WO2002058639A2

WO2002058639A2 - Pyranoflavonoid compounds and their use as estrogen receptor modulators

Info

Publication number: WO2002058639A2
Application number: PCT/US2002/003075
Authority: WO
Inventors: Michael A. Goetz; Susan P. Rohrer; Keith M. Witherup
Original assignee: Merck & Co., Inc.
Priority date: 2001-01-23
Filing date: 2002-01-18
Publication date: 2002-08-01
Also published as: WO2002058639A3

Abstract

The present invention relates to compounds derived from the plant Batocarpus costaricensis useful as estrogen receptor modulators. The compounds of the instant invention are ligands for estrogen receptors and as such may be useful for treatment or prevention of a variety of conditions related to estrogen functioning including: bone loss, bone fractures, osteoporosis, cartilage degeneration, endometriosis, uterine fibroid disease, hot flashes, increased levels of LDL cholesterol, cardiovascular disease, impairment of cognitive functioning, cerebral degenerative disorders, restinosis, gynacomastia, vascular smooth muscle cell proliferation, obesity, incontinence, and cancer, in particular of the breast, uterus and prostate.

Description

TITLE OF THE INVENTION

PYRANOFLAVONOID COMPOUNDS AND THEIR USE AS ESTROGEN

RECEPTOR MODULATORS

BACKGROUND OF THE INVENTION

Naturally occurring and synthetic estrogens have broad therapeutic utility, including: relief of menopausal symptoms, treatment of acne, treatment of dysmenorrhea and dysfunctional uterine bleeding, treatment of osteoporosis, treatment of hirsutism, treatment of prostatic cancer, treatment of hot flashes and prevention of cardiovascular disease. Because estrogen is very therapeutically valuable, there has been great interest in discovering compounds that mimic estrogen-like behavior in estrogen responsive tissues. Specifically, it has been found in the present invention that certain pyranoflavonoid-containing extracts from Batocarpus costaricensis, a tree indigenous to the Central American tropics, are useful as selective estrogen receptor beta (β) ligands, i.e. modulators.

For example, estrogen-like compounds would be beneficial in the treatment and prevention of bone loss. Bone loss occurs in a wide range of subjects, including women that are post-menopausal or have had a hysterectomy, patients who were or are currently being treated with corticosteroids, and patient's having gonadal dysgenesis. The current major bone diseases of public concern are osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget's disease, immobilization-induced osteopenia, and glucocorticoid-induced osteoporosis. All of these conditions are characterized by bone loss, resulting from an imbalance between bone resorption, i.e. breakdown, and bone formation, which continues throughout life at the rate of about 14% per year on the average. However, the rate of bone turnover differs from site to site, for example, it is higher in the trabecular bone of the vertebrae and the alveolar bone in the jaws than in the cortices of the long bones. The potential for bone loss is directly related to turnover and can amount to over 5% per year in vertebrae immediately following menopause, a condition which leads to increased fracture risk.

In the U.S., there are currently about 20 million people with detectable fractures of the vertebrae due to osteoporosis. In addition, there are about 250,000 hip fractures per year attributed to osteoporosis. This clinical situation is associated with a 12% mortality rate within the first two years, while 30% of the patients require nursing home care after the fracture.

Osteoporosis affects approximately 20 to 25 million post-menopausal women in the U.S. alone. It has been theorized that the rapid loss of bone mass in these women is due to the cessation of estrogen production of the ovaries. Since studies have shown that estrogen slows the reduction of bone mass due to osteoporosis, estrogen replacement therapy is a recognized treatment for post- menopausal osteoporosis.

In addition to bone mass, estrogen appears to have an effect on the biosynthesis of cholesterol and cardiovascular health. Statistically, the rate of occurrence of cardiovascular disease is roughly equal in postmenopausal women and men; however, premenopausal women have a much lower incidence of cardiovascular disease than men. Because postmenopausal women are estrogen deficient, it is believed that estrogen plays a beneficial role in preventing cardiovascular disease. The mechanism is not well understood, but evidence indicates that estrogen can upregulate the low density lipid (LDL) cholesterol receptors in the liver to remove excess cholesterol.

Postmenopausal women given estrogen replacement therapy experience a return of lipid levels to concentrations comparable to levels associated with the premenopausal state. Thus, estrogen replacement therapy could be an effective treatment for such disease. However, the side effects associated with long term estrogen use limit the use of this alternative.

Other disease states that affect postmenopausal women include estrogen-dependent breast cancer and uterine cancer. Anti-estrogen compounds, such as tamoxifen, have commonly been used as chemotherapy to treat breast cancer patients. Tamoxifen, a dual antagonist and agonist of estrogen receptors, is beneficial in treating estrogen-dependent breast cancer. However, treatment with tamoxifen is less than ideal because tamoxifen's agonist behavior enhances its unwanted estrogenic side effects. For example, tamoxifen and other compounds that agonize estrogen receptors tend to increase cancer cell production in the uterus. A better therapy for such cancers would be an anti-estrogen compound that has negligible or nonexistent agonist properties.

Although estrogen can be beneficial for treating pathologies such as bone loss, increased lipid levels, and cancer, long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine and endometrial cancers. These and other side effects of estrogen replacement therapy are not acceptable to many women, thus limiting its use.

Alternative regimens, such as a combined progestogen and estrogen dose, have been suggested in an attempt to lessen the risk of cancer. However, such regimens cause the patient to experience withdrawal bleeding, which is unacceptable to many older women. Furthermore, combining estrogen with progestogen reduces the beneficial cholesterol-lowering effect of estrogen therapy. In addition, the long term effects of progestogen treatment are unknown.

In addition to post-menopausal women, men suffering from prostatic cancer can also benefit from anti-estrogen compounds. Prostatic cancer is often endocrine-sensitive; androgen stimulation fosters tumor growth, while androgen suppression retards tumor growth. The administration of estrogen is helpful in the treatment and control of prostatic cancer because estrogen administration lowers the level of gonadotropin and, consequently, androgen levels.

The estrogen receptor has been found to have two forms: ERα and ERβ. Ligands bind differently to these two forms, and each form has a different tissue specificity to binding ligands. Thus, it is possible to have compounds that are selective for ERα or ERβ, and therefore confer a degree of tissue specificity to a particular ligand.

What is needed in the art are compounds that can produce the same positive responses as estrogen replacement therapy without the negative side effects. Also need are estrogen-like compounds that exert selective effects on different tissues of the body.

Lin, C.N. et al., Phytochemistry, 1992, 31, 2922 describes the isolation of three pyranoflavonoids from Artocarpus communis, i.e. compounds named cyclocommunol, cyclocommunin, and dihydroisocycloartomunin. However, there is no disclosure or suggestion of the use of any of these compounds as estrogen receptor modulators.

The compounds of the instant invention are ligands for estrogen receptors and as such may be useful for treatment or prevention of a variety of conditions related to estrogen functioning including: bone loss, bone fractures, osteoporosis, cartilage degeneration, endometriosis, uterine fibroid disease, hot flashes, increased levels of LDL cholesterol, cardiovascular disease, impairment of cognitive functioning, cerebral degenerative disorders, restinosis, gynacomastia, vascular smooth muscle cell proliferation, obesity, incontinence, and cancer, in particular of the breast, uterus and prostate. Specifically, the compounds of the present invention are obtained from an extract of the plant Batocarpus costaricensis, a plant indigenous to the Central American tropics. In particular, the compound cyclocommunol, 3 ,8 , 10-trihydroxy-6-(2-methyl- 1 -propenyl)-6H,7H- [l]benzopyrano[4,3-b]benzopyran-7-one, which is obtained from this plant source is useful as a selective estrogen receptor modulator.

SUMMARY OF THE INVENTION

The present invention relates to compounds isolated in crude or pure form from the plant Batocarpus costaricensis, useful as a selective estrogen receptor beta modulator. More particulary, the present invention relates to the compound cyclocommunol, 3,8,10-trihydroxy-6-(2-methyl-l-propenyl)-6H,7H- [l]benzopyrano[4,3-b]benzopyran-7-one, which is obtained from this plant source and which is useful as a selective estrogen receptor beta modulator.

The compound cyclocommunol has the following chemical structure.

Cyclocommunol

The present invention also relates to methods for isolating the compounds of the present invention.

The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.

The present invention also relates to methods for making the pharmaceutical compositions of the present invention.

The present invention also relates to methods for eliciting an estrogen receptor modulating effect in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention. The present invention also relates to methods for eliciting an estrogen receptor beta modulating effect in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.

The present invention also relates to methods for treating or preventing disorders related to estrogen functioning, bone loss, bone fractures, osteoporosis, cartilage degeneration, endometriosis, uterine fibroid disease, cancer of the breast, uterus or prostate, hot flashes, cardiovascular disease, impairment of cognitive function, cerebral degenerative disorders, restenosis, gynacomastia, vascular smooth muscle cell proliferation, obesity and incontinence in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.

The present invention also relates to methods for reducing bone loss, lowering LDL cholesterol levels and eliciting a vasodilatory effect, in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds isolated in crude or pure form from the plant Batocarpus costaricensis, useful as a selective beta agonist. More particulary, the present invention relates to the compound cyclocommunol, 3,8,10-trihydroxy-6-(2-methyl-l-propenyl)-6H,7H-[l]benzopyrano[4,3-b]benzopyran- 7-one, which is obtained from this plant source and which is useful as selective beta agonist.

The compound cyclocomunol has the following chemical structure.

Cyclocommunol

An embodiment of the invention is a method of eliciting an estrogen receptor modulating effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of any of the compounds or any of the above pharmaceutical compositions described above.

A second subclass of the embodiment is the method wherein the estrogen receptor is an ERβ receptor.

A second class of the embodiment is the method wherein the estrogen receptor modulating effect is an agonizing effect.

Another embodiment of the invention is a method of treating or preventing hot flashes in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.

Exemplifying the invention is a pharmaceutical composition comprising any of the compounds described above and a pharmaceutically acceptable carrier. Also exemplifying the invention is a pharmaceutical composition made by combining any of the compounds described above and a pharmaceutically acceptable carrier. An illustration of the invention is a process for making a pharmaceutical composition comprising combining any of the compounds described above and a pharmaceutically acceptable carrier.

Further exemplifying the invention is the use of any of the compounds described above in the preparation of a medicament for the treatment and/or prevention of osteoporosis in a mammal in need thereof. Still further exemplifying the invention is the use of any of the compounds described above in the preparation of a medicament for the treatment and/or prevention of: bone loss, bone resorption, bone fractures, cartilage degeneration, endometriosis, uterine fibroid disease, breast cancer, uterine cancer, prostate cancer, hot flashes, cardiovascular disease, impairment of congnitive functioning, cerebral degenerative disorder, restenosis, vascular smooth muscle cell proliferation, incontinence, and/or disorders related to estrogen functioning.

The present invention is also directed to combinations of any of the compounds or any of the pharmaceutical compositions described above with one or more additional pharmaceutical actives. For example, the compounds of the instant invention may be effectively administered in combination with effective amounts of other agents such as an organic bisphosphonate, and estrogen receptor modulators such as, e.g., raloxifene, tamoxifene, or iodoxifene. Nonlimiting examples of said organic bisphosphonates include alendronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, risedronate, piridronate, pamidronate, tiludronate, zoledronate, pharmaceutically acceptable salts or esters thereof, and mixtures thereof. Preferred organic bisphosphonates include alendronate and pharmaceutically acceptable salts and mixtures thereof. Most preferred is alendronate monosodium trihydrate.

For use in medicine, the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts." When the compounds of the present invention contain a basic group, salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.

The compounds of the present invention can have chiral centers and occur as racemates, racemic mixtures, diastereomeric mixtures, and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. Therefore, where a compound is chiral, the separate enantiomers, substantially free of the other, are included within the scope of the invention; further included are all mixtures of the two enantiomers. Also included within the scope of the invention are polymorphs, hydrates and solvates of the compounds of the instant invention.

The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds of this invention which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985, which is incorporated by reference herein in its entirety. Metabolites of these compounds include active species produced upon introduction of compounds of this invention into the biological milieu.

The term "therapeutically effective amount" shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician.

The term "bone resorption," as used herein, refers to the process by which osteoclasts degrade bone.

Representative compounds of the present invention typically display submicromolar affinity for the beta estrogen receptors. Compounds of this invention are therefore useful in treating mammals suffering from disorders related to estrogen functioning. Pharmacologically effective amounts of the compound, including the pharmaceutically effective salts thereof, are administered to the mammal, to treat disorders related to estrogen functioning, such as bone loss, hot flashes and cardiovascular disease.

The compounds of the present invention are available in racemic form or as individual enantiomers. For convenience, structures are graphically represented as a single enantiomer but, unless otherwise indicated, is meant to include both racemic and enantiomeric forms.

It is generally preferable to administer compounds as enantiomerically pure formulations since most or all of the desired bioactivity resides with a single enantiomer. Racemic mixtures can be separated into their individual enantiomers by any of a number of conventional methods. These include chiral chromatography, derivatization with a chiral auxiliary followed by separation by chromatography or crystallization, and fractional crystallization of diastereomeric salts.

The compounds of the present invention can be used in combination with other agents useful for treating estrogen-mediated conditions. The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating estrogen-mediated conditions includes in principle any combination with any pharmaceutical composition useful for treating disorders related to estrogen functioning.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The compounds of the present invention can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, topical (e.g., ocular eyedrop), subcutaneous, intramuscular or transdermal (e.g., patch) form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician, veterinarian or clinician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day. For oral administration, the compositions are preferably provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably, from about 1 mg to about 100 mg of active ingredient. Intravenously, the most preferred doses will range from about 0.1 to about 10 mg/kg/minute during a constant rate infusion. Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittant throughout the dosage regimen.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier materials") suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

The novel compounds of the present invention can be isolated according to the procedure of the following schemes and examples, using appropriate materials and are further exemplified by the following specific examples. The following examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted.

Isolation Procedure 1

The isolation of the compounds of the present invention proceeded from a crude methylene chloride extract prepared from the stems of Batocarpus costaricensis (Moraceae), a tree collected and extracted in Costa Rica. Approx. 1.29 gm of the extract was chromographed on an LH-20 column with methanol mobile phase to yield a 30 mg pool with potent beta-selective activity in the estrogen receptor ligand-binding assay. Fractionation of this 30 mg residue (3 x 10 mg) on a reverse phase preparative Zorbax RX-8 HPLC column employing a water (0.1 % TFA) / acetonitrile (0.1 % TFA) gradient gave 3.2 mg of a binary mixture containing 30% cyclocommunol and 70% 5'-methoxycyclocommunol. This binary mixture was resolved into the individual components, cyclocommunol and 5'- methoxycyclocommunol, by normal phase HPLC using an analytical Primesphere silica column and a hexane / ethyl acetate (linear gradient) mobile phase.

Isolation Procedure 2

Approx. 5.1 Kg (dry wt.) of Batocarpus costaricensis woody stem material (collected in Costa Rica) was extracted with 95 % ethanol to give 55.27 gm of dried residue. The residue was exhaustively extracted with acetone to give a cyclocommunol-enriched acetone soluble fraction. These acetone solubles were fractionated on a 5 cm x 90 cm (1.75 L) LH-20 column (x 3); the cyclocommunol- containing fractions were pooled to give a total of 360 mg residue. The residue was repetitively fractionated (30 mg x 12 runs) on a Zorbax RX-8 preparative HPLC column using a TFA-acidified water: acetonitrile linear gradient to give 21 mg of pure cyclocommunol.

In other alternative isolatation methods an organic solvent selected from the group consisting of C1-C4 straight or branched chain alcohols and C1-C2 di, tri- or tetra-chlorinated alkanes is used for extracting the material. Preferred solvents are ethanol and methylene chloride. In further steps the extracts are fractioned and the solvent removed.

NMR DATA FOR CYCLOCOMMUNOL (CϊriHi Oή):

The following lH and 13c NMR data were obtained in acetone-d6 on a VARIAN Spectrometer 500 MHz.

iHNMR (chemical shift, multiplicity)

1. 1.67 ppm, d

2. 1.92 ppm, d

3. 5.46 ppm, d

4. 6.16 ppm, d

5. 6.23 ppm, s

6. 6.41 ppm, s

7. 6.50 ppm, s

8. 6.60 ppm, dd

9. 7.66 ppm, d 10. 12.84 ppm, s

13QNMR (chemical shiff)

1. 18.592 ppm

2. 25.809 ppm

3. 70.256 ppm

4. 94.816 ppm 5. 99.721 ppm

6. 104.802 ppm

7. 105.416 ppm

8. 108.333 ppm

9. 109.834 ppm

10 110.729 ppm

11 122.125 ppm

12 126.198 ppm

13 138.686 ppm

14 156.403 ppm

15 158.031 ppm

16 158.954 ppm

17 163.267 ppm

18 163.951 ppm

19 164.620 ppm

20 179.018 ppm

Est rogen Rec eptor Binding Assav

The estrogen receptor ligand binding assays are designed as scintillation proximity assays employing the use of tritiated estradiol and recombinant expressed estrogen receptors. The full length recombinant human ER-α and ER-β proteins are produced in a bacculoviral expression system. ER-α or ER-β extracts are diluted 1:400 in phosphate buffered saline containing 6 mM α-monothiolgiycerol. 200 μL aliquots of the diluted receptor preparation are added to each well of a 96-well Flashplate. Plates are covered with Saran Wrap and incubated at 4° C overnight.

The following morning, a 20 μl aliquot of phosphate buffered saline containing 10% bovine serum albumin is added to each well of the 96 well plate and allowed to incubate at 4° C for 2 hours. Then the plates are washed with 200 μl of buffer containing 20 mM Tris (pH 7.2), 1 mM EDTA, 10% Glycerol, 50 mM KC1, and 6 mM α-monothiolglycerol. To set up the assay in these receptor coated plates, add 178 μl of the same buffer to each well of the 96 well plate. Then add 20 μl of a 10 nM solution of 3H-estradiol to each well of the plate.

Test compounds are evaluated over a range of concentrations from 0.01 nM to 1000 nM. The test compound stock solutions should be made in 100% DMSO at 100X the final concentration desired for testing in the assay. The amount of DMSO in the test wells of the 96 well plate should not exceed 1%. The final addition to the assay plate is a 2 μl aliquot of the test compound which has been made up in 100% DMSO. Seal the plates and allow them to equilibrate at room temperature for 3 hours. Count the plates in a scintillation counter equipped for counting 96 well plates. Samples of the extracts of the present invention are evaluated for their activity in these assays.

Pharmaceutical Composition

As a specific embodiment of this invention, 25 mg of a compound of the present invention is formulated with sufficient finely divided lactose to provide a total amound of 580 to 590 mg to fill a size 0, hard-gelatin capsule.

Claims

WHAT IS CLAIMED IS:

1. A compound or mixtures of compounds isolated from Batocarpus costaricensis.

2. A compound or mixtures of compounds according to Claim 1 in purified form.

3. A compound according to Claim 2 corresponding to the chemical structure .

and salts thereof, esters thereof, and mixtures thereof.

4. A method for isolating a compound corresopnding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof;

comprising the step of extracting a sample of Batocarpus costaricensis with an organic solvent.

5. A method according to Claim 4 wherein said organic solvent is selected from the group consisting of C1-C4 straight or branched chain alcohols and C1-C2 di-, tri- or tetra-chlorinated alkanes.

6. A method according to Claim 4 wherein said organic solvent is ethanol.

7. A method according to Claim 4 wherein said organic solvent is methylene chloride.

8. A method according to Claim 6 comprising the further step of removing the solvent.

9. A method according to Claim 7 comprising the further step of removing the solvent.

10. A pharmaceutical composition comprising a compound according to Claim 1 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition made by combining a compound according to Claim 1 and a pharmaceutically acceptable carrier.

12. A process for making a pharmaceutical composition comprising combining a compound according to Claim 1 and a pharmaceutically acceptable carrier.

13. A method of eliciting an estrogen receptor modulating effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound according to Claim 1.

14. A method of eliciting an estrogen receptor modulating effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound according to Claim 3.

15. The method according to Claim 14 wherein the estrogen receptor modulation effect is an estrogen receptor agonizing effect.

16. The method according to Claim 15 wherein the estrogen receptor antagonizing effect is an ERβ receptor agonizing effect.

17. A method of treating or preventing hot flashes in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound according to Claim 1.

18. A method of inhibiting bone resorption in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound according to Claim 1.

19. A method of treating osteoporosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound according to Claim 1.

20. A method of preventing osteoporosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound according to Claim 1.

21. A pharmaceutical composition comprising a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition made by combining a compound corresponding to the chemical structure

23. A process for making a pharmaceutical composition comprising combining a compound corresponding to the chemical structure

24. A method of eliciting an estrogen receptor modulating effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof.

25. The method according to Claim 24 wherein the estrogen receptor modulation effect is an estrogen receptor agonizing effect.

26. The method according to Claim 25 wherein the estrogen receptor antagonizing effect is an ERβ receptor agonizing effect.

27. A method of treating or preventing hot flashes in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof.

28. A method of inhibiting bone resorption in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof.

29. A method of treating osteoporosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof.

30. A method of preventing osteoporosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a compound corresponding to the chemical structure

and salts thereof, esters thereof, and mixtures thereof.