GB2303628A

GB2303628A - 5-Hydroxy- and 5-phenyl-substituted 1-[4-(aminoalkoxy)phenyl]-2-phenylbenzene derivatives for the treatment of post menopausal symptoms and intermediates

Info

Publication number: GB2303628A
Application number: GB9615378A
Authority: GB
Inventors: Henry Uhlman Bryant; Ken Matsumoto; Donald Richard Finley
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 1995-07-27
Filing date: 1996-07-22
Publication date: 1997-02-26
Also published as: GB9615378D0

Abstract

Compounds of Formula I: [wherein: n is 2 or 3; R is dimethylamino, diethylamino, 1-piperidinyl, 1-pyrrolidinyl, 4-morpholinyl, or 1-hexamethyleneimino; R 1 is hydroxy, phenyl, or mono or di-substituted phenyl (wherein each substituent is independently halo, methyl, hydroxy, C 1 -C 6 -alkoxy, benzyloxy, C 1 -C 6 -alkanoyloxy, benzoyloxy, substituted benzoyloxy (bearing 1 to 3 substituents each of which is independently halo, C 1 -C 4 -loweralkyl, or C 1 -C 4 -loweralkoxy), C 1 -C 5 -alkoxycarbonyloxy, or C 4 -C 6 -alkylsulfonyloxy); and R 2 is hydrogen, hydroxy, C 1 -C 6 -alkoxy, benzyloxy, C 1 -C 6 -alkanoyloxy, benzoyloxy, substituted benzoyloxy(bearing 1 to 3 substituents each of which is independently halo, C 1 -C 4 -loweralkyl, or C 1 -C 4 -loweralkoxy), C 1 -C 5 -alkoxycarbonyloxy, or C 4 -C 6 -alkylsulfonyloxy] and the pharmaceutically acceptable acid addition salts and solvates thereof, are useful for the treatment of various post menopausal symptoms such as osteoporosis, cardiovascular disease (eg. hyperlipidemia) estrogen-dependent cancer (eg. breast or uterine cancer), uterine fibroid disease, endometriosis, aortal smooth muscle cell proliferation and restenosis. Compounds of Formula IV and of Formula VIII: are also claimed per se where Rn are as defined above, and R 1a and R 2a have the same values as R 1 and R 2 respectively other than they may not be hydroxy (-OH) or contain a hydroxy group.

Description

BENZIS COPOUNDS, INTERMgDIATES, PROCESSES, COMPOSITIONS, AND SSSSODS This invention relates to the fields of pharmaceutical and organic chemistry and provides novel benzene compounds which are useful for the treatment of the various medical indications associated with post-menopausal syndrome. The present invention further relates to intermediate compounds and processes useful for preparing the pharmaceutically active compounds of the present invention, and pharmaceutical compositions.

Post-menopausal syndrome" is a term used to describe various pathological conditions which frequently affect women who have entered into or completed the physiological metamorphosis known as menopause. Although numerous pathologies are contemplated by the use of this term, three major effects of post-menopausal syndrome are the source of the greatest long-term medical concern: osteoporosis, cardiovascular effects such as hyperlipidaemia, and estrogen-dependent cancer, particularly breast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverse etiologies, but which are characterized by the net loss of bone mass per unit volume. The consequence of this loss of bone mass and resulting bone fracture is the failure of the skeleton to provide adequate structural support for the body. One of the most common types of osteoporosis is that associated with menopause. Most women lose from about 20% to about 60% of the bone mass in the trabecular compartment of the bone within 3 to 6 years after the cessation of menses. This rapid loss is generally associated with an increase of bone resorption and formation.

However, the resorptive cycle is more dominant and the result is a net loss of bone mass. Osteoporosis is a common and serious disease among post-menopausal women.

There are an estimated 25 million women in the United States, alone, who are afflicted with this disease.

The results of osteoporosis are personally harmful and also account for a large economic loss due its chronicity and the need for extensive and long term support (hospitalization and nursing home care) from the disease sequelae. This is especially true in more elderly patients. Additionally, although osteoporosis is not generally thought of as a life threatening condition, a 20% to 30% mortality rate is related with hip fractures in elderly women. A large percentage of this mortality rate can be directly associated with postmenopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausal osteoporosis is the trabecular bone. This tissue is often referred to as spongy or cancellous bone and is particularly concentrated near the ends of the bone (near the joints) and in the vertebrae of the spine. The trabecular tissue is characterized by small osteoid structures which inter-connect with each other, as well as the more solid and dense cortical tissue which makes up the outer surface and central shaft of the bone. This inter-connected network of trabeculae gives lateral support to the outer cortical structure and is critical to the biomechanical strength of the overall structure. In postmenopausal osteoporosis, it is, primarily, the net resorption and loss of the trabeculae which leads to the failure and fracture of bone.In light of the loss of the trabeculae in post-menopausal women, it is not surprising that the most common fractures are those associated with bones which are highly dependent on trabecular support, e.g., the vertebrae, the neck of the weight bearing bones such as the femur and the fore-arm. Indeed, hip fracture, collies fractures, and vertebral crush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the only generally accepted method for treatment of post-menopausal osteoporosis is estrogen replacement therapy. Although therapy is generally successful, patient compliance with the therapy is low primarily because estrogen treatment frequently produces undesirable side effects.

Throughout premenopausal time, most women have less incidence of cardiovascular disease than age-matched men.

Following menopause, however, the rate of cardiovascular disease in women slowly increases to match the rate seen in men. This loss of protection has been linked to the loss of estrogen and, in particular, to the loss of estrogen's ability to regulate the levels of serum lipids. The nature of estrogen's ability to regulate serum lipids is not well understood, but evidence to date indicates that estrogen can up regulate the low density lipid (LDL) receptors in the liver to remove excess cholesterol. Additionally, estrogen appears to have some effect on the biosynthesis of cholesterol, and other beneficial effects on cardiovascular health.

It has been reported in the literature that postmenopausal women having estrogen replacement therapy have a return of serum lipid levels to concentrations to those of the pre-menopausal state. Thus, estrogen would appear to be a reasonable treatment for this condition. However, the side-effects of estrogen replacement therapy are not acceptable to many women, thus limiting the use of this therapy. An ideal therapy for this condition would be an agent which would regulate the serum lipid level as does estrogen, but would be devoid of the side-effects and risks associated with estrogen therapy.

The third major pathology associated with postmenopausal syndrome is estrogen-dependent breast cancer and, to a lesser extent, estrogen-dependent cancers of other organs, particularly the uterus. Although such neoplasms are not solely limited to a post-menopausal women, they are more prevalent in the older, post-menopausal population. Current chemotherapy of these cancers has relied heavily on the use of anti-estrogen compounds such as, for example, tamoxifen.

Although such mixed agonist-antagonists have beneficial effects in the treatment of these cancers, and the estrogenic side-effects are tolerable in acute life-threatening situations, they are not ideal. For example, these agents may have stimulatory effects on certain cancer cell populations in the uterus due to their estrogenic (agonist) properties and they may, therefore, be contraproductive in some cases. A better therapy for the treatment of these cancers would be an agent which is an anti-estrogen compound having negligible or no estrogen agonist properties on reproductive tissues.

In response to the clear need for new pharmaceutical agents which are capable of alleviating the symptoms of, inter alia, post-menopausal syndrome, the present invention provides new benzene compounds, pharmaceutical compositions thereof, and methods of using such compounds for the treatment of post-menopausal syndrome and other estrogen-related pathological conditions.

Smooth aortal muscle cell proliferation plays an important role in diseases such as atherosclerosis and restenosis. Vascular restenosis after percutaneous transluminal coronary angioplasty (PTCA) has been shown to be a tissue response characterized by an early and late phase.

The early phase occurring hours to days after PTCA is due to thrombosis with some vasospasms while the late phase appears to be dominated by excessive proliferation and migration of aortal smooth muscle cells. In this disease, the increased cell motility and colonization by such muscle cells and macrophages contribute significantly to the pathogenesis of the disease. The excessive proliferation and migration of vascular aortal smooth muscle cells may be the primary mechanism to the reocclusion of coronary arteries following PTCA, atherectomy, laser angioplasty and arterial bypass graft surgery.See Intimal Proliferation of Smooth Muscle Cells as an Explanation for Recurrent Coronary Artery Stenosis after Percutaneous Transluminal Coronary Angioplasty," Austin et al., Journal of the American Colleae of Cardioloqv, 8: 369-375 (Aug. 1985).

Vascular restenosis remains a major long term complication following surgical intervention of blocked arteries by percutaneous transluminal coronary angioplasty (PTCA), atherectomy, laser angioplasty and arterial bypass graft surgery. In about 35% of the patients who undergo PTCA, reocclusion occurs within three to six months after the procedure. The current strategies for treating vascular restenosis include mechanical intervention by devices such as stents or pharmacologic therapies including heparin, low molecular weight heparin, coumarin, aspirin, fish oil, calcium antagonist, steroids, and prostacyclin. These strategies have failed to curb the reocclusion rate and have been ineffective for the treatment and prevention of vascular restenosis.See "Prevention of Restenosis after Percutaneous Transluminal Coronary Angioplasty: The Search for a 'Magic Bullet',1 Hermans et al., American Heart Journal, 122: 171187 (July 1991).

In the pathogenesis of restenosis excessive cell proliferation and migration occurs as a result of growth factors produced by cellular constituents in the blood and the damaged arterial vessel wall which mediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth aortal muscle cells are useful in the treatment and prevention of restenosis. The present invention provides for the use of compounds as smooth aortal muscle cell proliferation inhibitors and, thus inhibitors of restenosis.

The present invention is directed to compounds of Formula I:

wherein: n is 2 or 3; R is dimethylamino, diethylamino, l-piperidinyl, 1pyrrolidinyl, 4-morpholinyl, or 1-hexamethyleneimino; R1 is hydroxy, phenyl, or mono or di-substituted phenyl wherein each substituent is independently halo, methyl, hydroxy, C1-Cs-alkoxy, benzyloxy, Cl-C6-alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4-loweralkoxy, C1-C5-alkoxycarbonyloxy, or C4 -C6 -alkyl sul fonyloxy;; R2 is hydrogen, hydroxy, C1-C6-alkoxy, benzyloxy, C1 -C6- alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-Cq- loweralkyl, or Cl-C4-loweralkoxy, Cl-Cs-alkoxycarbonyloxy, or C4-C6-alkylsulfonyloxy; and the pharmaceutically acceptable acid addition salts thereof.

These compounds are useful for the treatment of post menopausal symptoms such as osteoporosis, cardiovascular conditions such as hyperlipidaemia, and the like.

Accordingly, the present invention is also directed to methods employing and compositions comprising these compounds, for the treatment of post menopausal syndrome.

The methods may additionally employ, and the compositions additionally comprise, an estrogen or progestin.

Preferred compounds are those with one or more of the following values, in any combination: n=2 R = l-piperidinyl R1 = hydroxy or 4-hydroxyphenyl R2 = hydroxy Particularly preferred compounds are 3-[4-[2-(1 piperidinyl)ethoxy]phenyl]-4-(4-hydroxyphenyl)-1- hydroxybenzene and 1- [4- [2- (1-piperidinyl) ethoxy]phenyl] -2,5- bis(4-hydroxphenyl)benzene and their pharmaceutically acceptable salts.

Also provided by the present invention are processes for preparing the compounds of formula I. One process, for the preparation of compounds wherein R1 = hydroxy, comprises reacting a compound of formula III

wherein R2a = R2 exclusive of unprotected hydroxy, with an alkyl chloride of the formula Cl-(CH2)n-R, in an inert solvent, in the presence of base, and at a temperature of from 0 to 100-C, to obtain a compound of formula IV

which is subsequently oxidized to obtain the final compounds of the present invention wherein R1= hydroxy. Optionally, a protected hydroxy group (R2a) can thereafter be deprotected.

Also provided by the present invention is a process for the preparation of compounds of formula I wherein R1= phenyl or substituted phenyl. In this process, a compound of formula VIII:

wherein Ria = phenyl or substituted phenyl as defined except that any OH is protected, and R2a = R2 exclusive of hydroxy, is reacted with an alkyl chloride of the formula Cl-(CH2)n-R, in an inert solvent, in the presence of base, and at a temperature of from 0 to 100.C. Optionally, a protected hydroxy group can thereafter be deprotected.

The benzene compounds of the present invention are synthesized by two different reaction routes, one for those compounds wherein R1 = hydroxy, and another for those compounds wherein R1 = phenyl or substituted phenyl.

Both routes employ, as starting material, compounds of formula II:

wherein X = a protected hydroxy group, such as C1-C6-alkoxy, benzyloxy, C1-Cs-alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substitutents each of which is independently halo, C1-Cq-loweralkyl, or Cl-C4-loweralkoxy, C1-Cs-alkoxycarbonyloxy, or C4-Cs-alkylsulfonyloxy; and R2Q = R2 other than hydroxy. Preparation 1 below illustrates the synthesis of a representative compound of formula II.

The foregoing starting materials are employed in a first synthetic route for the preparation of the compounds of the present invention wherein R1 = hydroxy. This route is as follows:

X OH Ij)- ( (CH2),-R NH4OOCH I ~~~~~~~~~~ 5% Pd/c (al latio ( deprotection ) I R2a I R2a I R2a II III IV III LiBr/CUBr2 (oxidation) O- (CH2) n~R I R2a R2a V (+ optional deprotection of protected OH at R2a) This synthetic sequence is illustrated by Preparations 2 and 3 and Examples 1 and 2.

The starting materials of formula II are also employed in a second synthetic route for the preparation of the compounds of the present invention wherein R1 = phenyl or substituted phenyl. This route is as follows:

X X - X I RiaLi A (oxidation) I N N I \ R2a I R2a I 2a Ria - Ria N II VI / VII H2 5% Pd/C (deprotection) OHo- O-(CH2)n-R Cl(CH2)nR N 'jazzN Rlb R1~ R2a \ R2a I R2a R1 N - R1 N VIII IX (+ optional deprotection of protected OH in Ria and at R2a) This synthetic route is illustrated by Preparations 4-6 and Examples 3 and 4.

The alkylation reaction by which the -O-(CH2)n-R group is introduced into the compounds is common to both synthetic routes. This reaction is conducted in accordance with known techniques. The reaction is conducted in an inert solvent, such as methyl ethyl ketone, acetone, THF, dimethylformamide, and DMS0. A base is supplied to serve as a hydrogen chloride acceptor. The reaction consumes the reactants and base in equimolar amounts; generally, the reactants are supplied in equimolar excess of an equimolar amount. The reaction goes forward under temperatures of a wide range, but is commonly conducted at temperatures of from 0 to 100-C. Isolation and purification are carried out in conventional manners.

In order to direct the alkylation to the desired location, any other hydroxy in the intended end product must be protected prior to the alkylation. Hydroxy protection is well known; see, for example, Protective Grounds in Oraanic Chemistry, Plenum Press (London and New York, 1973); Protectina Grounds in Oraanic Svnthesis, Wiley (New York, 1981); and The Pestides, Vol. I, Schrooder and Lubke, Academic Press (London and New York, 1965). Hydroxyl protection is also discussed in US-A-4418068. Preferred hydroxy protected groups are, for R1 = hydroxy substituted phenyl, a methoxy substituted phenyl, and for R2, methoxy.

Subsequent deprotection of the hydroxy protecting groups is also carried out in known procedures, which are discussed in the same references.

The conversion of the formula IV compounds to the formula V compounds is a known type of reaction. See U.S.

4,659,516, which is incorporated by reference, especially at column 9, lines 51-64. In the synthesis of the present compounds, a compound of formula IV is reacted with cupric bromide and lithium bromide to produce a compound of formula V. The reaction is conducted in an inert solvent, such as acetonitrile, THF, toluene, and the like, and at temperatures of from about room temperature to 100-C. Preferably, the reactants are employed in amounts representing equimolar amounts of the compound of formula IV and the lithium bromide, and two molar amounts of the cupric bromide. The resulting product, with R2a=protected hydroxy groups, can subsequently be deprotected to obtain the R2=hydroxy compounds.

Although the free-base form of formula I compounds can be used in the methods of the present invention, it is often preferred to prepare and use a pharmaceutically acceptable salt form. Thus, the compounds used in the methods of this invention primarily form pharmaceutically acceptable acid addition salts with a wide variety of organic and inorganic acids, and include the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention.

Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric, and the like. Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used.Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, B-hydroxybutyrate, butyne-1,4-dioate, hexyne-l, 4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2 -hydroxyethanesulfonate, methansulfonate, naphthalene-l-sulfonate, naphthalene-2-sulfonate, ptoluenesulfonate, xylenesulfonate, tartarate, and the like.

A preferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or ethyl acetate. The salt normally precipitates out of solution within about one hour to 10 days and can be isolated by filtration or the solvent can be stripped off by conventional means.

The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.

The following examples are presented to further illustrate the preparation of compounds of the present invention. It is not intended that the invention be limited in scope by reason of any of the following examples.

NMR data for the following Examples were generated on a GE 300 MHz NMR instrument, and anhydrous d-6 DMSO was used as the solvent unless otherwise indicated.

PreDaration 1 3-(4-Benzyloxyphenyl)-4-(4-methoxyphenyl)- cyclohe:-2-en-1-one

The 1- (4-benzyloxyphenyl) -2- (4-methoxyphenyl) hexan1,5-dione (91 g, 230 mmol) was stirred in 1.2 L of EtOH, a 2 N ethanolic KOH solution (200 mL, 400 mmol) was added, and the heterogeneous mixture was refluxed 18 h, then cooled to 0 OC, acidified with 5 N HCl, concentrated in vacuo to a smaller volume, diluted with brine, and extracted with EtOAc (4 x 150 mL).The EtOAc extracts were combined, washed with brine, dried (Na2SO4), concentrated to an amber oil, purified by prep chromatography (EtOAc/ toluene gradient), and triturated with Et20/ pet. ether to give 69.1 g (78.5% yield) of product as an off-white solid: mp 90 OC; 1H NMR (CDCl3) 8 2.12-2.54 (comp, 4H, CH2CH2), 3.78 (s, 3H, OCH3), 4.24-4.26 (m, 1H, CH), 5.05 (s, 2H, OCH2Ar), 6.66 (s, 1H, C=CH), 6.826.93 (m, 4H, ArH), 7.15-7.20 (d, 2H, J = 9 Hz, ArH), 7.337.47 (comp, 7H, ArH); MS(FD) 384(M+). Anal. Calcd for C26H2403: C, 81.22; H, 6.29. Found: C, 81.47; H, 6.43.

Prenaration 2 3-(4-Hydroxyphe~1)-4-(4-methoxDhenyl)cyclohex-2-en-1-one

The 3-(4-benzyloxyphenyl) -4- (4- methoxyphenyl)cyclohex-2-en-1-one (3.6 g, 9.3 mmol) was stirred in 150 mL MeOH, 5% Pd/ C (2.5 g, l.lmmol Pd) was carefully added, then ammonium formate (0.7 g, ll.lmmol) added and reaction mixture refluxed 30 min. The TLC (MeCN/ CH2C12, 1:3) indicated some starting material remained, so additional ammonium formate (0.4 g, 6.3 mmol) was added and reaction mixture refluxed another 30 min, then cooled, filtered, the filter pad washed twice with MeOH and CH2Cl2 once, and the filtrate concentrated to a brown foam.The crude product was purified by prep chromatography (MeCN/ CH2Cl2 gradient) to give 1.9 g(70% yield) of yellow foam: mp 70 OC; 1H NMR (CDCl3) 62.13-2.50 (comp, 4H, CH2CH2), 3.78 (s, 3H, OCH3), 4.24-4.26 (m, 1H, CH), 6.11 (bs, 1H, ArOH), 6.66 (s, 1H, C=CH), 6.77-6.86 (m, 4H, ArH), 7.15-7.18 (d, 2H, J = 9 Hz, ArH), 7.38-7.41 (d, 2H, J = 9 Hz, ArH); MS(FD) 294(M+). Anal. Calcd for C19H18O3: C, 77.53; H, 6.16. Found: C, 77.24; H, 6.55.

Prenaration 3 3-t4-t2-(1-PAperld≅yl)ethorylphenyll-4- (4 -methoxyphenyl ) cyclohsx-2-en-1-one

OH O/L"3 I citrate OCH3 OCH 0 0 A 200 mL methyl ethyl ketone solution of 3-(4hydroxyphenyl) -4- (4-methoxyphenyl) cyclohex-2-en-1-one (3.12 g, 10.6 mmol) was stirred as K2CO3 (7.32 g, 53 mmol) , then N-(2-chloroethyl)piperidine. HC1 (2.14 g, 11.6 mmol) were added in succession and the reaction mixture was refluxed for 6 h, then cooled, filtered, and the filtrate concentrated to a yellow gum. The crude product was purified by prep chromatography (i -PrOH/ CH2Cl2 gradient) to give 3.7 g (86% yield) of product as a pale yellow gum.An analytical sample was prepared by dissolving the gum (0.5 g, 1.2 mmol) in 50 mL acetone, adding citric acid monohydrate (0.23 g, 1.1 mmol), ref fluxing 5 min, concentrating, triturating with Et2O, filtering, and drying in vacuo to give 0.55 g (84% conversion) of the citrate salt as a white solid: mp 110 OC; 1H NMR (CDC13 + DMSO-d6) 61.48-1.77 (comp, 6H, piperidine), 2.07-2.51 (comp, 4H, CH2CH2), 2.61-2.80 (m, 4H, citrate), 2.97-3.07 (m, 4H, piperidine), 3.24 (bs, 2H, CH2N), 3.66 (s, 3H, OCH3), 4.14 (s, 1H, CH), 4.25 (bs, 2H, OCH2), 6.51 (s, 1H, C=CH), 6.71-6.76 (m, 4H, ArH), 7.03-7.08 (m, 2H, ArH), 7.33-7.37 (m, 2H, ArH); MS(FAB) 406 [M+1]+. Anal. Calcd for C26H31No3 C6H8O7: C, 64.31; H, 6.58; N, 2.34.Found: C, 64.36; H, 6.56; N, 2.46.

PreParation 4 1,4-Bin (4-nethoxyphenyl) -2- (4 -ben:yloxyphenyl) cyclohexa-1, 3 -diene

A 1.7 M pentane solution of tert-butyllithium (2 mL, 3.4 mmol) was syringed into a 20 mL ThF solution of 4bromoanisole (0.2 mL, 1.6 mmol) at -70 OC, stirred 15 min, then transferred via a double-tipped syringe needle into a 15 mL solution of 3- (4-benzyloxyphenyl) -4- (4-methoxyphenyl) cyclohex-2-en-1-one (0.6 g, 1.5 mmol) at -70 OC and stirred 30 min as it was allowed to warm to 0 OC. The reaction mixture was quenched with aqueous NH4Cl, extracted several times with EtOAc, the EtOAc extracts combined, washed with brine, dried (Na2SO4), concentrated, triturated with pet.

ether, filtered, and dried in vacuo to give 0.35 g (50% yield) of product: mp 137-141 OC; 1H NMR (CDC13) 52.79- 2.80 (m, 4H, CH2CH2), 3.78 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 5.03 (s, 2H, OCH2Ar), 6.50 (s, 1H, C=CH), 6.70-6.73 (d, 2H, J = 9 Hz, ArH), 6.81-6.84 (d, 2H, J = 9 Hz, ArH), 6.89-6.92 (d, 2H, J = 9 Hz, ArH), 7.02-7.06 (m, 4H, ArH), 7.40-7.50 (m, 5H, ArH); MS(FD) 474(M+). Anal. Calcd for C33H3003: C, 83.52; H, 6.37. Found: C, 83.33; H, 6.35.

Preparation 5 2-(4-Benzyloxyphenyl)-1,4-bls(4-motho honyl)bonzoue

The 1,4-bis (4-methoxyphenyl) -2 (benzyloxyphenyl)cyclohexa-1,3-diene ( 2.2 g, 4.8 mmol) and activated MnO2 ( 10.3 g, 119 mmol) were stirred in 120 mL xylenes and refluxed vigorously for 3 h in a flask fitted with a Dean-Stark trap to remove water. The reaction mixture was cooled, filtered, concentrated, purified by prep chromatography (EtOAc/ hexane gradient), and recrystallized from EtOAc/ hexane to give 2.2 g ( 95% yield) of yellow product.Elemental analysis was 1.5% high for C, but the MS and NMR were consistent for the structure of the product and material was carried on to the next step: mp 131-133 C; 1H NMR (CDCl3) 63.81 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.06 (s, 2H, OCH2Ar), 6.78-6.81 (d, 2H, J = 9 Hz, ArH), 6.87-6.90 (d, 2H, J = 9 Hz, ArH), 6.99-7.02 (d, 2H, J = 9 Hz, ArH), 7.10-7.13 (m, 4H, ArH), 7.41-7.47 (m, 6H, ArH), 7.59-7.63 (m, 4H, ArH); MS(FD) 472(M+). Anal. Calcd for C33H28O3: C, 83.87; H, 5.97. Found: C, 85.43; H, 6.13.

Prenaration 6 2- (4-Hydroxyphenyl) -1,4-bin (4-metbo:yphenyl)ben:ene

A 100 mL THF/ EtOH (1:1) solution of 2-(4 benzyloxyphenyl)-1,4-bis(4-methoxyphenyl)benzene (1.75 g, 3.7 mmol) was hydrogenated at 60 psi with 5% Pd/ C (0.4 g, 0.2 mmol Pd) at rt for 4 h, filtered, concentrated, and recrystallized from Et2O/ pet. ether to give 1.1 g (77% yield) of product. The material was slightly high for C in the elemental analysis, but the NMR and MS were consistent for the product and it was used without further purification: mp 189-190 OC; 1H NMR (CDCl3 + DMSO-d6) t.71 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 6.65-6.70 (m, 4H, ArH), 6.90-6.95 (m, 4H, ArH), 7.00-7.03 (d, 2H, J = 9 Hz, ArH), 7.36-7.54 (comp, 5H, ArH), 8.49 (s, 1H, ArOH); MS(FD) 382(M+). Anal.

Calcd for C26H2203: C, 81.65; H, 5.80. Found: C, 82.20; H, 5.95.

Example 1 3-l4-t2-(1-PAperidinyl)othoxylthenyll-4- (4 -methoxyphenyl) - 1 -hydroxybenzene, hydrochloride

Lithium bromide (0.3 g, 3.6 mmol), then cupric bromide (1.6 g, 7.2 mmol) were added to a 60 mL MeCN solution of 3-[4-[2-(l-piperidinyl)ethoxy]phenyl]-4-(4- methoxyphenyl)cyclohex-2-en-l-one (1.45 g, 3.6 mmol) and the green mixture was refluxed 30 min. The reaction mixture was cooled, poured into 100 mL saturated aqueous NaHCO3, extracted separately with EtOAc (3 x 75 mL) and CH2C12 (2 x 75 mL). The EtOAc extracts and the CH2C12 extracts were separately washed with brine, dried (Na2SO4), then recombined and concentrated to a brown foam.The crude product was purified by prep chromatography (EtOH/ CH2Cl2 gradient) to give 0.9 g (62% yield) of product as the free base. A 50 mL MeOH solution of the free base (0.7 g, 1.7 mmol) was stirred at 0 OC with excess HCL saturated MeOH for 10 min, concentrated, and the residue recrystallized from THF/ MeOH/ Et2O to give 0.55 g (72% conversion) of HCl salt as a tan solid: mp > 200 OC; 1H NMR (CDCl3 + DMSO-d6) & 70-2.08 (comp, 6H, piperidine), 2.67-2.74 (bs, 2H, piperidine), 3.26 (bs, 2H, CH2N), 3.45-3.51 (comp, 2H, piperidine), 3.63 (s, 3H, OCH3),4.37-4.38 (m, 2H, OCH2), 6.57-6.60 (d, 4H, J = 9 Hz, ArH), 6.70-6.73 (m , 2H, ArH), 6.82-6.91 (m, 4H, ArH), 7.02-7.05 (d, 1H, J = 9 Hz, ArH), 8.76 (s, 1H, ArOH); MS(FD) 404 (M+). Anal.Calcd for C26H29NO3 HCl: C,70.98; H, 6.87; N, 3.18. Found: C, 70.77; H, 6.93; N, 3.37.

Zza=1U2 3-[4-[2-(1-Piperidinyl)ethoxylphenyl]- 4-(4-hydrosyphenyl)-1-hydroxybenzene

The 3-[4-[2-(l-piperidinyl)ethoxy)phenyl]-4-(4- methoxyphenyl)-l-hydroxybenzene (2.5 g, 6.1 mmol) was stirred in 100 mL 1,2-dichloroethane (DCE) at 0 oC, a 2.45 M BCl3/ DCE solution (7 mL, 17 mmol) was added, the flask sealed, and the dark reaction mixture was stirred at rt for 18 h. The TLC (EtOH/ CH2Cl2, 1:4) of the reaction mixture indicated some starting material remained so additional BCl3/ DCE (3.5 mL, 8.6 mmol) was added to the reaction mixture, flask sealed, and was stirred another 18 h, which consumed the remaining starting material.The reaction mixture was slowly poured into stirring saturated aqueous NaHCO3/ ice and extracted with EtOAc several times, the EtOAc extracts combined, washed with brine, dried (Na2SO4), concentrated, triturated with Et2O, filtered, and dried in vacuo to give 2 g (84% yield) of an off-white product: mp 197-199 C; 1H NMR (CDCl3 + DMSO-d6) 61.31-1.49 (comp, 6H, piperidine), 2.36-2.39 (m, 4H, piperidine), 2.61-2.65(t, 2H, J = 6 Hz, CH2N), 3.91-3.93 (t, 2H, J = 6 Hz, OCH2), 6.52-6.61 (m , 4H, ArH), 6.69-6.77 (m, 4H, ArH), 6.88-6.91 (d, 2H, J = 8 Hz, ArH), 7.04-7.06 (d, 1H, J = 8 Hz, ArH), 8.42 (bs, 1H, ArOH), 8.65 (bs, 1H, ArOH); MS(FD) 390 [M+l]+. Anal. Calcd for C2sH27NO3: C, 77.09; H, 6.99; N, 3.60. Found: C, 76.84; H, 7.24; N, 3.39.

Example 3 2-t4-t2-(l-Piper4dinyl)ethoxylphenyll-1,4-bAs- (4-methoxyphenyl)benzene, hydrochloride salt

A heterogeneous mixture of 2-(4-hydroxyphenyl)-1,4bis(4-methoxyphenyl)benzene (1.7 g, 4.4 mmol), powdered K2CO3 ( 4 g, 29 mmol), and N-(2-chloroethyl)piperidine. HCl (0.8 g, 4.6 mmol) in 150 mL methyl ethyl ketone was refluxed 18 h, filtered, concentrated, and purified by prep chromatography (EtOH/ CH2Cl2 gradient) to give the product as a gum.The gum was dissolved in 50 mL MeOH, cooled to 0 OC, excess HCl saturated MeOH was added, stirred 10 min, concentrated, triturated with Et2O, filtered, and dried in vacuo to give 1.5 g (65% yield) of product as the HCl salt: mp > 220 OC; 1H NMR (CDC13 + DMSO-d6) 61.35-2.41 (comp, 6H, piperidine), 2.76-2.79 (m, 2H, piperidine), 3.32-3.33 (m, 2H, CH2N), 3.53-3.57 (m, 2H, piperidine), 3.72 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 4.44-4.47 (m, 2H, OCH2), 6.68-6.72 (m, 4H, ArH), 6.89-6.92 (d, 2H, J = 9 Hz, ArH), 6.98-7.05 (m 4H, ArH), 7.33-7.36 (d, 1H, J = 8 Hz, ArH), 7.46-7.52 (m, 4H, ArH); MS(FD) 493(M+). Anal. Calcd for C33H25NO3 . HCl: C, 74.77; H, 6.84; N, 2.64. Found: C, 75.03; H, 6.94; N, 2.72.

Example 4 1- [4-[2-(1-piperidinyl)ethoxy]phenyl]-2, 5-bin (4 -hydroxyphenyl ) benzene, hydrochloride

The 2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-1,4-bis- (4-methoxyphenyl)benzene HC1 (1 g, 1.9 mnol) was slurried in 75 mL of DCE, the BC13 (5 ml at 0 C, 58 mmol) was added, the flask sealed, and the reaction mixture stirred 18 h at rt. The TLC (MeOH/ CH2Cl2, 1:4) indicated that the reaction had not gone to completion, so additional BC13 (8 ml, 93 mmol) was added, the flask sealed, and reaction mixture stirred another 18 h at rt.The reaction mixture was carefully quenched with 25 mL MeOH at 0 OC, poured into 150 mt of aqueous NaHCO3, and extracted with CH2Cl2 (3 x 75 mL).

The CH2C12 extracts were combined, washed with brine, dried (Na2S04), and concentrated to give the product. The free base was dissolved in 25 mL MeOH, cooled to 0 OC, excess HCl saturated MeOH was added, stirred 10 min, concentrated, triturated with Et2O, filtered, and dried in vacuo to give 0.6 g (64% yield) of product as the HCl salt: mp > 230 OC; 1H NMR (CDCl3 + DMSO-d6) 61.39-1.95 (comp, 6H, piperidine), 3.02-3.18 (m, 2H, CH2N), 3.50-3.64 (m, 4H, piperidine), 4.42-4.52 (m, 2H, OCH2), 6.70-6.73 (d, 2H, J = 8 Hz, ArH), 6.93-7.06 (m, 6H, ArH), 7.20-7.23 (d, 2H, J = 8 Hz, ArH), 7.42-7.45 (d, 1H, J = 8 Hz, ArH), 7.53-7.70 (m , 4H, ArH), 9.56 (s, 1H, ArOH), 9.73 (s, 1H, ArOH); MS(FD) 466(M+).

Anal. Calcd for C31H21NO3 HCl: C, 74.16; H, 6.43; N, 2.79.

Found: C, 73.91; H, 6.67; N, 2.62.

Example 5 2- [4- -Pioetidiqyl) ethoxylpepyl] -1.

4-di-(4-methoxyphenyl)benzene, citrate

o~ND NI N N OCH N OCH3 N N I H300 H3CO A heterogeneous mixture of 2-(4-hydroxyphenyl)-1,4- bis(4-methoxyphenyl)benzene (1 g, 2.6 mmol), powdered K2C03 (1.9 g, 14 mmol), and N-(2-chloroethyl)piperidine HCl (0.6 g, 3.7 mmol) in 75 mL methyl ethyl ketone was refluxed 2.5 h, filtered, concentrated, and purified by prep chromatography (MeCN/ CH2Cl2 gradient) to give 1.1 g (86% yield) of product as the free base. The free base (1 g, 2mmol) was dissolved in 100 mL acetone, warmed on a steam bath, citric acid monohydrate (0.4 g, 1.9 mmol) was added, and the mixture was heated an additional 10 min, then concentrated, triturated with Et2O, filtered, and dried in vacuo to give 1.3 g (100% conversion) of the citrate salt as a white solid: mp 79-80 OC; 1H NMR (CDCl3) 61.87-1.90 (comp, 6H, piperidine), 2.822.87 (comp, 8H, piperidine and citrate), 3.44 (bs, 2H, CH2N), 3.77(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.33 (bs, 2H, OCH2), 6.75-6.79 (m, 4H, ArH), 6.98-7.13 (m , 6H, ArH), 7.41-7.44 (d, 1H, J = 8 Hz, ArH), 7.55-7.61 (m, 4H, ArH); MS(FAB) 495 lM+2]+. Anal. Calcd for C33H35NO3 C6H8O7: C, 68.30; H, 6.32; N, 2.04. Found: C, 68.04; H, 6.54; N, 2.02.

In the examples illustrating the methods, a post-menopausal model was used in which effects of different treatments upon circulating lipids were determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to 225g) were obtained from Charles River Laboratories (Portage, MI). The animals were either bilaterally ovariectomized (OVX) or exposed to a sham surgical procedure at Charles River Laboratories, and then shipped after one week. Upon arrival, they were housed in metal hanging cages in groups of 3 or 4 per cage and had ad libitum access to food (calcium content approximately 0.5%) and water for one week. Room temperature was maintained at 22.20 i 1.70 C with a minimum relative humidity of 40%. The photoperiod in the room was 12 hours light and 12 hours dark.

Dosina Regimen Tissue Collection. After a one week acclimation period (therefore, two weeks post-OVX) daily dosing with test compound was initiated. 17a-ethynyl estradiol or the test compound were given orally, unless otherwise stated, as a suspension in 1% carboxymethylcellulose or dissolved in 20% cyclodextrin.

Animals were dosed daily for 4 days. Following the dosing regimen, animals were weighed and anesthetized with a ketamine: Xylazine (2:1, V:V) mixture and a blood sample was collected by cardiac puncture. The animals were then sacrificed by asphyxiation with CO2, the uterus was removed through a midline incision, and a wet uterine weight was determined.

Cholesterol Analvsis. Blood samples were allowed to clot at room temperature for 2 hours, and serum was obtained following centrifugation for 10 minutes at 3000 rpm. Serum cholesterol was determined using a Boehringer Mannheim Diagnostics high performance cholesterol assay. Briefly the cholesterol was oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide was then reacted with phenol and 4-aminophenazone in the presence of peroxidase to produce a p-quinone imine dye, which was read spectrophotometrically at 500 nm. Cholesterol concentration was then calculated against a standard curve. The entire assay was automated using a Biomek Automated Workstation.

Uterine Eosinoshil Peroxidase (EPO) Assay. Uteri were kept at 40 C until time of enzymatic analysis. The uteri were then homogenized in 50 volumes of 50 mM Tris buffer (pH 8.0) containing 0.005% Triton X-100. Upon addition of 0.01% hydrogen peroxide and 10 mM O-phenylenediamine (final concentrations) in Tris buffer, increase in absorbance was monitored for one minute at 450 nm. The presence of eosinophils in the uterus is an indication of estrogenic activity of a compound. The maximal velocity of a 15 second interval was determined over the initial, linear portion of the reaction curve.

Source of Compound: 17a-ethynyl estradiol was obtained from Sigma Chemical Co., St. Louis, MO.

Influence of Formula I Compounds on Serum Cholesterol and Determination of Aaonist/Non-Aaonist Activity Data presented in Table 1 below show comparative results among ovariectomized rats, rats treated with 17aethynyl estradiol (EE2; an orally available form of estrogen), and rats treated with certain compounds of the present invention. Although EE2 caused a decrease in serum cholesterol when orally administered at 0.1 mg/kg/day, it also exerted a stimulatory action on the uterus so that EE2 uterine weight was substantially greater than the uterine weight of ovariectomized control animals. This uterine response to estrogen is well recognized in the art.

Not only did the compounds of the present invention generally reduce serum cholesterol compared to the ovariectomized control animals, but uterine weight was only minimally increased to slightly decreased with the formula I compounds tested.

As is expressed in the below data, estrogenicity also was assessed by evaluating the response of eosinophil infiltration into the uterus. The compounds of the present invention either did not cause any increase in the number of eosinophils observed in the stromal layer of ovariectomized rats, or caused only modest increases. Estradiol caused a substantial, expected increase in eosinophil infiltration.

The data presented in Table 1 below reflects the response of 5 to 6 rats per treatment. aNAl indicates that a compound was not active at the doses tested.

Table 1 Compound Dose Uterine Uterine Sern (nglkg) Wt. (% EPO (V. Cholesterol (effective inc. vs. mas) (% dec. vs. dose to OVX) OVX) dec. serum chol. by 50%) EE2 0.1 188.8* 252.0* 84.8* Example 1 1 -7.8 6.2 1 10 -2.2 5.6 -31.3 25 -12 5.6 -11.6 NA EE2 0.1 172.7* 288.9* 80.3* Example 2 0.1 15.4 9.3 -3.6 1 12 4.5 -37.8* 10 17.7 9.0 25 NA EE2 0.1 174.3* 160.2* 85.4* Example 2 1 5.3 0.0 4.2 10 -3.2 3.0 -16.1 25 16 3.6 27.6* > 25 EE2 0.1 194.6* 253.9* 87.8* Example 3 0.1 0.0 4.5 5.2 1 10.8 5.2 6.7 10 32.4 7.4 34.9* 66 EE2 0.1 147.5* 81.6* 84.7* Example 4 1 0.8 2.1 -5.8 10 61.9* 36.0 25.4* 25 63.4* 55.5* 52.8 7.6 *P = < 0.5 ANOVA with post-hoc Fisher's PLSD on raw data In addition to the demonstrated benefits of the compounds of the present invention, especially when compared to estradiol, the above data clearly demonstrate that compounds of Formula I are not estrogen mimetics.

Furthermore, no deleterious toxicological effects (survival) were observed with any treatment.

MCF-7 Proliferation Assav MCF-7 breast adenocarcinoma cells (ATCC HTB 22) were maintained in MEM (minimal essential medium, phenol redfree, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES ((N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid]10 mM), non-essential amino acids and bovine insulin (1 ug/mL) (maintenance medium). Ten days prior to assay, MCF-7 cells were switched to maintenance medium supplemented with 10% dextran coated charcoal stripped fetal bovine serum (DCC-FBS) assay medium) in place of 10% FBS to deplete internal stores of steroids. MCF-7 cells were removed from maintenance flasks using cell dissociation medium (Ca++/Mg++ free HBSS (phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA).Cells were washed twice with assay medium and adjusted to 80,000 cells/mL.

Approximately 100 gt (8,000 cells) were added to flat-bottom microculture wells (Costar 3596) and incubated at 370 C in a 5% CO2 humidified incubator for 48 hours to allow for cell adherence and equilibration after transfer. Serial dilutions of drugs or DMSO as a diluent control were prepared in assay medium and 50 AL transferred to triplicate microcultures followed by 50 WL assay medium for a final volume of 200 pL After an additional 48 hours at 370 C in a 5% CO2 humidified incubator, microcultures were pulsed with tritiated thymidine (1 uCi/well) for 4 hours. Cultures were terminated by freezing at -70 C for 24 hours followed by thawing and harvesting of microcultures using a Skatron Semiautomatic Cell Harvester.Samples were counted by liquid scintillation using a Wallac BetaPlace P counter. Results in Table 2 below show the IC50 for certain compounds of the present invention.

Table2 Compound (Example Reference) IC50 nM 1 390 2 100 3 1000 4 100 Uterine Fibrosis Test Procedures Test 1 Between 3 and 20 women having uterine fibrosis are administered a compound of the present invention. The amount of compound administered is from 0.1 to 1000 mg/day, and the period of administration is 3 months.

The women are observed during the period of administration, and up to 3 months after discontinuance of administration, for effects on uterine fibrosis.

Test 2 The same procedure is used as in Test 1, except the period of administration is 6 months.

Test 3 The same procedure is used as in Test 1, except the period of administration is 1 year.

Test 4 A. Induction of fibroid tumors in guinea pig.

Prolonged estrogen stimulation is used to induce leiomyomata in sexually mature female guinea pigs. Animals are dosed with estradiol 3-5 times per week by injection for 2-4 months or until tumors arise. Treatments consisting of a compound of the invention or vehicle is administered daily for 3-16 weeks and then animals are sacrificed and the uteri harvested and analyzed for tumor regression.

B. Implantation of human uterine fibroid tissue in nude mice.

Tissue from human leiomyomas are implanted into the peritoneal cavity and or uterine myometrium of sexually mature, castrated, female, nude mice. Exogenous estrogen are supplied to induce growth of the explanted tissue. In some cases, the harvested tumor cells are cultured in vitro prior to implantation. Treatment consisting of a compound of the present invention or vehicle is supplied by gastric lavage on a daily basis for 3-16 weeks and implants are removed and measured for growth or regression. At the time of sacrifice, the uteri are harvested to assess the status of the organ.

Test 5 A. Tissue from human uterine fibroid tumors is harvested and maintained, in vitro, as primary nontransformed cultures.

Surgical specimens are pushed through a sterile mesh or sieve, or alternately teased apart from surrounding tissue to produce a single cell suspension. Cells are maintained in media containing 10% serum and antibiotic. Rates of growth in the presence and absence of estrogen are determined.

Cells are assayed for their ability to produce complement component C3 and their response to growth factors and growth hormone. In vitro cultures are assessed for their proliferative response following treatment with progestins, GnRH, a compound of the present invention and vehicle.

Levels of steroid hormone receptors are assessed weekly to determine whether important cell characteristics are maintained in vitro. Tissue from 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds of the present invention are of potential in the treatment of uterine fibrosis.

Endometriosis Test Procedure In Tests 1 and 2, effects of 14-day and 21-day administration of compounds of the present invention on the growth of explanted endometrial tissue can be examined.

Test 1 Twelve to thirty adult CD strain female rats are used as test animals. They are divided into three groups of equal numbers. The estrous cycle of all animals is monitored. On the day of proestrus, surgery is performed on each female. Females in each group have the left uterine horn removed, sectioned into small squares, and the squares are loosely sutured at various sites adjacent to the mesenteric blood flow. In addition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receive intraperitoneal injections of water for 14 days whereas animals in Group 3 receive intraperitoneal injections of 1.0 mg of a compound of the present invention per kilogram of body weight for the same duration. Following 14 days of treatment, each female is sacrificed and the endometrial explants, adrenals, remaining uterus, and ovaries, where applicable, are removed and prepared for histological examination. The ovaries and adrenals are weighed.

Test 2 Twelve to thirty adult CD strain female rats are used as test animals. They are divided into two equal groups. The estrous cycle of all animals is monitored. On the day of proestrus, surgery is performed on each female.

Females in each group have the left uterine horn removed, sectioned into small squares, and the squares are loosely sutured at various sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1 receive intraperitoneal injections of water for 21 days whereas animals in Group 2 receive intraperitoneal injections of 1.0 mg of a compound of the present invention per kilogram of body weight for the same duration. Following 21 days of treatment, each female is sacrificed and the endometrial explants and adrenals are removed and weighed. The explants are measured as an indication of growth. Estrous cycles are monitored.

Test 3 A. Surgical induction of endometriosis Autographs of endometrial tissue are used to induce endometriosis in rats and/or rabbits. Female animals at reproductive maturity undergo bilateral oophorectomy, and estrogen is supplied exogenously thus providing a specific and constant level of hormone. Autologous endometrial tissue is implanted in the peritoneum of 5-150 animals and estrogen supplied to induce growth of the explanted tissue. Treatment consisting of a compound of the present invention is supplied by gastric gavage on a daily basis for 3-16 weeks, and implants are removed and measured for growth or regression.

At the time of sacrifice, the intact horn of the uterus is harvested to assess status of endometrium.

B. Implantation of human endometrial tissue in nude mice.

Tissue from human endometrial lesions is implanted into the peritoneum of sexually mature, castrated, female, nude mice. Exogenous estrogen is supplied to induce growth of the explanted tissue. In some cases, the harvested endometrial cells are cultured in vitro prior to implantation. Treatment consisting of a compound of the present invention supplied by gastric gavage on a daily basis for 3-16 weeks, and implants are removed and measured for growth or regression. At the time of sacrifice, the uteri are harvested to assess the status of the intact endometrium.

Test 4 A. Tissue from human endometrial lesions is harvested and maintained in vitro as primary nontransformed cultures.

Cells are assayed for their ability to produce complement component C3 and their response to growth factors and growth hormone. In vitro cultures are assessed for their proliferative response following treatment with progestins, GnRH, a compound of the invention, and vehicle. Levels of steroid hormone receptors are assessed weekly to determine whether important cell characteristics are maintained in vitro. Tissue from 5-25 patients is utilized.

nh1b1tlon of Aortal Smooth Cell Prol1feratlon/Restenosis Test Procedure Compounds of the present invention have capacity to inhibit aortal smooth cell proliferation. This can be demonstrated by using cultured smooth cells derived from rabbit aorta, proliferation being determined by the measurement of DNA synthesis. Cells are obtained by explant method as described in Ross, J. of Cell Blo. SQ: 172 (1971).

Cells are plated in 96 well microtiter plates for five days.

The cultures become confluent and growth arrested. The cells are then transferred to Dulbecco's Modified Eagle's Medium (DMEM) containing 0.5 - 2% platelet poor plasma, 2 mM Lglutamine, 100 U/ml penicillin, 100 mg ml streptomycin, 1 mC/ml 3H-thymidine, 20 ng/ml platelet-derived growth factor, and varying concentrations of the present compounds. Stock solution of the compounds is prepared in dimethyl sulphoxide and then diluted to appropriate concentration (0.01 - 30 mM) in the above assay medium. Cells are then incubated at 370 C. for 24 hours under 5% C02/95% air. At the end of 24 hours, the cells are fixed in methanol. 3H thymidine incorporation in DNA is then determined by scintillation counting as described in Bonin, et al., ExD. Cell Res. 181: 475-482 (1989).

Inhibition of aortal smooth muscle cell proliferation by the compounds of the present invention are further demonstrated by determining their effects on exponentially growing cells. Smooth muscle cells from rabbit aortae are seeded in 12 well tissue culture plates in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 24 hours, the cells are attached and the medium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and desired concentrations of the compounds. Cells are allowed to grow for four days. Cells are treated with trypsin and the number of cells in each culture is determined by counting using a ZM-Coulter counter.

Activity in the above tests indicates that the compounds of the present invention are of potential in the treatment of restenosis.

The present invention also provides a method of alleviating post-menopausal syndrome in women which comprises the aforementioned method using compounds of Formula I and further comprises administering to a woman an effective amount of estrogen or progestin. These treatments are particularly useful for treating osteoporosis and lowering serum cholesterol because the patient will receive the benefits of each pharmaceutical agent while the compounds of the present invention would inhibit undesirable side-effects of estrogen and progestin. Activity of these combination treatments in any of the post-menopausal tests, infra, indicates that the combination treatments are useful for alleviating the symptoms of post-menopausal symptoms in women.

As used herein, the term 0estrogen' includes steroidal compounds having estrogenic activity such as, for example, 17B-estradiol, estrone, conjugated estrogen (Premarin&commat;), equine estrogen, 17a-ethynyl estradiol, and the like. As used herein, the term 'progestinS includes compounds having progestational activity such as, for example, progesterone, norethynodrel, norgestrel, megestrol acetate, norethindrone, and the like.

Various forms of estrogen and progestin are commercially available. Estrogen-based agents include, for example, ethynyl estrogen (0.01 - 0.03 mg/day), mestranol (0.05 - 0.15 mg/day), and conjugated estrogenic hormones such as Premarins (Wyeth-Ayerst; 0.3 - 2.5 mg/day). Progestinbased agents include, for example, medroxyprogesterone such as Proverbs (Upjohn; 2.5 -10 mg/day), norethynodrel (1.0 10.0 mg/day), and norethindrone (0.5 - 2.0 mg/day). A preferred estrogen-based compound is Premarin, and norethynodrel and norethindrone are preferred progestin-based agents.

The method of administration of each estrogen- and progestin-based agent is consistent with that which is known in the art. For the majority of the methods of the present invention, compounds of Formula I are administered continuously, from 1 to 3 times daily. However, cyclical therapy may especially be useful in the treatment of endometriosis or may be used acutely during painful attacks of the disease. In the case of restenosis, therapy may be limited to short (1-6 months) intervals following medical procedures such as angioplasty.

As used herein, the term 1effective amount1 means an amount of compound of the present invention which is capable of alleviating the symptoms of the various pathological conditions herein described. The specific dose of a compound administered according to this invention will, of course, be determined by the particular circumstances surrounding the case including, for example, the compound administered, the route of administration, the state of being of the patient, and the pathological condition being treated.

A typical daily dose will contain a nontoxic dosage level of from about 5 mg to about 1000 mg/day of a compound of the present invention. Preferred daily doses generally will be from about 15 mg to about 500 mg/day.

The compounds of this invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.

These compounds preferably are formulated prior to administration, the selection of which will be decided by the attending physician. Thus, another aspect of the present invention is a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, optionally containing an effective amount of estrogen or progestin, and a pharmaceutically acceptable carrier, diluent, or excipient.

The total active ingredients in such formulations comprises from 0.1% to 99.9% by weight of the formulation.

By 1pharmaceutically acceptable" it is meant the carrier, diluent, excipients and salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations of the present invention can be prepared by procedures known in the art using well known and readily available ingredients. For example, the compounds of formula I, with or without an estrogen or progestin compound, can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like.Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes.

Additionally, the compounds are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular physiological location, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances or waxes.

Compounds of formula I, alone or in combination with a pharmaceutical agent of the present invention, generally will be administered in a convenient formulation.

The following formulation examples only are illustrative and are not intended to limit the scope of the present invention.

Formulations In the formulations which follow, 'active ingredient" means a compound of Formula I, or a salt or solvate thereof.

Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared using the following: Ingredient Quantity (mg/capsule) Active ingredient 0.1 - 1000 Starch, NF 0 - 650 Starch flowable powder 0 - 650 Silicone fluid 350 centistokes 0 - 15 The formulation above may be changed in compliance with the reasonable variations provided.

A tablet formulation is prepared using the ingredients below: Formulation 2: Tablets Ingredient Ouantity (ma/tablet) Active ingredient 2.5 - 1000 Cellulose, microcrystalline 200 - 650 Silicon dioxide, fumed 10 - 650 Stearate acid 5 - 15 The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5 - 1000 mg of active ingredient are made up as follows: Formulation 3: Tablets Ingredient Quantity (mg/tablet) Active ingredient 25 - 1000 Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as 10% solution in water) Sodium carboxymethyl cellulose 4.5 Magnesium stearate 0.5 Talc ~ ~ 1 The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.

The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50 -60 C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.1 - 1000 mg of medicament per 5 ml dose are made as follows: Formulation 4: Suspensions Ingredient Quantity (mg/5 ml) Active ingredient 0.1 - 1000 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mg Benzoic acid solution 0.10 mL Flavor q.v.

Color g.v.

Purified water to 5 mL The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.

An aerosol solution is prepared containing the following ingredients: Formulation 5: Aerosol Ingredient Quantity (% by weight) Active ingredient 0.25 Ethanol 25.75 Propellant 22 (Chlorodifluoromethane) ~ 70.00 The active ingredient is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to 300 C, and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remaining propellant. The valve units are then fitted to the container.

Suppositories are prepared as follows: Formulation 6: Suppositories Ingredient Quantity (mg/suppository) Active ingredient 250 Saturated fatty acid glycerides 2,000 The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimal necessary heat. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows: Formulation 7: Intravenous Solution Ingredient Quantity Active ingredient 50 mg Isotonic saline 1 000 mL The solution of the above ingredients is intravenously administered to a patient at a rate of about 1 mL per minute.

Formulation 8: Combination Capsule I Ingredient Quantity (mg/capsule) Active ingredient 50 Premarin 1 Avicel pH 101 50 Starch 1500 117.50 Silicon Oil 2 Tween 80 0.50 Cab-O-Sil 0.25 Formulation 9: Combination Capsule II Ingredient Quantity (mg/capsule) Active ingredient 50 Norethynodrel 5 Avicel pH 101 82.50 Starch 1500 90 Silicon Oil 2 Tween 80 0.50 Formulation 10: Combination Tablet Ingredient Quantity (mg/capsule) Active ingredient 50 Premarin 1 Corn Starch NF 50 Povidone, K29-32 6 Avicel pH 101 41.50 Avicel pH 102 136.50 Crospovidone XL10 2.50 Magnesium Stearate 0.50 Cab-O-Sil 0.50

Claims

CLAIMS 1. A compound of Formula I:

wherein: n is 2 or 3; R is dimethylamino, diethylamino, 1-piperidinyl, 1pyrrolidinyl, 4-morpholinyl, or 1-hexamethyleneimino; R1 is hydroxy, phenyl, or mono- or disubstituted phenyl wherein each substituent is independently halo, methyl, hydroxy, Cl-C6 -alkoxy, benzyloxy, C1-C6-alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4-loweralkoxy, C1-C5-alkoxycarbonyloxy, or C4-Cs-alkylsulfonyloxy;; R2 is hydrogen, hydroxy, C1-Cs-alkoxy, benzyloxy, C1-C6alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4-loweralkoxy, C1-Cs-alkoxycarbonyloxy, or C4-Cs-alkylsulfonyloxy; or a pharmaceutically acceptable acid addition salt thereof.
2. A compound of Claim 1 wherein n is 2, R is 1piperidinyl, R1 is hydroxy or 4-hydroxyphenyl, and R2 is hydroxy.
3. A compound of Claim 1 which is 3-[4-[2-(1- piperidinyl)ethoxy]phenyl]-4-(4-hydroxyphenyl)-1- hydroxybenzene, or 1- [4- [2- (l-piperidinyl)ethoxy]phenyl] -2,5- bis(4-hydroxyphenyl)benzene, or a pharmaceutically acceptable salt or solvate thereof.
4. A pharmaceutical composition comprising a compound according to Claim 1, or a pharmaceutically acceptable salt or solvate thereof, and optionally comprising an effective amount of an estrogen or progestin in combination with a pharmaceutically acceptable carrier, diluent, or excipient.
5. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of postmenopausal syndrome.
6. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of the postmenopausal syndrome pathological condition of osteoporosis.
7. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of the postmenopausal syndrome pathological condition related to a cardiovascular disease.
8. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of a cardiovascular disease related to hyperlipidemia.
9. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of the postmenopausal syndrome pathological condition of estrogen-dependent cancer.
10. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of breast or uterine cancer.
11. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of uterine fibroid disease.
12. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of endometriosis.
13. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of aortal smooth muscle cell proliferation.
14. A compound of formula I as claimed in any of Claims 1-3 for use in alleviating the symptoms of restenosis.
15. A process for preparing a compound of formula V:

wherein: n is 2 or 3; R is dimethylamino, diethylamino, 1-piperidinyl, 1pyrrolidinyl, 4-morpholinyl, or 1-hexamethyleneimino; and R2a is hydrogen, C1-C6-alkoxy, benzyloxy, C1-C6alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4-loweralkoxy, Cl-C5-alkoxycarbonyloxy, or C4-C6-alkylsulfonyloxy; which comprises reacting a compound of formula III:

with an alkyl chloride of the formula Cl-(cH2)n-Rt in an inert solvent, in the presence of base, and at a temperature of 0 to 100-C, to obtain a compound of Formula IV:

and thereafter oxidizing the compound of Formula IV to obtain the compound of Formula V.
16. A compound of Formula IV

wherein: n is 2 or 3; R is dimethylamino, diethylamino, 1-piperidinyl, 1pyrrolidinyl, 4-morpholinyl, or 1-hexamethyleneimino; and R2a is hydrogen, C1-C6-alkoxy, benzyloxy, C1-C6alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4 loweralkyl, or C1-C4-loweralkoxy, C1-Cs-alkoxycarbonyloxy, or C4-Cs-alkylsulfonyloxy.
17. A process for preparing a compound of Formula IX:

wherein: n is 2 or 3; R is dimethylamino, diethylamino, l-piperidinyl, 1pyrrolidinyl, 4 -morpholinyl, or 1-hexamethyleneimino; Rla is phenyl or mono- or disubstituted phenyl wherein each substituent is independently halo, methyl, C1-C6-alkoxy, benzyloxy, C1-Cs-alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, Cl-C4-loweralkyl, or C1-C4-loweralkoxy, C1-Cs-alkoxycarbonyloxy, or C4-C6-alkylsulfonyloxy;; and R2 is hydrogen, C1-C6-alkoxy, benzyloxy, C1-C6 alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4-loweralkoxy, Ci-Cs-alkoxycarbonyloxy, or C4-C6-alkylsulfonyloxy, which comprises reacting a compound of Formula VIII:

with an alkyl chloride of the formula Cl-(CH2)n-R, in an inert solvent, in the presence of base, and at a temperature of from 0 to 100-.
18. A compound of Formula VIII:

wherein: R1Q is phenyl or mono- or disubstituted phenyl wherein each substituent is independently halo, methyl, C1-C6-alkoxy, benzyloxy, C1-C6-alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4-loweralkyl, or C1-C4-loweralkoxy, C1-Cs-alkoxycarbonyloxy, or C4-Cs-alkylsulfonyloxy; and R2a is hydrogen, C1-C6-alkoxy, benzyloxy, C1-C6 alkanoyloxy, benzoyloxy, substituted benzoyloxy bearing 1 to 3 substituents each of which is independently halo, C1-C4loweralkyl, or C1-C4 -loweralkoxy, Cl-cs-alkoxycarbonyloxy or C4-C6 alkylsulfonyloxy.