ERp, where the ligand may exhibit different binding affinity and/or agonist/antagonist activity for ERa versus ERp.

[0007] ERa and ER agonists have a wide range of biological effects that implicate disease such as cancer and disorders of the central nervous system (CNS). Clinical studies have indicated that administering estradiol (E2) in posl-menopausal hormone replacement therapy (HRT) can lead to increased incidence of breast and endometrial cancer. (See Beral el ai, "Breast cancer and hormone-replacement therapy in the Million Women Study " Lancet. 2003;362(9382:4l9-27, Epub 2003/08/21 , PubMed PMID: 12927427; Gann el ai, "Combined hormone therapy and breast cancer: a single- edged sword," JAMA : the journal of the American Medical Association. United Stales 2003. p. 3304-6; Li el al., "Relationship between long durations and different regimens of hormone therapy and risk of breast cancer," JAMA : the Journal of the American Medical Association. 2003;289(24):3254-63. Epub 2003/0626. doi: I0.100l/jama.289.24.3254. PubMed PMID: 12824206; and Anderson el al., "Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial," JAMA : the journal of the American Medical Association. 2<M>4;2 1 ( 14): 1701-12. Epub 2004/04/15. doi: 10.1001 /jama.2 1.14.1701. PubMed PMID: 15082697). This effect is mediated predominantly by ERa, the dominant isoform present in the mammary gland and uterus. (See Song el al., "Estrogen receptor-beta agonist diary Ipropionitrile counteracts the estrogenic activity of estrogen receptor-alpha agonist propylpyra/ole-lriol in the mammary gland of ovariectomi/ed Sprague Dawley rats. The Journal of steroid biochemistry and molecular biology. 20l2;i30( i-2):26-35. Epub 2012/01/24. doi: I0.10l6/j.jsbmb.20l 1.12.018. PubMed PMID: 22266284).

[0008] The increased cancer risk has led to decreased usage of HRT in postmenopausal women. But, studies also have shown that HRT can provide a positive effect mediated primarily by ER(¾, which is a decrease in the risk of dementia in postmenopausal women. (See Leblanc el al., "U.S. Preventive Services Task Force Evidence Syntheses, formerly Systematic Evidence Reviews. Hormone Replacement Therapy and Cognition. Rockville (MD): Agency for Healthcare Research and Quality (US): 2002). As such, specific ERp agonists can provide the CNS benefits of E2 with minimal side effects. However, current SERMs such as tamoxifen and raloxifene, are not specific for ERp, have carcinogenic side effects, and provide little memory enhancement. (See Yaffe el al., "Cognitive function in postmenopausal women treated with raloxifene. New England Journal of Medicine. 2001 ;344: 1207-13; and Paganini-Hill el al., "Preliminary assessment of cognitive function in breast cancer patients treated with tamoxifen. Breast Cancer Research and Treatment. 2000;64: 165-76). Safer and more effective treatments can be developed by selectively targeting ERfV

[0009] Thus, new ligands for estrogen receptors are desirable. In particular, new ligands that exhibit selective agonist or antagonist activity for ERp versus ERa are desirable. These new ligands should be suitable for treating diseases and disorders associated wilh ER activity, such as cell proliferative diseases and disorders or psychiatric diseases and disorders. Such new ligands are disclosed herein in the form of substituted H'-hydroxylphenyl )cycloalkane compounds.

SUMMARY

[0010] Disclosed are substituted (4'-hydroxylphenyl)cycloalkane compounds and there use as selective agonists of the estrogen receptor beta (ERp). The disclosed compounds may be formulated as pharmaceutical compositions and administered to treat diseases associated wilh ERp agonist activity.

[0011 ] In some embodiments, the disclosed compounds have a Formula I:

where:

A-B is -CH _^ CH _^ -^CHjCH^CH^, -CH _; CH=CH-, or -CH=CHCH;>-;

A'-B' is -CH ₂ CH ₂ -, or-CH=CH-;

Z is a carbon atom;

X is hydroxy!, hydroxyalkyl, amino, or aminoalkyl;

Y is hydrogen, alkyl, or X and Y together form carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, aminoalkylidenyl, or oxime. or Y is - CH ₂ CH ₂ - and Y and Z form a bridge.

Optionally, when A-B is -CH ₂ CH ₂ -, then A'-B' is not -CH=CH- Optionally, when A-B is -CH2CH2- and A'-B' is -CH2CH2-, then X is not hydroxyethyl and X is not aminomethyl. Optionally, when X is hydroxyalkyl, X is hydroxyl-C( I -6)alkyl, preferably hydroxy-C( l-3)alkyl. Optionally, when X is aminoalkyl, X is amino-C(l-6)alkyl, preferably amino-C(l -3)alkyl. Optionally, when X and Y together form carboxyalkylidenyl, X and Y form carboxy-C( l-6)alkylidenyl, preferably carboxy-C(l- 3)alkylidenyl. Optionally, when X and Y together form esteralkylidenyl, X and Y form C( I -6)alkyl-esler-C( 1 -6)alkylidenyl, preferably C( I -3)alkyl-esler-C( I -3)alkylidenyl. Optionally, when X and Y together form hydroxyalkyldenyl, X and Y form hydroxy-C( 1- 6)alkylidenyi, preferably hydroxy-C( i-3)alkylidenyl. Optionally, when X and Y together form aminoalkylidenyl, X and Y form amino-C( l-6)alkylidenyl, preferably amino-C( l- 3)alkylidenyl.

[0012] The disclosed compounds may include 4-substiluted-(4'- hydroxyphenyl)cycloheptane compounds. In the disclosed compounds having Formula I, A-B may be -CH ₂ CH ₂ CH ₂ - and A'-B' may be -CH ₂ CH ₂ - and the disclosed compounds may have a Formula la:

where X and Y are as defined for Formula I.

[0013] The disclosed compounds may include ^subsliluted-i^- hydroxyphenyl)cycloheptene compounds. In the disclosed compounds having Formula I, A-B may be -CH ₂ CH=CH-, and A'-B' may be -CH ₂ CH ₂ - or -CH=CH- and the disclosed compounds may have a Formula Ia(i), a Formula Ia(ii). or a Formula Ia(iii):

where X and Y are as defined for Formula I.

[0014] The disclosed compounds may include 4-substituted-(4'- hydroxyphenyOcyclohexane compounds. For example, in the disclosed compounds having Formula I, A-B may be -CH ₂ CH ₂ -, and A'-B' may be -CH ₂ CH ₂ - and the compound may have a Formula lb:

where X and Y are as defined for Formula I.

[0015] In the disclosed substituted (4'-hydroxyphenyl)cycloalkane compounds, subsliluent Z is carbon and Y may be-CH ₂ CH ₂ - where Y and Z form a bridge. As such, the disclosed compounds may have Formula Ic:

where X and Y are as defined for Formula I.

[0016] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions. As such, also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the compounds disclosed herein. or pharmaceutically acceptable salts of any of the compounds disclosed herein, together with a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the disclosed compounds may be used for preparing a medicament for treating a disease or disorder associated with estrogen receptor β (ERP) activity, and in particular, a disease or disorder that may be treated with an agonist of ERp. As such, the disclosed compounds may exhibit ERp agonist activity, and preferable the compounds exhibit specificity as ERp agonists versus activity ERp antagonists and/or estrogen receptor a (ERa) agonists or antagonists.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. Structures of 17p-estradiol and raloxifene.

[0018] Figure 2. Lowest energy docking poses from clusters where ligands were predicted to bind in two modes (A-B). The human ERa estrogen receptor that was used was in the agonist conformation (PDB code I ere: chain A). Panel C shows the predicted binding orientation for 18 in ERp, agonist conformation (PDB code 2jj3; chain A). Panel D shows the predicted binding orientation for 18 in ERp, antagonist conformation (PDB code 112j; chain A).

[0019] Figure 3. Cell-based ERa assay data including regression for ligands that showed agonist activity.

[0020] Figure 4. Cell-based ERa agonist assay data for chemicals without sufficient quality data to determine activity.

[0021] Figure 5. Cell-based ERa antagonist assay data for chemicals without sufficient quality data to determine activity.

[0022] Figure 6. Cell-based ER agonist assay data, for ligands that showed antagonist activity.

[0023] Figure 7. Cell-based ERp assay data for chemicals that showed antagonist activity. [0024] Figure 8. Cell-based ERp assay data for chemicals that did not display antagonist activity.

[0025] Figure 9. Lowest energy docking poses for the ERa-binding compounds identified using fluorescence polarization. PDB file lere, chain A was used as the receptor to investigate the predicted affinity for binding in the ERot agonist conformation. E2 is estradiol and provided for comparison.

[0026] Figure 10. Lowest energy docking poses for the ERa-binding compounds identified using fluorescence polarization. PDB file lere, chain A was used as the receptor to investigate the predicted affinity for binding in the ERot agonist conformation. Chemical 13 was docked using both enantiomers from the racemic mixture.

DETAILED DESCRIPTION

[0027] The present invention is described herein using several definitions, as set forth below and throughout the application.

[0028] Unless otherwise specified or indicated by context, the terms "a", "an", and "the" mean "one or more." For example, "a substitution" should be interpreted to mean "one or more substitutions." Similarly, "a substituent group" should be interpreted to mean "one or more substituent groups."

[0029] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean plus or minus <10% of the particular term and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0030] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as being "open" transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms "consist" and "consisting of should be interpreted as being "closed" transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term ''consisting essentially of should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0031] Disclosed are substituted (4'-hydroxylphenyl)cycloalkane compounds and there use as selective agonists of the estrogen receptor beta isoform (ERp). Preferred embodiments of the disclosed compounds include (4'-hydroxylphenyl)cyclohepiane compounds and (4'-hydroxylphenyl)cyclohexane compounds. The disclosed compounds may alternatively be referred to as substituted 4-cycloalkylphenol compounds or p- cycloalkyl substituted phenol compounds that include one or more substitutions on the cycloalkyl substituent, which cycloalkyl substituent preferably is a cycloheptyl substituent or a cyclohexyl substituent.

[0032] In some embodiments, the disclosed compounds include one or more substitutions on the 4-carbon of the cycloalkyl substituent and have a Formula 1:

where:

A-B is -CH ₂ CH2-,-CH2CH ₂ CH2-, -CH;CH=CH-, or -CH=CHCH -;

A'-B' is-CH ₂ CH -, or-CH=CH-:

Z is a carbon atom;

X is hydroxyl, hydroxyalkyl (e.g., hydroxy-C(l-6)alkyl) or hydroxy-C(l-J)alkyl), amino, or aminoalkyl (e.g., amino-C(l-6)alkyl) or amino-C(l-3)alkyl): provided that when A-B is

-CH2CH2- and A'-B' is -CH2CH2-, then X is not hydroxyethyl and X is not aminomethyl;

Y is hydrogen, alkyl; or X and Y together form carboxyalkylidenyl (e.g., carboxy- C( l-6)alkylidenyl or carboxy-C( l-3)aikylidenyl): or X and Y together form esteralkylidenyl (e.g., (e.g., C(l-6)alkyl-ester-C( l-6)alkylidenyl or C( l- 3)alkyi-esler-C( l-3)aikylidenyl); or X and Y together form hydroxyalkylidenyl (e.g., hydroxy-C( l-6)alkylidenyl or hydroxy-C(l- 3)alkylidenyl): or X and Y together form aminoalkylidenyl (e.g., amino- C( I -6)alkylidenyl or amino-C( l-3)alkylidenyl); or X and Y together form oxime; or Y is -CH CHr- and Y and Z form a bridge.

[0033] The disclosed compounds may include 4-substiiuted-(4'- hydroxyphenyOcycloheptane compounds. For example, in the disclosed compounds having Formula I, subslituent A-B may be -CHiCH^CHj- and substituent A'-B' may be - CH;CH ₂ - and the disclosed compounds may have a Formula la:

where X and Y are as defined for Formula I. In some specific embodiments of compounds having Formula la, the subslituent X may be hydroxyl or hydroxyalkyl, and optionally Y may be hydrogen.

[0034] The disclosed compounds having Formula la may exhibit specific stereochemistry, for example, where X and Y are as defined for Formula I and the compounds have a formula selected from the group consisting of

[0035] The disclosed compounds may include 4-substiluled-(4'- hydroxyphenyDcycloheptene compounds. In the disclosed compounds having Formula I, A-B may be -CH ₂ CH=CH- and A'-B' may be -CH2CH2- or -CH=CH- and the disclosed compounds may have a Formula la(i), a Formula la(ii), or a Formula Ia(iii):

X X

where X and Y are as defined for Formula I. In some specific embodiments of compounds having Formula Ia(i), Formula la(ii). or Formula la(iii). the substituent X may be hydroxyl or hydroxyalkyl, and optionally Y may be hydrogen.

[0036] The disclosed compounds having Formula la(i). Formula Ia(ii), or Formula Ia(iii) may exhibit specific stereochemistry, for example, where X and Y are as defined for Formula I and the compounds have a formula selected from the group consisting of

where X and Y are as defined for Formula I. In some specific embodiments, the subsliluent X may be hydroxyalkyi, Y may be hydrogen, and the compound may have the formula:

[0037] The disclosed compounds may include 4-substituted-(4'- hydroxyphenyl)cyclohexane compounds. For example, in the disclosed compounds having Formula 1, A-B may be -CH ₂ CH ₂ -, A'-B' may be -CH ₂ CH ₂ -, and the compound may have a Formula lb

where X and Y are as defined Tor Formula I. In some embodiments of compounds having Formula lb, substituent X may be hydroxymethyl and Y optionally may be hydrogen. In even further embodiments of compounds having Formula lb, substituent X may be hydroxyalkyi and Y optionally may be alkyl.

[0038] The disclosed compounds having Formula lb may exhibit specific stereochemistry, for example, where X and Y are as defined for Formula I and the compounds have a formula selected from the group consisting of

[0040] In some embodiments of compounds having Formula lb, the substituents X and Y together may form carboxymethyiidenyi, esteralkylidenyl, hydroxyelhylidenyi, aminoethylidenyl, or oxime. For example, in some embodiments of the disclosed compounds having Formula lb, X and Y together may form a alkylidenyl or an iminyl group which optionally is substituted and where the compounds have Formula Ib(i):

and V is carbon or nitrogen, and W is alkyl, hydroxy), hydroxyalkyl, amino, aminoalkyl, carboxyl, alkylcarboxyl, or ester. For example, in some embodiments of the disclosed compounds having Formula lb, X and Y together may form carboxymethyiidenyi, ethyleslermethylidenyl, hydroxyelhylidenyi. or oxime, where the compounds have a formula selected from the following formulas, respectively.

[0039] In the disclosed substituted (4 ^, -hydroxyphenyl)cycloalkane compounds, substituent Z is carbon and Y may be-d^CH -, where Y and Z form a bridge. As such, the disclosed compounds may have Formula Ic:

where X and Y are as defined for Formula I. Specific compounds having Formula Ic may include but are not limited to compounds having a formula selected from the group consisting of

[0040] The compounds disclosed herein (e.g., compounds having any of Formula I, la, Ia(i), Ia(ii), ia(iii), lb, Ib(i), or Ic may have several chiral centers, and stereoisomers, epimers, and enantiomers of the disclosed compounds are contemplated. The compounds may be optically pure with respect to one or more chiral centers (e.g., some or all of the chiral centers may be completely in the S configuration; and/or some or all of the chiral centers may be completely in the R configuration; etc.). Additionally or alternatively, one or more of the chiral centers may be present as a mixture of configurations (e.g., a racemic or another mixture of the R configuration and the S configuration). Compositions comprising substantially purified stereoisomers, epimers, or enantiomers of compound having any of Formula I, la, Ia(i), la(ii), la(iii), lb, lb(i), or Ic are contemplated herein (e.g., a composition comprising at least about 90%, 95%, or 99% pure stereoisomer, epimer, or enantiomer.

[0041] The compounds disclosed herein may exhibit binding and agonist and/or antagonist activity for estrogen receptors. As used herein, "ERa" refers to estrogen receptor-alpha, and in particular, human estrogen receptor-alpha. As used herein, "ERJT refers to estrogen receptor-beta, and in particular human estrogen receptor-beta. Agonists and antagonists for ERa and ERp are known in the art as are assays for determining the binding affinity of a compound for ERa and ERp and determining whether a bound compound is an agonist or antagonist for ERa and ERp. (See e.g., McCullough ei ai, "Probing the human estrogen receptor-a binding requirements for phenolic mono- and di- hydroxyl compounds: a combined synthesis, binding and docking study," Biorg. & Med. Chem. (2014) Jan l;22(l):303-10. doi: 10.1016/j.bmc.2013.1 1.024. Epub (2013) Nov 21, and the corresponding Supplementary Information, the contents of which are incorporated herein by reference in their entireties). Suitable assays for determining the binding affinity of a compound for ERa and ERP and determining whether a bound compound is an agonist or antagonist for ERa and ERP may include fluorescence polarization displacement assays and cell-based ERa and ERp luminescence activity assays.

[0042] As used herein, the term "selective agonist" may be used to refer to compounds that selectively bind to an estrogen receptor, and in particular, ERp, relative to another estrogen receptor, and in particular ERa. For example, a compound that is a selective agonist for ERp may have a binding affinity for ERp receptor (e.g., as measured by Kj (nM)) that is at least 3-fold greater (or at least 5-fold greater, at least 1 -fold greater, at least 20-fold greater, at least 50-fold greater, at least 100-fold greater, at least 500-fold greater, or at least 1000-fold greater) than a binding affinity for ERa. Preferably, a selective agonist for ERP has a j (nM) for ERP that is less than KM ⁾ nM, more preferably less than 10 nM, or even more preferably less than 1 nM; and preferably, a selective agonist for ERP has a Kj (nM) for ERa that is greater than 500 nM, more preferably greater than 1000 nM, or even more preferably greater than 2000 nM.

[0043] As used herein, the term "selective agonist" may be used to refer to compounds that selectively bind and agonize an estrogen receptor, and in particular ERp, relative to another estrogen receptor, and in particular ERa. For example, a compound that is a selective agonist for ERP may have an ICso (nM) in an assay for ERP receptor agonist activity that is less than 100 nM, preferably less than 1 nM, even more preferably less than 1 nM: and a compound that is that is a selective agonist for ERp may have an ICso (nM) in an assay for ERa receptor agonist activity that is greater than 100 nM, preferably greater than 500 nM, even more preferably greater than 1000 nM.

[0044] As used herein, the term "selective agonist" may be used to refer to compounds that selectively bind and agoni/e an estrogen receptor, and in particular ERp, instead of antagonizing an estrogen receptor, and in particular ΕΙ*β. For example, a compound that is a selective agonist for ERf$ may have an IC5 ₀ (nM) in an assay for ERp receptor agonist activity thai is less than 100 nM, preferably less than 10 nM, even more preferably less than 1 nM; and a compound that is that is a selective agonist for ER0 may have an IC50 (nM) in an assay for ER0 receptor antagonist activity that is greater than UK) nM, preferably greater than 500 nM, even more preferably greater than 1000 nM.

[0045] Pharmaceutically acceptable salts of the disclosed compounds also are contemplated herein and may be utilized in the disclosed treatment methods. For example, a substituent group of the disclosed compounds may be protonated or deprotonated and may be present together with an anion or cation, respectively, as a pharmaceutically acceptable salt of the compound. The term "pharmaceutically acceptable sail" as used herein, refers to salts of the compounds which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.

[0046] Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, hepianoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.l.4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phlhalate, xylenesulfonate, phenylacetate, phenylpropionate. phenylbutyrate, citrate. lactate, alpha-hydroxybulyrate. glycolate. tartrate, methanesuifonate, propanesuifonate, naphthalene- 1 -sulfonate, naphthalene-2-suifonate, mandelate, and the like.

[0047] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0048] It should be recognized that the particular counter-ion forming a part of any salt of a compound disclosed herein is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

[0049] It will be further appreciated that the disclosed compounds can be in equilibrium with various inner salts. For example, inner salts include salts wherein the compound includes a deprotonated subslitueni group and a protonated substituent group.

[0050] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions. As such, also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the compounds disclosed herein, or pharmaceutically acceptable salts of any of the compounds disclosed herein, together with a pharmaceutical excipieni. In some embodiments, the disclosed compounds may be used for preparing a medicament for treating a disease or disorder associated with estrogen receptor β (ERp) activity, and in particular, a disease or disorder that may be treated with a specific agonist of ERp. As such, the disclosed compounds may exhibit ERP agonist activity, and preferable the compounds exhibit specificity as an ERp agonist versus an ERp antagonist, an ERct agonist, and/or an ERot antagonist.

[0051] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions for treating diseases that are associated with estrogen ERP activity. Diseases and disorders associated with ERp activity may include, but are not limited to, cell proliferative diseases and disorders (e.g.. breast cancer, ovarian cancer, and endometrial cancer), psychiatric diseases and disorders (e.g., depression or anxiety), neurodegenerative diseases or disorders, bone metabolic diseases or disorders {e.g. osteoporosis), metabolic diseases or disorders (e.g.. obesity or insulin resistance), and cardiovascular diseases or disorders. The disclosed pharmaceutical compositions may be administered to patients in need thereof in methods for treating diseases and disorders associated with ERP activity.

[0052] The compounds and pharmaceutical compositions disclosed herein may be administered to a patient in need thereof to treat a disease or disorder. In some embodiments, the compounds disclosed herein may be administered at an effective concentration such that the compound functions as an agonist for ERP in order to treat a disease or disorder associated with ERp activity. In some embodiments, the amount of the disclosed compounds that is effective for the compound to function as an agonist of ERp is about 0.05 - 50 μ (or about 0.05 - 10 μΜ, or about 0.05 - I μΜ).

[0053] As used herein, a "patienf may be interchangeable with "subject" or "individual" and means an animal, which may be a human or non-human animal, in need of treatment. Suitable patients for the disclosed methods may include, for example mammals, such as humans, monkeys, dogs, cats, horses, rats, and mice. Suitable human patient include, for example, those who have a disease or disorder associated with ERP activity or those who have been determined to be at risk for developing a disease or disorder associated with ERp activity.

[0054] As used herein, a "patient in need of treatment" may include a patient having a disease, disorder, or condition that is responsive to therapy with an ERp agonist. For example, a "patient in need of treatment" may include a patient having a cell proliferative disease, disorder, or condition such as cancer {e.g., cancers such as breast cancer). In addition, a "patient in need of treatment" may include a patient having a psychiatric disease or disorder {e.g., depression or anxiety).

[0055] As used herein, the terms "treating" or "to treat" each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0056] As used herein the term "effective amount" refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a disease or disorder associated with ER0 activity in a patient, whereby the effective amount induces, promotes, or causes ER|) agonist activity in the patient.

[00S7] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its si e, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0058] In some embodiments, a daily dose of the disclosed compounds may contain from about O.OI mg kg to about KM ⁾ mg kg (such as from about 0.05 mg kg to about 50 mg kg and/or from about 0.1 mg/kg to about 25 mg kg) of each compound used in the present method of treatment. The dose may be administered under any suitable regimen (e.g., weekly, daily, twice daily).

[0059] The pharmaceutical compositions for use according to the methods as disclosed herein may include be a single compound as an active ingredient or a combination of compounds as active ingredients. For example, the methods disclosed herein may be practiced using a composition containing a single compound that is an ER agonist. Alternatively, the disclosed methods may be practiced using a composition containing two or more compounds that are ERfi agonists, or a compound that is an ERP. agonist together with a compound that is an ERa antagonist. [0060] Instead οΓ administering a pharmaceutical composition comprising a compound that is an ERp agonist together with a compound that is an ERcc antagonist, the disclosed methods may be practiced by administering a first pharmaceutical composition (e.g., a pharmaceutical composition comprising an ERP agonist) and administering a second pharmaceutical composition {e.g.. a pharmaceutical composition comprising an ERct antagonist), where the first composition may be administered before, concurrently with, or after the second composition. As such, the first pharmaceutical composition and the second pharmaceutical composition may be administered concurrently or in any order, irrespective of their names.

[0061] As one skilled in the art will also appreciate, the disclosed pharmaceutical compositions can be prepared with materials (e.g., actives excipients, carriers, and diluents etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non- human subjects, but not suitable for administration to humans.

[0062] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophili/ed dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof. Alternatively, the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in liquid form (e.g., an injectable liquid or gel)

[0063] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes an excipient, carrier, or diluent. For example, the excipient, carrier, or diluent may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. [0064] The compounds utilized in the methods disclosed herein also may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate. lactose anhydrous, and various starches: examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as AvicelK PHIol and Avicel PHI()2, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil<$200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are MagnasweetK (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxyben/oic acid such as butyl paraben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as ben/aikonium chloride.

[0065] Suitable diluents for the pharmaceutical compositions may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicelw PHI 01 and Aviceloo PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatosew DCL21 ; dibasic calcium phosphate such as Emcompressx; mannitol; starch; sorbitol; sucrose; and glucose.

[0066] The disclosed pharmaceutical compositions also may include disintegrants. Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross- povidone, sodium starch glycolate, and mixtures thereof. [0067] The disclosed pharmaceutical compositions also may include effervescent agents. Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, Aimaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonales include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0068] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carriers) or excipient(s).

[0069] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets: powders or granules: solutions or suspensions in aqueous or non-aqueous liquids: edible foams or whips: or oil-in-waier liquid emulsions or water-in-oil liquid emulsions.

[0070] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0071] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0072] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an oiniment, ihe compound may be employed with either a parafflnic or a waler-miscibie ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0073] Pharmaceutical compositions adapted for topical administration in the mouth include lo enges, pastilles and mouth washes.

[0074] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0075] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle si/e (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0076] Pharmaceutical compositions adapted for administration by inhalation include fme particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0077] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0078] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antt-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a free/e-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, Tor example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

EXAMPLES

[0079] The following examples are illustrative and should not be interpreted to limit the claimed subject matter.

[0080] Example I . Probing the human estrogen receptor-a binding requirements for phenolic mono- and di-hvdroxyl compounds: a combined synthesis, binding and docking study.

[0081] Reference is made to McCullough et ai, Biorg. & Med. Chem. (2 14) Jan I ;22( 0:303-10. doi: 10.101 /j.bmc.2013.1 1.024. Epub (2013) Nov 21, and the corresponding Supplementary Information, the contents of which are incorporated herein by reference in their entireties.

[0082] Abstract

[0083] Various estrogen analogs were synthesized and tested for binding to human ERa using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg393 Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxy! group interacting with His524 in ERa. One nonsteroid-based dihydroxyl compound was 1.000-fold specific for ERp over ERa, and was also 20-fold specific for agonist ERP versus antagonist conformations. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERp, versus with Thr2 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERct with high affinity, via hydroxyl hydrogen bonding interactions with the ERa Arg3 3/Glu353/water triad, and van der Waals interactions with the rest of the molecule.

[0084] I . Introduction

[0085] Estrogen receptor-ct (ERa) is a 595-residue, 66 kDa protein with a ligand binding domain of 245 residues (28 kDa). ERa, along with estrogen receptor-β (ERP), belongs to the nuclear hormone family of intracellular receptors. It is one of the two principal receptors responsible for binding the endogenous estrogen, 17p-eslradiol (E2), shown in Figure I .' In the nucleus, ER binds to DNA as a dimer, recruiting coactivators or corepressors that will result in activating or repressing the transcription of different genes Binding of E2 activates the ER, regulating activity. Both ERa and ERp forms are found in different tissue types. However, ERa is expressed more in breast tissue and is also known to be involved in the pathway that regulates breast cancer development. ^' ' ⁴ ERa antagonists such as raloxifene (Figure I) can bind to ER in the same ligand-binding domain as E2, and disrupt normal ER cellular function. ⁴ ' ⁵ (See Figure 1).

[0086] A key structural feature of E2 is the presence of two hydroxyl groups that are separated by 1 1 A, which permits interaction with conserved binding site residues Arg394/Glu353 and His 524. But, the receptor is capable of binding many other compounds whose structures resemble that of the E2 hormone/' Some of these compounds are endogeneous, such as estrone and other human estrogens; and, some are exogeneous, like the drugs raloxifene (Figure 1) or tamoxifen that are used to treat breast cancer and osteoporosis. ⁷ In addition to drugs, there exist other exogeneous compounds, some naturally occurring like phytoestrogens and some synthetic such as organochlorines, that have measurable estrogenic activity. ⁵ Many of these latter compounds have been shown to be linked to breast cancer as well as birth defects. ⁸ ' ⁹ Through the National Institutes of Environmental Health Sciences, the BSB (Biomolecular Screening Branch), and other federal agencies, the government has developed a program to test many of the chemicals currently in our environment, to see if they have estrogenic activity. ¹⁰

[0087] Because of the estrogen receptor's prominent role as a breast cancer drug target, along with the threat posed by the potentially large number of estrogen agonists and antagonists in our environment (e.g. endocrine disruptors), it is essential to gain a better understanding of the binding requirements of the ERct ligand pocket. This understanding will allow for the design of better breast cancer drugs that interfere with the carcinogenic activity of estrogen agonists, and improve our ability to predict which pollutants might bind to ERa. Such predictions are strengthened by a better definition of the molecular features that trigger agonist or antagonist effects, as well as a validation of the docking methods used to predict binding.

[0088] One technique that can provide a quick and reliable experimental measurement of binding affinity is fluorescence polarization." A fluorescence polarization displacement assay can be used to screen non-fluorescent molecules, by displacing a fluorescent probe with the molecule of interest. ¹² Such fluorescence polarization displacement assays have been developed previously for ERa and ERp, based on a fluorescein isothiocyanate (FITC)-tagged estradiol (F-E2). ^{I 4} One such assay is available from Invitrogen. ¹⁵ Subsequent studies in our lab improved the synthesis of F-E2 and examined the in vivo behavior of F-E2 in vivo, in fish. F-E2 was found to localize in cells that develop into reproductive organs, consistent with the proposed role of E2 in gender determination in fish. ¹⁶ An analogous fluorescence polarization method was developed using an intrinsically fluorescent nonsteroid estrogen. ¹⁷

[0089] Herein we present the synthesis of a series of phenolic mono- and di- hydroxyl estrogen analogs, which were tested for binding affinity for human ERa, using fluorescence polarization displacement assay based on F-E2. Estrogen (E2) is a phenolic compound comprised of a steroid core and a second hydroxyl group that is 1 1 A from the phenolic hydroxyl. Compounds synthesized herein have the phenolic core, but vary in terms of whether they: (a) are steroid-based, and (b) possess a second hydroxyl group, - 11 A from the phenol. In addition to binding affinity measurements for compounds, docking calculations were performed. Docking is the process of positioning a ligand into the binding site of a protein and calculating a binding energy for each pose. ¹ * It has become an important early-stage method for finding molecules likely to bind to a protein, allowing for many chemicals to be rapidly screened as potential drug leads. ^{18 20} Docking has also proven useful for identifying compounds as targets for pollutant bioremediation. ^"1 Besides predicting relative binding affinity, docking is used to predict the orientation or pose of a known ligand bound to a protein. ²² Comparison of docking predictions with experimental affinity measurements allows one to rationalize binding site requirements, and also provides validation of the predictive ability of the docking calculations for a given target (e.g. ERa) and class of compounds (phenolic mono- and di-hydroxyl compounds). This is important because such experimental validation provides greater confidence in the docking calculations when they are done on larger sets of compounds, where experimental verification might not be feasible.

[0090] 2. Results and Discussion

[0091] 2.1 Synthesis

[0092] Wittig olefination of estrone benzyl ether, ²³ followed by epoxidation with mCPBA gave the known ²⁴ epoxide 1 as a mixture of diastereomers (Scheme I). Deprotonation of 1 with lithium diisopropylamine, followed by cleavage of the ben/yl ether under dissolving metal conditions gave the allylic alcohol 2. Palladium catalyzed alkoxycarbonylation of the vinyl inflate derived from estrone benzyl ether, according to the literature procedure, ²⁵ gave n-propyl (20.V)-3-(phenylmethoxy)-estra- 1,3,5( 10), 1 - teiraene-17-carboxylate (3). which upon reduction in the presence of Raney- i gave the saturated ester 4. The skipped diene (20.S>3-(phenylmethoxy)-l9,24-dinorchola- 1 ,3,S( 10), 1 ,22-peniaene (5) was prepared by the literature procedure. ²⁵ Hydrogenation of the less substituted olefin in the presence of Wilkinson's catalyst, followed by deben/ylation gave 7. Hydroboration-oxidalion of 5, by the literature procedure ²⁶ gave (20.V)-3-(phenylmethoxy)-l ,24-dinorchola-l,3,5( IO),l6-tetraen-23-ol (8). Subjecting 8 to acid resulted in the spirocyclic tetrahydrofuran 9 in quantitative yield, which upon catalytic hydrogenolysis gave 10. Alternatively, debenzylaiion of 8 afforded 11. Oxidation of 11 gave the aldehyde 12. Reaction of 12 with an excess of methyl Grignard, followed by work-up with saturated aqueous ammonium chloride proceeded by cyclization to afford the spirocyclic tetrahydrofuran 13 as a mixture of diastereomers. 3] Scheme I

[0094] A series of /^-substituted phenols were also prepared (Scheme 2). Reduction of 4-(4 ^* -hydroxyphenyl)cyclohexanone gave a separable mixture of trans-4- (4'-hydroxy-cyclohexyl)phenol 15 (86%) and its cis- diastereomer 14 (10%). The stereochemical assignments for each were made by comparison to their literature spectral data. ³⁷ Reaction of 4-(4 ^* -hydroxyphenyl)cyclohexanone with hydroxylamine- hydrochloride gave the oxime 16. [4-((4'-Hydroxyphenyl)cyclohepta-2,6- dienyDmethanol 17 was prepared from />-aceioxysiyrene according to the literature procedure. ²⁸ This involved cross metathesis with (l-methoxycarbonyl-2-vinyl-3-pentene- l,5-diyl)Fe(CO)j (21), followed by oxidativelly induced reductive elimination. Reduction of the resultant cyclopropanecarboxylate and concomitant Cope 13, 31 -rearrangement gave the cycloheptadiene 17. Catalytic reduction of 17 gave the saturated cycloheptane 18. Finally, Heck-type coupling of methyl 5-bromo-2-ftiranoate with p-acetoxystyrene gave the /ram-sty rylfuranoale 19, which upon reduction with lithium aluminum hydride gave the furfuryl alcohol 20.

[0095] Scheme 2.

[0096] 2.2 Fluorescence Polarization Displacement and Cell-Based ERot and ERR Luminescence Activity Assays

[0097] Twelve compounds from Schemes I and 2 were screened using fluorescence polarization, for their ability to bind ERo (Table 1). Only six compounds showed any significant affinity for the receptor at concentrations as high as 1 μΜ. These compounds include five of the six steroid-core compounds— 2, 4, 7, 11, and 13— and one bicyclic compound— 18. Of the remaining six compounds which did not bind to ERot, one has the steroid core while the others contain the linked ring cores containing a flanking hydroxyl group— a structure whose hydrophobic interior and hydrophilic exterior resembles that of estrogen itself. The highest affinity ERct ligand was 2, with a _d (32 nM) approaching that of E2 (3 nM). 18 is the only non-steroid core compound with measurable ERa binding affinity, but an accurate _d could not be obtained (estimated to be > I μΜ).

[0098] Tabic I . Dissociation constants (K _< i) from the fluorescence polarization displacement assay and ICjo data from cell-based ERa and ER0 agonist assays and ERp antagonist assays. ERct antagonist behavior was not observed. NA indicates data was not of sufficient quality to measure activity. Assay data for E2 binding to ERa ¹⁵ , and ERa agonist and ERp agonist and antagonist activity in cellular assays' ⁷ , were previously reported.

1Mi antagonist behavior was nut observed. ΝΛ indicates data was nol ol sul ^' licient quality to measure activity. Assay data lor E2 bi ding to ER¾. and ER? agonist and ER|t agonist and antagonist activity in cellular assays, were previously teported.

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[0099] Cell-based ERot and ERp luminescence assays were performed to determine whether the ERa ligands were acting as agonists or antagonists, and whether they had specificity for the a isoform (Table I , Figures 3-8). Three compounds, 4, 13, and 2, showed agonist activity in the ERa assay; and, all six compounds showed ERp agonist activity, with 4, 2, and 18 being the most potent; 18 is unique in its selectivity for ERp over ERa, and is 20-fold more potent as an antagonist, versus agonist. 11, 7, and 18 displayed ERp antagonist activity, with 7 being the most potent.

[00100] 2.3 Docking

[00101] Compounds were computationally docked into human ERa and ERP in agonist and antagonist conformations. Poses for ERa are shown in Figures 9 and 10. Initial control docking studies were performed with E2, to validate the docking method by demonstrating an ability to reproduce the known binding mode from the crystal structure. Interestingly. E2 docked with similar predicted affinity in two distinct poses for the ERa agonist conformation (Table 3), essentially flipping the positioning of the two hydroxyl groups with regard to interactions with Arg394/Glu353 and His524, located on opposite sides of the pocket. The predicted pose with the phenolic hydroxyl near Arg394/Glu353 is referred to as the "normal" mode, and that with the phenolic hydroxyl near His524 as the "reversed" mode. But, if docking is performed on receptor that has the tightly bound water present near Arg394/Glu353, then only the expected pose is obtained; and, E2 is the ligand with highest predicted affinity (Table 2), as expected. Thus, all docking was performed with the Arg394/Glu353 water present. This binding mode has been studied previously using molecular dynamics, and illustrates the important role of active site water molecules in ligand binding/"

[00102] Docking results were rank ordered according to the lowest energy pose for binding to the ERa agonist conformation, from the cluster with the highest population (Table 2). Identifying the compounds with measurable Kd values from the fluorescence polarization displacement assay (shown as bold in Table 2) indicates that the docking procedure using Autodock4 was able to separate the binding ligands from the non-binding ligands. ER is a unique docking target, since the binding site is comprised of a nearly closed hydrophobic pocket, flanked by hydrogen bonding groups that could provide specificity." Care in analyzing docking results is needed due lo the large binding area in which ligands can potentially bind, and symmetry of the pocket. Three examples of reversed binding modes that are likely false are shown in Figure 2. 0103] Interestingly, while estradiol docked in only one orientation when the bound water is present, other compounds were slill predicted to bind in two orientations (Table 2; Figure 2), one normal (with the phenolic hydroxy! interacting with Arg3 2/Glu353/Water), and one "reversed," where the phenolic hydroxyl interacts with His524. This promiscuity in predicted binding mode may be due lo symmetry in di- hydroxyl molecules like 2 (Figure 2). Curiously, the mono-hydroxyl 4 also is predicted to bind in a reversed mode (Figure 2), but with much lower affinity relative to the normal mode. This is likely due to the fact that 4 has only one hydroxyl group, the phenol, which provides significant binding energy via interaction with the Arg392/Glu353/waier triad. It is also clear that the aliphatic hydroxyl interaction with His524 is not essential, since it is absent in 4 and 7, and yet both bind with reasonable affinity (ICJV = 160-320 nM). Indeed, this observation is consistent with the ability of phenolic endocrine disruptors, which contain only one hydroxyl group, to bind to ER.'* ^" ' (.See Figure 2).

0104] Tabic 2. Docking of compounds prepared in Schemes I and 2 into the agonist and antagonist conformations of KRa and ERp. Compounds identified as having ERa affinity in the fluorescence polarization displacement assay arc in bold.

Compounds identilied as having tKv allinity in the tluorescencc polarization displacement assay are in bold.

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[00105] The docking of compounds 10 and 13 in the ERp-agonist conformation displayed predicted binding energies that were weaker than expected in Table 2. Inspection of the binding site) showed that these ligands experience steric clashes with binding site sidechains. Additionally, for structures 10 and 13, the oxygen atom in the tetrahydrofuran ring was not positioned near His475 for 10 or (for reversed mode binding) near Arg3 6, Glu305 for 13, to allow for hydrogen bond formation hydrogen bonds.

[00106] Compound 18 is in a unique class, in that it is not based on the steroid core, is selective for the β over the a ER isofonn, and is 25-fold selective for ERp agonist versus ERP antagonist activity (Table I ). Docking pose predictions (Figures 2C and 2D) show that 18 could form two hydrogen bonds (one with His475) in the ER agonist conformation, whereas in the ERp antagonist conformation, hydrogen bonding is with Thr2 , rather than His475. A molecular overlay of E2 and 18 shows the oxygen atoms of the two molecules are well-aligned (data not shown).

[00107] Conclusion

[00108] Human ERa remains an important target for therapeutic interventions (cancer: osteoporosis). Estrogen has a key interaction between its phenolic hydroxy! and a binding site Arg394/Glu353/water triad, along with other important interactions including van der Waals interactions with the steroid core, and hydrogen bonding interactions between an aliphatic hydroxyl group and His524 (His475 in ERp). The two estradiol hydroxyls are located 1 1 A from each other. The studies presented herein probe the importance of interactions with the aliphatic hydroxyl and with the steroid core, using a series of novel mono- and di-hydroxyl compounds (Schemes I and 2).

[00109] The estrogen analog with highest measured affinity in the fluorescence polarization displacement assay(ICso = 32 nM) and second highest predicted affinity is the di-hydroxyl steroid 2, which has a single point of unsaturation in the D-ring, and (relative to estradiol) has its aliphatic hydroxyl extended by one methylene group. Nonetheless, this gives an 0-0 distance essentially equivalent to that for estradiol. Di-hydroxyl steroid 2 behaves as an ERa agonist, and has only modest selectivity for a versus β ER isoforms. Indeed. 2 is a potent ERp agonist and antagonist. In contrast, 18 binds weakly to ERa , yet has on 0-0 distance ( 1 1.1 A) that is similar to 2. Of particular interest is the fact that 18 has the expected interaction with His475 in the ERp agonist docking, whereas in the ERp antagonist docking this aliphatic hydroxyl group is predicted lo interact instead with Thr2 (Figure 2). This could explain why 18 is so selective (25-fold) as an ERp agonist, versus as an antagonist (Table 1 ). Most of the other compounds from Scheme 2 that lacked the steroid core did not bind to ERot, even though they possessed the phenolic hydroxyl. Compounds (4, 13, 2). which possessed ERot agonist activities, were also ERP agonists; but, not ER antagonists. And, these compounds were more selective for ERP over ERa.

[00110] In summary, several compounds have been identified that are potent ERa agonists, and also behave as ERp agonists and antagonists (Table 1). The most potent is the dihydroxyl steroid 2. Also, the non-steroid dihydroxyl compound 18 is 1 ,000-fold more selective for ERp over ERa, and appears to adopt a different binding mode in these two targets (Figure 2).

[00111] Experimental Section

[00112] 4.1 General Methods

[00113] The P-estradiol (min. 98%) and fluorescein (FITC) were purchased from Sigma. The ct-ER and a-ER screening buffer were from Invitrogen. The FITC -estradiol linked tracer used in the experiments was synthesized by as described previously. (1) cV,- DMSO was purchased from Cambridge Isotopes. The 96-well plates used were black, polystyrene, NBS (non-binding surface), flat-bottom plates obtained from Corning. A PolarStar Galaxy fluorescent plate reader was used and controlled with FLUOStar Galaxy software (version 4.30-0). Estrone ben yl ether* and compounds 3, ²⁵ S, ^2b S, ^2b and 17"* were prepared by the literature procedures. [00114] 4.2 Estrogen analog synthesis

[00115] 4.2.1 3-Hvdroxyestra-1.3.5( 10 6-tetraene-17-methanol (2)

[00116] To a solution of methyl iriphenylphosphonium bromide (589 mg, 1.6S mmol) in THF ( 10 mL) at -40 C under N ₂ , was added a solution of w-butyl lithium (0.66 mL, 2.5 M in hexanes, 1.7 mmol). The ylide solution was warmed to room temperature and a solution of estrone benzyl ether (200 mg, 0.556 mmol) in THF (7 mL) was added. The mixture was stirred for 12 h, and then heated at reflux for 5 h. The solution was cooled, and concentrated, and the residue was purified by column chromatography (Si(¼, hexanes-ethyl acetate = 4: 1 ) to afford the exocyclic methylene product ( 168 mg, 84%) as a colorless solid. This product was used in the next step without further characterization. To a solution of the olefin (100 mg, 0.279 mmol) in dichloromethane (6 mL) at 0 C, was added solid m-chloroperoxyben/oic acid (57.5 mg, 0.333 mmol). The reaction mixture was 4 h, and then quenched with aqueous NaHCO.i. The mixture was extracted several times with dichloromethane, dried and concentrated to afford the epoxide 1 (90 mg, 86%) as a colorless oil, which was used in the next step without further purification. To a solution of the epoxide (50 mg, 0.13 mmol) in hexanes (1 mL) and toluene (0.5 mL) was added HMPA ( I drop). The mixture was cooled to -78 C, and then a solution of lithium diisopropylamine in hexanes (0.73 mmol) was added. The solution was warmed to room temperature and stirred for 10 h. The mixture was quenched with saturated aqueous NH4CI, and the mixture extracted several times with ether. The combined extracts were dried (MgSQt) and concentrated, and the residue was purified by column chromatography (SiCh, hexanes-ethyl acetate = 3:2) to afford a colorless oil (29 mg, 58%) which was used without further characterization. To liquid ammonia (ca. 1 mL), at -78 C was added lithium metal (24 mg, 3.5 mmol), followed by /-butyl alcohol (0.05 mL). To this solution was added a solution of the allylic alcohol (20 mg, 0.053 mmol) in THF ( 1 mL). The reaction mixture was stirred at -78 C for 15 min, and then quenched with NH ₄ CI, and diluted with ether. The mixture was warmed to room temperature, and water ( 10 mL) was added. The mixture was extracted several times with ether followed by extraction with dichloromethane. The combined extracts were dried (MgSQt), concentrated and the residue was purified by column chromatography (S1O2, hexanes-ethyl acetate = 3;2) to afford 2 (9.0 mg, 60%) as a colorless solid, mp 192-194 "C; Ή NMR (CDCh. 400 MHz) δ 7.15 (d, J = 8.4 Hz, IH), 6.64 (dd, J = 2.8, 8.4 Hz, IH), 6.58 (d, J = 2.8 Hz, IH), 5.65 (dd,J= 1.2, 2.8 Hz, IH), 4.80 (br s, OH), 4.32-4.25 (m, 2H), 2.95-2.80 (m, 2H), 2.40-1.70 (m, I I H), 0.87 (s, 3H): ^{l ?} C NMR (CDCI?, 1(H) MHz) δ 155.2. 153.5, 138.5, 133.1 , 126.4, 124.3, 126.4, 124.3, 1 15.5, 1 12.8, 60.4, 56.8, 46.4, 44.6, 37.4, 34.8, 31.1, 29.7, 27.9, 26.6, 16.5.

[00117] 4.2.2 w-Propyl 3-hvdroxyestra-1.3.5( I0)-triene-l7-carboxylate (4)

[00118] To a solution of 3 ( 177 mg, 0.41 1 mmol) in ethanol ( 10 mL) was added an aqueous slurry of Raney-Ni (60%, 0.6 mL). The reaction mixture was stirred under a H ₂ gas (balloon pressure) for 24 h, afier which the mixture was filtered through a bed of Miter-aid. The filler bed was washed several times with ethyl acetate, and the filtrate was concentrated under reduced pressure to afford 4 as a colorless solid (129 mg, 92%): mp 151.5-153 ^" C, |a|„ ²⁰ +69.5 (c 0.388, CHC1.,); Ή NMR (CDCI.,, 300 MHz) δ 7.17 (d, J = 8.4 Hz, 1 H), 6.64 (dd, J = 2.8, 8.5 Hz, IH), 6.57 (d, ./= 2.7 Hz, 1H), 4.55 (br s, OH), 4.10 (dt, ./ = 10.8, 6.7 Hz, IH), 4.02 (dl, J = 10.8, 6.7 Hz, IH), 2.90-2.80 (m, 2H), 2.44 (I, J = 9.3 Hz, IH), 2.35-2.15 (m, 3H), 1.90-1.75 (m, 3H), 1.68 (sextet, J = 7.2 Hz, 2H), 1.55- 1.30 (m, 7H), 0.98 (l, ./ = 7.3 Hz, 3H), 0.71 (s, 3H); "C NMR (CDCb, 75 MHz) 5 174.5, 153.5, 138.4, 132.8, 126.7, 1 15.4, 1 12.8, 66.0, 55.6, 55.1, 44.3, 43.9, 39.0, 38.6, 29.8, 27.8, 26.7, 24.3, 23.7, 22.3, 13.7, 10.9. Anal, calcd. for C ₂₂ H ₃ oO l/2H ₂ 0: C, 75.18; H 8.89. Found: C, 75.36; H, 8.28.

[00119] 4.2.3 (20ΛΊ 3-(Phenylmelhoxy)-19.24-dinorchola-l.3.5( n.l6-telraene (6)

[00120] To a solution of 5 (0.20 g, 0.50 mmol) in benzene (10 mL) in a Schlenk flask was added Rh(PPh^)^CI (40 mg, 0.043 mmol). The reaction mixture was cooled with a dry ice-acetone bath, evacuated under high vacuum, and the system refilled to 1 atm with H ₂ gas. The mixture was stirred for 7 h at room temperature, and then the solvent was evaporated. The residue was extracted several times with ether, filtered, and concentrated. The residue was purified by column chromatography (SiO _: , hexanes- CH ₂ CI: = 1 : 1 ) to afford 6 ( 138 mg, 69%) as a colorless solid, mp 82-83.5 C, | OCID* +67 (c 0.74, acetone); Ή NMR (CDC1*. 300 MHz) δ 7.46-7.30 (m, 5H), 7.20 (d, J = 8.4 Hz, IH), 6.78 (br d, J = 8.4 Hz, IH), 6.74 (br s, IH), 5.35 (br s, IH), 5.04 (s, 2H), 2.94-2.84 (m, 2H), 2.40-2.08 (m, 4H), 2.00-1.87 (m, 3H). 1.65-1.28 (m, 7H), 1.09 (d, J = 6.6 Hz, 3H), 0.89 (l. ./ = 7.3 Hz, 3H), 0.83 (s, 3H): ^{1 ¾} C NMR (CDCU 75 MHz) δ 160.2, 155.9, 137.6, 136.7, 132.9, 128.0, 127.3, 127.0, 125.6, 120.4, 1 14.4, 1 1 1.8. 70.0, 56.4, 47.8, 44.7, 37.8, 35.4, 33.6, 31.3, 30.3, 30.2, 28.2, 27.0, 21.3, 17.1 , 12.4. Anal, calcd. for C ₂ 9H.*0: C, 86.95: H, 9.06. Found: C, 86.99: H. 9.12.

[00121] 4.2.4 (20.V) 3-Hvdroxy-l .24-dinorchola-l .3.5( I ). l -tetraene (7)

[00122] Cleavage of the benzyl ether 6 (73 mg, 0.18 mmol) with sodium metal in n- butanol was carried out in a fashion similar to the cleavage of 8. Purification of the residue by column chromatography (SiO^, hexanes-ethyl acetate gradient = 5: 1) gave unreacted starting material ( 17 mg) followed by 7 (46 mg, 81%) as a colorless solid, mp 92-95 ^" C, |a|„ ²⁰ +86.3 (c 0.32, acetone): Ή NMR (d ₆ -acetone) δ 7.05 (d, ./ = 8.4 Hz, IH), 6.56 (dd, J = 2.1, 8.4 Hz, I H), 6.51 (d, J = 2.1 Hz, I H). 5.35 (br s, I H), 2.82-2.73 (m, 2H), 2.37- 2.28 (m, I H), 2.22-2.05 (m, 2H), 1.97-1.85 (m, 4H), 1.60-1.26 (m, 8H), 1.07 (d, J = 7.2 Hz, 3H), 0.87 (t, J = 7.5 Hz, 3H), 0.82 (s, 3H): ^IJ C NMR (oVacetone) δ 162.5, 1 6.7, 139.3, 133.2, 127.7, 122.7, 1 17.1, 1 14.7, 58.8, 50.0, 47.1, 40.4, 37.7, 35.8, 33.4, 32.5, 32.2, 30.6, 29.3, 23.2, 19.0, 14.1. Anal, calcd. for C22H3 ₀ O 1/6H ₂ 0: C, 84.28: H, 9.75. Found: C, 84.28: H, 9.82.

[00123] 4.2.5 f20.V> 3-Hvdroxy-19.24-Dinorchola-1.3.5n0).16-tetraen-23-ol ( 1 Π

[00124] To a solution of 8 (394 mg, 0.947 mmol) in n-butanol (20 mL), at 70 C, was added sodium metal (0.87 g, 38 mmol) in small pieces. After all of the sodium had reacted, the reaction mixture was cooled to room temperature and quenched with water, followed by saturated aqueous NH4CI. The reaction mixture was extracted several limes with ether, the combined extracts were dried (MgS0 ₄ ) and concentrated. The residue was purified by column chromatography (S1O ₂ , hexanes-ethyl acetate gradient = 4: 1 to 2: 1 ) to afford unreacted starting material (91 mg) followed by 11 ( 150 mg. 49%) as a colorless solid, mp 174.5-176 ^" C, (a]„ ^:o +77.5 (c 1.50, acetone): Ή NMR (oVacetone) δ 8.15 (s, phenol OH), 7.04 (d, J = 8.4 Hz, 1 H), 6.56 (dd, J = 2.7, 8.4 Hz, 1 H), 6.51 (d, J = 2.7 Hz, IH), 5.38 (br s, IH), 3.64-3.52 (m, 3H), 2.84-2.74 (m, 2H), 2.42-2.28 (m, 2H), 2.20-2.08 (m, I H), 1.96-1.70 (m, 4H). 1.60- 1.30 (m, 7H), 1.10 (d, ./ = 7.2 Hz. 3H), 0.82 (s. 3H): "C NMR (de-aceione) δ 162.8, 156.6, 139.2, 133.0, 127.6, 122.6, 1 17.0, 1 14.6, 61.4, 58.7, 49.9, 47.0, 43.0, 40.3, 37.5, 33.2, 32.0, 30.9, 30.5, 29.2, 23.7, 19.0. Anal, calcd. for Create: C, 80.94; H, 9.26. Found: C, 80.67; H, 9.32.

[00125] 4.2.6 17.23-Epoxy-3-( phenylmethoxy)- 19.24-dinorchola- 1.3.5( 1 O riene

[00126] To a solution of 8 (56 mg. 0.14 mmol) in CHC (2 mL) was added a drop of concentrated HCl. The mixture was allowed to stand stirred for 24 h at room temperature, and then passed through a short column of silica gel using hexanes-ethyl acetate as eluent. Concentration of the eluent gave 9 (50 mg, 89%) as a colorless oil. |a|,> ²⁰ +36 (c 1.0, CH ₂ Cb); Ή NMR (CDCIj, 300 MHz) δ 7.46-7.28 (m, 5H), 7.22 (d. J = 8.4 Hz, IH), 6.87 (dd, ./= 2.7, 8.4 Hz. 1H), 6.73 (d, J= 2.7 Hz, 1H), 5.04 (s, 2H), 3.87 (dl, J = 4.5, 7.8 Hz, I), 3.62 (dt, ./ = 6.4, 7.8 Hz, IH), 2.92-2.82 (m, 2H), 2.38-I .20 (m, I6H), 1.10 (d, J = 6.9Hz, 3H), 0.74 (s, 3H); ^W C NMR (CDC1<, 75 MHz) 6 155.8, 137.6, 136.7, 132.8, 128.2, 127.3, 126.9, 125.8, 1 14.4, 1 1 1.8, 95.5, 70.0, 66.0, 50.0, 48.2, 44.0, 39.3, 36.9, 35.1, 31.3, 31.0, 30.3, 28.1, 26.6, 23.6, 19.0, 15.8. Anal, calcd. for Cy H _i6 0 _; : C, 83.61; H 8.71. Found: C, 83.35; H, 8.75.

[00127] 4.2.7 17.23-Epoxy-3-hvdroxy- 19.24-dinorchola- 1.3.5( I 0)-triene ( 10)

[00128] To a solution of 9 (48.9 mg, 0.1 18 mmol) in melhanol/CHCh (1 : 100, 6 mL) was added 10% Pd on carbon (5.6 mg). The mixture was stirred under H; (ca. 46 psi) in a Paar hydrogenation apparatus for 3 h. The catalyst was removed by filtration through filter-aid and the filler bed was washed with copious CH ₂ CI ₂ and the combined nitrates were concentrated. The residue was purified by chromatography (SiO>, hexanes-ethyl acetate = 3:1) to afford 10 as a colorless solid (37.8 mg, 99%). mp 172-174 C: Ή NMR (CDCb. 300 MHz) 5 7.15 (d, J = 8.4 Hz, IH), 6.62 (dd, J = 2.7, 8.4 Hz, IH), 6.55 (d, J= 2.7 Hz, I H), 3.87 (dt, ./ = 4.5, 7.8 Hz, I H), 3.60 (dl, = 6.3. 8.1 Hz, I H), 2.85-2.75 (m, 2H), 2.35-1.20 (m, I6H), 1.07 (d, ./ = 6.9 Hz, 3H), 0.70 (s, 3H); ^,3 C NMR (CDCIj, 75 MHz) δ 153.3, 138.6, 133.2, 126.6, 1 15.4, 1 12.7, 96.0, 66.1, 50.0, 48.2, 43.9, 39.3, 36.8, 35.0, 31.2, 30.8, 30.0, 27.9, 26.4, 23.4, 18.8. 15.6. Anal, calcd. for C ₂ 2Hu)02 l/4H ₂ 0: C, 79.83; H 9.29. Found: C, 80.12; H, 9.33. [00129] 4.2.8 (2Q.V) 3-Hvdroxy- 1 .24-dinorchola- 1 3.5( 10). I (>-tetraen-23-al ( 12)

[00130] To a solution of 11 (100 mg, 0.2% mmol) in THF (4 mL) was added a solution of ethyl magnesium bromide in THF (0.67 mL, 1.0 M, 0.67 mmol). The solution was stirred at room temperature for IS min. and then solid 1.Γ- (azodicarbonyl)dipiperidine (0.17 g, 0.67 mmol) was added. The reaction mixture was stirred for I h, and then quenched with saturated aqueous NH ₄ CI and extracted several times with ether. The combined ethereal extracts were dried (MgS0 ₄ ), concentrated and the residue was purified by column chromatography (S1O2, hexanes-ethyl acetate = 5: 1 ) to afford 12 as a colorless solid (66 mg, 66%). mp 168.5-171 C, [α]»* +78 (c 0.80, acetone); Ή N R (oVacetone, 3(X) MHz) δ 9.66 (t, J = 2.1 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1 H), 6.57 (dd, .7 = 2.5, 8.4 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 5.46 (br s, 1H), 2.90-2.75 (m, 4H), 2.62 (ddd, J = 1.8, 5.7, 16.2 Hz, 1 H), 2.44-2.30 (m, 2H), 2.26-2.10 (m, 2H), 1.98- 1.86 (m, 3H), 1.60-1.34 (m, 5H), 1.1 (d, .7 = 7.2 Hz, 3H), 0.88 (s, 3H); ^,J C NMR (d,,- acetone, 75 MHz) δ 203.2, 161.4, 156.8, 139.5, 133.3, 127.9, 124.6, 1 17.2, 1 14.8, 59.2, 53.1, 50.2, 47.2, 40.5, 37.7, 33.6, 32.3, 30.7, 29.7, 29.4, 23.8, 19.3. Anal, calcd. for C22H28O2: C, 81.44; H, 8.70. Found: C, 81.21 : H, 8.54.

[00131] 4.2.9 17-23-EDOxy-3-hvdroxy-19-norchola-1.3-5n0¾-triene Π3)

[00132] To a solution of 12 (45.9 mg, 0.142 mmol) in THF (7 mL) at 0 C was added a solution of methyl magnesium bromide in ether (0.10 mL, 3.0 M, 0.30 mmol). The reaction mixture was stirred for 3 h, and then quenched with saturated aqueous NH4CI (15 mL). The mixture was extracted several times with CHjCb and the combined extracts were dried (MgSQ and concentrated. The residue was purified by chromatography (S1O2, hexanes-ethyl acetate = 5: 1) to afford 13 as a colorless solid (44 mg, 92%). Analysis of the product by Ή NMR spectroscopy indicated this to be a 1 : 1 mixture of diastereomers. mp 248-251 ^* C, Ή NMR (CDCI>, 300 MHz) δ 7.15 (d, J = 8.4 Hz, 1H), 6.62 (dd, J = 2.7, 8.4 Hz, 1 H), 6.56 (d, ,7 = 2.7 Hz, 1H), 4.18-4.07 (m, 1H), 3.85-3.74 (m, 1H), 2.85-2.75 (m, 2H), 2.35-1.20 (m, 15H), 1.23 & 1.20 (2 x d, .7 = 5.7 Hz, 3H total), 1.07 & 1.05 (2 x d, .7 = 6.9 Hz, 3H), 0.72 & 0.66 (2 x s, 3H total); "C NMR (CDCI3, 75 MHz) δ 153.3, 138.6, 133.2, 126.6, 1 15.4, 1 12.7, 97.1 [95.8], 73.6 [71.3], 49.85 [49.80], 48.8, 47.1 , 45.4, 43.9 |43.8|, 43.5, 39.3 |39.2|, 36.2, 34.5, 32.3, 31.2 |30.9|, 30.6 |30.1 |, 27.8, 26.5 [26.4]. 23.5 (23.4]. 21.6. 19.2 [ I8.9J, 16.3 [14.91. Anal, calcd. for Ci,H, ₂ 0 ₂ - i/2H ₂ 0: C, 79.04; H 9.52. Found: C, 79.34; H, 9.57.

[00133] 4.2.10 cis- and //vym-^M'-HvdroxycvclohexynDhenol ( 14)

[00134] To a solution of 4-(4 ^, -hydroxyphenyl)cyclohexanone (50 mg, 0.26 mmol) in methanol ( I mL) was added NaBH ₄ (15 mg. 4.0 mmol). The reaction mixture was stirred for 30 min, and then diluted with water. The mixture was extracted several limes with ethyl acetate and the combined extracts were concentrated and purified by column chromatography (SiO:, hexanes-ethyl acetate = 2:1) to afford /.v-14 (5.0 mg, 10%) followed by irans-iS (43 mg, 86%) both as colorless solids. O.v-14: Ή NMR (CD ₃ OD, 400 MHz) 6 7.04-6.69 (ΑΑ'ΒΒ', J, _H = 8 8 Hz. 4H), 4.02 (narrow t, «/ = 2.8 Hz, 1H), 2.50- 2.40 (m, 1 H), 1.91- 1.79 (m, 4H), 1.69-1.52 (m, 4H): "C NMR (CDjOD, 75 MHz) δ 156.5, 140.1, 128.8, 1 16.1, 66.5, 44.5, 34.0, 29.4. Trans-iS Ή NMR (CDjOD, 400 MHz) δ 7.01-6.68 (ΑΑ'ΒΒ', J _A = 8.4 Hz, 4H), 3.58 (tt, J = 4.4, 10.6 Hz, 1H), 2.39 (tt, J = 3.5, 1 1.8 Hz, tH), 2.06-1.99 (m, 2H), 1.87-1.79 (m, 2H), 1.56-1.33 (m, 4H).

[00135] 4.2.1 1 4-r4-Hvdroxvphenvl -cvclohexanone oxime n6)

[00136] To a solution of 4-(4'-hydroxyphenyl)cyclohexanone (50 mg, 0.26 mmol), hydroxylamine hydrochloride (36.6 mg, 0.526 mmol) in ethanol (5 mL) was added Amberlysl (56 mg). After stirring for 2 h, the mixture was filtered, and the filtrate concentrated. The residue was partitioned between water and ethyl acetate, and the organic layer was concentrated and dried to give (±)-16 (44 mg, 82%) as a colorless solid, mp 172-175 C. Ή NMR (CDiOD, 400 MHz) 6 7.03-6.69 (ΑΑ'ΒΒ', J _M = 8.8 Hz, 4H), 4.02 (narrow t, J = 2.8 Hz, IH), 2.0-2.40 (m. 1H), 1.91-1.79 (m, 4H), 1.69-1.52 (m, 4H); "C NMR (CD.iOD, 75 MHz) 6 161.0, 156.8, 138.4, 128.7, 1 16.3, 44.3, 36.0, 34.7, 33.0, 25.2. HRMS (ESI): m/z calcd for C^H^NOj+Na' |M+Na| ^' 228.0995, found 228.0997.

[00137] 4.2.12 m-l-Hvdroxvmethvl-4-r4'-hvdroxvDhenvl)-cvcloheDtane n8)

[00138] To a solution of (±)-17 (75 mg, 0.35 mmol) in methanol ( 15 mL) in a heavy walled reaction vessel, was added a catalytic amount of 20% Pd/C. The mixture was stirred under H ₂ pressure (45 psi) for 75 min and then the reaction mixture was filtered through the pad of celite. The filtrate was concentrated and the residue was purified by column chromatography (SiO;, hexanes-ethyl acetate = 65:35) to afford (±)-18 (38 mg, 50%) as a colorless solid, mp 60-61 C; Ή NMR (CDCh, 300 MHz) δ 7.06 and 6.75 (ΑΑΈΒ', J _AB = 9.0 Hz, 4H), 3.48 (d, ./ = 6.3 H/, I H), 2.59-2.58 (m, I H), 1.95-1.08 (m, I3H); ^l3 C NMR (CD ₃ OD, 75 MHz) δ 127.9, 1 15.3, 8.6, 46.1, 1.4, 38.8, 33.1, 31.6, 28.5, 27.5. HRMS (ESI): w/r calcd for CuH^C -Na |M+Na|' 243.1356, found 243.1356.

[00139] 4.2.13 5-l l/ -2- 4-HvdroxyDhenyl)ethenyll-2-furanmethanol (20)

[00140] A solution of methyl 5-bromo-2-furanoate (1.03 g, 5.02 mmol), 4- aceloxystyrene (0.97 g, 6.0 mmol), palladium acetate (0.01 g, 0.05 mmol), vri-o- tolylphosphine (0.03 g, 0.2 mmol), and triethylamine (3 mL) was heated under nitrogen in a sealed heavy-walled Pyrex tube at 100 C for 24 h. The reaction mixture was cooled, diluted with water and dichloromethane. The dichloromethane layer was separated, washed with water, and dried (MgSC , and the residue was purified by column chromatography (S1O2, hexanes-ethyl acetate = 4: 1) to afford 19 (350 mg, 24%), a pale yellow solid, mp 110.5-1 12 "C; Ή NMR (CDCh, 300 MHz) δ 7.51 (d, J = 8.1, 2H), 7.27 (d, J = 16.5 Hz, I H), 7.20 (d, J = 3.6 Hz, 1 H), 7.10 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 16.5 Hz, IH), 6.45 (d, J = 3.6 Hz, IH), 3.92 (s, 3H, OMe), 2.32 (s, 3H, OAc). This product was used in the next step without further characterization. To a solution of diester (50 mg, 0.17 mmol) in anhydrous ether ( 1 mL) at 0 C, was slowly added a solution of lithium aluminium hydride (0.52 mL, 1.0 M in THF, 0.52 mmol). Solution was stirred for 3 h at 0 C and then saturated aqueous sodium bicarbonate (2 mL) was added follow by dilute sodium hydroxide. The mixture was warmed to room temperature, extracted several times with ethyl acetate. The combined extracts were dried (MgS0 ₄ ), concentrated and the residue was purified by column chromatography (SiO , hexanes-ethyl acetate = 1: 1) gave 20 (28 mg, 74%) as a colorless solid, mp 129-131 C; Ή NMR (d ₆ -acetone, 300 MHz) δ 8.59 (br s, IH), 7.40 (d, J= 9.0 Hz, 2H), 6.97-6.79 (m, 4H), 6.30 (s, 2H), 4.57 (br s, 2H), 3.05 (br s, I H); "C NMR (d ₆ -acetone, 75 MHz) δ 158.2, 155.9, 1 4.1 , 129.7, 128.6, 127.4, 1 16.5, 1 14.9, 109.9, 109.4, 57.4. HRMS (ESI): m/z calcd for CjH^Oj+ a | M+Na|' 239.0679, found 239.0681. [00141] 4.3 Fluorescence Polarization

[00142] Assay was developed based on a commercially available kit from Invitrogen. ¹⁵ Assays were run on a BMG POLARstar Galaxy reader with acquisition parameters as follows: 200 flashes, positioning delay 1.0 s. K factor < 1.1 and > 0.9, excitation filter of 485 ± 5 nm and emission filler of 520 ± 15 nm. For the IC ₀ determinations the | ER-ct| was 30 nM and the | FITC-estradiol tracer| (|Tr|) was 10 nM. Sample volume was 150 pL. For each experiment the polarization was calibrated with a sample of FITC set at 20 mP. All proper blanks were used, including water for the FITC samples and blank samples containing only 30 nM ERot protein for the remaining data points. All protein samples contained 1% oVDMSO, the maximum amount tolerated as stated by the supplier of the ERa protein, Invitrogen, to ensure the solubility of all hydrophobic compounds investigated. The K _< i of the FITC-lagged estradiol for ER-a was determined by non-linear least squares fitting of the titration curve data to the following equation:

[00143] 4 4 Cell-Based ERa and ERfi Assays

[00144] ERa and ERP assay kits for cell-based assays (Indigo Biosciences) allowed for investigation into the functional activity (i.e. agonist and/or antagonist) of the ligands identified to bind based on the initial fluorescence polarization displacement assay. Briefly, the cells contained a luciferase reporter gene that was functionally linked to either the ERa or ERP-responsive promoter. By quantifying the luciferase expression via luminescence, the change in ER activity could be quantified. I -2 mM stocks of the ligands were prepared in de-DMSO and diluted to final concentrations ranging from 3.2 nM to 2 μΜ, using the Compound Screening Medium provided in the kit. For the agonist assay, the cells were prepared by warming to 37°C, plated, then the chemicals added. For the antagonist assay, the cells were prepared as above with the addition of E2 (for ERa 3.2 nM was added, approximating an IC ₇₅ ; and, for ERP 160 pM was added, approximating an ICso). The cells were then plated, and the chemicals added. All plates were incubated in a cell culture incubator at 37°C and 5% CO for 22 h. Each assay was performed in duplicate. Luminescence was characterized after removal of the incubating media and introduction of the Detection Substrate using a Molecular Devices SpectraMax M5 microplate reader. Data was fitted using Graph Pad Prism and 111 to the dose-response (four paramler) equation as follows.

[00145] 4.5 Molecular Docking

[00146] Ligand structures were drawn in PC Spartan Plus (Wavefunction) and three dimensional (3D) conformation was then optimized using semiempirical Austin Model I (AM I ) calculations. Since compound 13 was afforded as a pair of diastereomers both were modeled and docked. The AMI calculations provided geometries and bond distances for subsequent docking. AutoDock Tools (ADT) was used prepare the ligand files according to AutoDock requirements and assign Gasteiger charges.

[00147] The ER receptor for agonist (pdb code I ere) ⁴ and antagonist (pdb code I err) ⁵ * conformations were prepared for docking calculations using the 'A' chain. The ERp receptor for agonist (pdb code 2jJ3) ^u and antagonist (pdb code ll2j) ^,s conformations were prepared for docking calculations using the ^* A' chain. ADT was used to further prepare the ER receptor files by adding hydrogen atoms and adding partial charges to each atom of the protein. The grid box was centered on the co-crystallized ligand, drawn to a box to incorporate amino acids Arg394, Glu353, and His524 for ERa and Arg346, Glu305, and Hi $475 for ERp, then the estradiol ligand was removed. ³⁶ AutoDock (v. 4.2) calculations were performed with default parameters, except with 100 genetic algorithmic runs and 2,500.000 evaluations per run. ^*40

0148] Table 3. Docking results for the agonist formation of E ot in the absence of water molecules.

Lowest Calculated

Number of Energy Binding

Compound Clusters

Cluster Energy Mode (2.0A rmsd)

Population (kcal mol ¹ )

estradiol 69 -10.74 reversed estradiol 31 -10.72 normal

4 - 64 -11.09 reversed

4 36 -10.71 normal

2 1 100 -10.98 reversed

7 2 56 -9.93 reversed

7 44 -9.79 normal

11 69 -10.35 reversed

11 29 -9.28 normal

11 2 -9.16 reversed

10 96 -9.48 reversed

10 4 -9.08 normal

13a 1 100 -7.44 normal

13b 1 100 -9.13 reversed

17 3 22 -7.27 reversed

17 76 -7.21 reversed

17 -7.12 normal

20 1 100 -7.57 reversed

18 2 85 -7.42 reversed

18 15 -7.34 normal

14 2 97 -6.71 normal

14 3 -6.39 reversed

15 2 73 -6.85 normal

15 27 -6.77 reversed

16 71 -7.42 reversed

16 28 -7.33 normal 16 1 -7.17 normal 0149] Table 4. Docking results Tor the agonist formation of ER in the presence of a single water molecule near Arg294 and Glu353 as observed in the crystal structure. Chemicals 20 and 14 were not predicted to bind similarly to the normal or reversed modes as otherwise noted.

Calculated

Number of Lowest

Binding

Clusters Energy

Compound Energy Mode

(2.0A Cluster

(kcal mol ^"

rnisd) Population

')

estradiol 1 100 -10.36 normal

4 2 97 -10.29 normal

2 2 42 -10.16 reversed

2 2 58 -9.82 normal

11 1 100 -9.80 normal

7 1 100 -9.74 normal

10 1 100 -s.s: normal

13b 1 100 -8.73 normal 3a 1 100 -8.39 normal

4 2 3 -7.73 reversed

18 2 72 -7.56 reversed

18 2 28 -7.46 normal

IT 1 -7.46 reversed

17 · S7 -7.37 normal

16 97 -7 "*7 normal

15 ·> 7 -7.00 reversed

16 ·» -6.94 reversed

:o 4 76 -6.93 other

15 -6.S5 normal 14 3 ?9 -6.41 other

[00150] References and Notes

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[00177] 27. Frigo!i, M.; Mehl, G. H. Ear. J. Org. Chan. 2004, 636-642. DeOrazio, R. J.: Nikam, S. S.; Scott, I. L.: Sherer, B. A.: Wise, L. D. PCT Int. Appl. WO 01/81295 A 1, 2001.

[00178] 28. Indigo Biosciences, Human Estrogen Receptor Technical Manual.

[00179] 29. Pandey, R. .: Wang, L.; Wallock, N. J.: Lindeman, S.: Donaldson, W. A. J. Org. hem. 2008, 73, 7236-7245.

[00180] 30. van Lip/.ig, M.M.H.; ter Laak, A.M.; Jongegan, A.; Vermeulen, N.P.E.; Wamelink, M.; Geerke, D.; Meerman, J.H.N../. Med. Chem. 2004, 47, 1018-I030.

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[00183] 33. Norman, B.H.; Richardson, T.I.; Dodge, J.A.; Pfeifer, L.A.; Durst, G.L.; Wang, Y.; Durbin, J.D.; rishnan, V.; Dinn, S.R.; Liu, S.; Reilly, J.E.; Ryter, .T. Buwrg. Med. Chem. leu. 2007, 17, 5082-5085.

[00184] 34. Shiau, A.K.; Barstad, D.; Radek, J.T.; Meyers. M.J.; Nettles, K.W.; Kat/enellenbogen, B.S.: at/ellenbogen, J.A.: Agard, D.A.: Greene. G.L. Nat. Struct. Biol. 2002, 9, 359-364.

[00185] 35. Tuccinardi, T.; Bertini, S.; Martinelli, A.: Minutolo, F.; Ortore, G.; Placanica, G.; Prota, G.: Rapposelli, S.: Carleson, K.E.; Kat/enellenbogen, J.A.; Macchia, M..A Med. Chem. 2006, 49, 5001-50I2. [00186] 36. Morris. G. M: Huey, R : Lindstrom, W.: Sanner. . F.; Belew, R. .:

Goodsell, D. S.: Olson, A. i. J. ompui. hem. 2009, 30, 2785-2791.

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[00200] Example 2. Synthesis and analysis of additional substituted (4'- hvdroxyphenylkvcloalkane compounds.

[00201] 4-i4 ^, -Hvdroxyphenvh- 1 -methylcvclohexanol

OH

[00202] To a solution of 4-(4'-hydroxyphenyl)-cyclohexanone (250 mg, 1.31 mmoi) in THF (5 mL) al -78 °C under nitrogen, was added a solution of methylmagnesium bromide (1.76 mL, 3.0 M in ether, 5.3 mmol). The reaction mixture was stirred at -78 °C for I h, then warmed to room temperature and quenched with water. The resulting mixture was extracted several times with CH _^ Cl; and the combined extracts were washed with brine, dried and concentrated. The residue was recrystallized from acetone/hexanes to give 4-(4'-hydroxyphenyl)-l-meihylcyclohexanol ( loo mg, 38%) as a colorless solid, mp 140-142 °C; Ή NMR (di-acetone, 300 MHz) δ 8.06 (s, I H), 7.06 and 6.74 (AA'BB\ ./ _A n = 8.7 Hz, 4H), 2.37 (U, = 3.3, 12.0 Hz, IH), 1.91 (dd, ./ = 4.2, 12.9 Hz, I H), 1.82 (dd, ./= 4.2, 12.9 Hz, IH), 1.75-1.41 (m, 6H, 1.19 (s, 3H).

[00203] 4-1 ί 4-t-ButvldiDhenvlsilvloxvphenvncvclohexvlidene l-acetic acid ethvl ester

[00204] Imidazole (0.537 g. 7.90 mmol) was added to a stirring solution of 4-(4'- hydroxyphenyOcyclohexanone (0.500 g, 2.63 mmol) in dry DMF (8 mL). After 30 min /- butylchlorodiphenylsilane (1.37 mL, 1.45 g, 5.27 mmol) was added and the reaction mixture was stirred at room temperature for 14 h. Water (30 mL) was then added and the mixture extracted with CH _^ Ch, dried and concentrated. The excess DMF was removed under high vacuum and the residue was purified by column chromatography (Si0 ₂ , hexanes-ethyl acetate = 85: 15) to give 4-(4'-/- butyldiphenylsilyloxyphenyl)cyclohexanone ( 1.02 g, 90%) as a colorless solid, mp = 85- 86 T. Sodium hydride (43 mg, 55% in mineral oil 0.981 mmol) was added to a stirring solution of triethyl phosphonoacetate (0.1 3 mg, 0.816 mmol) in dry THF (5 mL) at 0 "C. After 30 min, a solution of 4-(4 ^* -/-butyldiphenylsilyloxyphenyl)-cyclohexanone (350 mg, 0.816 mmol) in dry THF (5 mL) was added and the reaction mixture was stirred at room temperature for 2 h. After this time, the mixture was diluted with water (25 mL) and the resulting mixture was extracted with ether, dried and concentrated. The residue was purified by column chromatography (S1O2. hexanes-ethyl aceiale = 95:05) to give 4-[(4-t- bulyidiphenyisilyioxyphenyi)cyciohexyiidene|-acelic acid ethyl ester (372 mg, 91%) as a colorless gum.

[00205] 4-l(4-HvdroxyDhenyl)cvclohexylidenelacetic acid elhyl ester

[00206] To a stirring solution of 4-|(4-/- butyldiphenylsilyloxyphenyl)cyclohexylidene|acetic acid ethyl ester (60 mg, 0.12 mmol) in dry THF (I mL) was added a solution of tetrabutylammonium fluoride (0.247 ml_, 1.0 M in THF, 0.247 mmol). The solution was stirred at room temperature after I h, and then the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with brine, dried and concentrated. The residue was purified by preparative TLC (S1O ₂ , hexanes-ethyl acetate = 90:10) to give 4-|(4- hydroxyphenyl)cyclohexylidenejacetic acid ethyl ester (20 mg, 64%) as a colorless solid, mp 92-94 °C: Ή N R (CDC , 300 MHz) δ 7.08 and 6.77 (ΑΑ'ΒΒ', J _M = 8.4 Hz, 4H), 5.68 (s, 1H) _? 4.58 (s, 1H), 4.17 (q, J = 7.1 Hz, 2H). 4.00-3.90 (m, 1H) _? 2.80-2.68 (m _? 1H). 2.45-1.97 (m, 6H), 1.30 (l, ./ = 7.3 Hz, 3H). "C NMR (CDClj, 75 MHz) δ 167.0, 162.2, 154.0, 138.6, 128.1, 115.4, 1 13.9, 59.8, 43.4, 37.9, 36.0, 35.2, 29.7, 14.5

[00207] 4-(4'-Hvdroxyphenyl)(2-hvdroxyethylidene)cvclohexane

[00208] To a solution of 4-|(4-/-bui l- dipheny!silyioxypheny!)cyciohexy!idene|acetic acid ethyl ester (275 mg, 0.551 mmol) in dry dichloromethane (2 mL) under nitrogen at -40 °C was added a solution of diisobulylaluminum hydride (1.41 mL, 1.0 M in CH;CI;, 1.41 mmol). After 90 min. saturated aqueous potassium sodium tartrate was added and reaction mixture warmed to room temperature. After 2 h the layers were separated and the aqueous layer was extracted several times with CH C . The combined organic layers were dried, filtered through a pad of celite and concentrated to give 4-(4'-i-butyldiphenyIsilyloxyphenyl)(2- hydroxyethylidene)cyclohexane (254 mg, quantitative) as a colorless gum. To a solution of 4-(4'-i-butyldiphenylsilyloxyphenyl)(2-hydroxyethylidene)-cy clohexane (235 mg, 0.514 mmol) in dry THF ( I mL) under nitrogen was added a solution of tetrabulylammonium fluoride in THF (1.03 mL, 1.0 M, 1.03 mmol). The solution was stirred for 3 h and then diluted with water and the resultant mixture extracted several times with ethyl acetate. The combined extracts were washed with brine, dried and concentrated. The residue was purified by column chromatography (SiO , hexanes-elhyl acetate = 80:20) to give 4-(4'-hydroxyphenyl)(2-hydroxyethylidene)cyclohexane (90 mg, 80%) as a colorless solid, mp 165-166 °C: Ή NMR (aVacetone, 3(H) MHz) δ 8.10 (s, 1H), 7.04 and 6.74 (ΑΑ'ΒΒ', J _M = 8.4 Hz, 4H), 5.36 (t, ,/ = 6.6 Hz, 1H), 4.17-4.02 (m, 2H), 2.78-2.70 (m, IH), 2.64 (tt, ./ = 3.3, 12.0 Hz, 1H), 2.35-2.10 (m, 2H), 1.98-1.80 (m, 4H), 1.54-1.37 (m, 2H). ^W C NMR (d ₆ -acetone, 75 MHz) δ 156.5, 141.1, 138.6, 128.5, 123.6, 1 16.0, 58.5, 44.6, 37.5, 37.0, 36.2. 29.2. Anal, calcd. for CwH^O,: C, 77.03; H 8.31. Found: C, 77.20; H, 8.28.

[00209] 4-[4-(2-Hvdroxyethyl)cvclohexyllphenol and 4-(4-ethylcvclohexyl)phenol

[00210] A solution of 4-(4'-hydroxyphenyl)(2-hydroxyethylidene)cyclohexane (50 mg, 0.23 mmol) in methanol (15 mL) with small pinch of 20% Pd/C was stirred under H (30 psi) for 12 h. The reaction mixture was filtered through a pad of celite, concentrated and the residue was purified by preparative TLC (SiO , hexanes-ethyl acetate = 65:35) to give 4-(4-elhylcyclohexyl)phenol (28 mg, 60%), followed by 4-[4-(2- hydroxyethyl)cyclohexyl jphenol product (7 mg, 14%) both as colorless solids. [00211] t/.v- and /ra/;v-4-(4-Elhylcyclohexyl)phenol: mp 80-81 °C: Ή NMR (CDCI ₃ , 3(M) MHz) δ 7.08 and 6.76 (ΑΑ'ΒΒ', J _M = X.I Hz, 4H). 4.55 (s, IH), 2.54-2.35 (m, IH), 1.92-1.82 (m, 2H). 1.70-1.50 (m, 3H), 1.45-1.00 (m, 6H), 0.91 (t, J = 7.2 H/, 3H). Anal, calcd. forC^oO: C, 82.30; H 9.87. Found: 81.06: H _; 9.52.

[00212] CM- and /ra«.v-4-|4-(2-Hydroxyethyl)cyclohexyl|phenol: mp 120-125 °C;

Ή NMR (d _f ,-acetone, 300 MH/) δ 8.02 (s, IH), 7.08-7.01 (m, 2H), 6.77-6.71 (m, 2H), 3.65-3.5 and 3.43-3.37 (m, 3H lotal), 2.52-2.33 (m, I H), 1.91-1.00 (m, I 1H).

[00213] 4-(4'-Hvdroxyphenyltevcloheptanol

[00214] To magnesium turnings (3.654 g, 0.1503 mol) and dry THF (30 mL) in a flame dried three-necked flask was added dropwise a small amount of a solution of 4- bromobut-l-ene (7.72 mL, 10.2 g, 0.0756 mol) in THF (20 mL). The reaction mixture was heated to reflux and once the Grignard formation was started, the remaining bromide was added drop-wise maintaining a gentle reflux. The reaction was stirred until most of the magnesium had reacted. A solution of methyl 4-methoxybenzoate (2.528 g, 0.01523 mmol) in THF (30 mL) was added drop-wise over 30 min. After stirring overnight at ambient temperature, saturated aqueous NH4CI (30 mL) was added to quench the reaction. The resultant emulsion was stirred for 2 h and extracted several times with ether. The combined extracts were washed with water, followed by brine, dried and concentrated to give S^'-methoxyphenyD-LS-nonadien-S-ol (3.182 g, 85%) as a yellow oil. Ή NMR (CDClj, 400 MHz) 6 7.28 (dd, J = 2.6, 9.0 Hz, 2H), 6.88 (dd, J = 2.5, 8.9 Hz, 2H), 5.84- 5.73 (m, 2 H), 4.98-4.88 (m, 4H), 3.81 (s, 3H), 1.96-1.84 (m, 8H). C NMR (CDCIj, 100 MH/) δ 158.1 , 138.9, 126.4, 1 14. 6, 1 13.4, 76.9, 55.2, 42.1, 28.1. To a solution of 5-(4'- methoxyphenyl)-l.8-nonadien-5-ol (3.20 g. 13.0 mmol) in dry CH2CI2 ( 130 mL. 0.01 ) was added Grubbs I ^st generation catalyst (0.043 g, 0.052 mmol, 4 mol%) and the resultant mixture was heated at 40 °C for 12 h. The mixture was concentrated to dryness and the residue was purified by column chromatography (SiO;, ether-hexanes = 80:20) to give I- (4-meihoxyphenyl)-4-cyclohepien-]-ol (1.56 g, 55%) as a green oil. Ή NMR (CDC1?, 4(M) MHz) δ 7.43 (dd, ./ = 2.2, 9.0 Hz, 2H), 6.87 (dd, ./ = 2.2, 9.0 Hz, 2H), 5.86-5.83 (m, 2H), 3.80 (s, 3H), 2.55-2.44 (m, 2H), 2.10-1.97 (m, 4H), 1.90-1.82 (m, 2H). ^,3 C NMR (CDCI3, 100 MHz) δ 158.3, 142.3, 132.1, 125.8, 1 13.5, 76.5, 55.2, 40.1, 23.0. To a solution of l-(4-methoxyphenyl)-4-cycloheplen-l-ol ( 1.720 g, 7.879 mmol) in dry CH ₂ CI ₂ (50 mL) was added triethylsilane ( 1.35 mL, 8.45 mmol) followed by trifluoroacetic acid (6.20 mL, 80.9 mmol). The mixture was stirred at room temperature for 48 h. After complete disappearance of the starling material, the solution was concentrated and purified by column chromatography (Si0 ₂ , hexanes- ethyl acetate = 1 : 1) to give 4-(4- met hoxyphenyDcycloheptene ( 1.433 g, 86%) as a brown oil. Ή NMR (CDCh, 400 MHz) 6 7.1 1 (dd, J = 1.4, 8.7 Hz, 2H), 6.84 (dd, J = 1.6. 8.8 Hz, 2H), 5.91-5.87 (m, 2H), 3.79 (s, 3H), 2.69 (tt, J = 3.2, 1 1.4 Hz, 1H) 2.35-2.25 (m, 2H), 2.23-2.13 (m, 2H), 1.91-1.83 (m, 2H), 1.54-1.43 (m, 2H); ^,3 C NMR (CDC1 _? , 100 MHz) δ 157.6, 141.5, 132.5, 125.5, 113.7, 55.2, 49.4, 34.9, 27.9. To a solution of 4-(4-met hoxyphenyDcycloheptene (0.551 g, 2.72 mmol) in freshly distilled CH ₂ CI ₂ (20 mL), under nitrogen, was added drop-wise a solution of mCPBA (1.008 g, 70% wt, 4.09 mmol) in freshly distilled CH ₂ Cfe (10 mL). Afier the disappearance of starting olefin, as indicted by TLC analysis, the solvent was evaporated and residue was treated with saturated NaHCO.i solution (20 mL) with stirring for 30 min. The mixture was extracted several times with CH ₂ C1 , and the combined extracts were concentrated. The residue was purified by column chromatography (SiO;, hexane-ethyl acetate = 1 :1) to give 4-(4-methoxyphenyl)cycloheptene oxide (0.441 g, 74%) as yellow oil. This was revealed to be an equimolar mixture of exo- and endo- stereoisomers. Ή NMR (CDCb. 400 MHz) δ 7.1 1-7.06 (m, 4H), 6.86-6.80 (m, 4H), 3.78 (s, 3H), 3.77 (s, 3H), 3.16-3.19 (m, 2H), 3.13-3.07 (m, 2H), 2.55 (tl, ./= 3.3, 1 1.4 Hz, 1 H), 2.40-2.29 (m, 4H), 2.14 (it../ = 2.3, 1 1.2 Hz, I H), 1.93-1.84 (m, 2H), 1.83-1.77 (m, 2H), 1.75-1.67 (m, 2H), 1.66-1.57 (m, 4H). 1.50-1.40 (m, 2H); "C NMR (CDCb, 100 MHz) δ 157.8/157.6, 141.2, 139.9, 127.6/127.3, 1 13.8/1 13.7, 56.1, 55.1, 49.2, 48.0, 32.6, 32.0, 28.8, 27.5. To a solution of 4-(4-methoxyphenyl)cycloheptene oxide (0.100 g, 0.458 mmol) in dry THF (10 mL), under nitrogen, was added LiAIH (48.0 mg, 1.26 mmol) and AlClj (56 mg. 0.42 mmol). Afier stirring for 12 h, the mixture was treated with 15 drops of water and diluted with aqueous OH (3 mL) and water (10 mL). The mixture was then filtered through ceiile and extracted several times with ether, and the combined extracts were dried and concentrated. The residue was purified by column chromatography (SiO?, ethyl acetate-hexanes = 4:1) to give 4-(4-methoxyphenyl)cycloheptanol (32 mg. 32%) as a yellow oil. This was determined to be a mixture of c/.v- and trans- stereoisomers by NMR spectroscopy. Ή NMR (CDCh, 400 MHz) δ 7.10 (t, J= 7.8 Hz, 2H), 6.83 (d, J= 8.2 Hz, 2H), 3.90-3.98/4.00-4.05 (m, 1H), 3.78 (s, 3H), 2.72-2.55 (m. 1H), 2.16-1.48 (m. UH). "C NMR (CDCIj, 4(K) MH/) 6 ppm 157.6, 141.4, 127.5, 1 13.7, 72.7, 71.6, 55.2, 46.2, 38.2, 37.6, 36.9, 35.7, 31.7, 29.6, 23.3. 21.3. To a solution of 4-(4- methoxyphenyDcycloheptanol (28 mg, 0.13 mmol) in anhydrous CH2CI2 (30 mL) cooled to at -78 C, was added drop-wise a solution of boron tribromide (0.25 mL, 1.0 M in CH;CI:, 0.025 mmol). After the addition was complete, the reaction mixture was stirred for 30 min and then warmed to room temperature over a 2 h period. The mixture was quenched with water (10 mL) and mixture extracted several times with CH^C . The combined extracts were washed with brine, dried and concentrated to give 4-(4- hydroxyphenyl)cycloheptanol (24 mg, 90%) as a yellow solid. This was determined to be a mixture of c .v- and iruns- stereoisomers by NMR spectroscopy. Ή NMR (CDCI.?, 400 MHz) δ 7.07-6.98 (m, 2H), 6.77-6.70 (m, 2H), 4.84 (s, OH), 4.55-4.46 and 4.41-4.31 (m, 1H), 2.75-2.57 (m, I H), 2.51-1.3 (m, 13H). ^,3 C NMR (CDClj, 100 MHz) δ 153.5, 141.0, 127.7, 1 15.9, 56.1 , 55.7, 45.9, 45.3, 40.0, 39.4, 39.2, 37.7, 37.6, 36.3, 34.2, 31.3, 25.2, 23.5. 0215] In the foregoing description, it will be readily apparent to one skilled in the an that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.