A TTX Mwashout Vehicle Or E2 M"Washout Patent Application Publication May 26, 2005 Sheet 1 of 16 US 2005/0113453 A1

US 20050113453A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0113453 A1 Scanlan et al. (43) Pub. Date: May 26, 2005

(54) DEVELOPMENT OF NEW SELECTIVE Related U.S. Application Data ESTROGEN RECEPTOR MODULATORS (60) Provisional application No. 60/513,235, filed on Oct. (76) Inventors: Thomas S. Scanlan, San Francisco, CA 21, 2003. (US); Martin J. Kelly, Portland, OR (US); Jian Qiu, Portland, OR (US); Publication Classification Sandra Tobias, San Francisco, CA (US); Oline K. Ronnekleiv, Portland, (51) Int. Cl." ...... A61K 31/195; A61K 31/137 OR (US) (52) U.S. Cl...... 514/567; 514/649; 562/442; 564/338 Correspondence Address: WOODCOCKWASHIBURN LLP (57) ABSTRACT ONE LIBERTY PLACE, 46TH FLOOR The present disclosure concerns a new class of Selective 1650 MARKET STREET estrogen receptor modulators (SERMs). The disclosure also PHILADELPHIA, PA 19103 (US) includes the identification of a previously unknown mem brane associated estrogen receptor. Methods for making and (21) Appl. No.: 10/970,242 using the disclosed SERMs are disclosed, including phar maceutical formulations of the disclosed novel compounds (22) Filed: Oct. 20, 2004 in useful compositions.

Baclofen TTX Baclofen

A TTX MWashout Vehicle or E2 M"Washout Patent Application Publication May 26, 2005 Sheet 1 of 16 US 2005/0113453 A1

A Baclofen TTX + Baclofen

TTX Washout Vehicle or E2 Washout R1 R2 (vehicle) , V R2 (E2) Y WS -Time 10 2O 30 40

F.G. 1 Patent Application Publication May 26, 2005 Sheet 2 of 16 US 2005/0113453 A1

FIG 2 Patent Application Publication May 26, 2005 Sheet 3 of 16 US 2005/0113453 A1

Voltage (mV) - 160 - 140 - 120 - 100

ra O

O Control O Baclofen FIG. 3A

D Voltage (mV) - 160 - 140 - 120 -100 -80

-8 O

O Control O Baclofen

FG. 3B Patent Application Publication May 26, 2005 Sheet 4 of 16 US 2005/0113453 A1

FIG. 4A

Patent Application Publication May 26, 2005 Sheet 5 of 16 US 2005/0113453 A1

FIG. 4C

FIG. 4D Patent Application Publication May 26, 2005 Sheet 6 of 16 US 2005/0113453 A1

FIGS Patent Application Publication May 26, 2005 Sheet 7 of 16 US 2005/0113453 A1

120

100

20 Patent Application Publication May 26, 2005 Sheet 8 of 16 US 2005/0113453 A1

B 12O G E. 100 8O 9. (i) s 60 () E 40 th g 2O O Oil EB STX STX 21g 5ug FIG. 7B Patent Application Publication May 26, 2005 Sheet 9 of 16 US 2005/0113453 A1

A Baclofen i

h 10 pA 2 min Patent Application Publication May 26, 2005 Sheet 10 of 16 US 2005/0113453 A1

Patent Application Publication May 26, 2005 Sheet 11 of 16 US 2005/0113453 A1

Baclofer E2 A BS Sr. 888883: S.S. S. as 3. 8s. &rs: :

- 20 pA 2 min

20 pA 2 min

C Baclof i

20 pA 2 in

D BISE is

- 20 pA 2 min Patent Application Publication May 26, 2005 Sheet 12 of 16 US 2005/0113453 A1

Patent Application Publication May 26, 2005 Sheet 13 of 16 US 2005/0113453 A1

A E2 Baclofen "

B E2 Baclofen Gs peptide. SESses 20 pA 2 min

E2 C Baclofen

" Patent Application Publication May 26, 2005 Sheet 14 of 16 US 2005/0113453 A1

140

120

100 Patent Application Publication May 26, 2005 Sheet 15 of 16 US 2005/0113453 A1

A Arcuate h Tissue Single Neurons Control 2 3 4 5 6 I (bp) TH 223

2O7 GAD

PKCS 251

AC VI 235

GAPDH 212

F.G. 14A

B Arcuate Tissue Single Neurons Control

F.G. 14B Patent Application Publication May 26, 2005 Sheet 16 of 16 US 2005/0113453 A1

Endoplasmic reticulum Itered Cell Functions Protein Synthesis RNA Synthesis

FIG 15 US 2005/0113453 A1 May 26, 2005

DEVELOPMENT OF NEW SELECTIVE SERMS may act as either agonists or antagonists to Selec ESTROGEN RECEPTOR MODULATORS tively modulate one or more estrogen receptorS. Certain SERM compounds have mixed estrogenic and anti-estro CROSS-REFERENCE TO RELATED genic activities and act as estrogen receptor agonists in Some APPLICATION tissues and as estrogen receptor antagonists in other tissues. 0001) This application claims benefit under 35 USC 0008 Estrogens, such as 17(3-estradiol, have been 119(e) of U.S. Provisional Application No. 60/513,235, filed thought to exert their effects by binding to one of two Oct. 21, 2003, the entirety of which is herein incorporated by nuclear estrogen receptors, (ER).C. or (ER)?. Because (ER)Cl. reference. and (ER)? are found in different tissues and have been demonstrated to have different biological roles, researchers ACKNOWLEDGEMENT OF GOVERNMENT have focused on the development of SERMs that effect a SUPPORT response by selectively binding to either (ER).C. or (ER) B. 0002 This invention was made with Government support 0009. Two examples of nonsteroidal SERMs that exhibit under Grant Nos. NS 35944, NS 38809, DA 05158, DA different activities towards (ER)C. and (ER)? are tamoxifen 10703, and DK57574. The Government has certain rights in and raloxifene. Tamoxifen and raloxifene have been devel this invention. oped for the treatment and/or prevention of Osteoporosis, cardiovascular disease and breast cancer in addition to the FIELD treatment and/or prevention of a variety of other disease 0003. This disclosure concerns novel compounds and states. Both compounds have been shown to exhibit an methods for their use, including Selective estrogen receptor Osteoprotective effect on bone mineral density combined modulators and methods for making Such Selective estrogen with a positive effect on plasma cholesterol levels and a receptor modulators. greatly reduced incidence of certain types of cancer. Unfor tunately, tamoxifen and raloxifene both induce Side effects BACKGROUND Such as hot flushes and tamoxifen promotes life-threatening disorders, Such as endometrial cancer. 0004 Estrogens are an important class of steroidal hor mones that Stimulates the development and maintenance of 0010. The present disclosure includes novel nonsteroidal fundamental Sexual characteristics in humans. In addition, SERMs that elicit the desired effects of hormone replace estrogens have been demonstrated to affect a variety of ment therapy without inducing the negative effects of hor diverse biological processes. Many of the incidental effects mone replacement therapy or those of known Selective of estrogens are positive, including the maintenance of bone estrogen receptor modulators. density, central nervous System function and preservation of memory. However, estrogens also have been demonstrated SUMMARY to have Serious negative effects, including promoting the 0011. The present disclosure includes novel compounds, development of breast and endometrial cancers. compositions and methods for making and using the com 0005 Based upon a life expectancy of nearly eighty years pounds and compositions. Generally, the disclosed com in the United States, a woman can expect to spend about a pounds function as Selective estrogen receptor modulators third of her lifetime in a post-menopausal State. A woman's (SERMs). In one aspect, the compounds have Formula 1 and estrogen levels drop dramatically during menopause and include hydrates and pharmaceutically acceptable prodrugs menopausal women often experience many Side affects and Salts thereof. Moreover, all chiral, diastereomeric and asSociated with the reduction in estrogen production. To geometric isomeric forms of the disclosed Formulas are treat these conditions, physicians often prescribe hormone intended. replacement therapy, which primarily consists of the admin istration of estrogen in combination with progestin. Formula 1 0006. In light of the more serious side effects associated R M-Air-Y-G-Z. with current hormone replacement therapy, including increased risk of ischemic Stroke, myocardial infarction, thromboembolism, cerebrovascular disease, and endome trial carcinoma, a significant amount of research has been carried out to identify effective nonsteroidal estrogen and antiestrogenic compounds. N/ 0007. A large number of compounds have been described that either mimic or block the effects of the most potent estrogen, 17B-estradiol. Compounds that bind to an estrogen 0012. With reference to Formula 1, R can be E or Z with receptor and Stimulate many of the same biological effects as respect to M and R represents a hydrogen or a lower 17B-estradiol are termed "estrogen receptor agonists.” Com aliphatic group, X is an ortho, meta or para positioned pounds that inhibit the binding of 17 B-estradiol to an estro hydrogen, hydroxyl, protected hydroxy, alkoxy, Sulfhydryl, gen receptor or interfere with the effects of 17,3-estradiol thioether, amino (-NRR wherein R and Rare indepen binding to an estrogen receptor are referred to as “estrogen dently hydrogen or lower alkyl) or a halogen; M comprises receptor antagonists.” Compounds that affect different estro a ketone, amide or Sulfonamide group. Ar represents an gen receptors with different potencies are typically referred aromatic group and can be any aromatic group, including, to as Selective estrogen receptor modulators (SERMs). without limitation, phenyl, biphenyl, 1-napthyl, 2-napthyl, US 2005/0113453 A1 May 26, 2005 and including heteroaryl groups, Such as, by way of example, furan, thiophene, pyrrole, imidazole, oxazole, thia Zole, pyridine, and quinoline groups. Y can be attached at - V Y-G Formula 3 any position, Such as at the ortho, meta or para position x1 Y, relative to M. In one aspect, Y is a heteroatom, Such as a \ / nitrogen or oxygen. In certain embodiments of the disclosed SERMs according to Formula 2 Y represents a phenyl group. R Q-N V 1. R

Formula 2

0015 Thus, compounds according to Formula 3 can have, for example, Formula 4 or 5. With respect to Formulas 4 and 5, R, X, R', and Y are defined as above with respect to Formula 1. 0013 With continued reference to Formulas 1 and 2, G Formula 4 includes a linker group, Such as a lower alkyl group, a hydrocarbon chain, an oligoethylene glycol chain or the like. In one embodiment of Formula 2 wherein G includes a lower alkyl group, G is an ethyl group. Z typically includes a heteroatom. In certain embodiments Z includes a charged moiety, for example an anionic group, Such as a boronate, Sulfate, Sulfonate, phosphate, phosphonate or a carboxy group. In examples wherein Zincludes a cationic group, the cationic group can be an amino group, Such as a dimethy lamino or piperidino group. In other examples Z includes a hydroxyl group. Examples of groups represented by the formula Y-G-Z include those wherein Y is a heteroatom, Formula 5 Such as oxygen and the formula-G-Z represents, without limitation, one of the following:

COH CO2H COH

NMe2 NMe2

rOH OH 0016 AS indicated in the formulas above, the disclosed compounds include Vinyl ketone derivatives, acrylamide derivatives and vinyl sulfonamide derivatives. Moreover, as OH illustrated in the Structures above, the configuration about the Vinyl group can be E or Z. rt 0017. One aspect of the disclosure includes methods for Synthesizing novel Selective estrogen receptor modulators. In another embodiment, the method includes providing a 0.014 With reference to Formulas 1 and 2, M typically is Starting material according to Formula 6, wherein G is a one of an amide ketone or a Sulfonamide group and more carboxy group, an activated carboxy group, a Sulfonic acid typically represents one of -C(O)-, -C(O)-NR'-; or group or an activated Sulfonic acid group. The activated -SO2NR'-; wherein R' is hydrogen, a lower alkyl group carboxy and Sulfonic acid groups include any groups that or aralkyl group. Thus, examples of Such compounds can be can react with an amine or amine equivalent to form an represented by Formula 3 wherein Q typically forms an amide or Sulfonamide according to Formula 3 or Formula 4. amide or Sulfonamide linkage (Q represents -SO2- or Typically activated carboxy groups include acyl halides and CO). activated esters. In particular examples, Y is a carboxy US 2005/0113453 A1 May 26, 2005

group, which has been activated for reaction with nucleo philes in situ. Similarly, typical activated Sulfonic acid groups include Sulfonyl halides, Such as Sulfonyl chlorides. Scheme 1 Such activated Sulfonic acid groups can be used to acylate a R Y variety of amines, including primary and Secondary amines.

o He Formula 6

X Formula 7 HO R1R2N N/ R O R O

o -- o 0.018 For example, one embodiment of a method for Synthesizing novel estrogen receptor modulators is illus trated in Scheme 1. With reference to Scheme 1, Formula 7, X X variable Y indicates an anion or anion equivalent, Such as a Formula 8 Formula 9 halogen, which can be Subjected to lithium-halogen eXchange conditions to give the corresponding carbanion. The carbanion can be used to introduce a carboxy group as 0020. Another aspect of the method is illustrated in illustrated in Formula 8. The carboxy group can be intro Scheme 2. With reference to Scheme 2, the method includes duced, for example, as a protected carboxy equivalent, Such providing a vinyl Sulfonic acid according to Formula 11 and as a protected chloroformate derivative. One example of coupling a primary or secondary amine (R'R''NH) to pro Such a reagent is allyl chloroformate, which can be used to vide a Sulfonamide according to Formula 12. The coupling introduce the carboxy group of Formula 8. Compounds reaction can be accomplished in the presence of, for according to Formula 8 are versatile Synthetic intermediates example, DCC, DIC, EDCI, HBTU, TBTU, PyBop or because Such compounds can be coupled with any primary PyBrop. The conversion also optionally can employ addi or Secondary amine to provide compounds according to tives, such as DMAP and/or HOBt. Formula 9. Thus, compounds according to Formula 9, wherein R and R independently are selected from hydro Scheme 2 gen, aliphatic, aryl or aralkyl groups, can be prepared by reacting compounds according to Formula 8 with the cor RRN 2 O responding primary or Secondary amine having the formula: R SO3- R SS R'R''NH. Typically at least one of R and R includes an aryl O group, and examples of Suitable amines including aryl groups include those wherein at least one of R and R is a phenyl group or a phenyl derivative. 0.019 Generally, the conversion of a compound having Formula 8 to a compound having Formula 9 according to the X X presently disclosed method includes using a coupling Formula 10 Formula 11 reagent to activate the carboxy group for reaction. Examples of Such coupling reagents include, without limitation, car 0021 Particular novel compounds included in the present bodiimides, uronium Salts, phosphonium Salts, and the like. disclosure bind to a previously unidentified, membrane Examples of carbodiimides include, without limitation, asSociated estrogen receptor. Moreover, Specific examples of dicyclohexylcarbodiimide, diisopropylcarbodiimide and these compounds Selectively bind to the membrane associ 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydro ated estrogen receptor with greater affinity than they bind to chloride (EDCI). Examples of uronium salts include 2-(1H benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro the classical nuclear receptors, (ER).C. and (ER)?. Therefore, phosphate (HBTU) and 2-(1H-benzotriazole-1-yl)-1,1,3,3- one aspect of the disclosure includes a method for providing tetramethyluronium tetrafluoroborate (TBTU). Examples of a Selective agonist or antagonist of a membrane associated phosphonium Salts include benzotriazole-1-yl-oxy-tris-pyr eStrogen receptor. rolidino-phosphonium hexafluorophosphate (PyBop) and 0022. The disclosure also includes methods and compo bromo-tris-pyrrolidino-phosphonium hexafluorophosphate Sitions for treating a variety of disorders, particularly those (PyBrop). The conversion also optionally can employ addi characterized by an estrogen deficiency Such as those asso tives, such as 4-dimethylaminopyridine (DMAP) and/or ciated with Ovarectomy, ovarian failure or menopause. Such N-hydroxybenzotriazole (HOBt). In one embodiment, the conditions and disorders include, generally, those described disclosed synthesis, HBTU can be used in the presence of as autonomic dysfunctions, cognitive decline, motor dyS DMAP to couple amines to compounds having Formula 8. functions, mood disorders, eating disorders and cardiovas US 2005/0113453 A1 May 26, 2005

cular diseases, as well as different types of disorders. In one inhibitors and demonstrates that 17B-estradiol attenuation of aspect, the disclosed compounds exert prophylactic effects the GABAB response involves protein kinase A. against certain types of injuries. For example, the com pounds can be used as neuroprotectants. Indeed, compounds 0037 FIG. 9 is a bar graph Summarizing the effects of that agonize the membrane-associated estrogen receptor protein kinase A (PKA) drugs on the baclofen response. identified herein act as neuroprotectants to reduce neuronal 0038 FIG. 10 includes representative traces of the cell death in response to ischemic Stroke and inhibit reper baclofen responses in the presence of PKC inhibitors. fusion injury. 0039 FIG. 11 is a bar graph summarizing the effects of 0023 Particular compositions include at least one com protein kinase C(PKC) inhibitors on the baclofen response. pound according Formula 1 as well as a Second therapeutic 0040 FIG. 12 includes representative traces of the compound. The Second compound also can bind to an baclofen responses in the presence of phospholipase C estrogen receptor. For example, in one embodiment the second compound is a SERM and in another embodiment (PLC) and Gc, inhibitors. the Second compound is an estrogen, Such as 173-estradiol, 0041 FIG. 13 is a bar graph summarizing the effects of or a progesterone, Such as progestin. the PLC and Gc, inhibitors. 0042 FIG. 14A is a representative gel illustrating that BRIEF DESCRIPTION OF THE DRAWINGS single arcuate neurons express mRNA for TH, GAD, PKC6, 0024 FIG. 1 is a schematic illustrating the protocol for AC VII and GAPDH. drug administration in the whole-cell patch, Voltage clamp 0043 FIG. 14B is a representative gel illustrating that Studies. single arcuate neurons express mRNA for POMC, GAD, 0.025 FIG. 2 is an image showing an example of a PKCS, AC VII and GAPDH. biocytin-filled neuron after whole-cell patch-clamp record 0044 FIG. 15 is a schematic overview showing the rapid ing for 13 min illustrating the extent of the biocytin labeling. Versus delayed estrogen receptor mediated modulation of neurotransmitter regulated, G protein-coupled receptors via 0.026 FIG. 3A is a graph illustrating the pre- and post a membrane-associated estrogen receptor in hypothalamic baclofen (5uM) I/V relationships from a dopamine neuron. CUOS. 0027 FIG. 3B is a graph of the pre- and post-baclofen I/V relationships following 17(3-estradiol treatment in DETAILED DESCRIPTION another cell illustrating the Same reversal potential for the 004.5 The following explanations of terms and methods baclofen response. are provided to better describe the present compounds, 0028 FIG. 4A is an image showing the biocytin-strepta compositions and methods, and to guide those of ordinary Vidin-Cy2 labeling of fusiform arcuate dopamine neuron skill in the art in the practice of the present disclosure. It is that responded to estrogen. also to be understood that the terminology used in the disclosure is for the purpose of describing particular 0029 FIG. 4B is an image showing the immunocy embodiments and examples only and is not intended to be tochemical staining of tyrosine hydroxylase (TH) in the limiting. neuron imaged in FIG. 4A. 0046) The specification includes the following acronyms 0030 FIG. 4C is an image showing the biocytin-strepta and abbreviations: vidin-Cy2 labeling of a small pyramidal arcuate POMC 0047 BAPTA: 1,2-bis-(o-aminophenoxyethane)-N, CUO. N,N',N'-tetraacetic acid 0.031 FIG. 4D is an image showing the immunocy tochemical Staining of B-endorphin in the neuron imaged in 0048 CTX: cholera toxin FIG. 4C. 0049 DAG: diacylglycerol 0032 FIG. 5 includes representative traces of the 0050 DCM; dichloromethane GABA responses before and after steroid treatment. 0051 ddHO: deionized distilled water 0.033 FIG. 6 includes bar graphs summarizing the effects of 17 B-estradiol and SERMs on the baclofen response. 0.052 DMF: N,N-dimethylformamide 0034 FIG. 7A compares uteri size following treatment 0053) EGTA: ethylene glycol tetraacetic acid with 17 B-estradiol versus an exemplary SERM disclosed 0054) EtOAc: ethyl acetate herein and shows that in 17B-estradiol-treated mice, there was a noticeable increase in uterine size after estradiol 0055) ERK: extracellular-signal related kinase benzoate (EB), compared with oil-vehicle treatment or treat 0056 GIRK: G protein-coupled, inwardly-rectify ment with an exemplary SERM disclosed herein. ing K channel 0035 FIG. 7B is a bar graph recording the uterine weights for EB treated mice versus oil and SERM treated 0057 Hex: hexanes mice (n=3-5 mice/group). 0.058 MAPK or MAP kinase: mitogen activated 0036 FIG. 8 includes representative traces of the protein kinase baclofen responses in the presence of PKA activators or 0059) PLC: phospholipase C US 2005/0113453 A1 May 26, 2005

0060) PKA: protein kinase A or more of the Secondary effects associated with estrogen insufficiency, Such as Osteoporosis, heart disease, hot flushes 0061 PKC: protein kinase C and mood disorders. 0062) POMC: proopiomelanocortin 0072 “Derivative” refers to a compound or portion of a 0063) SERM: selective estrogen receptor modulator compound that is derived from or is theoretically derivable from a parent compound. 0.064 TTX: tetrodotoxin 0073. The term “alkyl group” refers to a branched or 0065 Ranges can be expressed herein as from “about” unbranched Saturated hydrocarbon group of 1 to 24 carbon one particular value, and/or to “about another particular atoms, Such as methyl, ethyl, n-propyl, isopropyl, n-butyl, value. When Such a range is expressed, another embodiment isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetrade includes from the one particular value and/or to the other cyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower particular value. Similarly, when values are expressed as alkyl group is a Saturated branched or unbranched hydro approximations, by use of the qualifier “about,” it will be carbon having from 1 to 10 carbon atoms. understood that the particular value forms another embodi ment. It will be further understood that the endpoints of each 0074 The term “alkenyl group” refers to a hydrocarbon of the ranges are significant both in relation to the other group of 2 to 24 carbon atoms and Structural formula endpoint, and independently of the other endpoint. containing at least one carbon-carbon double bond. 0.066 The disclosed compounds include chiral com 0075. The term “alkynyl group” refers to a hydrocarbon pounds, Such as those that contain an asymmetrically tetra group of 2 to 24 carbon atoms and a structural formula Substituted carbon atom. Such compounds can be isolated in containing at least one carbon-carbon triple bond. racemic or in optically active forms. It is known to those of 0076. The term “halogenated alkyl group” refers to an ordinary skill in the art how to Synthesize compounds in alkyl group as described above with one or more hydrogen optically active form. It also is well known how to prepare atoms present on these groups Substituted with a halogen (F, optically active compounds by resolution of racemic mix Cl, Br, I). tures of compounds. All chiral, diastereomeric and geomet 0077. The term “cycloalkyl group” refers to a non-aro ric isomeric forms of the disclosed Structures are intended matic carbon-based ring composed of at least three carbon unless Specifically indicated otherwise. atoms. Examples of cycloalkyl groups include, but are not 0067. In this specification and in the claims which follow, limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, reference will be made to a number of terms which shall be etc. The term "heterocycloalkyl group' is a cycloalkyl group understood to have the following meanings: as described above wherein at least one of the carbon atoms 0068 “Optional” or “optionally” means that the Subse of the ring is Substituted with a heteroatom Such as, but not quently described event or circumstance can but need not limited to, nitrogen, oxygen, Sulfur, or phosphorous. occur, and that the description includes instances wherein 0078. The term “ethylene glycol chain” or “oligoethylene Said event or circumstance occurs and instances wherein it glycol chain” refers to a group having a repeating ethylene does not. glycol unit. The ethylene glycol chain can be any length, however typically it includes from 2 to about 100 and more 0069 Variables, such as R, G, M, Q, X, Y and Z, used typically from 2 to about 70 ethylene glycol units. Most throughout the disclosure are the same variables as previ typically an oligoethylene glycol chain includes from about ously defined unless Stated to the contrary. 2 to about 20 ethylene glycol units. 0070 The term “estrogen receptor agonist” refers to a 0079 The term “aliphatic group” includes alkyl, alkenyl, compound that acts at an estrogen receptor and has at least alkynyl, halogenated alkyl and cycloalkyl groups as Some of the same biological effects as 173-estradiol. Com described above. A “lower aliphatic' group is a branched or pounds that act at an estrogen receptor to block the effects of 173-estradiol are called “estrogen receptor antagonists.” unbranched Structure having from 1 to 10 carbon atoms. Typically, the compounds disclosed herein are partial ago 0080. The term “aryl group” refers to any carbon-based nists or antagonists, which means that they mimic or block aromatic group including, but not limited to, benzene, naph Some of the effects of 17 B-estradiol, but not others. In some thalene, etc. The term “aromatic” also includes “heteroaryl cases the compounds exhibit mixed agonist/antagonist activ group,” which refers to an aromatic group that has at least ity, wherein the compounds act as an estrogen receptor one heteroatom incorporated within the ring of the aromatic agonist in certain tissues, but act as an estrogen receptor group. Examples of Such heteroatoms include, but are not antagonist in other tissues. Examples of Such compounds limited to, nitrogen, oxygen, Sulfur, and phosphorous. The disclosed herein exhibit partial agonist activity by binding aryl group can be Substituted with one or more groups Selectively to one estrogen receptor with higher affinity than including, but not limited to, alkyl, alkynyl, alkenyl, aryl, to other estrogen receptorS. Compounds exhibiting Such halide, nitro, amino, ester, ketone, aldehyde, hydroxy, car Selectivity are termed “Selective estrogen receptor modula boxylic acid, or alkoxy, or the aryl group can be unsubsti tors or “SERMS. tuted. 0.071) “Hormone replacement therapy” refers to treat 0081. The term “aralkyl” refers to an aryl group having ment given in response to reduced or insufficient estrogen an alkyl group, as described above, attached to the aryl production in a Subject, for example as Seen in menopause. group. An example of an aralkyl group is a benzyl group. Hormone replacement therapy often is undertaken in 0082 The term “alkyl amino” includes alkyl groups as response to aging, ovarectomy or premature ovarian failure. described above wherein at least one hydrogen atom is Hormone replacement therapy is often used to help treat one replaced with an amino group. US 2005/0113453 A1 May 26, 2005

0083) The term “heteroatom” is understood by those of Salts and/or internal Salts. The Salts are generally pharma ordinary skill in the art to refer to an atom other than a ceutically-acceptable Salts that are non-toxic. Examples of carbon atom. Examples of heteroatoms include, without Salt-forming acidic groups include, but are not limited to, a limitation, boron, nitrogen, oxygen, phosphorus and Sulfur. carboxyl group, a phosphonic acid group or a boronic acid group, that can form Salts with Suitable bases. These Salts 0084. The term “hydrocarbon chain” as used herein can include, for example, nontoxic metal cations which are therefore typically refers to a chain of carbon atoms, typi derived from metals of groups IA, IB, IIA and IIB of the cally comprising from 2 to about 22 carbon atoms. The chain periodic table of the elements. In one embodiment, alkali can comprise aliphatic and aryl groupS and can comprise metal cations Such as lithium, Sodium or potassium ions, or Straight chain, branched chain and/or cyclic groups. alkaline earth metal cations Such as magnesium or calcium 0085. The term “hydroxyl group” is represented by the ions can be used. The Salt can also be a Zinc or an ammonium formula -OH. The term “alkoxy group” is represented by cation. The Salt can also be formed with Suitable organic the formula -OR, wherein R can be an alkyl group, amines, Such as unsubstituted or hydroxyl-Substituted optionally Substituted with an alkenyl, alkynyl, aryl, aralkyl, mono-, di- or tri-alkylamines, in particular mono-, di- or cycloalkyl, halogenated alkyl, or heterocycloalkyl group as tri-alkylamines, or with quaternary ammonium compounds, described above. for example with N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, bis- or tris-(2-hydroxy-lower alky 0.086 The term “hydroxyalkyl group” refers to an alkyl l)amines, Such as mono-, bis- or tris-(2-hydroxyethyl)amine, group that has at least one hydrogen atom Substituted with 2-hydroxy-tert-butylamine or tris(hydroxymethyl)methy a hydroxyl group. The term “alkoxyalkyl group' refers to an lamine, N,N-di-lower alkyl-N-(hydroxy-lower alky alkyl group that has at least one hydrogen atom Substituted l)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine with an alkoxy group described above. Where applicable, or tri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or the alkyl portion of a hydroxyalkyl group or an alkoxyalkyl quaternary ammonium compounds Such as tetrabutylammo group can have aryl, aralkyl, halogen, hydroxy, alkoxy nium Salts. 0087. The term “amine group” is represented by the 0097 Particular compounds possess at least one basic formula -NRR", wherein R and R' can be, independently, group that can form acid-base Salts with inorganic acids. hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, Examples of basic groups include, but are not limited to, an cycloalkyl, halogenated alkyl, or heterocycloalkyl group amino group or imino group. Examples of inorganic acids described above. that can form salts with Such basic groups include, but are 0088. The term “amide group” is represented by the not limited to, mineral acids Such as hydrochloric acid, formula -C(O)NRR", wherein R and R' independently can hydrobromic acid, Sulfuric acid or phosphoric acid. Basic be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, groups also can form Salts with organic carboxylic acids, cycloalkyl, halogenated alkyl, or heterocycloalkyl group Sulfonic acids, Sulfo acids or phospho acids or N-Substituted described above. Sulfamic acid, for example acetic acid, propionic acid, 0089. The term “ester” is represented by the formula glycolic acid, Succinic acid, maleic acid, hydroxymaleic -OC(O)R, wherein R can be an alkyl, alkenyl, alkynyl, acid, methylmaleic acid, fumaric acid, malic acid, tartaric aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocy acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, Salicylic cloalkyl group described above. acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, 0090 The term “carbonate group” is represented by the embonic acid, nicotinic acid or isonicotinic acid, and in formula -OC(O)OR, wherein R can be an alkyl, alkenyl, addition, with amino acids, for example with a-amino acids, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or and also with methaneSulfonic acid, ethaneSulfonic acid, heterocycloalkyl group described above. 2-hydroxymethaneSulfonic acid, ethane-1,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzeneSulfonic acid, 0091. The term “carboxylic acid” is represented by the naphthalene-2-Sulfonic acid, 2- or 3-phosphoglycerate, glu formula –C(O)OH. cose-6-phosphate or N-cyclohexylsulfamic acid (with for 0092. The term “aldehyde" is represented by the formula mation of the cyclamates) or with other acidic organic -C(O)H. compounds, Such as ascorbic acid. 0093. The term “keto group” is represented by the for 0098. The term “ether group” is represented by the for mula-C(O)R, wherein R is an alkyl, alkenyl, alkynyl, aryl, mula R(O)R', wherein R and R' can be, independently, an aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated group described above. alkyl, or heterocycloalkyl group described above. 0094. The term “carbonyl group” is represented by the 0099] The term “halide” refers to F, Cl, Br, or I. formula C=O. 0100. The terms “urethane' and carbamate are repre 0.095 “Cationic group” or “cationic moiety” refers to a sented by the formula -OC(O)NRR", wherein R and R' can group that is positively charged or can be positively charged. be, independently, hydrogen, an alkyl, alkenyl, alkynyl, aryl, For example, a cationic group can be an amine that is aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl capable of being protonated at a physiologically relevant group described above. pH. A Second example of a cationic group is a positively 0101 The groups described above can be optionally charged quaternary amine. Substituted with one or more Substituents. The definition of 0096. The disclosed compounds also encompass salts any Substituent or variable at a particular location in a including, if Several Salt-forming groups are present, mixed molecule is independent of its definitions elsewhere in that US 2005/0113453 A1 May 26, 2005

molecule. Examples of Suitable Substituents include but are 0106 The ability of the disclosed compounds to inhibit or not limited to alkyl, alkenyl, alkynyl, aryl, halo, trifluorom ameliorate hot flushes can be determined, for example, in a ethyl, trifluoromethoxy, hydroxy, alkoxy, OXO, alkanoyl, Standard assay that measures the ability of an agent to blunt alkanoyloxy, aryloxy, amino, amido, alkanoylamino, aroy the increase in tail skin temperature that occurs when lamino, aralkanoylamino, Substituted alkanoylamino, aroy morphine-addicted rats undergo acute withdrawal from mor lamino, aralkanoylamino, Substituted alkanoylamino, Sub phine using naloxone. See, Merchenthaler, et al. The effect Stituted arylamino, Substituted aralkanoylamino, thiol, of estrogens and antiestrogens in a rat model for hot flush. Sulfide, thiono, Sulfonyl, Sulfonamide, nitro, cyano, carboxy, Maturitas 1998, 30, 307-316, which is incorporated herein carbamyl, Substituted carbamyl and the like. by reference. See also, Berendsen et al. Effect of tibolone and raloxifene on the tail temperature of oestrogen-deficient 0102) The term “prodrug” is intended to include any rats. Eur: J. Pharmacol. 2001, 419, 47-54, and Pan et al. A covalently bonded carriers that release an active parent drug comparison of oral micronized estradiol with Soy phy of the present invention in Vivo when the prodrug is admin toestrogen effects on tail skin temperatures of ovariecto istered to a Subject. Since prodrugs are known to enhance the mized rats. Menopause 2001, 8, 171-174. Both the Ber properties of pharmaceuticals, Such as, Solubility and bio endsen and Pan publications are incorporated herein by availability, the compounds disclosed herein can be deliv ered in prodrug form. Thus, also contemplated are prodrugs reference. of the presently claimed compounds, methods of delivering 0107 Certain disclosed compounds are useful for the prodrugs and compositions containing Such prodrugs. Pro treatment of multiple Sclerosis. Preferred compounds for drugs of the disclosed compounds typically are prepared by treating or Suppressing the Symptoms of multiple Sclerosis modifying functional groups present in the compound in can be Selected by using the experimental autoimmune Such a way that the modifications are cleaved, either in encephalomyelitis mouse model described by Polanczyk et routine manipulation or in Vivo, to yield the parent com al. Am. J. Pathol. 2003, 163, 1599-1605. The Polanczyk pound. Prodrugs include compounds having a hydroxy, publication is incorporated herein by reference. amino, or Sulfhydryl group functionalized with any group that is cleaved to yield the corresponding hydroxyl, free 0.108 Disclosed compounds that are useful for treating amino, or free Sulfhydryl group, respectively. Examples of eating disorders, Such as anorexia nervosa and/or bulimia prodrugs include, without limitation, compounds having a nervosa can be identified using a simple feeding assay as is hydroxy, amino and/or Sulfhydryl group acylated with an known to those of ordinary skill in the art. acetate, formate, and/or benzoate group. 0109) The effect of the disclosed compounds on learning and memory can be assessed using the Morris water maZe 0103 Protected derivatives of the disclosed compound and object recognition assays according to the protocols also are contemplated. A variety of Suitable for use with the described by Stackman et al. J. Neuroscience 2002, 22, disclosed compounds is disclosed in Greene and Wuts 10163-10171. The Stackman et al. publication is incorpo Protective Groups in Organic Synthesis; 3rd Ed., John rated herein by reference. Wiley & Sons, New York 1999. 0110. The neuroprotective activity of the disclosed com 0104. It is understood that substituents and Substitution pounds also can be evaluated, for example, in a Standard in patterns of the compound described herein can be Selected Vitro pharmacological assay using glutamate challenge. See, by one of ordinary skill in the art to provide compounds that Zaulyanov, et al. Cellular cc Molecular Neurobiology 1999, are chemically stable and that can be readily Synthesized by 19: 705-718; and Prokai, et al. Journal of Medicinal Chem techniques known in the art and further by the methods Set istry 2001, 44, 110-114. Both the Zaulyanov et al. and Prokai forth in this disclosure. Reference will now be made in detail et al. publications are incorporated herein by reference. to the present preferred embodiments. 0111. The disclosed compounds also can be evaluated for I. SELECTION OF PREFERRED COMPOUNDS their ability to protect neurons from damage associated with global cerebral ischemia. Compounds can be evaluated for 0105 Preferred compounds for modulating an estrogen their ability to reduce Such damage using the cardiac arrest receptor typically are Selected for their potency and Selec and cardiopulmonary resuscitation assay described by Vogel tivity in agonizing or antagonizing one or more estrogen et al. Anesthesiology 2003, 99, 112-121. In one aspect, the receptors. Typically the disclosed compounds Selectively compounds can reduce the damage to neurons even when bind to a membrane associated estrogen receptor and bind to administered after resuscitation. The Vogel et al. publication the nuclear estrogen receptors (ER).C. and (ER)? with lower is incorporated herein by reference. affinity, if at all. As described in the Methods and Examples Section below, and as is generally known, efficacy of par 0112 Neuroprotection also can be evaluated using a ticular compounds against a target estrogen receptor can be middle cerebral artery occlusion procedure in mice accord determined in Vitro, and the disclosed in vitro methods can ing to the protocol described by Dubal et al. in Proc. Natl. be used to screen and identify novel SERMs. Moreover, Acad. Sci. U.S.A. 2001, 98, 1952-1957; and in J. Neurosci. particular compounds are provided for treating or protecting 1999, 6385-6393. Additional assays for evaluating the dis against various conditions and disorders, including condi closed compounds in different models of disorders charac tions that are associated with menopause or other conditions terized by estrogen insufficiency are described in the Meth characterized by estrogen insufficiency, Such as those asso ods and Examples Section below. ciated with Ovarectomy, ovarian failure or menopause. 0113 Use of the compounds is not necessarily limited to Examples of Such conditions include, without limitation, hot conditions involving estrogen insufficiency. Techniques and flushes, cognitive decline, osteoporosis, depression, assays for characterizing the efficacy of therapeutics for ischemic brain damage and atherosclerosis. treating or preventing Such conditions and disorders are well US 2005/0113453 A1 May 26, 2005

known and are described, for example, by Malamas et al. like in addition to the molecule of choice. Pharmaceutical and Mewshaw et al. in U.S. patent publication Nos. 2003/ compositions can also include one or more additional active 0171412 A1 and 2003/0181519 A1, respectively. Both the ingredients Such as antimicrobial agents, anti-inflammatory Malamas et al. and Mewshaw et al. publications are incor agents, anesthetics, and the like. Pharmaceutical formula porated herein by reference. tions can include additional components, Such as carriers. The pharmaceutically acceptable carriers useful for these II. PHARMACEUTICAL COMPOSITIONS formulations are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, 0114. Another aspect of the disclosure includes pharma Pa., 15th Edition (1975), describes compositions and for ceutical compositions prepared for administration to a Sub mulations Suitable for pharmaceutical delivery of the com ject and which include a therapeutically effective amount of one or more of the currently disclosed compounds. The pounds herein disclosed. therapeutically effective amount of a disclosed compound 0119). In general, the nature of the carrier will depend on will depend on the route of administration, the type of the particular mode of administration being employed. For mammal that is the Subject and the physical characteristics instance, parenteral formulations usually contain injectable of the Subject being treated. Specific factors that can be fluids that include pharmaceutically and physiologically taken into account include disease Severity and Stage, acceptable fluids Such as water, physiological Saline, bal weight, diet and concurrent medications. The relationship of anced Salt Solutions, aqueous dextrose, glycerol or the like as these factors to determining a therapeutically effective a vehicle. For Solid compositions (for example, powder, pill, amount of the disclosed compounds is understood by those tablet, or capsule forms), conventional non-toxic Solid car of ordinary skill in the art. riers can include, for example, pharmaceutical grades of 0115 The therapeutically effective amount of the com mannitol, lactose, Starch, or magnesium Stearate. In addition pound or compounds administered can vary depending upon to biologically-neutral carriers, pharmaceutical composi the desired effects and the factors noted above. Typically, tions to be administered can contain minor amounts of dosages will be between about 0.01 mg/kg and 100 mg/kg non-toxic auxiliary Substances, Such as wetting or emulsi of the Subject's body weight, and more typically between fying agents, preservatives, and pH buffering agents and the about 0.01 mg/kg and 10 mg/kg of the subject's body like, for example Sodium acetate or Sorbitan monolaurate. weight. In one aspect the therapeutically effective amount 0120 In some embodiments, sustained release of the can be Selected to achieve an in Vivo concentration of the pharmaceutical preparation that comprises an effective therapeutic agent in a target tissue of a Subject of about the amount of a disclosed SERM can be beneficial. Slow-release concentration found to be effective in vitro. formulations are known to those of ordinary skill in the art. 0116. In one embodiment, the disclosed SERM is used in By way of example, polymerS Such as bis(p-carboxyphe combination with additional compounds disclosed herein noxy)propane-Sebacic-acid or lecithin Suspensions can be and/or other therapeutic agents, Such as other SERMs, used to provide Sustained release. anti-cancer agents or anti-proliferative agents. For example 0121. It is specifically contemplated in some embodi the disclosed compounds may be used with chemotherapeu ments that SERM delivery is via an injected and/or tic agents, Such as tamoxifen, taxol, epothilones, methotr implanted drug depot, for instance comprising multi-vesicu exate, and the like. In one aspect, a disclosed SERM is used lar liposomes Such as in DepoFoam (SkyePharma, Inc, San in combination with a steroid hormone, Such as an estrogen, Diego, Calif.) (see, for instance, Chamberlain et al. Arch. including 17 B-estradiol, a progesterone or the like. The Neuro. 1993, 50, 261-264; Katri et al. J. Pharm. Sci. 1998, estrogen or progesterone can be a naturally occurring or 87, 1341-1346; Ye et al., J. Control Release 2000, 64, Synthetic estrogen or progesterone. When different thera 155-166; and Howell, Cancer J. 2001, 7, 219-227). peutic agents are used in combination, the therapeutic agents can be administered together or Separately. The therapeutic III. METHODS FOR USING THE DISCLOSED agents can be administered alone, but more typically are COMPOUNDS administered with a pharmaceutical carrier Selected on the basis of the chosen route of administration and Standard 0.122 The disclosed compounds can be used to selec pharmaceutical practice. tively modulate an estrogen receptor in a Subject and thus are useful for treating a variety of disorders, including those 0117. Any of the SERMs described herein can be com characterized by an estrogen deficiency. Moreover, because bined with a pharmaceutically acceptable carrier to form a certain disclosed compounds exhibit Selectivity for one or pharmaceutical composition. Pharmaceutical carriers are more estrogen receptors, the compounds can be used to treat known to those skilled in the art. These most typically would conditions including but not limited to those described as be standard carriers for administration of compositions to autonomic dysfunctions, cognitive decline, motor dysfunc humans, including Solutions Such as Sterile water, Saline, and tions, mood disorders, eating disorders and cardiovascular buffered Solutions at physiological pH. The compositions disorders, as well as different types of disorders. Generally, could also be administered intramuscularly, Subcutaneously, the compounds are useful for hormone replacement therapy or in an aerosol form. Other compounds will be administered without inducing the same incidence of Serious Side effects according to Standard procedures used by those skilled in the associated with the Steroidal hormones (such as estrogen or art. Synthetic estrogens) used in current hormone replacement 0118 Molecules intended for pharmaceutical delivery therapies. The disclosed compounds also avoid Side effects can be formulated in a pharmaceutical composition. Phar Such as hot flushes encountered in treatment with currently maceutical compositions can include carriers, thickeners, known SERMs, such as tamoxifen or raloxifene. More diluents, buffers, preservatives, Surface active agents and the Specifically, the disclosed compounds can be used to treat US 2005/0113453 A1 May 26, 2005

disorders including, without limitation, ischemia-induced methods are generalizable to the Synthesis of compounds not neuronal death, head trauma, Alzheimer's disease, disorders explicitly described below upon consideration of the func of temperature regulation, Such as hot flushes, sleep cycle tionality of the molecule in View of the reagents and reac disruptions, Parkinson's disease, tardive diskinesia, depres tions disclosed. In view of the disclosed conditions, a perSon Sion, Schizophrenia, anorexia nervosa, bulimia nervosa, car diovascular disease, atherOSclerosis, long QTL Syndromes, of ordinary skill in the art will recognize alternate methods such as Romano-Ward or Torsades de Pointes syndromes, for preparing analogous compounds that may have func Osteoporosis, rheumatoid arthritis, osteoarthritis, bone frac tional groups that are incompatible with the Specific condi tures and multiple Sclerosis. In one embodiment particular tions disclosed herein. compounds can be used to promote vasodilation by modu lating fluid balance, Such as by blocking epithelial transport 0127 Depending upon the functional groups present in a of sodium or chloride. Without limitation to theory, the given transformation, protecting groups for various groups compounds appear to function by activating maxi-K chan may be preferred for masking the group during the trans nels. See, Valverde et al. Science 1999, 285, 1929-1931. formation. Suitable protecting groups for various function alities are described in Greene and Wuts Protective Groups 0123. In another embodiment, the disclosed compounds in Organic Synthesis, 3rd Ed.; John Wiley & Sons, New can be used to treat autoimmune diseases, particularly autoimmune diseases that occur more frequently in Women York (1999). than in men. Examples of Such diseases include, without 0128. One embodiment of a method for synthesizing the limitation, multiple Sclerosis, rheumatoid arthritis, Grave's disease, Systemic lupus erythematosus and myasthenia disclosed compounds is illustrated in Scheme 3, below. With gravis. In another embodiment the disclosed compounds reference to Scheme 3, the method includes providing a function to maintain or enhance immune competency in a compound 2 and converting compound 2 to compound 4. Subject. Moreover, the disclosed compounds exert prophy Compound 2 was prepared according to the protocol lactic effects against certain types of injuries. For example, reported by Weatherman et al. Chemistry cc Biology 2001, 8, the compounds can be used as neuroprotectants. Indeed, 427-436. In working examples compound 2 was Subjected to compounds that agonize the membrane-associated estrogen lithium-halogen exchange conditions, followed by reaction receptor identified herein act as neuroprotectants in response with allyl chloroformate to give compound 4. Compound 6 to ischemic Stroke and inhibit reperfusion injury. was prepared via palladium-catalyzed cleavage of the allyl 0.124 Moreover, because of the ability of the disclosed ester group. In working examples, catalytic Pd(PPh3) was compounds to Selectively modulate one or more specific used along with PhSiH to produce compound 6. Compound types of estrogen receptor, they can be used to identify the 6 is a versatile intermediate that can be used to prepare a contribution of different estrogen receptors that mediate variety of N-Substituted acrylamide derivatives. For different physiological effects. The disclosed compounds example, any primary or Secondary amine can be incorpo also can be used to bind to and identify the particular class rated via an amide bond forming reaction with compound 6. of membrane bound receptors at which these agents act. The method illustrated in Scheme 3 produces both the E and Indeed, a working embodiment of the disclosed Selective estrogen receptor modulators had a ten-fold higher potency Z isomers, compounds 8 and 9, respectively. than 17 B-estradiol for the membrane-associated receptor and a million-fold lower affinity than 17B-estradiol for the nuclear estrogen receptors (ER)C. and (ER)?. 0.125. Another application of the disclosed compounds is affinity chromatography. Because examples of the presently disclosed compounds bind to a novel, membrane-associated estrogen receptor, the compounds can be used to purify the receptor, or remove the receptor from a Sample. To use the compounds, they typically are attached to a Solid Support as is known to those of ordinary skill in the art. The compounds can be attached directly or via a linker molecule. Exemplary affinity chromatography techniques Suitable for use with the disclosed compounds are disclosed in Current Protocols in Protein Science, John Wiley & Sons (J. E. Coligan et al., Eds.). IV. SYNTHESIS 0.126 The compounds disclosed herein, as well as ana logs of Such compounds that will be readily apparent to those of ordinary skill in the art of medicinal chemistry upon consideration of this disclosure, can be prepared in a number ways using techniques well known to those of ordinary skill in the art. Exemplary methods for making particular com pounds are described below. It is understood by those of ordinary skill in the art of organic Synthesis that these US 2005/0113453 A1 May 26, 2005 10

0.130 Disubstituted acrylamide derivatives can be pre -continued pared according to the method illustrated in Scheme 5. With HO reference to Scheme 5, bis-allylation of 18 yields 20. Aldol O reaction of 20 with benzaldehyde yields 22, which under goes elimination to yield 24, as a mixture of E and Z isomers. Cleavage of the allyl protecting groups yields 26, followed by amide bond formation to form E and Z isomers, 28 and 30, respectively.

Scheme 5 OH

He O HO 18

O N-1s He O 8 (E) S-1, 9 (Z) 2O

0129. Additional SERMs can be prepared according to Scheme 4. With reference to Scheme 4, compound 10 can be prepared from diphenylacetylene. A variety of alkyl groups (in addition to ethyl) can be introduced in this step by OH various techniques. The iodo group introduced in compound -- 10 can be used to introduce a carboxy group, Such as the O carboxy methyl ester 12, which is readily converted to the N-1s corresponding carboxylic acid 7. Carboxylic acid 7 can be O reacted with a variety of primary and Secondary amines to N-1- yield the corresponding amides, Such as 8 and the corre 22 sponding Z isomer 9.

Scheme 4 P h Ph H Ph-S-Ph. -- o ------I O 1. O N-1S Ph Ph Ph Ph O o o N-1------24 A O\ A OH 12 7

O3 --- OH

O HO 26 US 2005/0113453 A1 May 26, 2005 11

V. METHODS AND EXAMPLES -continued 0132) The foregoing disclosure is further explained by the following non-limiting examples.

Example 1 0133) This example describes the synthesis of 2-(4-tert butoxyphenyl)-3-phenylpent-2-enoic acid allyl ester, 4. HO

1. n-BuLi 28 (E) THF, -78° C. 30 (Z) 2. O 0131 Scheme 6 depicts an embodiment of a method for synthesizing vinylketone derived SERMs. With reference to -(- --~ Scheme 6, alkylation of compound 32 is a versatile reaction that can be used to introduce a variety of groups. For example, R can include, with limitation, a hydroxy group, a protected hydroxy group, a carboxy group, a protected carboxy group, an amine or a protected amino group. In working examples R was a dimethyl amino group or a \ . piperidino group. Intermediate 34 is reacted with the lithium anion of compound prepared from compound 2 (Scheme 3), to yield, after deprotection, E and Z isomers 38 and 40, respectively. ( ) K) -(-

HO 32 0134) n-Butyl lithium (6.68 mL, 16.7 mmol; 2.5 M) was OMe added dropwise to a solution of vinyl bromide 2 (3.00 g, 8.35 mmol) in anhydrous THF (75 mL) that was cooled to -78 Ho R O C. After stirring for 15 min, allyl chloroformate (4.43 mL, N-1\, 41.8 mmol) was added neat and dropwise. This was allowed 34 to stir from -78 C. to room temperature over 4 h. Saturated Sodium bicarbonate was added, extracted with ethyl acetate, NS1 dried over anhydrous magnesium Sulfate, filtered and con O He centrated under reduced pressure to give a yellow oil. The R O crude product was purified by column chromatography (5% N-1\, EtOAc/Hex) to give 4 as a light yellow oil (1.80 g, 4.94 36 mmol) in 59% yield. Rf of E-isomer=0.20 and of Z-isomer= OH 0.14 (5% EtOAc/Hex). H NMR (CDC1): 8 E-isomer: 1.01 (t, 3H); 1.25 (s, 9H); 2.68 (q, 2H); 4.72 (d. 2H); 5.25 (m, 2H); 5.94 (m, 1H); 6.69 (d. 2H); 6.87 (d. 2H); 7.00 (m, 2H); 7.11 (m, 3H). & Z-isomer: 0.82 (t, 3H); 1.35 (s, 9H); 2.37 (q, 2H); 4.27 (d. 2H); 4.96 (m, 2H); 5.43 (m, 1H); 7.00 (d. 2H); 7.26 (m, 7H). HRMS (EI) mass calculated for CHOs: 364.2038, found: 364.2038. CNMR (CDC1): 8 Z-isomer: O 1N1 R 12.71, 14.10, 23.32, 25.66, 27.98, 28.87, 38.98, 65.12, 38 (E) 74.35, 78.57, 117.82, 123.73, 127.36, 127.64, 128.06, 40 (Z) 129.60, 131,74, 131.80, 131.90, 141.17, 149.25, 154.93, 169.18. US 2005/0113453 A1 May 26, 2005

Example 2 0135) This example describes the synthesis of 2-(4-tert -continued

butoxyphenyl)-3-phenylpent-2-enoic acid, 6.

a Pd(PPh3)4 PhSiH, ( ) ( ) ---THF

HO 0139 HBTU (25.7 mg, 0.0617 mmol) and DMAP (0.4 mg, 3.09 umol) were added to a solution of compound 3 (20.0 mg, 0.0617 mmol) in dichloromethane (1 mL) that was pre-cooled to 0°C. The reaction was allowed to stir at 0°C. for 30 min and at room temperature for an additional 30 min. It was then re-cooled to 0 C. and the amine hydrochloride salt (14.7 mg, 0.0679 mmol) was added followed by drop wise addition of N,N-diisopropylethylamine (DIEA) (0.04 mL, 0.247 mmol). The reaction was stirred at room tem perature for 30 min then quenched with Saturated ammo nium chloride and extracted with dichloromethane. The 0136 To a solution of compound 4 (0.16g, 0.439 mmol) combined organic layers were washed 1X with Saturated in 10 mL of anhydrous dichloromethane, was added PhSiHa Sodium bicarbonate, dried over magnesium Sulfate, filtered (0.11 mL, 0.878 mmol) followed by Pd(PPh) (10 mg, 8.78 and concentrated under reduced pressure. The crude product almol). The reaction was stirred at room temperature under was purified by column chromatography (10% MeOH/ argon for 30 min. It was then quenched with ddHO, the pH CHCI3) to give a white foam (25.4 mg., 0.519 mmol) in 84% was decreased to 2 with 1M HCl, extracted with dichlo yield. Rf of E-isomer=0.15 and of Z-isomer=0.33 (10% romethane, dried over magnesium Sulfate, filtered and con MeOH/CHCl). H NMR (CDC1): 8 E-isomer: 1.02 (t,3H); centrated under reduced pressure. The crude yellow oil was 1.25 (s, 9H); 2.35 (s, 6H); 2.82 (t, 2H); 2.79 (q, 2H); 4.02 (t, purified by column chromatography (35% EtOAC/Hex) to 2H); 6.72 (d. 2H); 6.85 (d. 2H); 6.92 (d. 2H); 7.01 (m, 3H); give a white solid (0.14g, 0.432 mmol) in 99% yield. 7.10 (m,3H); 7.38 (d. 2H). & Z-isomer: 0.91 (t, 3H); 1.38 (s, 0137 Rf of E-isomer=0.33 and of Z-isomer=0.49 (10% 9H); 2.40 (s, 6H); 2.45 (q, 2H); 2.82 (t, 2H); 3.96 (t, 2H); MeOH/CHCl). H NMR (CDC1): 8 E-isomer: 1.02 (t,3H); 6.63 (d. 2H); 6.82 (m, 3H); 7.01 (d. 2H); 7.30 (m, 6H). 2.80 (q, 2H); 6.70 (d. 2H); 6.90 (d. 2H); 6.98 (m, 3H); 7.10 (m, 2H). & Z-isomer: 0.84 (t,3H); 2.35 (q, 2H); 7.00 (d. 2H); 7.22 (d. 2H); 7.27 (m, 7H). HRMS (EI) mass calculated for Example 4 CHOs: 324.1725, found: 324.1570. 0140. This example describes the synthesis of (E,Z)-2- Example 3 (4-hydroxyphenyl)-3-phenylpent-2-enoic acid 4-(2-dim 0138. This example describes the synthesis of 2-(4-tert ethylaminoethoxy)-phenyl)amide (8 and 9). butoxyphenyl)-3-phenylpent-2-enoic acid 4-(2-dimethy lamino ethoxy) phenylamide, 7.

o O 1. HBTU, DMAP TFA/TFE/CHCI CHCl2, 25 C. 0-10 C. 2. DIEA, 25° C. -er

6 US 2005/0113453 A1 May 26, 2005 13

0143 Diethyl aluminum chloride (9.78 g., 0.0393 mol) -continued was added to a Solution of bis(cyclopentadienyl)titanium dichloride (21.8 mL, 0.0393 mol; 1.8M) in anhydrous dichloromethane (200 mL) and the mixture was allowed to Stir at room temperature for 10 min under an atmosphere of argon. Diphenylacetylene (5.0 g, 0.0281 mol) was added

slowly and stirred for 5 h. The reaction mixture was cooled to -78 C. and diluted with 70 mL of dichloromethane. N-Iodosuccinimide (NIS) (14.5 g., 0.0645 mol) was added slowly so that the temperature remained at -78. C., after addition, the reaction was stirred at room temperature over night. The next day, it was poured into hexanes (100 mL), 5% NaSO in 3N NaOH (200 mL) was added and filtered. After filtration, the two layers were separated, the organic layer was washed with 5% NaSO (200 mL), followed by 3N HCl (200 mL) and saturated NaHCO (100 mL). The organic layer was dried over magnesium Sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography in hexanes to give 10 as a light brown oil (7.15 g, 0.0214 mol) in 76% yield. H NMR (CDC1) 8: 1.03 (t, 3H); 2.82 (q, 2H); 7.05 (m, 9H). 'C NMR (CDC1): 8 11.77, 38.58, 98.89, 126.58, 126.92, 127.50, 127.78, 128.22, 128.32, 129.05, 129.88, 131.59, 139.60, 144.51, 150.24. 0141 Compound 7 (25.3 mg, 0.0514 mmol) was dis solved in anhydrous dichloromethane (0.47 mL) and cooled Example 6 to 0°C. 2.2.2-Trifluoroethanol (0.37 mL) and trifluoroacetic 0144. This example describes the synthesis of 2,3-Diphe acid (0.74 mL) were added and the reaction mixture was nylpent-2-enoic acid methyl ester, 12. stirred from O C. to 10° C. over 1.5 h. After this time, the solution was poured into 1M HCl, extracted with CHCl, dried over magnesium Sulfate, filtered and the Solvent was removed under reduced pressure. The crude product was purified by column chromatography (15% MeOH/CHCl) to Ph Ph PdCl2(PPh3)2 Ph Ph -- yield a white foam (21.5 mg, 0.499 mmol) in 97% yield. Rf MeOH of E-isomer 8=0.18 and of Z-isomer 9=0.22 (15% MeOH/ I DIEA, DMF O CHCl). H NMR (MeOD): & E-isomer: 0.90 (t, 3H); 2.49 1O W \ (s, 6H); 2.58 (q, 2H); 2.95 (t, 2H); 4.10 (t, 2H); 6.43 (d. 2H); O 6.86 (m, 4H); 7.10 (m, 5H); 7.47 (d. 2H). 'C NMR 12 (MeOD): 13.20, 30.40, 45.18, 58.66, 65.68, 115.69, 115.82, 123.41, 127.93, 129.11, 130.42, 171.77, 133.29, 136.06, 14148,143.52, 156.74, 157.57, 171.96. H NMR (MeOD): 0145 Methanol (5 mL), DIEA (0.17 mL, 0.987 mmol) & Z-isomer: 0.90 (t, 3H); 2.49 (s, 6H); 2.50 (q, 2H); 2.92 (t, and PdCl(PPh3) (107 mg, 0.153 mmol) were added to 2H); 4.05 (t, 2H); 6.75 (d. 2H); 6.83 (d. 2H); 7.01 (d. 2H); 7.27 (m, 6H); 7.39 (d. 2H). 'C NMR (MeOD): 13.21, vinyl iodide 10 (300 mg, 0.898 mmol) that was dissolved in 28.22, 45.35, 58.75, 65.78, 117.2, 116.5, 123.8, 128.5, anhydrous DMF. Carbon monoxide was bubbled for 5 min 129.2, 129.5, 131.2, 132.5, 137.2, 142.2, 147.8, 156.5, and the reaction mixture was allowed to stir at 80 C. under 158.4, 171.8. HRMS (EI) mass calculated for CHNO: atmosphere of CO for 3 days. The cooled reaction mixture 430.2256, found: 430.2263. was diluted with ethyl acetate, washed with ddHO, the layers were Separated, and the organic layer was dried over Example 5 magnesium Sulfate, and filtered and concentrated under 0142. This example describes the synthesis of 1-iodo-1, reduced pressure. The crude product was purified by column 2-diphenylbut-1-ene, 10. chromatography in chloroform to give the product (198 mg, 0.743 mmol) in 83% yield. Rf of E-isomer=0.47 and of Z-isomer=0.53 (CHCl). H NMR (CDC1) 8: 0.86 (t, 2H, Ph Ph Z-isomer); 1.01 (t, 3H, E-isomer); 2.38 (q, 1H, Z-isomer); Ph Ph - 2.69 (q, 2H, E-isomer); 3.38 (s, 1H, Z-isomer); 3.78 (s, 3H, 2. NIS, CH2Cl2, room temp. E-isomer); 7.07 (m, 7H); 7.36 (m, 4H). 'C NMR (CDC1) 8: 12.72, 12.80, 27.99, 29.96, 51.57, 51.94, 126.75, 126.97, 127.45, 127.57, 127.73, 127.84, 128.08, 128.37, 128.93, 128.99, 129.75, 130.19, 132.20, 137.05, 140.07, 149.91. US 2005/0113453 A1 May 26, 2005

Example 7 (0.08 mL, 0.459 mmol) to give the desired product 17 (19 mg, 0.0488 mmol) in 32% yield and 16 (29 mg, 0.070 mmol) 0146 This example describes the synthesis of 2,3-diphe in 60% yield. Rf of E-isomer=0.15 16 and of Z-isomer nylpent-2-enoic acid, 14. 17=0.10 (5% MeOH/CHCl). E-isomer H NMR (CDC1) 8: 1.05 (t, 3H); 2.32 (s, 6H); 2.70 (t, 2H); 2.82 (q, 2H); 4.05 (t, Ph Ph KOH 2H); 6.85 (d. 2H); 7.10 (m, 8H); 7.42 (m, 4H). C NMR MeOHAHO/THF (CDC1) 8: 13.09, 18.01, 29.69, 45.77, 58.17, 66.16, 114.86, He reflux 121.59, 126.93, 127.14, 127.92, 128.17, 128.99, 129.97, 131.07, 137.23, 140.50, 148.77, 155.66, 167.72. E-isomer A7-0 \ "H NMR (CDC1) 8: 0.90 (t,3H); 2.39 (s, 6H); 2.41 (q, 2H); 12 2.81 (t, 2H); 4.00 (t, 2H); 6.62 (d. 2H); 6.82 (d. 2H); 7.39 (m, Ph Ph 10H). Example 9 y-on 0150. This example describes the synthesis of (4-allylox O yphenyl)acetic acid allyl ester, 20. 14 OH allyl bromide 0147 To a solution of compound 12 (50 mg, 0.188 mmol) Her NaH, DMF in methanol (4 mL) and THF (9 mL), a 0.2M solution of O room temp. KOH (9.4 mL, 1.88 mmol) was added drop wise. The HO reaction was then heated to reflux for 2 days. Next day it was 18 poured into 10 mL of 1N HCl, stirred for 10 min, extracted with CHCl, dried organic layer over magnesium Sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (5% S-1, OrrO MeOH/CHCl) to yield a white solid (32 mg, 0.127 mmol) in 68% yield. Rf of E-isomer=0.33 and of Z-isomer=0.47 (5% MeOH/CHCl). H NMR (CDC1) 8: E-isomer 1.02 (t, 0151 Sodium hydride (1.90 g, 0.0789 mmol) was sus 3H); 2.80 (q, 2H); 7.02 (m, 4H); 7.11 (m, 6H). Z-isomer 0.84 pended in anhydrous DMF (70 mL) and cooled to 0° C. (t, 3H); 2.33 (q, 2H); 7.04 (d. 2H); 7.33 (m, 8H). 4-Hydroxy phenyl acetic acid 18 (5.00 g, 0.329 mmol) was dissolved in DMF (30 mL), added to the cooled sodium Example 8 hydride Suspension and allowed to Stir at room temperature for 2 h. After this time, the reaction was re-cooled to 0 C. 0.148. This example describes the synthesis of E and Z and allyl bromide (11.3 mL, 0.131 mmol) was added. After 2,3-Diphenylpent-2-enoic acid 4-(2-dimethylaminoet Stirring for 4.5 h at room temperature, it was poured into hoxy)-phenyl)amide, 16 and 17. Saturated brine, extracted with ether, washed organic layer Ph Ph with 10% KOH then brine. The organic layer was dried over 1. HBTU, DMAP magnesium Sulfate, filtered and concentrated under reduced CHCl2, 25°C. preSSure. The crude product was purified by column chro OH 2. DIEA, 25° C. matography (CHCl) to yield an oil (4.97g, 0.0214 mmol) in 65% yield. R0.68 (CHCl). H NMR (CDC1,) 8:3.58 (s, 2H); 4.51 (d. 2H); 4.58 (d. 2H); 5.24 (m, 2H); 5.90 (m, 1H); 6.02 (m, 1H); 6.87 (d. 2H); 7.19 (d. 2H). CNMR (CDC1) 8: 40.39, 65.96, 68.80, 114.81, 117.60, 118.15, 126.16, 13026, 132.05, 133.26, 157.72, 171.50. HRMS (EI) mass calculated for CHOs: 232.1100, found (M+H): 2321104. Example 10 0152 This example describes the synthesis of 2-(4-ally loxy-phenyl)-3-hydroxy-3-phenyl propionic acid allyl ester, 22. 1N1 NS O 16 (E) 17 (Z) Orr n-1S O O LHMDS, THF, -78° C. --- O 0149 Compounds 16 and 17 were synthesized as described for compounds 8 and 9 (Example 4) using com pound 14 (as a mixture of E and Z isomers) (29 mg, 0.115 mmol), HBTU (48 mg, 0.126 mmol), DMAP (0.7 mg, 5.7 umol), amine hydrochloride (27 mg, 0.126 mmol) and DIEA US 2005/0113453 A1 May 26, 2005

O155 To a solution of 22 (1.00g, 2.96 mmol) in dichlo -continued romethane (10 mL) that was cooled to 0°C., triethylamine (TEA) (1.2 mL, 8.87 mmol) was added followed by mesyl chloride (MSC) (0.46 mL, 5.91 mmol). The reaction was stirred for 2 h, then diluted with ether, filtered through a plug O OH of celite and concentrated under reduced pressure. The crude mesylate was diluted in anhydrous THF (40 mL), cooled to O nu-1S 0° C. and added 1,8-diazabicylco5.4.0]undec-7-ene (DBU) (1.3 mL, 8.87 mmol). After stirred for 2 hat room tempera O O ture, added 10% HCl, extracted with chloroform, washed O organic layer with Saturated Sodium bicarbonate, and brine then dried organic layer with magnesium Sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (15% EtOAC/Hex) to 22 yield 10 (0.90 g, 2.81 mmol) in 95% yield. Rf 0.59 (15% EtOAc/Hex). H NMR (CDC1) 8: 4.53 (d, 2H); 4.69 (d. 0153 LHMDS (9.47 mL, 9.47 mmol) was added to a 2H); 5.28 (m, 4H); 5.93 (m, 1H); 5.07 (m, 1H); 6.90 (d. 2H); solution of 20 (2.00 g, 8.61 mmol) in anhydrous THF (120 7.13 (m, 7H); 7.81 (s, 1H). 'C NMR (CDC1) 8: 65.63, mL) that was pre-cooled to -78° C. Benzaldehyde (0.88 mL, 68.76, 114.83, 117.72, 117.77, 128.03, 128.17, 128.90, 8.61 mmol) was dissolved in THF and added to the cooled 130.55, 131.01, 132.08, 132.24, 133.17, 134.81, 140.21, mixture. The reaction was warmed to -20° C. and stirred 158.22, 167.65. LRMS (+ESI) mass calculated for overnight. The next day it was quenched with Saturated CHOs: 320.1, found (M--H): 321.2. ammonium chloride, extracted acqueous layer with EtOAc, Example 12 combined organic layers, dried over magnesium Sulfate, filtered and concentrated under reduced pressure. The crude 0156 This example describes the synthesis of 2-(4-hy product was purified by column chromatography (10% droxyphenyl)-3-phenyl acrylic acid, 26. EtOAc/Hex) to yield the compound 22 (2.01 g, 5.94 mmol) in 69% yield. R 0.16 (10% EtOAc/Hex). H NMR (CDC1) 8: 3.07 (d. 1H); 3.84 (d. 1H); 4.44 (d. 2H); 4.64 (m, 2H); 5.24 (m, 4H); 5.85 (m, 1H); 6.01 (m, 1H); 6.71 (d. 2H); 6.99 (d. 2H); 7.09-7.18 (m, 5H). Example 11 0154) This example describes the synthesis of compound 2-(4-allyloxyphenyl)-3-phenylacrylic acid allyl ester, 24.

O OH 1. TEA, MsCl 'n-1s CHCl2, O C. 2. DBU, THF O O C. O

22 O157 PhSiH (0.78 mL, 6.24 mmol) was added to a solution of 24 in THF (35 mL) followed by the addition of Pd(PPh) at room temperature. The reaction was stirred at room temperature under argon for 15 min. It was quenched by adding did H2O, added 1N HCl to decrease the pH to 2, extracted with dichloromethane, dried over magnesium Sul fate, filtered and concentrated under reduced pressure. The crude mixture was purified by column chromatography (1:1 EtOAc/Hex) to yield 26 (266.9 mg, 1.11 mmol) in 71% yield. R 0.33 (5% EtOAc/Hex). H NMR (MeOD) 8: 6.71 (d. 2H); 6.91 (d. 2H); 7.02 (m, 2H); 7.11 (m, 9H); 7.71 (s, 24 1H). LRMS (+ESI) mass calculated for CHOs: 240.1, found (M+H): 241.0. US 2005/0113453 A1 May 26, 2005

Example 13 chloride (2.84 g., 19.7 mmol), cesium carbonate (34.78 g, 0158. This example describes the synthesis of E and Z 9.86 mmol), potassium iodide (65.5 mg, 0.394 mmol) were N-4-(2-Dimethylamino-ethoxy)-phenyl-2-(4-hydroxy added and Stirred with reflux overnight. After cooling to phenyl)-3-phenyl-acrylamide, 28 and 30, respectively. room temperature, ddIO was added, extracted with chlo roform, dried over magnesium Sulfate, filtered and concen trated under reduced pressure. The crude product was puri fied by column chromatography (10% MeOH/CHCl) to O H 1. HBTU, DMAP yield 12a (3.47 g, 15.5 mmol) in 79% yield. Rf 0.31 (10% CHCl2, 25°C. MeOH/CHCl). H NMR (CDC1) 8: 2.35 (s, 6H); 6.68 (t, OH 2. DIEA, 25 C. 2H); 3.90 (s, 3H), 6.95 (d. 2H); 8.00 (d. 2H). 'C NMR (CDC1) 8: 45.90, 51.80, 58.11, 66.18, 114.12, 122.60, O O HN O HO 131.51, 162.58, 166.28. LRMS (EI) mass calculated for 26 \ N - CH7NO. 223.1, found: 223.1. Example 15 0162 This example describes the synthesis of 4-(2-pip eridin-1-ylethoxy)benzoate, 44.

1n 1C

HO O O OMe HeCsCO3, KI THF HO 32 1 N. 28 (E) 30 (Z) ON n1nO OrrO 0159 Compounds 28 and 30 were synthesized as 44 described for compounds 8 and 9 (Example 4) using com pound 26 (266 mg, 1.11 mmol), HBTU (462 mg, 1.22 mmol), DMAP (6.8 mg, 55.4 umol), amine hydrochloride 0163 Procedure same as for compound 42 (example 14) (264 mg, 1.22 mmol) and DIEA (0.77 mL, 4.43 mmol) to using methyl-4-hydroxybenzoate (4.00g, 2.62 mmol), 1-(2- give the desired product (72 mg, 0.178 mmol) in 16% yield. chloroethyl) piperidine hydrochloride (4.76 g, 2.62 mmol), R 0.27(10% MeOH/CHCl). H NMR (CDC1) 8: 2.36 (s, potassium carbonate (18.16 g., 13.14 mmol), potassium 6H); 2.77 (t, 2H); 4.06 (t, 2H); 6.80 (d. 2H); 6.86 (d. 2H); iodide (43.6 mg, 0.526 mmol) and DMF (100 mL). The 7.19 (m, 10H); 7.80 (s, 1H);8.06 (s, 1H). CNMR (CDC1) crude product was purified by column chromatography (5% 8: 45.60, 58.03, 65.76, 114.74, 116.06, 121.82, 123.29, MeOH/CHCl) to yield compound 44 (5.54g, 2.10 mmol) 126.71, 127.94, 128.62, 130.40, 130.82, 131.16, 133.74, in 80% yield. "H NMR (CDC1) 8: 1.45 (m, 2H); 1.62 (m, 151.71, 155.92, 156.84, 166.63. 4H); 2.50 (m, 4H); 2.80 (t, 2H); 3.84 (s.3H); 4.19 (t, 2); 6.98 (d. 2H); 7.99 (d. 2H). 'C NMR (CDC1) 8: 24.15, 25.94, Example 14 51.82, 55.09, 57.75, 66.23, 114.16, 131.53. 0160 This example describes the synthesis of 4-(2-dim ethylaminoethoxy)benzoate, 42. Example 16 0164. This example describes the synthesis of 4-(2-dim ethylaminoethoxy)-N-methoxy-N-methylbenzamide, 46. O N-1-

OMe OMe 1. MeO(Me)NH2Cl CsCO3, KI THF THF Ho HO N O 2. i-PrMgCl 32 1. n-1N OMe 42 JuC N 42 1N-1- OrrO no-1 46 0.161 To a solution of methyl-4-hydroxybenzoate 32 (3.00 g, 19.7 mmol) in THF (80 mL), dimethylaminoethyl US 2005/0113453 A1 May 26, 2005

0165 Compound 44 (2.69 g, 12.0 mmol) and methyl methoxy amine hydrochloride (1.82 g, 18.7 mmol) were -continued suspended in anhydrous THF (100 mL) and cooled to -20° C. Isopropyl magnesium chloride (17.6 mL) was added dropwise while maintaining the temperature below -10° C. The reaction was warmed to room temperature and Stirred overnight. Saturated ammonium chloride was added, -(- extracted with ether, dried over magnesium Sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (10% MeOH/ ( ) ( ) CHCl) to yield compound 46 (2.04 g, 8.09 mmol) in 67% yield. R 0.26 (10% MeOH/CHCl). H NMR (CDC1) 8: 2.35 (s, 6H); 2.78 (t, 2H); 3.35 (s, 3H); 3.58 (s, 3H); 4.10 (t, 2H); 6.90 (d. 2H); 7.70 (d. 2H). 'C NMR (CDC1) 8:34.0, O O 1)N-1N 46.0, 58.0, 61.2, 66.0, 114.0, 126.0, 130.0, 1610, 169.0. 50 LRMS (EI) mass calculated for CHNO: 252.1, found: 252.1 Example 17 0169. A solution of vinyl bromide 2 (200 mg, 0.556 0166 This example describes the synthesis of N-meth mmol) in THF (10 mL) was cooled to -78° C. and t-butyl oxy-N-methyl-4-(2-piperidin-1-ylethoxy)benzamide, 48. lithium (0.69 mL, 1.16 mmol) was added. After stirring for 45 min, compound 46 (140 mg, 0.556 mmol) was added and stirred from -78 C. to room temperature over 3 h. The OMe 1. MeO(Me)NH2Cl reaction was quenched with Saturated ammonium chloride, Ho extracted with chloroform, dried over magnesium Sulfate ON O 2. THFi-PrMgCl S. n-1N and concentrated under reduced pressure. The crude mixture 44 was purified by column chromatography (10% MeOH/ CHCl) to yield compound 50 (22 mg, 0.0466 mmol) as a mixture of isomers in 10% yield. Rf 0.49 (10% MeOH/ CHCl). H NMR (CDC1) 8: 0.85 (t,3H); 0.92 (t, 2H); 1.25 ON-1- OrrO (s, 6H); 1.35 (s, 9H); 2.29 (s.3H); 2.31 (s, 6H); 2.37 (q, 2H); 48 2.54 (q, 1H); 2.69 (t, 1H); 2.74 (t, 2H); 4.00 (t, 1H): 4.09 (t, 2H); 6.63 (d. 2H); 7.71 (d. 1H); 6.92 (m, 4H); 7.12 (m, 8H); 7.73 (d. 1H); 7.99 (d. 1H). 'C NMR (CDC1) 8: 12.87, 0167 The procedure followed was the same as for com 13.22, 27.28, 28.74, 28.51, 29.23, 45.81, 58.01, 65.96, pound 46, using compound 44 (2.00 g, 7.60 mmol), meth 66.14, 113.84, 114.39, 123.46, 123.99, 126.58, 126.83, ylmethoxyamine hydrochloride (1.15g, 11.77 mmol), iso 127.21, 128.14, 128.30, 128.53, 129.38, 129.48, 129.90, propyl magnesium chloride (11.36 mL, 2.0MTHF) and THF (80 mL). The crude product was purified by column chro 130.03, 130.16, 131.75, 131.95, 132.17, 132.62, 136.97, matography (10% MeOH/CHCl) to yield 48 (2.01 g, 6.88 137.88, 139.86, 140.36, 142.85, 144.46, 154.15, 154.78, mmol) in 91% yield. R0.41 (10% MeOH/CHCl). H NMR 162.26, 162.93, 197.11, 197.37. (CDC1) 8: 1.45 (m, 2H); 1.60 (m, 4H); 2.30 (m, 4H); 2.79 (t, 2H); 3.35 (s, 3H); 3.58 (s, 3H); 4.10 (t, 2H); 6.85 (d. 2H); 7.70 (d. 2H). C NMR (CDC1) 8: 24.16, 25.92, 55.07, Example 19 57.80, 60.86, 66.05, 113.84, 130.51, 160.10. 0170 This example describes the synthesis of 2-(4-tert Example 18 butoxyphenyl)-3-phenyl-1-4-(2-piperidin-1-ylethoxy)-phe 0168 This example describes the synthesis of 2-(4-tert nylpent-2-en-1-one 52. butoxyphenyl)-1-4-(2-dimethylaminoethoxy)-phenyl-3- phenylpent-2-en-1-one, 50. -(- 1. t-BuLi, THF 1. t-BuLi, THF -78°C., 45 min. -78°C., 45 min. -- He 2. compound 48 c) CS' 2. compound 46 K) ( ) Br Br US 2005/0113453 A1 May 26, 2005 18

0173 The same procedure was used for deprotection of -continued

50 as was used to prepare 8 and 9 (example 4). The following amounts of reagents were used: 50 (22 mg, 0.0464 mmol), trifluoroethanol (0.34 mL), trifluoroacetic acid (0.66 mL), and dichloromethane (0.66 mL). The crude product was purified using preparatory TLC (10% MeOH/CHCl) to yield 54 as a mixture of isomers (11 mg, 0.265 mmol) in 58% yield. R0.26 (10% MeOH/CHCl). H NMR (CDC1) 8: 0.86 (t,3H); 0.93 (t, 2H); 2.31 (s.3H); 2.33 (s, 6H); 2.37 (q, 2H); 2.56 (q, 1H); 2.72 (t, 1H); 2.76 (t, 2H); 4.00 (t, 1H): 4.08 (t, 2H); 6.43 (d. 2H); 6.65 (d. 1H); 6.72 (d. 1H); 6.83 -O (dd,3H); 7.15 (m,8H); 7.71 (d. 1H); 7.94 (d. 1H). CNMR (CDC1) 8: 12.94, 13.27, 27.284, 29.35, 45.53, 57.80, 65.65, 113.84, 114.34, 115.23, 115.65, 126.81, 127.18, 127.92, 128.07, 128.32, 128.54, 129.19, 129.41, 129.87, 130.20, 0171 The procedure used to prepare compound 50 was 130.78, 131.23, 13.99, 132.16, 136.89, 138.40, 140.03, followed, using vinyl bromide (200 mg, 0.556 mmol), THF 140.39, 140.43, 142.16, 144.30, 155.33, 155.98, 162.01, (10 mL), t-butyl lithium (0.65 mL, 1.11 mmol) and com 162.63, 197.40, 198.82. pound 48 (163 mg, 0.556 mmol). The crude compound was purified by column chromatography (10% MeOH/CHCl) to Example 21 yield compound 52 as a mixture of isomers in 10% yield. Rf 0.44 (10% MeOH/CHCl). H NMR (CDC1) 8: 0.85 (t, 0174) This example describes the synthesis of 2-(4-hy 2H); 0.91 (t, 3H); 1.20 (s, 6H); 1.31 (s, 9H); 1.41 (m, 3H); droxy-phenyl)-3-phenyl-1-4-(2-piperidin-1-yl-ethoxy)- 1.57 (m, 6H); 2.40 (q, 1H); 2.48 (m, 6H); 2.56 (q, 2H); 2.72 phenyl-pent-2-en-1-one, 56. (t, 2H); 2.77 (t, 2H); 4.04 (t, 2H); 4.11 (t, 1H); 6.64 (d. 1H); 6.66 (d. 2H); 6.75 (d. 2H); 6.89 (dd, 2H); 7.04 (d. 2H); 7.12 (m,9H); 7.61 (d. 1H), 7.72 (d. 2H); 7.98 (d. 1H). ''C NMR (CDC1) 8: 12.01, 13.22, 18.03, 23.97, 25.61, 27.29, 28.38, O TFA, 28.85, 54.93, 57.58, 60.83, 65.76, 113.86, 114.35, 115.01, TFE 123.46, 123.82, 127.22, 127.91, 128.56, 129.38, 129.48, CHCl2 130.03, 130.74, 131.98, 132.19, 140.36, 144.46, 162.22. Ho LRMS (+ESI) mass calculated for CHNO: 511.3, found (M+H): 512.0. Example 20 O O1N1 O 0172 This example describes the synthesis of 1-4-(2- 52 Dimethylaminoethoxy)-phenyl-2-(4-hydroxyphenyl)-3- OH phenylpent-2-en-1-one, 54. Olu O 56

0.175. The same procedure was used for deprotection of O O1N-N 52 as was used to prepare 8 and 9 (example 4). The 50 following amounts of reagents were used: 52 (28 mg, 0.0547 OH mmol), trifluoroethanol (0.40 mL), trifluoroacetic acid (0.78 mL), and dichloromethane (0.78 mL). The crude product was purified using prep TLC (10% MeOH/CHCl) to yield 56 as a mixture of isomers (13 mg, 0.286 mmol) in 54% yield. R 0.11 (10% MeOH/CHCl). H NMR (CDC1) 8: 0.88 (t, 2H); 0.95 (t, 3H); 1.45 (m, 3H); 1.62 (m, 6H); 2.40 (q, 1H); 2.54 (m, 6H); 2.60 (q, 2H); 2.75 (t, 2H); 2.80 (t, 2H); 4.06 (t, 2H); 4.13 (t, 1H); 6.46 (d. 1H); 6.62 (d. 2H); 6.74 (d. 2H); 6.83 (t, 2H); 7.13 (m, 9H); 7.72 (d. 2H); 7.96 (d. 1H). 'C NMR (CDC1) 8: 12.92, 13.28, 23.85, 25.28, 27.28, 29.33, 54.89, 57.56, 65.30, 113.85, 114.36, 115.31, 115.71, 126.81, 127.18, 127.91, 128.06, 128.56, 129.31, US 2005/0113453 A1 May 26, 2005

129.41, 129.81, 130.13, 130.18, 130.75, 132.02, 132.18, and the MEK1 inhibitor PD98059 (50 mM) were dissolved 137.91, 140.44, 14426, 155.22, 162.06, 162.70, 197.52, in DMSO. Protein kinase A inhibitory peptide 6-22 Amide 1978O. (1 mM), the protein kinase Ainhibitor: Rp-cAMPS (50 mM) and Cholera toxin A Subunit (1 lug?u) were dissolved in Example 22 HO. The Gq binding protein designed to mimic the C terminus of the Gq C. Subunit and GS C. binding protein 0176) This example describes the identification and char designed to mimic the C terminus of the Gs a Subunit were acterization of a previously unidentified membrane associ synthesized by PeptidoGenic Research (Livermore, Calif.). ated estrogen receptor and an assay for the identification and The peptide Sequence for Gq peptide was Ac-LGLN evaluation of SERMs. LKEYNLV-OH and for Gs peptide was CRMHLROYELL. The peptides were also dissolved in H.O. BAPTA tetraso 0177 General Methods for SERM Assays and Screening dium salt (1,2-bis-(o-aminophenoxyethane)-N,N,N',N'-tet 0178 Female Topeka guinea pigs (400-600g), bred in the raacetic acid) was dissolved in the internal Solution at a 10 Oregon Health Sciences University institutional breeding mM concentration. Aliquots of the Stock Solutions were facility and female multicolor guinea pigs (400-500 g, Elm Stored as appropriate until needed. Hill, Mass.) were used. The guinea pigs were maintained under constant temperature (26 C.) and light (on between 0182 Statistical Analyses: 06:30-20:30 h). Animals were housed individually, with 0183 Statistical analyses for comparing between groups food and water provided ad libitum. They were ovariecto were performed using a one-way analysis of variance (with mized under ketamine/xylazine anesthesia (33 mg/kg & 6 post hoc (Newman-Keuls) paired analysis). Differences mg/kg, respectively; Subcutaneous) 5-7 days prior to experi were considered Statistically Significant if the probability of mentation, and given Sesame oil vehicle (0.1 mL Subcuta error was less than 5%. neous) 24 h prior to experimentation. Serum estrogen con centrations were determined by radioimmunoassay (Wagner 0184 Tissue Preparation: et al., 2001) from trunk blood collected on the day of 0185. On the day of experimentation the animal was experimentation and were less than 10 pg/mL. An additional decapitated, its brain removed from the skull and the hypo group of animals (n=6) were ovariectomized and after one thalamus dissected. The resultant hypothalamic block was week injected with oil vehicle, estradiol benzoate (25 ug in mounted on a plastic cutting platform that was then Secured oil) or compound 8 (example 4, 25 ug in oil) 24 h prior to in a vibratome well filled with ice-cold, oxygenated (95% Sacrifice. O, 5% CO) artificial cerebrospinal fluid (aCSF, in mM: 0179 Wild-type C57BL/6 mice in these studies were NaCl, 124; NaHCO 26; dextrose, 10; HEPES, 10; KCl, 5; obtained from Jackson laboratories. All animals were main NaH2PO, 2.6; MgSO, 2; CaCl, 1). Four coronal slices tained under controlled temperature (25 C.) and photope (350 um) through the arcuate were cut. The slices were riod conditions (14-h light, 10-h dark; lights on between transferred to a multi-Well auxiliary chamber containing 0700 and 2100) with food and water ad libitum. Adult mice oxygenated aOSF, and kept there until electrophysiological were ovariectomized under isoflurane anesthesia, and recording after approximately 2 h. allowed to recover for 1 week. At this time the animals were 0186 Electrophysiology: injected daily for 2 days with oil vehicle, estradiol benzoate 0187 Whole-cell patch recordings in voltage clamp were (EB; 1 ug) or compound 8 (2 or 5 lug), and anesthetized and performed as previously described (Wagner et al., 2001). killed by decapitation after 24 h. The uteri were collected, Briefly, slices were maintained in a chamber perfused with weighed and fixed in 4% paraformaldehyde for later histo warmed (35° C), oxygenated aOSF containing the same logical analysis (data not shown). constituents and respective concentrations except for CaCl, 0180 Commercially Available Drugs: which was raised to 2 mM. Artificial CSF (aCSF) and all drug Solutions were perfused via a peristaltic pump at a rate 0181 Drugs were purchased from Calbiochem (LaJolla, of 1.5 mL/min. Drug solutions were prepared in 20 mL Calif., USA) unless otherwise specified. Tetrodotoxin (TTX; Syringes by diluting the appropriate Stock Solution with Alomone, Jerusalem, Israel) was dissolved in Milli-Q HO and further diluted with 0.1% acetic acid (final concentration aCSF, and the flow was controlled via a three-way stopcock. 1 mM; pH 4-5). 17B-estradiol (17B-estradiol) was purchased 0188 For whole-cell recordings, electrodes were fabri from Steraloids (Wilton, N.H., USA), recrystallized to cated from borosilicate glass (World Precision Instruments, ensure purity and dissolved in 100% ethanol to a stock Inc., Sarasota, Fla., USA; 1.5 mm O.D.). Resultant elec concentration of 1 mM. 17o-estradiol (1 mM, Steraloids), trodes were then filled with an internal Solution containing anti-estrogen: ICI 182,780 (10 mM, Tocris Cookson, Ball 0.5% biocytin and consisting of the following in mM: K' win, Mo.) and the Selective estrogen receptor modulators gluconate, 128; NaCl, 10; MgCl2, 1; EGTA, 11; HEPES, 10; 4-OH-tamoxifen (10 mM, Steraloids), raloxifene (10 mM, ATP 1.2; GTP, 0.4; the pH was adjusted to 7.3-74 with 1 N Eli Lilly, Indianapolis, Ind.) and compound 8 (10 mM) were KOH, 272-315 mC)sm. Voltage pulses were amplified and also dissolved in 100% ethanol. 17 B-estradiol 17-hemisuc passed through the electrode using an AXopatch ID pream cinate: BSA (17B-estradiol-BSA, 1 mM, Steraloids) was plifier (Axon Instruments, Union City, Calif.). The resultant dissolved in HO. The protein kinase A inhibitor, H-89 current deflections were monitored using a digital oscillo dihydrochloride (10 mM), the protein kinase A activator scope (Tektronix 2230, Beaverton, Oreg., USA). Upon the forskolin (50 mM), the protein kinase C inhibitors bisin reduction of the current deflection, negative pressure was dolylmaleimide I hydrochloride (BIS, 100 uM), Gö6976 (2 applied via a 5 mL Syringe connected by polyethylene tubing mM) and rottlerin (10 mM), the phospholipase C inhibitor to the electrode in order to form a seal (>1 GS2). Following U73122 (20 mM), the less active analog U73343 (20 mM) formation of a Seal, intracellular access was achieved by US 2005/0113453 A1 May 26, 2005

Suction, followed by perfusion with 1 uM TTX for at least by spinning at 2000xg for 4 min at room temperature. The 4-6 min to block Spontaneous firing and Synaptic potentials filtrate was then added to 2.5 mL of Scintillant and counted before applying the GABA receptor agonist baclofen (FIG. in a liquid Scintillation counter. A binding curve was fitted 1). All the responses to baclofen were measured in Voltage using a Single binding site competition model with the Prism clamp as outward currents (V=-60 mV), and only those (GraphPad Software, San Diego, Calif.) statistical analysis cells that showed less than 10% change in acceSS resistance Software package. The Standard deviation was determined to (access resistances ranged from 20-30 MS2) throughout the be less than 0.2 log units from the ECso value. Percent recording were included in this study. Membrane currents relative binding affinity was then determined by dividing the underwent analog-digital conversion via a Digidata 1200 ICso determined for unlabeled estradiol by the ligand ICso interface coupled to pClamp 7.0 (Axon Instruments, Union and multiplying by 100. City, Calif.). Low-pass filtering of the currents was con ducted at a frequency of two KHZ. The liquid junction 0193 Dispersed Single-Cell RT-PCR: potential was -10 mV, and was corrected for in Subsequent 0194 Guinea pig 350 um coronal hypothalamic slices data analysis. were cut on a vibratome from caudal to rostral and placed in 0189 Post-Hoc Identification of Hypothalamic Arcuate an auxiliary chamber containing oxygenated aOSF. The Neurons: slices were allowed to recover for 1-2 h in the chamber before dispersion. The arcuate nucleus of the hypothalamus 0190. Following electrophysiological recording, the was microdissected and incubated in 2-3 mL of Hank's slices were fixed with 4% paraformaldehyde in Sorensen's balanced salt solution (HBSS in mM; CaCl, 1.26; MgSO, phosphate buffer (pH 7.4) for 120 min, immersed overnight 1; KC1, 5.37; KHPO, 0.44; NaCl, 136.89; Na HPO, 0.34; in 20%. Sucrose dissolved in Sorensen's buffer and then D-glucose, 5.55; Hepes, 15 in DEPC-treated water (pH 7.3, frozen in O.C.T. embedding medium and prepared for 300 mOsm) containing 1 mg/mL protease XIV (Sigma, St. immunocytochemistry as previously described (Kelly and Louis, Mo.) for approximately 15 min at 37° C. The tissue Ronnekleiv, 1994). Briefly, coronal sections (20 um) were was then washed four times in one volume low calcium cut on a cryostat (Leitz Model 1720 Digital Cryostat) and aCSF and two times in HBSS. The cells were isolated by mounted on Fisher SuperFrost Plus slides. Sections were trituration with flame-polished Pasteur pipettes, dispersed on washed for 5 min with 0.1 M sodium phosphate buffer (pH a dish and continuously perfused with HBSS at a rate of 1.5 7.4), and then Streptavidin-Cy2 (Jackson ImmunoResearch mL/n. Cells were visualized using a Nikon inverted micro Laboratories, Pa.) (1:1000) was applied for 2 h. The reaction Scope and individual neurons were patched and harvested was terminated by washing with buffer. The slices were into the patch pipette by applying negative pressure. The scanned for the injected neuron with a Nikon Eclipse 800 content of the pipette was expelled into a Siliconized micro fluorescence microscope (Nikon Instruments, Melville, centrifuge tube containing 5 till of the following Solution: N.Y.). After localization of the biocytin-filled neurons, the 0.5uL of 10x buffer (100 mM Tris-HCL,500 mM KCl, 1% Slides containing the appropriate Sections were processed for Triton-X 100; Promega, Madison, Wis.), 15 U RNasin the presence of tyrosine hydroxylase (TH) or B-endorphin (Promega), 0.5 till 100 mM DTT and DEPC-treated water. using fluorescence immunohistochemistry as described pre viously (Kelly and Rønnekleiv, 1994). Briefly, the sections 0.195. In addition, hypothalamic tissue was homogenized with the biocytin-identified neurons were incubated over and total RNA extracted using the RNeasy kit (Qiagen, night with a monoclonal TH antibody at 1:10,000 (Diasorin, Valencia, Calif.) according to the manufacturer's protocol. Stillwater, Minn.), or with a polyclonal B-endorphin anti The harvested cell solution and 25 ng of hypothalamic total body at 1:5,000 (Dave et al., 1985), washed in 0.1 M RNA in 1 it were denatured for 5 min at 65 C., cooled on phosphate buffer followed by incubation with a goat anti ice for 5 min, and then single stranded cDNA was synthe mouse IgG-Cy3 at 1:500 or donkey anti-rabbit IgG-Cy3 at sized from cellular RNA by adding 50 U MuLV reverse 1:500, respectively (Jackson ImmunoResearch Laborato transcriptase (Applied BioSystems, Foster City, Calif.), 1.5 ries, Pa.). The Sections were washed with Sodium phosphate till 10x buffer, 2 uM MgCl2, 0.2 till deoxynucleotide triph buffer, and coverslips were applied using a glycerolglycine osphates (dNTPs), 15 U RNasin, 10 mM DTT, 100 ng buffer containing 5% N-propylgallate. Immunostained cells random hexamers and DEPC-treated water to a final volume were photographed using a Nikon microScope. See FIG. 2. of 20 till. Cells and tissue RNA used as negative controls, were processed as described above but without reverse 0191) Estrogen Receptor Binding Assays: transcriptase. The reaction mixtures were incubated at 42 C. for 60 min, denatured at 99 C. for 5 min and cooled on 0.192 The relative binding affinity of compounds for the ice for 5 min. estrogen receptors (ER)C. and (ER)? was determined using a Spin column assay with commercially available full-length 0196). PCR was performed using 3 ul of cDNA template forms of both (ER).C. and (ER)(3 (Pan Vera Corp, Madison, from each RT reaction in a 30 ul PCR reaction volume Wis., USA). Receptor was added to a final concentration of containing: 3 ul. 10x buffer, 2.4 uL MgCl2 (2 mM final 15 nM to a solution containing 10 mM Tris, pH 7.5, 10% concentration for TH, POMC, GABA-R2, PKC8, adenylyl glycerol, 2 mM DTT and 1 mg/mL BSA and 3 nM 2,4,6, cyclase VII and GAPDH) or 3.6 uL MgCl, (3 mM final 7,16,17-Hestradiol at 4° C. 100 ul of the solution was concentration for GAD), 0.2 mM dNTPs, 0.2 uM forward added to 1 till of the ligand in ethanol, mixed gently by and reverse primers, 2 Units Taq DNA polymerase pipetting and incubated at 4 C. overnight. The mixture was (Promega) and 0.22 ug TaqStart Antibody (Clontech, Palo then applied to a micro Spin column containing G-25 Sepha Alto, Calif.). Taq DNA polymerase and TaqStart Antibody dex (Harvard Apparatus Inc.) equilibrated in binding buffer were combined and incubated at room temperature for 5 (minus tritiated estradiol) according to the manufacturers min, the remainder of the reaction contents were added to instructions. Bound estradiol was Separated from free ligand the tube and incubated at 94 C. for 2 min. Then, each US 2005/0113453 A1 May 26, 2005 reaction went through 60 cycles (35 cycles for GAPDH) of physiology analysis, only cells with gigaohm or better Seals amplification according to the following protocols: 94 C., were included in this study. The mean resting membrane 45 sec; 55° C. (GAD), 57°C. (PKCS), 58° C. (GABA-R2), potential was -54.3+0.4 mV at a 0 pA holding current, and 60° C. (TH and adenylyl cyclase VII), 61° C. (POMC), 63 the mean input resistance was 1.9-0.3 GS2. Moreover, fifty C. (GAPDH) 45 sec; 72° C., 1 min 10 sec; with a final 72 percent of A12 dopamine neurons exhibited a T-type Ca" C. extension for 5 min. Ten microliters of the PCR products current and a hyperpolarization-activated, cation current (I) were visualized with ethidium bromide on a 1.5% agarose (Loose et al., 1990). Seventy-one percent of the POMC gel. neurons exhibited I and a transient outward K current (IA) 0.197 All of the primers were synthesized by Invitrogen (Kelly et al., 1990). Therefore, the passive membrane prop (Carlsbad, Calif.) and were as follows: Guinea pig GAD65; erties measured with whole-cell patch recording is similar to 207 bp product, forward primer 5'-GGCTCTGGTGATG results obtained using Single electrode Voltage clamp record GAATA-3', reverse primer 5'-CAGAATCACGCTGTCT ings (Loose et al., 1990; Kelly et al., 1990). GTT-3', Guinea pig TH; 223 bp product, forward primer 0200. The whole-cell recording method was used to 5'-TCCACGTTATACTGGTTCAC-3', reverse primer measure the rapid effects of 17 B-estradiol on the activation 5'-TTGCATCACTGAAGCTCTC-3'; Guinea pig GABA of the G protein-coupled, inwardly-rectifying K' channel R2; 241 bp product, forward primer 5'-TGTTTGTGC (GIRK) conductance by the GABAB receptor agonist CAAAGCTCATC-3' reverse primer 5'-GTGTCTTGCAGT baclofen. 173-estradiol rapidly attenuates both u-opioid and TGCATAGT-3', Guinea pig POMC (accession number GABA receptor-mediated responses in hypothalamic arcu S78260); 344 bp product, forward primer (bases 40-60) ate neurons (Lagrange et al., 1994, Lagrange et al., 1996; 5'-CTGGCCTTGCTGCTTCAGAT-3' reverse primer (bases Lagrange et al., 1997). Therefore, for measuring 17 B-estra 383-363) 5-ATGGAGTAGGAGCGCTTGTC-3'; Guinea diol modulation of the GABAB response an ECso concen pig GAPDH; 212 bp product (accession number tration (5 uM) of baclofen was used according to the CPU51572), forward primer 5-CATCCACTGGTGCTGC protocol depicted in FIG. 1. A robust outward current was CAAG-3', reverse primer 5'-GTCCTCGGTGTAGC measured in response to baclofen that Subsided after wash CCAAGA-3'. Human protein kinase C6; 251 bp product out (FIG. 5 graph A and FIG. 6). The application of (accession number L07861) forward primer (bases 1127 baclofen 20 min later elicited the same robust response, 1147) 5'-AAAGGCAGCTTCGGGAAGGT-3', reverse Suggesting that desensitization and run-down were not primer (bases 1377-1357) 5'-TGGATGTGGTACATCAG occurring in response to Successive applications of 5 uM GTC-3'. Guinea pig adenylyl cyclase VII; 235 bp product, baclofen. However, if 17 B-estradiol (100 nM) was applied forward primer 5'-CTGTTCGGCAAGTTTGACCAG-3', during the interim period (i.e. after the washout of the first reverse primer 5'-TGACGCCACACAGCACATT3'. application of baclofen), there was a significant (p<0.005) decrease of 41% in the response to a Second application of 0198 17B-Estradiol and SERMs Rapidly Attenuate the baclofen (FIG. 5 graph B and FIG. 6). Current/voltage GABA Response in Hypothalamic Dopamine and Pro relationships generated before and during the application of Opiomelanocortin (POMC) Neurons: 100 nM 17 B-estradiol showed that this steroid did not 0199 The following data demonstrate that 17 B-estradiol change the reversal potential for the baclofen-mediated and SERMs rapidly attenuate a baclofen-induced GABA response: control E=-88.8-3.6 mV, n=13; verSuS response in hypothalamic neurons, which indicates that after 17 B-estradiol E=-85.4+3.9 mV, n=12 (FIGS. these compounds affect neurotransmission through non 3A and 3B). The effects of 17B-estradiol were stereospecific transcriptional events. Whole cell recordings were made in Such that the biologically inactive Stereoisomer 173-estra arcuate neurons (n=195) from ovariectomized female guinea diol (100 nM) had no effect on the baclofen response (FIG. pigs. FIG. 1 is a Schematic illustrating the protocol for drug 5 graph C and FIG. 6). Furthermore, the effects of 17 B administration in the whole-cell patch, Voltage clamp Studies estradiol were blocked by the anti-estrogen ICI 182, 780, (V=-60 mV). After seals were formed and the whole-cell when co-perfused with 17B-estradiol (FIG. 6). Treatment configuration was obtained, slices were perfused with TTX with ICI 182, 780 alone had no effect on the baclofen (1 uM) for 5 min. The first GABA receptor-mediated response (data not shown). response was generated by perfusing the GABA agonist baclofen (at ECso concentration of 5 uM) until a steady-state 0201 This previously unidentified estrogen receptor was outward current was obtained (first response, R1). After determined to be membrane associated by using the mem baclofen wash out, the current returned to its predrug resting brane impermeable estrogen conjugate 17B-estradiol-BSA. level. The cells were then treated with 17? 3-estradiol and/or 17 B-estradiol-BSA (100 nM) was fully efficacious in inhib other drugs for 15 min, and baclofen (5 uM) was perfused iting the baclofen response indicating that this estrogen again, and a second response (R2) was measured. The effects receptor-mediated response is initiated at the plasma mem of 17B-estradiol and/or other drugs on the baclofen response brane (FIG. 5 graph D and FIG. 6). The integrity of the are expressed as a percentage of R2 over R1. A Subgroup of 17 B-estradiol-BSA preparation was verified by performing a these neurons (n=55) was identified using dual labeling 17 B-estradiol radioimmunoassay of the slice perfusate. No immunocytochemistry (data and images not shown). This unbound 17B-estradiol was found in the media (data not revealed that 41% of the cells were TH-positive (i.e. dopam shown) indicating that 17B-estradiol-BSA conjugate did not ine neurons) and 39% were B-endorphin-positive (i.e. contain contaminating free 17B-estradiol. POMC neurons). Moreover, based on dual immunocy 0202) The previously unidentified, membrane associated tochemical Staining and in Situ hybridization for GADs a estrogen receptor was characterized by using Several Subgroup of arcuate dopamine neurons co-express GABA SERMs. Tamoxifen (1 uM) was inactive (p-0.05 versus (Rónnekleiv, unpublished findings), which was Substanti control) and did not attenuate the effects of 17 B-estradiol on ated by the scRT-PCR data (see below). For the electro the baclofen activation of GIRK (R2/R1 for 17B-estradiol: US 2005/0113453 A1 May 26, 2005 22

58.6+3.4%, n=10; versus tamoxifen--17B-estradiol: 0205 The Rapid Effect of 17B-Estradiol on the GABA 60.4+6.6%, n=5). However, 4-OH tamoxifen (1 uM) did Response Involves Protein Kinase A(PKA): partially mimic the actions of 17,3-estradiol by blocking the 0206. The involvement of specific signaling proteins in baclofen response by 25% (FIG. 6). With reference to FIG. the 17B-estradiol mediated modulation of GABA was 6, 17B-estradiol (100 nM) attenuated the GABA receptor determined by blocking different pathways. For example, if mediated outward current by 41%. The inhibitory effects of activation of the PKA pathway is involved, then the effect of 17 B-estradiol on the baclofen response were blocked by the 17 B-estradiol on GABA responses should be blocked by estrogen receptor antagonist ICI 182, 780 (1 uM). Bovine inhibiting PKA and mimicked by stimulating PKA. Thus, Serum Albumin-conjugated Estrogen (17B-estradiol-BSA, Selective PKA inhibitors and activators were used to dem 100 nM), 4-OH tamoxifen (1 uM), raloxifene (1 uM), onstrate that the PKA is involved. For example, as shown in GW5638 (1 uM) and compound 8 (10 nM) also inhibited the FIG. 8 graph A and FIG. 9, forskolin (10 uM) could mimic baclofen response, but 17o-estradiol (1 uM) had no effect. the actions of 17f8-estradiol to attenuate the GABA Bars represent the mean+S.E.M. of 4-11 cells tested per response. On the other hand, the specific PKA inhibitor H89 group (***p-0.005, **p-0.01, p<0.05, versus vehicle (10 uM) blocked the 17B-estradiol induced suppression of control group). Because 4-OH tamoxifen always exists as an the GABA response (FIG. 8 graph B and FIG. 9). E/Z mixture of olefin isomers (Katzenellenbogen et al., 0207. The involvement of PKA in 17B-estradiol modu 1985), it is possible that only one of the isomers is active at lation of GABA responses was further confirmed by the mediating this novel estrogen response. Raloxifene (1 uM), effect of the specific PKA inhibitory peptide PKI (protein another SERM with a hydroxylated aromatic ring, com kinase A Inhibitor 6-22 Amide, 20 uM) or the non-hydro pletely mimicked the actions of 17f8-estradiol in terms of lyzable cAMP analog Rp-cAMP (200 uM) that blocks PKA efficacy in the suppression of the baclofen response (FIG. activation on neurons. After ~15 min of dialysis with PKI or 6). In contrast, the non-hydroxylated SERM GW-5638, Rp-cAMP, the 17 B-estradiol-induced reduction of the which structurally resembles the triphenylethylene core of GABA response was abolished (FIG. 8 graph C and FIG. tamoxifen, was found to be significantly more efficacious 9). Cholera toxin (CTX), which is a bacterial exotoxin than 17 B-estradiol at inhibiting GIRK channel activation by secreted by vibrio cholerae, elevates intracellular cAMP levels in a variety of tissues by ADP-ribosylating the G-pro baclofen (FIG. 6). tein Gs thereby Stimulating adenylyl cyclase activity in an 0203 Compound 8 is a SERM Devoid of Nuclear ER apparently irreversible manner. Intracellular dialysis with Activity that Selectively Attenuates Rapid Responses: the active unit of CTX into individual cells occluded the rapid inhibition of GABA response by estrogen (FIG. 8 0204 All of the above mentioned compounds are high graph D and FIG. 9). These results indicate that the Sup affinity ligands for the nuclear estrogen receptors, which pression of the GABA response by 17B-estradiol requires complicates the interpretation of the observed pharmacology the activation of PKA. FIG. 9 Summarizes the effects of the and makes it difficult to exclude unequivocally a role for PKA drugs tested. The PKA activator forskolin could mimic nuclear estrogen receptors. However, compound 8 (example the effects of 17 B-estradiol, the specific PKA inhibitors, H89 4) has approximately one million-fold reduced binding (10 uM), Rp-cAMPS and PKAI could block the effects. affinity for the nuclear estrogen receptor (ER).C. or ER? CTX-A could occlude the attenuation of the baclofen compared to that of 17B-estradiol (Table 1). Moreover, response by 17 B-estradiol, but the denatured CTX-A could unlike 4-OH tamoxifen, compound 8 is geometrically stable not. Bars represent the meant S.E.M. of 4-11 cells tested per and does not exist as a mixture of E/Z olefin isomers. In group (*** p<0.005, * and "p-0.05, versus vehicle-control; addition, compound 8 has no uterotropic actions even at five CTX-A+17 B-estradiol versus denatured CTX-A+17 B-estra times the dose of 17B-estradiol (FIGS. 6A and 6B), con diol, p<0.05). firming in vivo that 8 has no 4-OH tamoxifen-like estrogenic 0208 Attenuation of the GABA Response Involves Pro activity mediated by the nuclear estrogen receptors. How tein Kinase CD(PKC8) ever, in the whole cell electrophysiological assay, 10 nM 8 was as efficacious as 100 nM 17B-estradiol in attenuating the 0209 PKC also is involved in 17B-estradiol modulation GABA response (FIG. 6). of the GABAB response, as demonstrated by the effects of several selective PKC inhibitors. The first inhibitor, bisin TABLE 1. dolymaleimide (BIS), is a selective inhibitor of PKC that does not distinguish between the conventional, novel and Relative binding affinities (RBA) of ligands atypical isoforms of PKC. The second, Gö6976, is a selec to full length (ERC or (ER). tive inhibitor of the conventional PKC isoforms (Martiny Relative binding affinity Relative binding affinity Baron et al., 1993; Way et al., 2000). Treatment of neurons Ligand (ER)c. (ER)|B with BIS nearly eliminated the effects of 17 B-estradiol (FIG. 10 graph A and FIG. 11). In contrast, Gö6976 treatment was 17B-estradiol 1OO 1OO 4-hydroxytamoxifen 36 43 without effect (FIG.11). Indeed, after replacing intracellular GW-5638 5 8 EGTA with 10 mM 1,2-bis-(o-aminophenoxyethane)-N,N, Compound 8 4.3 x 10 9.0 x 10 N',N'-tetraacetic acid (BAPTA), a calcium buffer with simi (example 4) lar Cat" affinity as EGTA but a much faster on rate, the *Relative binding affinities are expressed as a percentage of the potency of estrogen inhibition of the GABAB response was still 17B-estradiol. Under the conditions described in Example 22, 17f-estra observed (FIG. 10 graph B and FIG. 11). Because conven diol was found to have an ICs of 5 nM for (ER)C and 3 nM for (ER)?. tional isoforms of PKC are unlikely to be active with this level of calcium buffering, these results further Support a US 2005/0113453 A1 May 26, 2005 23 role for a Ca"-independent, novel PKC isoform in mediat 0215 Expression of GABA Receptor, PKCö and Ade ing the effects of estrogen. Finally, the selective PKCö nylate Cyclase VII Transcripts in Arcuate (GABA, Dopam inhibitor rottlerin (5 uM) completely blocked 17B-estradi ine and POMC) Neurons: ol’s ability to inhibit the GABA response in hypothalamic 0216) The expression of GABA receptor, PKCö and neurons (FIG. 10, graph C and FIG. 11). FIG. 11 summa adenylate cyclase VII transcripts in arcuate neurons was rizes the effects of the PKC inhibitors tested. Bars represent determined using single-cell RT-PCR from seventy-five the meantS.E.M. of 4-11 cells tested per group (*** acutely dispersed arcuate neurons. The results demonstrated p<0.005, **p-0.01, * p-0.05 versus vehicle-control). that 90% of the neurons expressed GADs transcripts includ ing TH-expressing and POMC-expressing neurons (data not 0210 Inhibition of the GABA Response by 17B-Estra shown). Most importantly, 92% of the neurons expressed diol Involves Go: GABA R2 transcripts, which correlates with the 90% response rate to baclofen. Furthermore, single cell RT-PCR 0211 Additional results demonstrate that the inhibition results demonstrate that dopamine and POMC neurons of the baclofen induced GABA response by 17B-estradiol express PKCö and adenylyl cyclase VII transcripts. In one involves the messenger protein GC, Although the specific group of cells (n=22), PKCö and adenylyl cyclase VII PKC inhibitor BIS blocked the 17B-estradiol effect, forsko transcripts were expressed in the majority (70%) of TH lin (10 uM) was found to mimic the effects of estrogen in the neurons (FIG. 14A), including those that co-express presence of BIS blockade (FIG. 10 graph D and FIG. 11). GADs. TH and GAD were co-localized in 60% of this Thus, the action of PKC is upstream of the activation of population of neurons. Due to limited amount of cDNA from PKA. Indeed, the estrogen receptor-mediated inhibition of individual neurons, POMC expression was determined in the GABA response depended on the activation of Go, as another group of cells (n=29), demonstrating that PKCö and indicated by treating arcuate neurons with a peptide (11 adenylyl cyclase VII transcripts were expressed in the amino acids) that mimics the C-terminal binding site of Go, majority (75%) of POMC neurons, including those that (Akhter et al., 1998). This peptide blocks the interaction co-express GADs (FIG. 14B). POMC and GAD were between G protein coupled receptors and GC, proteins. In co-localized in 28% of this population of neurons. There cells dialyzed with this peptide (200 uM), the 17B-estradiol fore, the single cell RT-PCR data confirm the electrophysi mediated reduction of the GABAB response was signifi ological findings that dopamine and POMC neurons express cantly blocked (FIG. 12 graph A and FIG. 13) when the critical transcripts for rapid eStrogen Signaling. GAPDH compared to cells dialyzed with a control peptide (11 amino transcripts were analyzed in the same cells as an internal control for the reverse transcriptase (RT) reaction. One cell acids) that mimics the C-terminal domain of Go (FIG. 12 contained no RT as a negative control (-RT). Basal hypo graph B and FIG. 13). Therefore, Go, plays a primary role thalamic tissue RNA was also reverse transcribed in the in 17 B-estradiol-mediated rapid inhibition. presence of RT (tissue controls, +). A tissue control without 0212 Moreover, the activation of phospholipase C RT (-) was included in each trial. In addition, the following (PLC), a well known Go, effector, also plays a role. Indeed, controls were included: Hank's balanced salt Solution the activation of PLCB is required for the estrogen-induced (HBSS) from the dispersed cellular milieu and a water inhibition of GABAB response as indicated by the treatment blank, both of which were negative following RT-PCR (data of neurons with the broad spectrum PLC inhibitor U73122 not shown). (10 uM). When U73122 (10 uM) was perfused in the 0217. A Unique Membrane Estrogen Receptor Mediates extracellular-bathing media, the estrogen-mediated reduc the Rapid Effects of 17 B-Estradiol: tion of GABA response was blocked (FIG. 12 graph Cand 0218. The foregoing results demonstrate that a membrane FIG. 13), however the less active PLC inhibitor U73343 at estrogen receptor has been identified. For example, estrogen the same concentration had no effect (FIG. 12 graph D and Suppresses the action of the GABAB receptor agonist FIG. 13). With reference to FIG. 13, Bars represent the baclofen to activate GIRK channels in GABA, POMC, and meani-S.E.M. of 4-11 cells tested per group (*** p<0.005, dopamine neurons. This 17 B-estradiol effect is rapid, with ** p-0.01 versus vehicle-control; U73122+17B-estradiol measurable Suppression occurring within minutes after addi versus U73343+17 B-estradiol, p<0.05; Gq peptide--17B-es tion of 17 B-estradiol. The kinetics of this response indicates tradiol versus Gs peptide+17B-estradiol, p<0.05). mediation of the response by a membrane 17B-estradiol receptor instead of one of the classical nuclear estrogen 0213 The Attenuation of the GABA Response does not receptors (ER)C. or (ER)f3, which operate by transcription Involve Mitogen Activated Protein (MAP) Kinase: regulation. 0214. Inhibition of MAP kinase activity does not prevent 0219. The pharmacology observed for this rapid estrogen estrogen modulation of the baclofen response. Recent Stud response further Supports the involvement of a novel trans ies have shown that 17,3-estradiol rapidly activates the MAP membrane estrogen receptor. The membrane impermeable kinase pathway in primary neuronal cortical cultures and in 17B-estradiol-BSA conjugate gives an identical response to organotypic cerebrocortical explant cultures (Watters et al., free 17f-estradiol, as would be expected for a membrane 1997; Singh et al., 1999; Singh et al., 2000). However, asSociated receptor wherein the hormone-binding Site of the treatment with MAP kinases inhibitors PD98059 (10 uM, in receptor is accessible from the extracellular Surface of the the pipette) or U0126 (5uM), did not affect 17B-estradiol plasma membrane. The 17B-estradiol response is Stereospe inhibition of baclofen responses (R2/R1 for 17B-estradiol: cific with respect to the configuration of the D-ring hydroxyl 58.6+3.4%, n=10; versus PD98059+17B-estradiol: group; 17 B-estradiol elicits the rapid response whereas 17O 66.1+11.8%, n=5). estradiol is inactive. This is notable because 17o-estradiol US 2005/0113453 A1 May 26, 2005 24 functions as an agonist of the nuclear estrogen receptors, protein kinase C Suppresses the GABA receptor-activation albeit with slightly reduced potency compared to 17B of GIRK channels in the hippocampal CA1 pyramidal estradiol (Barkhem et al., 1998). The SERMs 4-OH tamox neurons (Dutar and Nicoll, 1988) and attenuates the GABA ifen, raloxifene, and GW-5638 all behave like 17B-estradiol receptor-mediated inhibition of norepinephrine release from in mediating this response whereas the Steroidal antiestrogen cerebellar slices (Taniyama et al., 1992). ICI-182, 780 antagonizes the 17f8-estradiol response. Most 0224 Currently, there are 12 known members of the PKC importantly, the novel SERM8 that is devoid of estrogen (or family (Way et al., 2000). The family is divided into three antiestrogen) activity with the nuclear estrogen receptors is groups based on Sequence homology and biochemical regu a stronger activator of this rapid 173-estradiol response than lation. Class A, or conventional PKCs (PKCC, BI, B II and 17 B-estradiol even at a 10-fold lower concentration. More Y) are the well-known, Ca"-dependent PKCs. Class B, or over, this membrane ER has a subnanomolar affinity for novel PKCs (PKCö, e, 0 and m), are Ca" independent. estrogen as indicated by pharmacological (Schild) analysis Finally, Class C PKCS, or atypical PKCs (PKC and T/), (Lagrange et al., 1997). These results demonstrate that the are the most divergent class. Atypical PKCs are also Cat" pharmacology of this rapid response is different, and in the independent and do not require diacylglycerol for activation case of 8, Separable from that of the nuclear estrogen (Way et al., 2000). The rapid, GABA suppressing effects of receptors (Razandi et al., 1999; Levin, 2001; Chambliss and estrogen in hypothalamic neurons were Sensitive to the Shaul, 2002). broad spectrum PKC inhibitor BIS, but not to Gö6976, 0220 Recently, Toran-Allerand and colleagues (Toran implicating the involvement of a PKC not belonging to the Allerand et al., 2002) have identified a high affinity, satu conventional PKC class. In addition, estrogens inhibition of rable estrogen receptor, “ERX,” that is associated with the GABA response was not altered by inclusion of 10 mM caveloar-like microdomains in developing neocortical neu BAPTA in the intracellular recording patch pipette, provid rons. This membrane-associated receptor is coupled to the ing confirming that the Ca'-dependent, conventional PKCs activation of mitogen-activated protein kinases (MAPKs), are not involved. However, the selective PKC6 inhibitor extracellular-signal related kinase (ERK)1 and ERK2, which rottlerin blocked the actions of 17 B-estradiol Suggesting that appear to be important for the development and Survival of this novel class PKC is a mediator of the rapid 17 B-estradiol neurons (Watters et al., 1997; Singh et al., 1999; Singh et al., response. Moreover, the scRT-PCR data on the expression of 2000; Fitzpatrick et al., 2002). ERX also has a distinct PKCS transcripts in arcuate neurons described herein further pharmacology in that 17C-estradiol is equipotent as 17B implicate PKCö in the 17f-estradiol-mediated inhibition of estradiol in activating the MAP kinase pathway (Toran the GABA response. Likewise, PKCô is involved in the Allerand et al., 2002; Wade et al., 2001). However, as estrogen-mediated inhibition of K channels and fluid reten demonstrated herein no effects were observed for 17o tion in female distal colonic epithelial cells, although the estradiol on the GABA response, or the u-opioid response, upstream signaling pathway is not known (Doolan et al., which is coupled to the same family of GIRK channels in 2000). hypothalamic neurons (Lagrange et al., 1997). Similarly, Gu 0225 PKC Activation is Upstream of PKA Activation: and Moss (Gu and Moss, 1996) found that 17o-estradiol did not mimic the actions of 17B-estradiol in the hippocampus 0226 PKC activation in the 17,3-estradiol mediated inhi to potentiate the glutamate (kainate)-mediated currents in bition of the GABA is upstream of PKA activation. For CA1 pyramidal neurons. Likewise, Mermelstein et al. example, internal perfusion of BIS completely blocked the (1996) found that 17o-estradiol was much less efficacious inhibition of the baclofen response by 17 B-estradiol, but did than 173-estradiol in reducing L-type calcium currents in not attenuate the inhibition of the baclofen response by neostriatal neurons. Therefore, the membrane estrogen forskolin applied via bath perfusion. PKC is known to receptor that modulates channel activity in neurons via the activate adenylyl cyclases (Jacobowitz et al., 1993; PKC-PKA pathway is pharmacologically distinct from the Yoshimura and Cooper, 1993; Lin and Chen, 1998); more receptor that is coupled to activation of ERK% that promotes over, when adenylyl cyclase (AC) is activated by PKC, growth and Survival. instead of by GC, or forskolin, it is resistant to inhibition by GC (Pieroni et al., 1993). To date, nine AC isozymes have 0221) 17B-Estradiol Activates PKCö and PKA to Alter been cloned (AC types I-IX). Notably, AC VII has a poten the Coupling of GPCRs to K' Channels in Hypothalamic tial binding site for PKCö that is not present in the sequences Neurons: of the other adenylyl cyclases, which would allow PKCö to 0222. The data detailed herein demonstrate that the signal directly phosphorylate AC VII (Nelson et al., 2003). GABA transduction pathway for the rapid response to estrogen in neurons in the cortex, hippocampus, Striatum and cerebel hypothalamic neurons follows that depicted in FIG. 15. lum are immunoreactive for ACVII (Mons et al., 1998), and, With reference to FIG. 15, the sequence of events are: (1) as disclosed herein, hypothalamic GABA, TH and POMC 17B-estradiol binds to a novel transmembrane estrogen neurons express AC VII transcripts. receptor; (2) ligand binding activates Gol; (3) activated Go. 0227) Go, Mediates the Inhibition of the GABA, in turn activates PLC; (4) activated PLC liberates DAG; (5) Response by 17 B-Estradiol Through PLC: free DAG stimulates PKCö; (6) PKCö activates adenylyl cyclase (VII); (7) cAMP levels are elevated; (8) cAMP 0228. The results described herein show that a membrane stimulates PKA; (9) PKA phosphorylates membrane targets ER is specifically coupled to the messenger GC, and that critical for K channel function. this messenger protein is involved in the inhibition of the GABA response by 1713-estradiol. This conclusion is con 0223). Further support for the pathway shown in FIG. 15 firmed by results observed when intracellular dialysis with a is found in that protein kinase pathways affect GABA peptide fragment of Go, blocked the receptor interaction receptor-mediated Signaling in CNS neurons. Activation of with G protein. This GC, peptide has been used to block Go, US 2005/0113453 A1 May 26, 2005 25

Signaling pathways in cortical pyramidal neurons (Carr et perfused in situ, first with Saline and then with neutral al., 2002). In addition, the estrogen-mediated reduction of buffered 10% formalin Solution. the GABA response was significantly reduced by the 0232 Total cholesterol and triglycerides are determined phospholipase C inhibitor U73122 compared to cells per using enzymatic methods with commercially available kits fused with the less active inhibitor U73343. Thus, the from Boehringer Mannheim and Wako Biochemicals, messenger GC, is involved in the inhibition of the GABA, respectively. Separation and quantification of plasma lipo response by 17,3-estradiol. proteins are performed, and each lipoprotein fraction is 0229. Most PKCs are activated by diacylglycerol and quantified by multiplying the total cholesterol value by the some require the presence of Ca". Thus, PKCs are down relative percent area of each respective chromatogram peak. stream of the phospholipase C (PLC)-inositol triphosphate/ 0233 Aortic atherosclerosis is quantified by carefully diacylglycerol Signaling cascade. Because different forms of isolating the aortas and placing the vessels in formalin PLC can be activated by various messengers including GC, fixative for 48-72 h before handling. Atherosclerotic lesions GBY (PLCB), and tyrosine kinases (PLCY), the PKC family are identified using Oil Red O staining. The vessels are is involved in a diverse array of Signaling cascades (Tanaka briefly destained, and then imaged. The lesions are quanti and Nishizuka, 1994; Battaini, 2001). fied en face along the aortic arch and lesion assessment is Example 23 performed on the vessels, Specifically on the region con tained within the aortic arch from the proximal edge of the 0230. This example describes the identification of estro brachio-cephalic trunk to the distal edge of the left Subcla gen receptor antagonists and the determination of antagonist vian artery. Aortic atherOSclerosis data can be expressed as potency using Schild analysis. The general protocol for this percent lesion involvement strictly within this defined lumi analysis is disclosed by Lagrange et al., 1997. Cumulative nal area. concentration-response curves are generated by applying increasing concentrations of the Selective estrogen receptor Example 25 agonist, compound 8, in the whole cell electrophysiological 0234. This example describes the evaluation of prophy assay described above. Increasing concentrations of lactic neuroprotection using the disclosed compounds in baclofen are applied until the drug-induced outward current response to oxygen deprivation/reperfusion, and provides a reaches a new steady level. The ECso values for both method for identifying novel neuroprotective Selective baclofen and compound 8 are calculated using SigmaPlot estrogen receptor modulators. Female Mongolian gerbils (Jandel Scientific, Costa Madre, Calif.) Software to deter (available from, for example, Charles River Laboratories, mine the best fit to the logistic equation. Cells are then Kingston, N.Y.) after acclimation, are anesthetized with Superfused with 1 nM Solution of the potential estrogen isoflurane and ovariectomized (day 0). Beginning the fol receptor antagonist. Increasing concentrations of compound lowing morning (day 1), gerbils are treated Subcutaneously 8 (as applied in generating the initial concentration-response with either vehicle (10% EtOH/corn oil), 17B-estradiol (1 curve above) are then applied to generate a second response mg/kg, Sc) or a compound disclosed herein. Prior to day 6, curve. The cells are then treated with a 2 nm solution of the the gerbils are fasted overnight (to facilitate consistent potential antagonist and the concentration response curve ischemic injury) and then Subjected to global ischemia repeated. This process is repeated with increasing concen Surgery. The Surgery proceeds by anesthetizing the gerbils trations of the potential antagonist to generate multiple with isoflurane, Visualizing the common carotid arteries via concentration response curves. Linear regression fit of the a mid-line neck incision and Simultaneously occluding both data (log(Dose Ratio-1) versus-log antagonist concentra arteries for 5 minutes with micro-aneurysm clips. Following tion)} yields a slope of -1.0 when compound 8 and the occlusion, the clipS are removed to allow cerebral reperfu proposed antagonist bind to the previously unidentified Sion and the neck incision is closed with wound clips. On membrane associated estrogen receptor disclosed herein. day 12, gerbils are exposed to a lethal dose of CO, the brains frozen on dry ice and stored at -80 C. Example 24 0235. The degree of neuronal protection is evaluated by 0231. This example describes the evaluation of the car in situ hybridization analysis of neurogranin mRNA. Briefly, dioprotective activity of the disclosed compounds and pro 20 uM coronal cryostat Sections are collected on gelatin vides a method for identifying novel cardioprotective Selec coated slides, dried and stored at -80 C. At the time of tive estrogen receptor modulators. Apollipoprotein processing, the desiccated slide boxes are warmed to room E-deficient C57/B1J (apo E KO) mice (available from temperature, the slides postfixed in 4% paraformaldehyde, Taconic Farms), 4-7 weeks of age, are ovariectomized. The treated with acetic anhydride and then delipidated and animals are randomized by weight into groups (n=12-15 dehydrated with chloroform and ethanol. Processed section mice per group). The mice are treated with the disclosed mounted slides are then hybridized with 200 uL (6x10' compound or 17B-estradiol Sulfate (at 1 mg/kg/day) in the DPM/slide) of an antisense or sense (control) riboprobe for diet using a protocol wherein the amount of consumed is Neurogranin (S-UTP-labeled NG-241; bases 99-340) in a measured weekly, and the dose adjusted accordingly, based 50% formamide hybridization mix and incubated overnight on animal weight. The diet used is a Western-style diet at 55 C. in a humidified slide chamber without coverslip (57U5) that is prepared by Purina and contains 0.50% ping. The following morning, the slides are collected in cholesterol, 20% lard and 25 IU/KG Vitamin E. The mice are racks, immersed in 2XSSC (0.3 M NaCl, 0.03 M sodium dosed/fed using this paradigm for a period of 12 weeks. citrate; pH 7.0)/10 mM DTT, treated with RNase A (20 Control animals are fed the Western-style diet and receive no ug/ml) and washed (2x30 min) at 67° C. in 0.1xSSC to compound. At the end of the Study period, the animals are remove nonspecific label. After dehydration, the Slides are euthanized and plasma Samples obtained. The hearts are opposed to BioMax (BMR-1; Kodak) X-ray film overnight. US 2005/0113453 A1 May 26, 2005 26

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coupled to activation of the mitogen-activated pro effects of steroids in neuroblastoma cells: Effects of tein kinase pathway. Endocrinology 142: 2336-2342. estrogen on mitogen activated protein kinase signal 0306 Wagner EJ, Bosch MA, Kelly M J, Ronnek ling cascade and c-foS immediate early gene tran leiv OK (1999) A powerful GABA receptor-medi scription. Endocrinology 138: 4030-4033. ated inhibition of GABAergic neurons in the arcuate 0312) Way KJ, Chou E, King G L (2000) Identifi nucleus. NeuroReport 10: 2681-2687. cation of PKC-isoform-specific biological actions 0307 Wagner EJ, Manzanares J, Moore KE, Look using pharmacological approaches. Trends Pharma ingland K J (1994) Neurochemical evidence that col Sci 21: 181-187. estrogen-induced Suppression of kappa-opioid-re 0313 Wetzel C H., Hermann B, Behl C, Pestel E, ceptor-mediated regulation of tuberoinfundibular Rammes G, Zieglgansberger W, Holsboer F, Rup dopaminergic neurons is prolactin-independent. precht R (1998) Functional antagonism of gonadal Neuroendo 59: 197-201. Steroids at the 5-hydroxytryptamine type 3 receptor. 0308 Wagner EJ, Reyes-Vazquez C, Ronnekleiv O Mol Endocrinol 12: 1441-1451. K, Kelly M J (2000) The role of intrinsic and 0314) Yoshimura M, Cooper D M (1993) Type agonist-activated conductances in determining the Specific Stimulation of adenylyl cyclase by protein firing patterns of preoptic area neurons in the guinea kinase C. J. Biol Chem 268: 4604-4607. pig. Brain Res 879: 29-41. 0315 Zhu Y, Rice CD, Pang Y, Pace M, Thomas P 0309 Wagner E J, Ronnekleiv OK, Bosch M A, (2003) Cloning, expression, and characterization of a Kelly M J (2001) Estrogen biphasically modifies membrane progestin receptor and evidence it is an hypothalamic GABAergic function concomitantly intermediary in meiotic maturation of fish oocytes. with negative and positive control of luteinizing Proc Natl Acad Sci USA 100: 2231-2236. hormone release. J Neurosci 21: 2085-2093. 0316. It will be apparent to those skilled in the art that 0310 Watson RE, Langub MC, Landis JW (1992) various modifications and variations can be made in the Further evidence that most luteinizing hormone present compounds, compositions and methods without releasing hormone neurons are not directly estrogen departing from the Scope or Spirit of the disclosure. Other responsive: Simultaneous localization of luteinizing embodiments of the compounds, compositions and methods hormone-releasing hormone and estrogen receptor will be apparent to those skilled in the art from consideration immunoreactivity in the guinea-pig brain. J Neu of the Specification and practice of the procedures disclosed roendocrinol 4: 311-317. herein. It is intended that the Specification and examples be 0311 Watters JJ, Campbell JS, Cunningham M.J, considered as exemplary only, with a true Scope and Spirit of Krebs E G, Dorsa D M (1997) Rapid membrane the invention being indicated by the following claims.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 16 <210> SEQ ID NO 1 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 1

ggctotggtg atggaata 18

<210> SEQ ID NO 2 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 2

cagaatcacg citgtctgtt 19

<210> SEQ ID NO 3 &2 11s LENGTH 20 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 3 US 2005/0113453 A1 May 26, 2005 30

-continued tocacgittat actggttcac 20

<210> SEQ ID NO 4 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 4 ttgcatcact gaagctcitc 19

<210 SEQ ID NO 5 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 5 tgtttgttgcc aaagct catc 20

<210> SEQ ID NO 6 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia

<400 SEQUENCE: 6 gtgtc.ttgca gttgcatagt 20

<210 SEQ ID NO 7 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia &300s PUBLICATION INFORMATION: &3O8> DATABASE ACCESSION NUMBER ST8260 &309s DATABASE ENTRY DATE: 2000-08-14 <313> RELEVANT RESIDUES: (40) . . (59) <400 SEQUENCE: 7 citggccttgc tigottcagat 20

<210 SEQ ID NO 8 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia &300s PUBLICATION INFORMATION: &3O8> DATABASE ACCESSION NUMBER ST8260 &309s DATABASE ENTRY DATE: 2000-08-14 <313> RELEVANT RESIDUES: (363) ... (382) <400 SEQUENCE: 8 atggagtagg agcgcttgtc 20

<210 SEQ ID NO 9 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia porcellus <400 SEQUENCE: 9 catccactgg togctgccaag 20

<210> SEQ ID NO 10 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Cavia porcellus US 2005/0113453 A1 May 26, 2005 31

-continued SEQUENCE: 10 gtoctoggtg tagcc.caaga 20

SEQ ID NO 11 LENGTH 2.0 TYPE DNA ORGANISM: Homo sapiens PUBLICATION INFORMATION: DATABASE ACCESSION NUMBER LO 861 DATABASE ENTRY DATE: 1993-11-02 RELEVANT RESIDUES: (1127) . . (1146) <400 SEQUENCE: 11 alaaggcagct tcgggalaggt 20

SEQ ID NO 12 LENGTH 2.0 TYPE DNA ORGANISM: Homo sapiens PUBLICATION INFORMATION: DATABASE ACCESSION NUMBER LO 861 DATABASE ENTRY DATE: 1993-11-02 RELEVANT RESIDUES: (1357) . . (1376)

<400 SEQUENCE: 12 tggatgtggit acatcaggto 20

SEQ ID NO 13 LENGTH 21 TYPE DNA ORGANISM: Cavia

<400 SEQUENCE: 13 citgttcggca agtttgacca g 21

SEQ ID NO 14 LENGTH 19 TYPE DNA ORGANISM: Cavia

<400 SEQUENCE: 14 tgacgc.caca cago.acatt 19

SEQ ID NO 15 LENGTH 11 TYPE PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Synthetic Construct

<400 SEQUENCE: 15 Leu Gly Lieu. Asn Lieu Lys Glu Tyr Asn Lieu Val 1 5 10

SEQ ID NO 16 LENGTH 11 TYPE PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Synthetic Construct US 2005/0113453 A1 May 26, 2005 32

-continued <400 SEQUENCE: 16 Cys Arg Met His Leu Arg Glin Tyr Glu Lieu Lieu 1 5 10

What is claimed: 1. A compound according to the formula:

R M-Air-Y-G-Z.

6. The compound according to claim 1, wherein the compound has the formula wherein R is H or a lower aliphatic group; M comprises a ketone, amide or Sulfonamide group; Ar is an aro matic group; X is a hydroxy, alkoxy, or a halogen, Y comprises a heteroatom; G is a linker group; and Z is a carboxy, hydroxy or amino group. 2. The compound according to claim 1, wherein the compound has the formula

R M-Ar-Y-G-Z. 7. The compound according to claim 1, wherein M comprises an amide group. 8. The compound according to claim 7, wherein the compound has the formula

3. The compound according to claim 1, wherein the compound has the formula

M-Ar-Y-G-Z.

wherein R is hydrogen, lower alkyl or an aralkyl group. 9. The compound according to claim 1, wherein G is a lower alkyl group. 10. The compound according to claim 1, wherein Z includes a charged group. 4. The compound according to claim 1, wherein Ar is a 11. The compound according to claim 1, wherein Z phenyl group. includes a cationic group. 5. The compound according to claim 1, wherein the 12. The compound according to claim 1, wherein Z compound has the formula includes an amino group. US 2005/0113453 A1 May 26, 2005 33

13. The compound according to claim 1, wherein Y is oxygen and -G-Z has a formula Selected from the group consisting of

CO2H CO2H COH

NMe2 NMe2 20. The compound according to claim 1, wherein the compound has the formula OH OH

O g-()-r- N N. OH o

14. The compound according to claim 1, wherein R is hydrogen or lower alkyl. OH 15. The compound according to claim 1, wherein X is a hydroxy group. 21. A composition, comprising a pharmaceutically effec 16. The compound according to claim 1, wherein X is tive amount of a first compound or a pharmaceutically hydrogen. acceptable Salt of the compound according to the formula: 17. The compound according to claim 1, wherein the compound has the formula R M-Ar-Y-G-Z R M-Air-Y-G-Z. K.) ( ) ( ) l X wherein R is H or a lower aliphatic group; M comprises a ketone, amide or Sulfonamide group; Ar is an aro 18. The compound according to claim 1, wherein the matic group; X is a hydroxy, alkoxy, or a halogen; Y comprises a heteroatom; G is a linker group; and Z is compound has the formula a carboxy, hydroxy or amino group; and

a pharmaceutical carrier. 22. The composition according to claim 21, wherein at least one compound has the formula

R M-Air-Y-G-Z.

N/ 19. The compound according to claim 1, wherein the compound has the formula US 2005/0113453 A1 May 26, 2005 34

23. The composition according to claim 21, wherein the

compound has the formula

28. The composition according to claim 21, wherein the compound has the formula 24. The composition according to claim 21, wherein the compound has the formula O u-()-r-‘s

29. The composition according to claim 21, further com 25. The composition according to claim 21, wherein the prising a pharmaceutically effective amount of a Second compound has the formula compound. 30. The composition according to claim 29, wherein the Second compound has the formula

R M-Ar-Y-G-Z

N/ 26. The composition according to claim 21, wherein the compound has the formula wherein R is H or a lower aliphatic group; M comprises a ketone, amide or Sulfonamide group; Ar is an aro matic group; X is a hydroxy, alkoxy, or a halogen; Y comprises a heteroatom; G is a linker group; and Z is a carboxy, hydroxy or amino group. 31. A method for agonizing or antagonizing a membrane asSociated estrogen receptor, comprising contacting the receptor with a compound of claim 1. 32. A method for treating a condition characterized by estrogen insufficiency in a Subject, comprising administering an effective amount of at least a first compound of claim 1 to the Subject. 33. The method of claim 32, wherein the condition characterized by estrogen insufficiency is ovarian failure. 34. The method of claim 33, wherein the condition wherein R is hydrogen, lower alkyl or an aralkyl group. characterized by estrogen insufficiency is menopause. 27. The composition according to claim 21, wherein the 35. The method according to claim 32, wherein the compound has the formula condition comprises an autonomic dysfunction, Sleep cycle US 2005/0113453 A1 May 26, 2005 35 disruption, cognitive disorder, motor dysfunction, mood 38. The method according to claim 37, wherein the second disorder, eating disorder or cardiovascular disorder. compound is a Steroid hormone. 36. The method according to claim 32, wherein the 39. The method according to claim 37, wherein the second condition is Selected from the group consisting of ischemia compound is a Selective estrogen receptor modulator. induced neuronal death, head trauma, Alzheimer's disease, 40. The method according to claim 32, wherein the second hot flushes, Parkinson's disease, tardive diskinesia, depres compound has the formula of a compound of claim 1. Sion, Schizophrenia, anorexia nervosa, bulimia nervosa, car 41. The method according to claim 32, wherein the diovascular disease, atherOSclerosis, long QTL Syndrome, method comprises agonizing or antagonizing a membrane Romano-Ward syndrome or Torsades de Pointes syndrome, asSociated estrogen receptor. Osteoporosis, rheumatoid arthritis, osteoarthritis, bone frac ture, multiple Sclerosis or combinations thereof. 42. The method of claim 41, wherein the method com 37. The method according to claim 32, further comprising prises agonizing the membrane associated estrogen receptor. administering an effective amount of a Second compound to 43. A method for providing hormone replacement therapy, the Subject, different from the compound administered in comprising administering an effective amount of at least a claim 32, and wherein the Second compound provides a first compound of claim 1 to the Subject. therapeutic effect in combination with the compound admin istered in claim 32.