CN1946707A - Dibenzo chromene derivatives and their use as ERbeta selective ligands - Google Patents

Abstract

This invention provides estrogen receptor modulators of fomula (I), having the structure where R<1>, R<2>, R<3>, R<4>, R<5>, R<6>, R<7>, and R<8> are as defined in the specification.

Description

Dibenzochromene derivatives and their use as ERβ selective ligands

Cross References to Related Applications

This application claims priority to U.S. Application No. ______, filed February 23, 2005, which claims the benefit of U.S. Application 60/547,967, filed February 26, 2004, the disclosures of both applications being incorporated herein by reference in their entirety refer to.

technical field

The present invention relates to novel substituted 5H-dibenzo[c,g]chromene derivatives, their use as estrogen preparations and methods for their preparation.

Background technique

The pleiotropic effects of estrogen in mammalian tissues have been documented, and estrogen is now found to affect many organ systems [Mendelsohn and Karas, New England Journal of Medicine 340:1801-1811 (1999), Epperson et al., Psychosomatic Medicine 61: 676-697 (1999), Crandall, Journal of Womes Health & Gender Based Medicine 8:1155-1166 (1999), Monk and Brodaty, Dementia & Geriatric Cognitive Disorders 11:1-10 (2000), Hurn and Macrae, Journal February Blood of Cerebral & Metabolism 20: 631-652 (2000), Calvin, Maturitas 34: 195-210 (2000), Finking et al., Zeitschrift fur Kardiologie 89: 442-453 (2000), Brincat, Maturitas 35: 107-117 (2000) , Al-Azzawi, Postgraduate Medical Journal 77: 292-304 (2001)]. There are several ways estrogen acts on tissues. Probably the most characteristic mechanism of action is its interaction with estrogen receptors, resulting in changes in gene transcription. Estrogen receptors are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Other members of this family include progesterone, androgen, glucocorticoid, and mineralocorticoid receptors. Upon ligand binding, these receptors dimerize and activate gene transcription either directly by binding to specific sequences on DNA (called response elements) or through interaction with other transcription factors such as AP1. The latter directly binds to specific sequences on DNA [Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall et al., Journal of Biological Chemistry 276: 36869-36872 (2001), McDonnell, Principles of Molecular Regulation, pp. 351-361 (2000)]. A class of "co-regulatory" proteins can also interact with ligand-binding receptors to further modulate their transcriptional activity [McKenna et al., Endocrine Reviews 20:321-344 (1999)]. Estrogen receptors have been shown to inhibit NFκB-mediated transcription in a ligand-dependent and ligand-independent manner [Quaedackers et al., Endocrinology 142: 1156-1166 (2001), Bhat et al., Journal of Steroid Biochemistry & Molecular Biology 67:233-240 (1998), Pelzer et al., Biochemical & Biophysical Research Communications 286:1153-7 (2001)].

Estrogen receptors can also be activated by phosphorylation. This phosphorylation is mediated by growth factors such as EGF, causing changes in gene transcription in the absence of ligand [Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall et al., Journal of Biological Chemistry 276: 36869-36872 (2001)].

Another, less specific way in which estrogen affects cells is through so-called membrane receptors. The existence of this receptor is controversial, but estrogen has been reported to elicit a very rapid non-genomic response from cells. The molecular nature of the transduction of these effects is unclear, but there is evidence that it is at least related to the nuclear form of the estrogen receptor [Levin, Journal of Applied Physiology 91:1860-1867 (2001), Levin, Trends in Endocrinology & Metabolism 10 : 374-377 (1999)].

Two types of estrogen receptors have been discovered so far. The first estrogen receptor was cloned about 15 years ago and is now called ERα [Green et al., Nature 320:134-9 (1986)]. A second receptor was discovered later and is called ERβ [Kuiper et al., Proceedings of the National Academy of Sciences of the United States of America 93:5925-5930 (1996)]. Early studies on ERβ focused on determining its affinity for various ligands, which was found to be different from ERα. The tissue distribution of Erβ in rodents has been mapped, inconsistently with that of ERα. For example, the expression of ERα in the uterus of mice and rats is dominant, while the expression of ERβ in the lungs of mice and rats is dominant [Couse et al., Endocrinology 138: 4613-4621 (1997), Kuiper et al., Endocrinology 138 : 863-870 (1997)]. Expression of ERα and Erβ is compartmentalized even in the same organ. For example, in the mouse ovary, Erβ is highly expressed in granulosa cells, while ERα is restricted to follicular endometrial cells and mesenchymal cells [Sar and Welsch, Endocrinology 140:963-971 (1999), Fitzpatrick et al., Endocrinology 140: 2581-2591 (1999)]. However, there is evidence that the receptors can be co-expressed and there is evidence from in vitro studies that ERα and Erβ can form heterodimers [Cowley et al., Journal of Biological Chemistry 272: 19858-19862 (1997)].

The most potent endogenous estrogen is 17β-estradiol. A large number of compounds have been described that mimic or block the activity of 17[beta]-estradiol. Compounds having approximately the same biological effects as 17[beta]-estradiol are referred to as "estrogen receptor agonists". Compounds that block the effects of 17[beta] estradiol when bound to it are known as "estrogen receptor antagonists". Indeed, there is a continuum between estrogen receptor agonist and estrogen receptor antagonist activity, with some compounds acting as estrogen receptor agonists in some tissues but estrogen receptors in others. Hormone receptor antagonists. These compounds with mixed activity are called selective estrogen receptor modulators (SERMS) and are very useful therapeutically (e.g. EVISTA) [McDonnell, Journal of the Society for Gynecologic Investigation 7: S10-S15 (2000), Goldstein et al., Human Reproduction Update 6:212-224 (2000)]. The exact reason why the same compounds have cell-specific effects has not been elucidated, but may be due to differences in receptor conformation and/or the environment surrounding coregulatory proteins.

It has been known for some time that the estrogen receptor adopts different conformations when bound to a ligand. But the consequences and nuances of these changes have only recently been revealed. The three-dimensional structures of ERα and ERβ have been resolved by co-crystallization with various ligands, clarifying the orientation of helix 12 in the presence of estrogen receptor antagonists (sterically hindering the protein sequence required for receptor-co-regulatory protein interactions). Recast [Pike et al., Embo 18:4608-4618 (1999), Shiau et al., Cell 95:927-937 (1998)]. In addition, phage display technology has been used to identify peptides that interact with the estrogen receptor in the presence of different ligands [Paige et al., Proceedings of the National Academy of Sciences of the United States of America 96:3999-4004 (1999)] . For example, peptides distinct from ERα that bind to the full estrogen receptor agonist 17[beta]-estradiol and diethylstilbestrol were identified. Another peptide differs from Chlorazol which binds to ERα and ERβ. These data suggest that each ligand can cause the receptor to assume a unique, unpredictable conformation with distinct biological activities.

As mentioned above, estrogens affect a variety of biological processes. Furthermore, where sex differences are involved (eg, morbidity, response to stress, etc.), their explanation may involve differences in estrogen levels between males and females.

Summary of the invention

The present invention relates to a compound of the following formula or a pharmaceutically acceptable salt thereof:

in

R ¹ , R ² , R ³ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently selected from hydrogen, hydroxyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or halogen;

R ⁴ is hydrogen, C ₁ -C ₆ alkyl, halogen, C ₁ -C ₆ alkoxy, -CN, -C ₂ -C ₈ alkenyl, -CHO, aryl, furyl, thienyl, pyrimidine base and pyridyl;

with the proviso that at least one of R ¹ -R ⁸ is not H.

In another aspect, the present invention relates to pharmaceutical compositions comprising at least one compound as described above.

In another aspect, the present invention relates to the use of the above compounds in the treatment or prevention of diseases.

Detailed description of the invention

The present invention provides compounds of formula I.

in

R ¹ , R ² , R ³ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently selected from hydrogen, hydroxyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or halogen;

R ⁴ is hydrogen, C ₁ -C ₆ alkyl, halogen, C ₁ -C ₆ alkoxy, -CN, -C ₂ -C ₈ alkenyl, -CHO, aryl, furyl, thienyl, pyrimidine base and pyridyl;

with the proviso that at least one of ^R1 - ^R8 is not H;

In some embodiments, aryl is optionally substituted phenyl or naphthyl. In some embodiments, R ⁵ , R ⁷ and R ⁸ are preferably each independently hydrogen or halogen; R ⁴ is hydrogen, an alkyl group containing 1-6 carbon atoms, an alkoxy group containing 1-6 carbon atoms , halogen, alkenyl having 2-8 carbon atoms, -CN, furyl, thienyl or pyridyl. In some preferred embodiments, R ⁵ , R ⁷ and R ⁸ are each independently hydrogen or halogen; R ⁴ is hydrogen, alkyl containing 1-6 carbon atoms, alkoxy containing 1-6 carbon atoms , halogen, alkenyl containing 2-7 carbon atoms, -CN, furyl, thienyl or pyridyl; and R ¹ , R ² , R ³ and R ⁶ are each independently hydrogen, halogen or hydroxyl. Most preferably, in some embodiments, R ⁵ , R ⁷ and R ⁸ are each independently hydrogen or halogen; R ⁴ is hydrogen, alkyl containing 1-6 carbon atoms, alkyl containing 1-6 carbon atoms Alkoxy, halogen, alkenyl containing 2-7 carbon atoms, -CN, furyl, thienyl or pyridyl; R ¹ , R ² , R ³ and R ⁶ are each independently hydrogen, halogen or hydroxyl ; and at least one of R ¹ , R ² , R ³ and R ⁶ is a hydroxyl group.

When the compound of the present invention contains a basic moiety, pharmaceutically acceptable salts can be formed from organic or inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic , mandelic acid, malic acid, phthalic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphorsulfonic acid, and similar Acceptable acid formation is known. When the compounds of the present invention contain acidic moieties, salts can also be formed from organic and inorganic bases, such as alkali metal salts (for example, sodium, lithium or potassium), alkaline earth metal salts, ammonium salts, alkyl groups containing 1-6 carbon atoms ammonium salts or dialkylammonium salts containing 1 to 6 carbon atoms in each alkyl group, and trialkylammonium salts containing 1 to 6 carbon atoms in each alkyl group.

As used herein, the term "alkyl", whether used alone or as part of another group (e.g. alkoxy, aralkyl, alkoxycarbonyl), unless expressly stated otherwise, refers to substituted or unsubstituted Aliphatic hydrocarbon chains, which include, but are not limited to, straight and branched chains containing 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl are included in the term "alkyl". Specifically included within the definition of "alkyl" are those optionally substituted aliphatic hydrocarbon chains. The carbon number used in the definitions herein refers to carbon skeleton and carbon branch, but does not include carbon atoms of substituents (such as alkoxy substitution, etc.).

The term "alkenyl" as used herein, whether used alone or as part of another group, refers to substituted or unsubstituted aliphatic hydrocarbon chains, including but not limited to containing 2 to 8 carbon atoms and containing Straight and branched chains with at least one double bond. Preferably, the alkenyl moiety contains 1 or 2 double bonds. Such alkenyl moieties can exist in the E or Z configuration, and the compounds of the invention include both configurations. Specifically included within the definition of "alkenyl" are those aliphatic hydrocarbon chains which are optionally substituted. A heteroatom (eg O, S or NR ₁ ) attached to an alkenyl group should not be attached to a carbon atom to which a double bond is bonded.

"Aryl" as used herein as a group or part of a group refers to an optionally substituted aromatic 5- to 13-membered mono- or bis-carbocyclic ring, such as phenyl or naphthyl. Preferably, groups containing an aryl moiety are monocyclic rings containing 5 to 7 carbon atoms in the ring. Phenyl is a preferred aryl group. In some embodiments, the phenyl moiety is optionally replaced by C ₁ -C ₆ alkyl, C ₂ -C ₇ alkenyl, halogen, hydroxy, C ₁ -C ₆ alkoxy, -CN, -NO ₂ , amino , C ₁ -C ₆ alkylamino, dialkylamino each containing 1-6 carbon atoms, sulfur, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkylsulfinyl, C ₁ -C ₆ alkylsulfonyl, C ₂ -C ₇ alkoxycarbonyl containing 2-7 carbon atoms, alkylcarbonyl containing 2-7 carbon atoms, trifluoroalkoxy, benzylnitrile or benzene Formyl substitution.

Heteroaryl as a group or part of a group refers to an aromatic 5- to 13-membered mono- or bicarbocyclic ring having one to five heteroatoms (independently mono, oxygen or sulfur). Preferably, the heteroaryl moiety-containing group is a monocyclic ring having 5 to 7 members in the ring, wherein one to two ring members are independently selected from nitrogen, oxygen or sulfur. Groups containing an aryl or heteroaryl moiety are optionally substituted or unsubstituted as described below. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl , pyridazinyl, indolyl, indazolyl, benzofuryl, isobenzofuryl, benzothienyl, isobenzothienyl, quinolinyl, isoquinolyl, quinoxalinyl, or Quinazolinyl.

The term "halogen" includes bromo, chloro, fluoro and iodo.

Optionally substituted substituents described herein, eg, alkyl, alkenyl, aryl, or heteroaryl, may be substituted with one or more (eg, 1-5 or 1-3) substituents. Suitable optional substituents may be independently selected from nitro, cyano, -N(R ₁₁ )(R ₁₂ ), halogen, hydroxy, carboxy, alkyl, alkynyl (e.g. containing 2-7 carbon atoms) , cycloalkyl (for example, containing 5-8 carbon atoms), aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylalkoxy, alkoxycarboxyl, alkoxy Alkoxy, perfluoroalkyl, perfluoroalkoxy, aralkyl, alkaryl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, -S (O) ₂ -N(R ₁₁ )(R ₁₂ ), -C(=O)-N(R ₁₁ )(R ₁₂ ), (R ₁₁ )(R ₁₂ )N-alkyl, (R ₁₁ )( R ₁₂ ) N-alkoxyalkyl, (R ₁₁ ) (R ₁₂ ) N-alkylaryloxyalkyl, -S(O) _s -aryl (wherein s=0-2) or -S( O) _s -heteroaryl (wherein s=0-2); wherein R ₁₁ and R ₁₂ are each independently hydrogen, unsubstituted (C ₁ -C ₆ ) alkyl, unsubstituted (C ₃ -C ₇ )cycloalkyl, aryl, aryl-(C ₁ -C ₃ )alkyl, alkoxy-(C ₁ -C ₃ )alkyl, arylthio-(C ₁ -C ₃ )alkyl, hetero Aryl, heteroaryl-(C ₁ -C ₃ )alkyl, heteroaryloxy-(C ₁ -C ₃ )alkyl or heteroarylthio-(C ₁ -C ₃ )alkyl; or if any They can be connected together in the form of an alkylene group to form a ring, such as a 3-8 membered ring. In certain embodiments of the invention, preferred substituents for alkyl, alkenyl, alkynyl or cycloalkyl include nitro, cyano, -N(R ₁₁ )(R ₁₂ ), halogen, hydroxyl , aryl, heteroaryl, alkoxy, alkoxyalkyl and alkoxycarboxyl. In certain embodiments of the invention, preferred substituents for aryl and heteroaryl include -N(R ₁₁ )(R ₁₂ ), alkyl, halogen, perfluoroalkyl, perfluoroalkoxy, Aralkyl and alkaryl. Examples of substituted alkyl and alkenyl moieties include 1-bromovinyl, 1-fluorovinyl, 1,2-difluorovinyl, 2,2-difluorovinyl, 1,2,2-trifluorovinyl Vinyl, 1,2-dibromoethane, 1,2-difluoroethane, 1-fluoro-2-bromoethane, CF ₂ CF ₃ , CF ₂ CF ₂ CF ₃ and the like.

The term "lower alkyl" refers to an alkyl group containing 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms in some embodiments.

"Alkoxy" as used herein refers to the group R-O-, wherein R is an alkyl group as described above. As used herein, the term "lower alkoxy" refers to the group R-O-, wherein R is an alkyl group containing 1 to 6 carbon atoms. In some embodiments, R containing 1 to 3 carbon atoms is preferred.

As used herein, the term "providing" with respect to providing a compound or substance encompassed by the present invention means administering such compound or substance directly, or administering a prodrug, derivative, or analog.

The compounds of the present invention are useful as estrogen receptor modulators for the treatment or suppression of signs, disorders or conditions mediated at least in part by estrogen deficiency or excess or which are treatable or suppressable with estrogen preparations. The compounds of the present invention are especially useful in the treatment of perimenopausal, menopausal or postmenopausal patients in whom the level of endogenous estrogen produced in the body is greatly reduced. Menopause, usually defined as the last natural menstrual period, is characterized by the cessation of ovarian function, resulting in a marked decrease in circulating estrogen in the bloodstream. As used herein, menopause also includes a state of reduced estrogen production, which may be caused by surgery, chemistry, or diseases that result in a reduction or cessation of ovarian function before maturation.

Accordingly, the compounds of the present invention are useful in the treatment or inhibition of osteoporosis and in inhibiting bone demineralization which may result from a disturbance in the independent formation of new bone tissue and resorption of old tissue, resulting in a net loss of bone. This bone loss occurs in many individuals, especially in postmenopausal women, women with bilateral oophorectomy, individuals receiving or who have received long-term corticosteroid therapy, individuals with gonadal dysgenesis, and individuals with Cushing's syndrome. The compounds may also be used to address specific needs for bone (including dental and oral bone) replacement in individuals with fractures, defective bone structure, and in individuals undergoing bone-related surgery and/or implantation of prostheses. In addition to the above-mentioned problems, these compounds are useful for the treatment or inhibition of osteoarthritis, hypocalcemia, hypercalcemia, Paget's disease, osteomalacia, osteopenia, multiple myeloma, and other forms of cancer.

The compounds of the present invention are also useful in the treatment or inhibition of benign or malignant abnormal tissue growth including prostatic hypertrophy, uterine leiomyoma, breast cancer, endometriosis, endometrial cancer, polycystic ovary syndrome, Endometrial polyps, benign breast disease, adenomysis, ovarian cancer, melanoma, prostate cancer, colon cancer, CNS cancer such as glioma or astioblastomia.

The compounds of the present invention are cardioprotective and can be used to lower cholesterol, triglycerides, Lp(a) and LDL levels; inhibit or treat hypercholesterolemia, hyperlipidemia, cardiovascular disease, atherosclerosis, peripheral Vascular disease, restenosis, and vasospasm; inhibits immune-mediated vascular injury resulting from cellular event-induced damage to the vessel wall. These cardiovascular protective properties are important when treating postmenopausal patients with estrogen to suppress osteoporosis and when estrogen therapy is performed in men.

The compounds of the present invention are also antioxidants and are therefore useful in the treatment or inhibition of free radical induced conditions. Specific conditions for antioxidant therapy are cancer, central nervous system disorders, Alzheimer's disease, bone disease, aging, inflammation, peripheral vascular disease, rheumatoid arthritis, autoimmune disease, respiratory distress, emphysema , Protection against reperfusion injury, viral hepatitis, chronic active hepatitis, tuberculosis, psoriasis, systemic lupus erythematosus, adult respiratory distress syndrome, central nervous system injury and stroke.

The compounds of the present invention are also useful for providing cognitive enhancement, and treating or inhibiting senile dementia, Alzheimer's disease, cognitive decline, neurodegenerative diseases, providing neuroprotection or cognitive enhancement.

The compounds of the present invention are also useful in the treatment or inhibition of inflammatory bowel disease, ulcerative proctitis, Crohn's disease and colitis; symptoms associated with menopause, such as vasomotor syndrome, including hot flashes, vaginal or Inflammation, vaginal dryness, itching, dyspareunia, dysuria, urinary frequency, urinary incontinence, urinary tract infection, vasomotor syndrome, including hot flushes, myalgia, arthralgia, insomnia, allergies, etc.; male pattern hair loss; skin atrophy; acne ; Type II diabetes; dysfunctional uterine bleeding and infertility.

The compounds of the invention are useful in conditions where amenorrhea is beneficial, such as leukemia, endometrial ablation, chronic kidney and liver disease or coagulation diseases or disorders.

The compounds of the invention are useful as contraceptive preparations, especially in combination with progesterone.

It is to be understood that when administered for the treatment or prophylaxis of a particular condition or disorder, effective dosages will depend on the particular compound employed, the mode of administration, the symptoms and their severity, the symptoms being treated, and various physical factors associated with the individual being treated. rather different. The compounds of this invention may be administered orally at an effective dose of about 0.1 mg/day to about 1,000 mg/day. Preferably the administration is from about 10 mg/day to about 600 mg/day, more preferably from about 50 mg/day to about 600 mg/day, in a single dose or in two or more divided doses. The expected planned daily dosage will vary with the route of administration.

These doses may be administered by any means that introduces the active compounds of this invention into the recipient's bloodstream, including orally, via an implant, parenterally (including intravenous, intraperitoneal, and subcutaneous injection), rectally, intranasally. , vaginal and transdermal.

Oral formulations containing the active compounds of this invention may include any conventionally used oral dosage forms, including tablets, capsules, troches, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain one or more active compounds in admixture with inert fillers and/or diluents, such as pharmaceutically acceptable starches (for example, corn starch, potato starch, or tapioca starch), sugars, and/or diluents. , artificial sweeteners, powdered cellulose (such as crystalline cellulose and microcrystalline cellulose), flour, gelatin, gums, etc. The tablet preparations used can be carried out by conventional compression, wet granulation or dry granulation, and use pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), Suspending agents or stabilizers, which include but are not limited to magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carmellose calcium, polyvinylpyrrolidone, gelatin, brown algae Acid, Gum Arabic, Xanthan Gum, Sodium Citrate, Complex Silicates, Calcium Carbonate, Glycine, Dextrin, Sucrose, Sorbitol, Dicalcium Phosphate, Calcium Sulfate, Lactose, Kaolin, Mannitol, Sodium Chloride, Talc, dry starch and powdered sugar. Preferred surface modifiers include nonionic and anionic surface modifiers. Representative examples of surface modifiers include, but are not limited to, Poloxamer 188, Benzalkonium Chloride, Calcium Stearate, Cetostearyl Alcohol, Cecilitol Emulsifying Wax, Sorbitan Ester, Colloidal Silica, Phosphate, Sodium Lauryl Sulfate, Magnesium Aluminum Silicate, and Triethanolamine. Oral formulations of the invention may utilize standard sustained or timed release formulations to alter the absorption of the active compounds. Oral formulations may also consist of administering the active ingredient in water or fruit juice containing suitable solubilizers or emulsifiers as required.

In some cases, the compound may be administered directly into the respiratory tract in aerosol form.

The compounds of the invention may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as free base or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutically acceptable forms suitable for injection include sterile aqueous solutions or dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under ordinary conditions of manufacture and storage and must be resistant to the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

For purposes of this disclosure, transdermal administration is considered to include all administrations across the body surface and linings of bodily passages, including epithelial and mucosal tissues. Such administrations can be carried out using the compound, or a pharmaceutically acceptable salt thereof, in emulsions, creams, foams, patches, suspensions, solutions and suppositories (rectal and vaginal).

Transdermal administration may utilize a transdermal patch comprising the active compound and a carrier that is inert to the active compound, nontoxic to the skin, and enables systemic absorption of the formulation through the skin into the bloodstream. Carriers may take any form, such as creams and ointments, pastes, gels and occlusive devices. These creams and ointments may be viscous liquids or semisolid emulsions of the oil-in-water or water-in-oil type. Pastes comprising absorbent powder dispersed in petroleum or hydrophilic petroleum containing the active ingredient are also suitable. Various occlusal devices may be used to release the active ingredient into the bloodstream, such as an active ingredient-containing depot covered with a semipermeable membrane, with or without a carrier, or an active ingredient-containing matrix. Other articulation arrangements are known in the literature.

Suppositories may be made from traditional materials, including cocoa butter, waxes with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

The reagents used in the preparation of the compounds of the invention are either commercially available or can be prepared by standard methods described in the literature.

The preparation of some representative examples of this invention is described in the following Reaction Schemes 1-6.

Reaction Scheme 1

Reaction Scheme 2

Reaction Scheme 3

Reaction scheme 4

Reaction scheme 5

Reaction Scheme 6

X=phenyl Example 23

X＝2-Methylphenyl Example 24

X＝3-Methylphenyl Example 25

X=4-methylphenyl Example 26

X＝4-Methoxyphenyl Example 27

X＝4-Chlorophenyl Example 28

X = 4-fluorophenyl Example 29

X＝2-thiophene Example 30

X＝3-thiophene Example 31

X = 3-fluorophenyl Example 32

X＝3-Chlorophenyl Example 33

X＝3-Methoxyphenyl Example 34

X＝2-Chlorophenyl Example 35

X=3,4-Difluorophenyl Example 36

X＝4-pyridyl Example 37

X = 2-fluorophenyl Example 38

X=3,4-Dimethylphenyl Example 39

X＝4-cyanophenyl Example 40

X = 3-fluoro-4-methylphenyl Example 41

X=3,4-Dimethoxyphenyl Example 42

X=3-trifluoromethylphenyl Example 43

X＝3,5-Difluorophenyl Example 44

X＝3,5-Dichlorophenyl Example 45

X = 3-methyl-4-fluorophenyl Example 46

X＝3-furyl Example 47

Reaction Scheme 7

The invention will now be illustrated with reference to the following specific, non-limiting examples.

Example 1

                                                                           

The title compound was prepared by reacting 3-methoxybenzyl alcohol (13.82 g, 0.1 mol) and NBS (19.58 g, 0.11 mol) in acetonitrile (250 mL) at room temperature for 3 hours. The solvent was removed and the resulting material was slurried in dichloromethane (250 mL) and filtered to remove insoluble succinimide by-product. The crude material was purified by chromatography (20% EtOAc-hexanes) to afford a white solid (17.69 g, 81%), mp 57-58°C.

Example 2

    2-[(2-Bromo-5-methoxybenzyl)oxy]-7-methoxynaphthalene

In the absence of To a solution in THF (500 mL) in water, a solution of DEAD (26.10 g, 0.15 mol) in THF (100 mL) was added dropwise over 0.5 h. The solution was stirred overnight at room temperature, and after evaporation of half the volume, a highly pure product precipitated. The solid was filtered and rinsed with THF, then dried to yield 32.96 g (59%) of a white solid: mp 156-157°C.

¹ H NMR (DMSO-d ₆ ): δ3.77 (3H, s), 3.86 (3H, s), 5.18 (2H, s), 6.92 (1H, dd, J=3.2Hz, J=8.7Hz), 7.00 (1H, dd, J=2.4Hz, J=8.7Hz), 7.09 (1H, dd, J=2.4Hz, J=8.9Hz), 7.22 (1H, d, J=3.2Hz), 7.25 (1H, d, J = 2.4Hz), 7.35 (1H, d, J = 2.4Hz), 7.59 (1H, d, J = 8.7Hz), 7.74 (1H, d, J = 8.7Hz), 7.76 (1H, d, J=8.7Hz); MS m/z 373/375 ([M+H]+).

Analysis of C ₁₉ H ₁₇ BrO ₃ :

Calculated: C: 61.14; H: 4.59

Measured value: C: 61.29; H: 4.21

Example 3

     3,9-Dimethoxy-5H-dibenzo[c,g]chromene

2-[(2-Bromo-5-methoxybenzyl)oxy]-7-methoxynaphthalene (9.35 g, 25 mmol), dichlorobis(triphenylphosphine)palladium (1.75 g, 2.5 mmol mol) and sodium acetate (6.15 g, 75 mmol) in anhydrous dimethylacetamide (500 mL) was stirred at 130°C under nitrogen for 2 days. After cooling, the catalyst was filtered off and the dimethylacetamide was removed in vacuo. The residue was slurried in methanol (200 mL) and the desired product was isolated by filtration, yielding 2.9 g (40%) of a tan solid: mp 190-191 °C;

¹ H NMR (DMSO-d ₆ ): δ3.81 (3H, s), 3.85 (3H, s), 5.14 (2H, s), 6.92 (1H, d, J=2.6Hz), 7.00-7.03 (2H , m), 7.18 (1H, d, J = 2.4Hz), 7.29 (1H, s), 7.79 (1H, d, J = 9.0Hz), 7.92 (1H, d, J = 8.7Hz), 8.26 (1H , s); MS m/z 293 ([M+H]+).

Analysis of C ₁₉ H ₁₆ O ₃ :

Calculated: C: 78.06; H: 5.52

Measured value: C: 77.95; H: 5.34

The above crystallization also produced an unwanted regioisomer, which was obtained from the methanol filtrate. The undesired regioisomer was not obtained in pure form and was demethylated as described for Example 4 below.

Example 4

        5H-Dibenzo[c,g]chromene-3,9-diol

To pyridinium hydrochloride salt (30 g, 0.26 mol) was added 3,9-dimethoxy-H-dibenzo[c,g]chromene (7.0 g, 23.9 mmol) at 190°C. The solution was stirred at 190°C for 3.5 hours, and the mixture was cooled to near room temperature. The mixture was poured into water (300 mL) and stirred to precipitate a solid. The solid was filtered, washed well with water and dried. Purification by chromatography (50% ethyl acetate-hexanes) yielded 5.0 g (79%) of a white solid: mp 231-233°C;

¹ H NMR (DMSO-d ₆ ): δ5.05 (2H, s), 6.68 (1H, d, J=2.3Hz), 6.83 (1H, dd, J=2.4Hz, J=8.4Hz), 6.90 ( 1H, dd, J=2.3Hz, J=8.7Hz), 6.93(1H, d, J=2.3Hz), 6.96(1H, d, J=2.1Hz), 7.13(1H, s), 7.71(1H, d, J = 8.8Hz), 7.78 (1H, d, J = 8.5Hz), 9.70 (2H, s); MS m/z 265 ([M+H]+).

Analysis of C ₁₇ H ₁₂ O ₃ :

Calculated: C: 77.26; H: 4.58

Measured value: C: 77.00; H: 4.31

Example 5

     5H-Dibenzo[c,f]chromene-3,11-diol 4

A mixture of other regioisomer and desbromo-coupling products was subjected to the same demethylation conditions as in Example 2. The crude product was purified by chromatography (50% ethyl acetate-hexanes) to give 0.45 g (1%) of a brown solid: mp 199-201 °C;

¹ H NMR (DMSO-d ₆ ): δ4.93 (2H, s), 6.83 (1H, d, J=2.5Hz), 6.89 (1H, dd, J=2.5Hz, J=8.4Hz), 6.95- 6.99(2H, m), 7.63(1H, d, J=8.7Hz), 7.73-7.81(3H, m), 9.75(2H, s); MS m/z 265([M+H]+).

HPLC at 254 nm indicated a purity of 98.9%.

Analysis of C ₁₇ H ₁₂ O ₃ ·0.2H ₂ O:

Calculated: C: 76.22; H: 4.67

Measured value: C: 76.43; H: 4.49

Example 6

     8-Chloro-5H-dibenzo[c,g]chromene-3,9-diol

H-Dibenzo[c,g]chromene-3,9-diol (0.20 g, 0.76 mmol) and N-chlorosuccinimide (0.12 g, 0.91 mmol) were dissolved in THF (20 mL) The solution in was stirred at room temperature for 48 hours. The reaction solution was concentrated onto Florosil and purified by silica chromatography (30% ethyl acetate-hexanes) to yield 0.14 g (62%) of a tan solid. This material was further purified by reverse phase preparative HPLC to yield the title compound as an off-white solid: mp 212-214 °C;

¹ H NMR (DMSO-d ₆ ): δ5.12 (2H, s), 6.70 (1H, d, J=2.37Hz), 6.85 (1H, dd, J=2.43Hz, J=8.46Hz), 7.13( 1H, d, J = 9.06Hz), 7.39 (1H, s), 7.73 (1H, d, J = 8.91Hz), 7.82 (1H, d, J = 8.53Hz), 8.24 (1H, s), 9.81 ( 1H, s), 10.39 (1H, s); MS (ESI) m/z 297/299 ([MH]-).

Analysis of C ₁₇ H ₁₁ ClO ₃ :

Calculated: C: 68.35; H: 3.71

Measured value: C: 68.17; H: 3.65

Example 7

  3-(Acetoxy)-5H-dibenzo[c,g]chromen-9-yl acetate

To a mixture of pyridine (35 mL) and acetic anhydride (35 mL) was added (H-dibenzo[c,g]chromene-3,9-diol) (5.0 g, 18.9 mmol), and the mixture Stir at room temperature. After about 1 hour a precipitate formed and stirring was continued for a further 5 hours. The mixture was filtered and the product was washed with ethyl acetate. The crude product was purified by chromatography (25% ethyl acetate-hexanes) to give a white solid (5.0 g, 76%): mp 194-195 °C;

¹ H NMR (DMSO-d ₆ ): δ2.31 (3H, s), 2.33 (3H, s), 5.20 (2H, s), 7.16 (1H, d, J=1.9Hz), 7.19 (1H, dd , J=2.4Hz, J=8.8Hz), 7.25(1H, dd, J=2.5Hz, J=8.3Hz), 7.44(1H, s), 7.54(1H, d, J=2.4Hz), 7.95( 1H, d, J=9.3Hz), 8.09(1H, d, J=8.3Hz), 8.50(1H, s); MS m/z 349([M+H]+).

Analysis of C ₂₁ H ₁₆ O ₅ :

Calculated: C: 72.41; H: 4.63

Measured value: C: 72.06; H: 4.48

Example 8

  3-(Acetoxy)-7-bromo-5H-dibenzo[c,g]chromen-9-yl acetate

To a mixture of N-bromosuccinimide (3.1 g, 17.2 mmol) in anhydrous acetonitrile (400 mL) was added 3-(acetoxy)-H-dibenzo[c,g]chromene-9 -Ethylacetate (5.0 g, 14.4 mmol). The reaction mixture was stirred overnight at room temperature. The solid was filtered, washed with acetonitrile and dried to yield 5.9 g (96%) of a beige solid. A sample of this solid was purified by chromatography (dichloromethane) to yield a white solid: mp 224-226°C;

¹ H NMR (DMSO-d ₆ ): δ2.32 (3H, s), 2.35 (3H, s), 5.35 (2H, s), 7.19 (1H, d, J=2.4Hz), 7.28 (1H, dd , J=2.4Hz, J=8.3Hz), 7.31 (1H, dd, J=2.0Hz, J=8.8Hz), 7.75 (1H, d, J=2.4Hz), 8.05 (1H, d, J=8.8 Hz), 8.12(1H, d, J=8.8Hz), 8.59(1H, s); MS m/z 427/429([M+H]+).

Analysis of C ₂₁ H ₁₅ BrO ₅ :

Calculated: C: 59.04; H: 3.54

Measured value: C: 58.87; H: 3.34

Example 9

                                                                         

To a solution of 7-methoxy-2-naphthol in acetonitrile (100 mL) was added N-chlorosuccinimide (1.61 g, 12.07 mmol) at ice bath temperature. The reaction mixture was allowed to stir at ice bath temperature for 2 hours, and an additional 0.1 equivalent of N-chlorosuccinimide was added. The reaction was allowed to warm to room temperature over 1 hour and poured into water (180 mL), extracted with ethyl acetate (2 x 300 mL). The organic layer was washed with brine and dried over anhydrous magnesium sulfate, filtered and the solvent was removed under vacuum. Ether was added and insoluble impurities were filtered. The mother liquor was concentrated and chromatographed (1:4, ethyl acetate-hexanes) to yield a light yellow solid: mp 76-78°C. HPLC indicated a product purity of 98.0% (230 nm).

Analysis of C ₁₁ H ₉ ClO ₂ :

Calculated: C, 63.32 H, 4.35

Found: C, 63.12 H, 4.25

Example 10

  2-[(2-Bromo-5-methoxy-benzyl)oxy]-1-chloro-7-methoxynaphthalene

In Example 7 (3.05 g, 14.6 mmol), 2-bromo-5-methoxybenzyl alcohol (3.49 g, 16.08 mmol) and triphenylphosphine (4.22 g, 16.08 mmol) in anhydrous THF (50 mL), DEAD (2.8 g, 16.08 mmol) was slowly added at room temperature over 15 minutes. After stirring for 40 minutes, the THF was evaporated and the crude product was triturated with methanol to yield 5.31 g of product (89%) as a white solid: mp 126-127°C.

Analysis of C ₁₉ H ₁₆ BrClO ₃ :

Calculated: C, 55.98 H, 3.96

Found: C, 55.86 H, 4.01

Example 11

  7-Chloro-3,9-dimethoxy-5H-dibenzo[c,g]chromene

Example 8 (3.12 g, 7.66 mmol), sodium acetate (1.88 g, 23.0 mmol) and dichlorobis(triphenylphosphine) palladium (1.61 g, 2.3 mmol) in dimethylacetamide (220 ml ) was heated at 130°C for 3 hours. The solvent was removed under high vacuum and the catalyst was filtered through florisil using dichloromethane. The solid was washed with dichloromethane-methanol (1:2) to yield 2.59 g of product. The mother liquor was concentrated and chromatographed (10% ethyl acetate-hexanes) and methanol to yield an additional 160 mg of product as a white solid. Total yield 2.75 g (74.5%): mp 166-168°C.

Analysis of C ₁₉ H ₁₅ ClO ₃ :

Calculated: C, 69.84 H, 4.63

Found: C, 69.59 H, 4.32

Example 12

  7-Chloro-5H-dibenzo[c,g]chromene-3,9-diol

A mixture of Example 9 (2.25 g, 6.92 mmol) and pyridinium hydrochloride (21.9 g, 162 mmol) was heated to 184°C for 2 hours. The reaction mixture was cooled, poured into water (300 mL) and extracted with ethyl acetate (2 x 200 mL), washed with brine and dried over anhydrous magnesium sulfate. Removal of solvent and chromatography of product (40% ethyl acetate-hexanes) gave a white solid which was triturated with dichloromethane (25 mL) to yield 1.2 g (58%); mp 230-231 °C;

Analysis of C ₁₆ H ₁₁ ClO ₃ :

Calculated: C, 68.35 H, 3.71

Found: C, 68.02 H, 3.57

Example 13

     7-Bromo-5H-dibenzo[c,g]chromene-3,9-diol

In a mixture of 7-bromo-3-(acetoxy)-H-dibenzo[c,g]chromen-9-yl acetate (427 mg, 1 mmol) in methanol (25 mL), 25% sodium methoxide in methanol (6 mL) was added. The reaction was stirred at room temperature for 0.5 h, then diluted with ethyl acetate (300 mL) and 1N HCl (150 mL). The ethyl acetate fraction was washed with water (2 x 100 mL) and brine (150 mL), dried over anhydrous magnesium sulfate and filtered. The solvent was removed under vacuum and the resulting solid was purified by chromatography (25% ethyl acetate-hexanes) to afford a yellow solid (286 mg, 83%): mp (decomposition) at 260 °C;

¹ H NMR (DMSO-d ₆ ): δ5.19 (2H, s), 6.70 (1H, d, J = 2.4Hz), 6.86 (1H, dd, J = 2.4Hz, J = 8.5Hz), 6.99 ( 1H, dd, J=2.3Hz, J=8.8Hz), 7.31 (1H, d, J=2.3Hz), 7.79 (1H, d, J=6.3Hz), 7.81 (1H, d, J=6.0Hz) , 8.22 (1H, s), 9.97 (2H, br s); MS m/z 343/345 ([M+H]+).

Analysis of C ₁₇ H ₁₁ BrO ₃ :

Calculated: C: 59.50; H: 3.23

Measured value: C: 59.22; H: 3.29

Example 14

      3,9-Dihydro-5H-dibenzo[c,g]chromene-7-carbonitrile

In 3-(acetoxy)-7-bromo-H-dibenzo[c,g]chromen-9-yl acetate (855 mg, 2 mmol) and anhydrous dimethylformamide (20 mL), copper cyanide (1.0 g, 10 mmol) was added. The mixture was heated to 150°C overnight. The mixture was cooled and suspended between ethyl acetate and water. The layers were separated and the ethyl acetate portion was washed with water (3 x 50 mL) and brine (50 mL). Ethyl acetate was dried over anhydrous magnesium sulfate, the solvent was removed, and the resulting solid was dissolved in methanol (10 mL) and 25% sodium methoxide (3 mL) was added. After stirring for 0.5 h, ethyl acetate (50 mL) and 1N HCl (20 mL) were added, the layers were separated and the organic portion was washed with water (3 x 30 mL) and brine (30 mL). After drying over anhydrous magnesium sulfate, the solvent was removed to give a dark yellow solid, which was purified by chromatography (25% ethyl acetate-hexane) to give a light yellow solid (208 mg, 36%): mp > 280 °C;

¹ H NMR (DMSO-d ₆ ): δ5.32 (2H, s), 6.71 (1H, d, J = 2.4Hz), 6.87 (1H, dd, J = 2.4Hz, J = 8.3Hz), 7.06 ( 1H, dd, J=2.4Hz, J=8.9Hz), 7.18 (1H, d, J=2.4Hz), 7.84 (1H, d, J=8.8Hz), 7.87 (1H, d, J=8.8Hz) , 8.50(1H, s), 9.90(1H, br s), 10.32(1H, br s); MS m/z 288([MH]-).

HPLC at 254 nm indicated a purity of 98.5%.

Analysis of C ₁₈ H ₁₁ NO ₃ ·0.5H ₂ O:

Calculated: C: 72.48; H: 4.05; N: 4.70

Measured value: C: 72.31; H: 3.96; N: 4.55

Example 15

  7-Methoxy-5H-dibenzo[c,g]chromene-3,9-diol

In 3-(acetoxy)-7-bromo-H-dibenzo[c,g]chromen-9-yl acetate (427 mg, 1 mmol), CuBr (143 mg, 1 mmol) To a mixture of anhydrous DMF (6 mL), NaOMe/methanol (3 mL) was added. The mixture was heated to 120°C for 1 hour. The reaction mixture was cooled and ethyl acetate (100 mL) and 1N HCl (50 mL) were added. The layers were separated, and the organic portion was washed with water (3 x 50 mL) and brine (50 mL), then dried over anhydrous magnesium sulfate. The solvent was evaporated and the crude product was purified by chromatography (25% ethyl acetate-hexanes) to yield a yellow solid (260 mg, 88%): mp (decomposition) 240 °C;

¹ H NMR (DMSO-d ₆ ): δ3.88 (3H, s), 5.09 (2H, s), 6.70 (1H, d, J = 2.4Hz), 6.83 (1H, dd, J = 2.4Hz, J = 8.3Hz), 6.93 (1H, dd, J = 2.4Hz, J = 8.8Hz), 7.19 (1H, d, J = 2.4Hz), 7.72 (1H, d, J = 8.5Hz), 7.77 (1H, d, J=8.8Hz), 7.92(1H,s), 9.75(2H,s); MS m/z 295([M+H]+).

Analysis of C ₁₈ H ₁₄ O ₄ :

Calculated: C: 73.46; H: 4.79

Measured value: C: 73.19; H: 4.62

Example 16

    7-Vinyl-5H-dibenzo[c,g]chromene-3,9-diol

In 3-(acetoxy)-7-bromo-H-dibenzo[c,g]chromen-9-yl acetate (300 mg, 0.7 mmol), anhydrous toluene (2 mL) and tris To a mixture of butylvinyltin (254 mg, 0.8 mmol) was added tetrakis(triphenylphosphine)palladium (160 mg, 0.14 mmol). The mixture was heated to reflux for 4.5 hours. The reaction mixture was cooled and diluted with water and ethyl acetate, and the organic portion was washed with water and brine. After drying over anhydrous magnesium sulfate and removal of the solvent, the crude product was treated with methanol (10 mL) and 25% sodium methoxide (2 mL). The mixture was stirred for 0.5 h, then ethyl acetate (50 mL) and 1N HCl (20 mL) were added. The organic portion was separated and washed with water (2 x 20 mL) and brine (25 mL) and dried over anhydrous magnesium sulfate. Removal of solvent yielded a solid which was purified by chromatography (25% ethyl acetate-hexanes) to yield a brown solid (45 mg, 15%): mp (decomposition) 195-197 °C;

¹ H NMR (DMSO-d ₆ ): δ5.07 (2H, s), 5.66-5.76 (2H, m), 6.69 (1H, d, J = 2.4Hz), 6.84 (1H, dd, J = 2.5Hz , J=8.4Hz), 6.91-7.04(2H, m), 7.34(1H, d, J=2.4Hz), 7.74(1H, d, J=8.9Hz), 7.78(1H, d, J=8.6Hz ), 8.10(1H, s), 9.72(2H, s); MS m/z 291([M+H]+).

Analysis of C ₁₉ H ₁₄ NO ₃ ·0.2H ₂ O:

Calculated: C: 77.64; H: 4.94

Measured value: C: 77.63; H: 4.81

Example 17

3-(Acetoxy)-7-methoxy-5H-dibenzo[c,g]chromen-9-yl acetate

To a mixture of pyridine (8 mL) and acetic anhydride (8 mL), was added 7-methoxy-H-dibenzo[c,g]chromene-3,9-diol (1.37 g, 4.7 mmol ), and the mixture was stirred at room temperature for 2 hours. The insoluble solid was filtered and rinsed with 10% ethyl acetate-hexane to afford a brown solid (1.6 g, 90%). A portion of this solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to obtain a white solid: mp 165-167°C;

¹ H NMR (DMSO-d ₆ ): δ2.31 (3H, s), 2.33 (3H, s), 3.95 (3H, s), 5.24 (2H, s), 7.18 (1H, d, J=2.4Hz ), 7.22 (1H, dd, J = 2.4Hz, J = 8.8Hz), 7.25 (1H, dd, J = 2.4Hz, J = 8.8Hz), 7.67 (1H, d, J = 2.4Hz), 7.96 ( lH, d, J=8.8Hz), 8.08(1H, d, J=8.8Hz), 8.28(1H, s); MS m/z 379([M+H]+).

HPLC at 280 nm indicated a purity of 99.8%.

Analysis for C ₂₂ H ₁₈ O ₆ ·0.3H ₂ O:

Calculated: C: 68.85; H: 4.88

Measured value: C: 68.93; H: 4.72

Example 18

3-(Acetoxy)-12-bromo-7-methoxy-5H-dibenzo[c,g]chromen-9-yl acetate

3-(Acetoxy)-12-bromo-7-methoxy-H-dibenzo[c,g]chromen-9-yl acetate (570 mg, 1.5 mmol) in anhydrous acetonitrile (100 mL), N-bromosuccinimide (350 mg, 1.95 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed and the crude residue was purified by chromatography (25% ethyl acetate-hexanes) to obtain a pale yellow solid (500 mg, 73%): mp 176-177 °C;

¹ H NMR (DMSO-d ₆ ): δ2.33 (3H, s), 2.35 (3H, s), 3.96 (3H, s), 5.12 (2H, s), 7.30 (1H, d, J=2.9Hz ), 7.31 (1H, s), 7.42 (1H, dd, J=2.4Hz, J=9.3Hz), 7.80 (1H, d, J=2.4Hz), 8.35 (1H, d, J=9.3Hz), 8.54(1H, dd, J=2.0Hz, J=7.6Hz).

Analysis of C ₂₂ H ₁₇ BrO ₆ :

Calculated: C: 57.79; H: 3.75

Measured value: C: 57.54; H: 3.63

Example 19

   12-Bromo-7-methoxy-5H-dibenzo[c,g]chromene-3,9-diol

3-(Acetoxy)-12-bromo-7-methoxy-H-dibenzo[c,g]chromen-9-yl acetate (260 mg, 0.57 mmol) in methanol (25 mL), 25% sodium methoxide in methanol (3 mL) was added and the mixture was stirred for 0.5 h. The mixture was suspended between ethyl acetate (50 mL) and 1N HCl (20 mL), and the organic portion was washed with water (3 x 50 mL) and brine (50 mL). The ethyl acetate was dried over anhydrous magnesium sulfate and the solvent was removed to give a brown solid which was purified by silica gel chromatography (50% ethyl acetate-hexanes) to give 95 mg (45%) of a brown solid: mp (decomposition) Higher than 250°C;

¹ H NMR (DMSO-d ₆ ): δ3.89 (3H, s), 4.97 (2H, s), 6.82 (1H, d, J=2.4Hz), 6.88 (1H, dd, J=2.7Hz, J =8.5Hz), 7.12(1H, dd, J=2.4Hz, J=9.3Hz), 7.29(1H, d, J=2.4Hz), 8.12(1H, d, J=9.3Hz), 8.29(1H, d, J=8.3Hz), 9.93(1H,s), 10.06(1H,s); MS m/z 373/375([M+H]+).

Analysis of C ₁₈ H ₁₃ BrO ₄ :

Calculated: C: 57.93; H: 3.51

Measured value: C: 57.64; H: 3.34

Example 20

  12-Chloro-7-methoxy-5H-dibenzo[c,g]chromene-3,9-diol

3-(Acetoxy)-7-methoxy-H-dibenzo[c,g]chromen-9-yl acetate (290 mg, 0.77 mmol) in anhydrous THF (10 mL) To the mixture in , N-chlorosuccinimide (133 mg, 1.0 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The solvent was removed and methanol (10 mL) and 25% sodium methoxide-methanol (3 mL) were added. After stirring at room temperature for 0.5 h, ethyl acetate (25 mL) and 1N HCl (10 mL) were added, the mixture was shaken, the layers were separated, and the ethyl acetate was washed with water (3 x 10 mL) and brine (10 mL) . The mixture was dried over anhydrous magnesium sulfate, the solvent was removed, and the crude brown solid was purified by silica gel chromatography (25% ethyl acetate-hexanes) to give 166 mg (66%) of a white solid: mp (decomposition) 240 °C ;

¹ H NMR (DMSO-d ₆ ): δ3.89 (3H, s), 4.99 (2H, s), 6.82 (1H, d, J = 2.4Hz), 6.88 (1H, dd, J = 2.4Hz, J =8.8Hz), 7.12(1H, dd, J=2.4Hz, J=8.8Hz), 7.29(lH, d, J=2.4Hz), 8.11(1H, d, J=9.3Hz), 8.27(1H, d, J=8.3Hz), 10.00 (2H, br s); MSm/z 329/331 ([M+H]+),

HPLC at 280 nm gave 99.6% purity.

Analysis of C ₁₈ H ₁₃ ClO ₄ ·0.2H ₂ O:

Calculated: C: 65.05; H: 4.06

Measured value: C: 65.21; H: 4.12

Example 21

  7-Methyl-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), anhydrous dimethylformamide (10 mL) and dichlorobis(tri To a mixture of o-tolylphosphine)palladium (79 mg, 0.1 mmol), tetramethyltin (268 mg, 1.5 mmol) was added. The reaction mixture was heated to 80 °C for 3 hours. TLC and MS analysis indicated a 1:1 mixture of starting material and product. The mixture was cooled to room temperature and additional tetramethyltin (268 mg, 1.5 mmol) and catalyst (79 mg, 0.1 mmol) were added. The mixture was heated at 80 °C overnight. The reaction mixture was cooled and ethyl acetate (50 mL) and water (25 mL) were added. After the layers were separated, the organic portion was washed with water (3 x 20 mL), then brine (20 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the crude product was purified by chromatography (25% ethyl acetate-hexanes) to give a pale yellow solid (117 mg, 42%): mp 224-226 °C;

¹ H NMR (DMSO-d ₆ ): δ2.38 (3H, s), 5.06 (2H, s), 6.69 (1H, d, J=2.4Hz), 6.83 (1H, dd, J=2.5Hz, J = 8.4Hz), 6.93 (1H, dd, J = 2.3Hz, J = 8.7Hz), 7.08 (1H, d, J = 2.3Hz), 7.71 (1H, d, J = 8.8Hz), 7.77 (1H, d, J=8.6Hz), 8.01 (1H, s), 9.67 (2H, s); MS m/z 279 ([M+H]+).

HPLC at 280 nm gave 99.5% purity.

Analysis for C ₁₈ H ₁₄ O ₃ (0.2H ₂ O):

Calculated: C: 76.69; H: 5.15

Measured value: C: 76.61; H: 4.99

Example 22

  7-[2-(Hydroxymethyl)phenyl]-5H-dibenzo[c,g]chromene-3,9-diol

In 3-(acetoxy)-7-bromo-H-dibenzo[c,g]chromen-9-yl acetate (2.03 g, 4.8 mmol), (2-hydroxymethylphenyl) To a mixture of boric acid dihydrate (1.54 g, 13.3 mmol), potassium carbonate (2.07 g, 15 mmol), dimethoxyethane (100 mL) and water (10 mL), tetrakis(triphenylphosphine ) palladium (600 mg, 0.4 mmol). The mixture was heated to reflux for 1 hour. The reaction mixture was cooled and suspended between ethyl acetate (200 mL) and water (75 mL). Ethyl acetate was washed with water (2 x 75 mL), then brine (75 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting yellow liquid was dissolved in methanol (100 mL) and 25% sodium methoxide (10 mL) was added. The mixture was stirred at room temperature for 0.5 h, then diluted with ethyl acetate (200 mL) and 1N HCl (75 mL). Ethyl acetate was washed with water (3 x 75 mL), then brine (75 mL), and dried over anhydrous magnesium sulfate. Removal of solvent afforded a brown solid, which was purified by chromatography (35% ethyl acetate-hexanes) to afford a white solid (500 mg, 15%): mp 171-173 °C;

¹ H NMR (DMSO-d ₆ ): δ4.08 (1H, dd, J=3.7Hz, J=14.3Hz), 4.26 (1H, dd, J=3.6Hz, J=14.3Hz), 4.92 (2H, s), 4.94-4.99 (1H, m), 6.36 (1H, d, J=2.0Hz), 6.63 (1H, d, J=2.1Hz), 6.85 (1H, dd, J=2.3Hz, J=8.5 Hz), 6.89(1H, dd, J=2.3Hz, J=8.7Hz), 7.11(1H, d, J=7.4Hz), 7.36-7.39(1H, m), 7.45-7.49(1H, m), 7.67 (1H, d, J = 7.7Hz), 7.78 (1H, d, J = 8.9Hz), 7.83 (1H, d, J = 8.6Hz), 8.20 (1H, s), 9.53 (1H, br s) , 9.70 (1H, br s); MS m/z 369 ([MH]-).

HPLC at 280 nm gave 94.9% purity.

Analysis for C ₂₄ H ₁₈ O ₄ ·0.3H ₂ O:

Calculated: C: 76.71; H: 4.99

Measured value: C: 76.56; H: 4.95

Example 23

  7-Phenyl-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylacetamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added phenylboronic acid (366 mg, 3 mmol). The reaction mixture was heated to 120 °C for 3 hours. The reaction mixture was cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (5% acetonitrile-dichloromethane) to yield a brown solid (170 mg, 50%): mp 225-227 °C;

¹ H NMR (DMSO-d ₆ ): δ4.95 (2H, s), 6.63 (2H, s), 6.85 (1H, dd, J=2.2Hz, J=8.4Hz), 6.92 (1H, dd, J =2.2Hz, J=8.7Hz), 7.33(2H, d, J=7.0Hz), 7.40-7.43(1H, m), 7.49-7.53(2H, m), 7.77(1H, d, J=8.9Hz ), 7.83 (1H, d, J=8.6Hz), 8.19 (1H, s), 9.61 (1H, br s), 9.69 (1H, br s); MS m/z 341 ([M+H]+) .

HPLC at 280 nm gave 98.2% purity.

Analysis for C ₂₃ H ₁₆ O ₃ ·0.5H ₂ O:

Calculated: C: 79.07; H: 4.90

Measured value: C: 78.68; H: 4.64

Example 24

  7-(2-Methylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 2-tolylboronic acid (400 mg, 3 mmol). The reaction mixture was heated to 120°C for 2.5 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark solid was purified by chromatography (5% acetonitrile-dichloromethane) to yield a yellow solid (150 mg, 42%): mp 193-196 °C;

¹ H NMR (DMSO-d ₆ ): δ1.97 (3H, s), 4.92 (2H, s), 6.36 (1H, d, J=2.3Hz), 6.62 (1H, d, J=2.4Hz), 6.83-6.92(2H, m), 7.09-7.13(1H, m), 7.27-7.39(3H, m), 7.78(1H, d, J=8.9Hz), 7.84(1H, d, J=8.6Hz) , 8.20(1H,s), 9.52(1H,s), 9.72(1H,s); MS m/z 355([M+H]+).

HPLC at 280 nm gave 99.8% purity.

Analysis for C ₂₄ H ₁₈ O ₃ ·0.5H ₂ O:

Calculated: C: 79.32; H: 5.27

Measured value: C: 79.04; H: 5.15

Example 25

  7-(3-methylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-tolylboronic acid (400 mg, 3 mmol). The reaction mixture was heated to 120°C for 2 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark oil was purified by chromatography (5% acetonitrile-dichloromethane) to yield a white solid (264 mg, 75%): mp 219-222 °C;

¹ H NMR (DMSO-d ₆ ): δ2.39 (3H, s), 4.94 (2H, s), 6.61-6.64 (2H, m), 6.84 (1H, dd, J=2.4Hz, J=8.5Hz ), 6.90 (1H, dd, J=2.3Hz, J=8.8Hz), 7.09-7.12 (2H, m), 7.22 (1H, d, J=9.1Hz), 7.39-7.43 (1H, m), 7.77 (1H,d,J=8.9Hz), 7.82(1H,d,J=8.6Hz), 8.18(1H,s), 9.54(1H,br s), 9.72(1H,s); MS m/z 355 ([M+H]+).

HPLC at 254 nm gave 99.3% purity.

Analysis for C ₂₄ H ₁₈ O ₃ ·0.1H ₂ O:

Calculated: C: 80.93; H: 5.15

Measured value: C: 80.81; H: 5.08

Example 26

  7-(4-methylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 4-tolylboronic acid (400 mg, 3 mmol). The reaction mixture was heated to 120 °C for 1 hour. The reaction mixture was cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark oil was purified by chromatography (5% acetonitrile-dichloromethane) to yield a white solid (265 mg, 75%): mp 238-240 °C;

¹ H NMR (DMSO-d ₆ ): δ2.40 (3H, s), 4.93 (2H, s), 6.64 (2H, dd, J=2.3Hz, J=7.0Hz), 6.84 (1H, dd, J =2.4Hz, J=8.5Hz), 6.90 (1H, dd, J=2.3Hz, J=8.8Hz), 7.20 (2H, d, J=8.0Hz), 7.31 (2H, d, J=8.0Hz) MS m /z 355([M+H]+).

HPLC at 280 nm gave 99.9% purity.

Analysis for C ₂₄ H ₁₈ O ₃ ·0.3H ₂ O:

Calculated: C: 80.16; H: 5.21

Measured value: C: 79.99; H: 5.12

Example 27

 7-(4-methoxyphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 4-methoxyphenylboronic acid (500 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting brown solid was purified by chromatography (25% ethyl acetate-hexanes) to yield a white solid (215 mg, 58%): mp 200-203 °C;

¹ H NMR (DMSO-d ₆ ): δ3.85 (3H, s), 4.95 (2H, s), 6.64 (1H, d, J=2.4Hz), 6.68 (1H, d, J=2.4Hz), 6.85(1H, dd, J=2.6Hz, J=8.4Hz), 6.91(1H,dd, J=2.2Hz, J=8.8Hz), 7.08(2H, d, J=8.8Hz), 7.25(2H, d, J = 8.8Hz), 7.77 (1H, d, J = 8.7Hz), 7.82 (1H, d, J = 8.8Hz), 8.17 (1H, s), 9.50 (1H, s), 9.69 (1H, s); MS m/z 371 ([M+H]+).

HPLC at 254 nm gave 98.3% purity.

Analysis for C ₂₄ H ₁₈ O ₄ ·0.2H ₂ O:

Calculated: C: 77.07; H: 4.96

Measured value: C: 76.81; H: 5.00

Example 28

 7-(4-Chlorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 4-chlorophenylboronic acid (468 mg, 3 mmol). The reaction mixture was heated to 120°C for 1.5 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark oil was purified by chromatography (25% ethyl acetate-hexanes) to yield a white solid (215 mg, 58%): mp 239-242 °C;

¹ H NMR (DMSO-d ₆ ): δ4.96 (2H, s), 6.60 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 2.4Hz), 6.84 (1H, dd, J =2.3Hz, J=8.8Hz), 6.92(1H, dd, J=2.5Hz, J=8.5Hz), 7.36(2H, d, J=8.5Hz), 7.58(2H, d, J=8.4Hz) , 7.78(1H, d, J=8.9Hz), 7.83(1H, d, J=8.6Hz), 8.21(1H, s), 9.56(1H, s), 9.72(1H, s); MS m/z 375/377([M+H]+).

HPLC at 280 nm gave 99.6% purity.

Analysis of C ₂₃ H ₁₅ ClO ₃ 0.2H ₂ O:

Calculated: C: 73.00; H: 4.10

Measured value: C: 72.89; H: 4.01

Example 29

  7-(4-fluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 4-fluorophenylboronic acid (420 mg, 3 mmol). The reaction mixture was heated to 120°C for 2 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting brown solid was purified by chromatography (25% ethyl acetate-hexanes) to yield a white solid (215 mg, 62%): mp 229-232 °C;

¹ H NMR (DMSO-d ₆ ): δ4.96 (2H, s), 6.62 (2H, dd, J=2.4Hz, J=9.4Hz), 6.84 (1H, dd, J=2.5Hz, J=8.5 Hz), 6.92(1H, dd, J=2.4Hz, J=8.8Hz), 7.33-7.43(4H, m), 7.78(1H, d, J=8.8Hz), 7.83(1H, d, J=8.5 Hz), 8.20(1H, s), 9.56(1H, s), 9.72(1H, s); MS m/z 359([M+H]+).

HPLC at 280 nm gave 98.1% purity.

Analysis _{for C23H15FO3 0.3H2O :}

Calculated: C: 75.94; H: 4.32

Measured value: C: 76.02; H: 4.29

Example 30

  7-thiophen-2-yl-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), DMF (5 mL), water (1 mL), 2N sodium carbonate (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 2-thiopheneboronic acid (260 mg, 2 mmol). The reaction mixture was heated to 120 °C overnight. The mixture was cooled and ethyl acetate (50 mL) and 5% ammonium chloride (25 mL) were added. Ethyl acetate was washed with water (2 x 20 mL) and brine (20 mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the crude solid was purified by chromatography (25% ethyl acetate-hexanes) to yield a pale yellow solid (84 mg, 24%): mp 233-236 °C;

¹ H NMR (DMSO-d ₆ ): δ5.01 (2H, s), 6.65 (1H, d, J = 2.5Hz), 6.83-6.88 (2H, m), 6.93 (1H, dd, J = 2.3Hz , J=8.8Hz), 7.09(1H, dd, J=1.0Hz, J=3.4Hz), 7.24(1H,dd, J=3.5Hz, J=5.1Hz), 7.70(1H,dd, J=1.0 Hz, J=5.1Hz), 7.78(1H,d,J=8.8Hz), 7.83(1H,d,J=8.7Hz), 8.24(1H,s), 9.67(1H,s), 9.75(1H, s); MS m/z 347 ([M+H]+).

HPLC at 300 nm gave 96.8% purity.

Analysis of C ₂₁ H ₁₄ O ₃ S 0.2H ₂ O:

Calculated: C: 72.06; H: 4.15

Measured value: C: 71.82; H: 4.01

Example 31

  7-thiophen-3-yl-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-thiopheneboronic acid (300 mg, 3 mmol). The reaction mixture was heated to 120 °C for 1 hour. The reaction mixture was cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (5% acetonitrile-dichloromethane) to yield a yellow solid (188 mg, 54%): mp 159-161 °C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.65 (1H, d, J = 2.5Hz), 6.83-6.86 (2H, m), 6.92 (1H, dd, J = 2.4Hz , J=8.7Hz), 7.18(1H, dd, J=1.2Hz, J=4.9Hz), 7.50(1H,dd, J=1.2Hz, J=4.9Hz), 7.68(1H,dd, J=2.9 Hz, J=4.9Hz), 7.76(1H, d, J=8.8Hz), 7.82(1H, d, J=8.6Hz), 8.17(1H, s), 9.57(1H, s), 9.70(1H, s); MS m/z 345 ([MH]-).

HPLC at 280 nm gave 99.8% purity.

Analysis of C ₂₁ H ₁₄ O ₃ S 0.5H ₂ O:

Calculated: C: 70.97; H: 4.25

Measured value: C: 71.06; H: 3.98

Example 32

  7-(3-fluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-fluorophenylboronic acid (420 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (25% ethyl acetate-hexanes) to yield a white solid (196 mg, 55%): mp 225-227 °C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.62-6.64 (2H, m), 6.84 (1H, dd, J=2.4Hz, J=8.5Hz), 6.92 (1H, dd , J=2.3Hz, J=8.8Hz), 7.16-7.29(3H, m), 7.52-7.59(1H, m), 7.77-7.85(2H, m), 8.22(1H, s), 9.59(1H, s), 9.72(1H, s); MSm/z 359([M+H]+).

HPLC at 210 nm gave 99.2% purity.

Analysis of C ₂₃ H ₁₅ FO ₃ ·0.2H ₂ O:

Calculated: C: 76.32; H: 4.29

Measured value: C: 79.21; H: 4.22

Example 33

  7-(3-Chlorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-chlorophenylboronic acid (468 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (138.6 mg, 37%): mp 243-246 °C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.59 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 2.4Hz), 6.84 (1H, dd, J =2.5Hz, J=8.4Hz), 6.93(1H, dd, J=2.3Hz, J=8.8Hz), 7.29-7.32(1H, m), 7.38-7.41(1H, m), 7.45-7.58(2H , m), 7.77-7.85 (2H, m), 8.22 (1H, s), 9.61 (1H, s), 9.73 (1H, s); MS m/z 375/377, 1Cl, ([M+H] +).

HPLC at 280 nm gave 97.5% purity.

Analysis of C ₂₃ H ₁₅ ClO ₃ :

Calculated: C: 73.70; H: 4.03

Measured value: C: 73.84; H: 4.15

Example 34

 7-(3-methoxyphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-methoxyphenylboronic acid (456 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (248.3 mg, 67%): mp 220-223 °C;

¹ H NMR (DMSO-d ₆ ): δ3.79 (3H, s), 4.96 (2H, s), 6.64 (2H, dd, J=2.3Hz, J=7.0Hz), 6.82-6.92 (4H, m ), 6.98(1H, dd, J=1.5Hz, J=6.7Hz), 7.40-7.45(1H, m), 7.75-7.84(2H, m), 8.19(1H, s), 9.54(1H, s) , 9.70 (1H, s); MS m/z 371 ([M+H]+).

HPLC at 280 nm gave 98.5% purity.

Analysis for C ₂₄ H ₁₈ O ₄ ·0.3H ₂ O:

Calculated: C: 76.71; H: 4.99

Measured value: C: 76.88; H: 4.80

Example 35

  7-(2-Chlorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 2-chlorophenylboronic acid (468 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (85.1 mg, 22%): mp 220-222 °C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.37 (1H, d, J = 2.3Hz), 6.63 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =2.5Hz, J=8.5Hz), 6.92(1H, dd, J=2.4Hz, J=8.8Hz), 7.33-7.38(1H, m), 7.43-7.53(2H, m), 7.61-7.67(1H , m), 7.78-7.85 (2H, m), 8.24 (1H, s), 9.58 (1H, s), 9.72 (1H, s); MS m/z 375/377, 1Cl, ([M+H] +).

HPLC at 280 nm gave 97.6% purity.

Analysis of C ₂₃ H ₁₅ ClO ₃ ·0.3H ₂ O:

Calculated: C: 72.65; H: 4.14

Measured value: C: 72.63; H: 3.83

Example 36

7-(3,4-difluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3,4-difluorophenylboronic acid (474 mg, 3 mmol). The reaction mixture was heated to 120°C for 2 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting brown solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (162.1 mg, 43%): mp 235-237°C;

¹ H NMR (DMSO-d ₆ ): δ4.99 (2H, s), 6.63 (2H, dd, J=2.4Hz, J=8.8Hz), 6.84 (1H, dd, J=2.5Hz, J=8.4 Hz), 6.93(1H, dd, J=2.3Hz, J=8.8Hz), 7.16-7.21(1H, m), 7.42-7.49(1H, m), 7.53-7.62(1H, m), 7.77-7.84 (2H, m), 8.23(1H, s), 9.60(1H, s), 9.73(1H, s); MS m/z377([M+H]+).

HPLC at 280 nm gave 97.6% purity.

Analysis for C ₂₃ H ₁₄ F ₂ O ₃ ·0.1H ₂ O:

Calculated: C: 73.05; H: 3.78

Measured value: C: 72.98; H: 3.63

Example 37

  7-(4-pyridyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 4-pyridylboronic acid (370 mg, 3 mmol). The reaction mixture was heated to 120°C for 2 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The insoluble tan solid was filtered and dried, then dissolved in dichloromethane:methanol and filtered through filter aid to remove the catalyst. Removal of solvent gave a brown solid (60 mg, 18%): mp > 250 °C;

¹ H NMR (DMSO-d ₆ ): δ4.99 (2H, s), 6.60 (1H, d, J = 2.2Hz), 6.64 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =2.4Hz, J=8.5Hz), 6.94(1H, dd, J=2.3Hz, J=8.8Hz), 7.40(2H,dd, J=1.5Hz, J=4.4Hz), 7.79-7.85(2H, m), 8.26(1H,s), 8.71(2H,dd, J=1.5Hz, J=4.4Hz), 9.65(1H,s), 9.76(1H.s); MS m/z 342([M+ H]+).

HPLC at 280 nm gave 99.7% purity.

Analysis of C ₂₂ H ₁₅ NO ₃ ·HCl:

Calculated: C: 69.94; H: 4.27; N: 3.71

Measured value: C: 70.27; H: 4.08; N: 3.58

Example 38

  7-(2-Fluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 2-fluorophenylboronic acid (420 mg, 3 mmol). The reaction mixture was heated to 120°C for 2 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (120 mg, 30%): mp 240-242 °C;

¹ H NMR (DMSO-d ₆ ): δ4.97 (2H, d, J = 2.7Hz), 6.55 (1H, s), 6.64 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =2.5Hz, J=8.5Hz), 6.93(1H, dd, J=2.3Hz, J=8.8Hz), 7.33-7.43(3H, m), 7.45-7.54(1H, m), 7.78-7.85(2H , m), 8.25(1H, s), 9.62(1H, s), 9.72(1H, s); MS m/z 359([M+H]+).

HPLC at 280 nm gave 99.2% purity.

Analysis for C ₂₃ H ₁₅ FO ₃ ·0.5H ₂ O:

Calculated: C: 75.20; H: 4.39

Measured value: C: 75.09; H: 4.22

Example 39

7-(3,4-Dimethylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3,4-dimethylphenylboronic acid (450 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting brown solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (95 mg, 26%): mp 218-221 °C;

¹ H NMR (DMSO-d ₆ ): δ2.29 (3H, s), 2.31 (3H, s), 4.92 (2H, s), 6.62-6.65 (2H, m), 6.82-6.91 (2H, m), 7.01-7.05(1H, m), 7.08(1H, s), 7.25(1H, d, J=7.7Hz), 7.77-7.83(2H, m), 8.16(1H, s), 9.48(1H, s) , 9.70(1H,s); MS m/z 369([M+H]+).

HPLC at 280 nm gave 97.3% purity.

Analysis of C ₂₅ H ₂₀ O ₃ ·0.2H ₂ O:

Calculated: C: 80.71; H: 5.53

Measured value: C: 80.61; H: 5.43

Example 40

  7-(4-cyanophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (400 mg, 1.2 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (140 mg, 0.12 mmol), was added 4-cyanophenylboronic acid (514 mg, 3.5 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (282.3 mg, 66%): mp > 250 °C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.55 (1H, d, J = 2.2Hz), 6.64 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =2.5Hz, J=8.5Hz), 6.94(lH, dd, J=2.3Hz, J=8.8Hz), 7.57(2H, d, J=8.3Hz), 7.79-7.85(2H, m), 7.98( 1H, d, J=7.8Hz), 8.25(1H, s), 9.62(1H, s), 9.74(1H, s); MS m/z 364([MH]-).

HPLC at 280 nm gave 96.5% purity.

Analysis of C ₂₄ H ₁₅ NO ₃ ·0.3H ₂ O:

Calculated: C: 77.74; H: 4.24; N: 3.78

Measured value: C: 77.91; H: 4.07; N: 3.53

Example 41

7-(3-Fluoro-4-methylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-fluoro-4-methylphenylboronic acid (462 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (226 mg, 61%): mp 238-240 °C;

¹ H NMR (DMSO-d ₆ ): δ4.97 (2H, s), 6.63-6.65 (2H, m), 6.84 (1H, dd, J=2.5Hz, J=8.5Hz), 6.91 (1H, dd , J=2.4Hz, J=8.8Hz), 7.04-7.13(2H, m), 7.38-7.43(1H, m), 7.76-7.84(2H, m), 8.20(1H, s), 9.55(1H, s), 9.72(1H, s); MS m/z 373([M+H]+).

HPLC at 280 nm gave 98.2% purity.

Analysis for C ₂₄ H ₁₇ O ₃ ·0.3H ₂ O:

Calculated: C: 76.30; H: 4.70

Measured value: C: 76.04; H: 4.60

Example 42

7-(3,4-dimethoxyphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-fluoro-4-methylphenylboronic acid (546 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (261.1 mg, 65%): mp 245-248 °C;

¹ H NMR (DMSO-d ₆ ): δ3.75 (3H, s), 3,84 (3H, s), 4.96 (2H, s), 6.64 (1H, d, J=2.4Hz), 6.75 (1H , dd, J=2.3Hz), 6.83-6.92(4H, m), 7.09(1H, d, J=8.2Hz), 7.74-7.83(2H, m), 8.17(1H, s), 9.51(1H, s), 9.70(1H, s); MS m/z 401([M+H]+).

HPLC at 280 nm gave 99.4% purity.

Analysis of C ₂₅ H ₂₀ O ₅ ·0.5H ₂ O:

Calculated: C: 73.34; H: 5.17

Measured value: C: 73.24; H: 4.95

Example 43

  7-(3-Trifluoromethylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-fluoro-4-methylphenylboronic acid (570 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting brown solid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a white solid (214 mg, 52%): mp 228-231 °C;

¹ H NMR (DMSO-d ₆ ): δ4.99 (2H, d, J = 2.7Hz), 6.55 (1H, d, J = 2.2Hz), 6.64 (1H, d, J = 2.4Hz), 6.85 ( 1H,dd, J=2.5Hz, J=8.5Hz), 6.94(1H,dd, J=2.3Hz, J=8.8Hz), 7.66-7.86(6H,m), 8.25(1H,s), 9.62( 1H, s), 9.73 (1H, s); MSm/z 409 ([M+H]+).

HPLC at 280 nm gave 100% purity.

Analysis of C ₂₄ H ₁₅ FO ₃ :

Calculated: C: 70.59; H: 3.70

Measured value: C: 70.23; H: 3.79

Example 44

  7-(3,5-difluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3,5-difluorophenylboronic acid (475 mg, 3 mmol). The reaction mixture was heated to 120°C for 1.5 hours, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a brown solid (217.4 mg, 58%): mp 238-240 °C;

¹ H NMR (DMSO-d ₆ ): δ5.01 (2H, s), 6.63 (2H, dd, J=2.3Hz, J=6.4Hz), 6.84 (1H, dd, J=2.4Hz, J=8.5 Hz), 6.94(1H, dd, J=2.3Hz, J=8.8Hz), 7.08-7.14(2H, m), 7.27-7.35(1H, m), 7.78-7.84(2H, m), 8.24(1H , s), 9.65(1H, s), 9.75(1H, s); MS m/z 375([MH]-).

HPLC at 280 nm gave 97.5% purity.

Analysis for C ₂₃ H ₁₄ F ₂ O ₃ ·0.3H ₂ O:

Calculated: C: 72.36; H: 3.85

Measured value: C: 72.31; H: 3.64

Example 45

       7-(3,5-dichlorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3,5-dichlorophenylboronic acid (573 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a brown solid (178.9 mg, 44%): mp 252-255 °C;

¹ H NMR (DMSO-d ₆ ): δ5.01 (2H, s), 6.57 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 2.4Hz), 6.84 (1H, dd, J =2.5Hz, J=8.5Hz), 6.94(1H, dd, J=2.3Hz, J=8.8Hz), 7.42(2H, d, J=1.9Hz), 7.66-7.70(1H, m), 7.79- 7.85(2H, m), 8.25(1H, s), 9.68(1H, s), 9.76(1H, s); MS m/z 409/411/413, 2Cl([M+H]+).

HPLC at 280 nm gave 97.6% purity.

Analysis of C ₂₃ H ₁₄ Cl ₂ O ₃ :

Calculated: C: 67.50; H: 3.45

Measured value: C: 67.13; H: 3.63

Example 46

  7-(3-Methyl-4-fluorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

In 7-bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol), dimethylformamide (5 mL), 2M sodium carbonate (1 mL) , water (1 mL) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol), was added 3-methyl-4-fluoro-phenylboronic acid (486 mg, 3 mmol). The reaction mixture was heated to 120°C for 1 hour, then cooled and diluted with ethyl acetate (25 mL) and 5% ammonium chloride. The organic layer was washed with water (3 x 10 mL) and brine (10 mL), and dried over anhydrous magnesium sulfate. The solvent was removed and the resulting dark liquid was purified by chromatography (2.5% acetonitrile-dichloromethane) to yield a brown solid (223 mg, 60%): mp 185-188 °C;

¹ H NMR (DMSO-d ₆ ): δ2.31 (3H, d, J=1.2Hz), 4.95 (2H, s), 6.62 (2H, dd, J=2.3Hz, J=10.4Hz), 6.84 ( 1H, dd, J=2.4Hz, J=8.5Hz), 6.91(1H,dd, J=2.3Hz, J=8.8Hz), 7.13-7.30(3H, m), 7.76-7.84(2H, m), 8.19(1H, s), 9.55(1H, s), 9.73(1H, s); MS m/z 371([MH]-).

HPLC at 280 nm gave 99.5% purity.

Analysis of C ₂₄ H ₁₇ FO ₃ :

Calculated: C: 77.41; H: 4.60

Measured value: C: 77.17; H: 4.54

Example 47

  7-(3-furyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-5H-dibenzo[c,g]chromene-3,9-diol (342 mg, 1.0 mmol), furan-3-boronic acid (224 mg, 2 mmol) and tetrakis(tri A mixture of phenylphosphine)palladium(0) (116 mg, 0.1 mmol) in DMF (20 mL) was heated at 120° C. for 4 hours with stirring. The mixture was filtered through celite, extracted with ethyl acetate (×3), washed with brine, dried over sodium sulfate, filtered, stripped of solvent and passed through silica column chromatography (18%-25% ethyl acetate-hexane alkane) to yield 200 mg (61%) of the title compound as a pale yellow solid: mp 216-218°C;

¹ H NMR (DMSO-d ₆ ): δ5.00 (2H, s), 6.66 (2H, m), 6.84 (1H, dd, J=8.45Hz, J=2.35Hz), 6.93 (1H, dd, J =8.77Hz, J=2.35Hz), 7.04(1H, d, J=2.17Hz), 7.76(1H, d, J=8.83Hz), 7.80-7.85(3H, m), 8.16(1H, s), 9.61(1H, s), 9.71(1H, s); MS(ESI) m/z 329(MH) ^- , 331(M+H) ⁺ .

Analysis of C ₂₁ H ₁₄ O ₄ :

Calculated: C: 76.36; H: 4.27

Measured value: C: 75.81; H: 4.32

Example 48

  7-(2-furyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-5H-dibenzo[c,g]chromene-3,9-diol (450mg, 1.32mmol), 2-(tributylstannyl)furan (0.622ml, 1.97mmol ) and tetrakis(triphenylphosphine)palladium(0) (153 mg, 0.132 mmol) in toluene (26 mL) was heated at 110° C. for 23 hours with stirring. The mixture was filtered through celite, concentrated and purified by reverse phase preparative HPLC to yield 280 mg (64%) of the title compound as a light brown solid. It was further purified _by trituration with _CH2Cl2 to yield a gray solid: mp 158-163°C;

¹ H NMR (DMSO-d ₆ ): δ5.04 (2H, s), 6.61 (1H, d, J = 3.11 Hz), 6.67-6.69 (2H, m), 6.85 (1H, dd, J = 8.47 Hz , J=2.33Hz), 6.95 (1H, dd, J=8.72Hz, J=2.27Hz), 6.99 (1H, d, J=1.98Hz), 7.77 (1H, d, J=8.77Hz), 7.82 ( 1H, d, J = 8.53Hz), 7.85 (1H, d, J = 1.32Hz), 8.24 (1H, s), 9.70 (1H, s), 9.75 (1H, s); MS (ESI) m/z 329(MH) ^- , 331(M+H) ⁺ .

Analysis of C ₂₁ H ₁₄ O ₄ ·0.2CH ₂ Cl ₂ :

Calculated: C: 72.62; H: 4.06

Measured value: C: 72.67; H: 4.03

Example 49

    7-Butyl-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-5H-dibenzo[c,g]chromene-3,9-diol (420 mg, 1.23 mmol), 2-tributylstannylthiazole (0.95 mL, 3.07 mmol) and A mixture of dichlorobis(tri-o-tolylphosphine)palladium(II) (97 mg, 0.123 mmol) in DMF (12 mL) was heated at 110° C. for 18 hours with stirring. The mixture was filtered through celite, extracted with ethyl acetate (x3), washed with brine, dried over sodium sulfate, filtered, stripped of solvent and purified by silica column chromatography (25% ethyl acetate-hexanes) to yield 200 mg (51%) of the title compound as a yellow solid. The analytical sample was further purified by another silica column chromatography (10%-20% ethyl acetate-hexane) to yield a pale yellow solid: mp 187-189°C;

¹ H NMR (DMSO-d ₆ ): δ0.94 (3H, t, J = 7.17Hz), 1.37-1.44 (2H, m), 1.47-1.58 (2H, m), 2.92 (2H, t, J = 7.00Hz), 5.04(2H, s), 6.73(1H, d, J=2.36Hz), 6.82(1H, dd, J=8.44Hz, J=2.48Hz), 6.92(1H, dd, J=8.76Hz , J=2.18Hz), 7.12(1H, d, J=2.05Hz), 7.71(1H, d, J=8.88Hz), 7.76(1H, d, J=8.53Hz), 8.00(1H, s), 9.63(1H, s), 9.67(1H, s); MS(ESI) m/z 319(MH) ^- , 321(M+H) ⁺ .

Analysis of C ₂₁ H ₂₀ O ₃ :

Calculated: C: 78.73; H: 6.29

Measured value: C: 78.42; H: 6.27

Example 50

  7-(3-pyridyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (686 mg, 2 mmol) was mixed with 3-pyridylboronic acid (737 mg , 6 mmol) to prepare the title compound, resulting in a yellow solid (201 mg, 30%): melting point > 250 °C;

¹ H NMR (DMSO-d ₆ ): δ4.99 (2H, s), 6.60 (1H, d, J = 2.5Hz), 6.65 (1H, d, J = 2.5Hz), 6.86 (2H, dd, J =8.5Hz, J=2.6Hz), 7.54-7.58(1H, m), 7.78-7.85(3H, m), 8.25(1H, s), 8.55(1H, d, J=1.4Hz), 8.63(1H , dd, J=4.8Hz, J=1.6Hz), 9.62(1H, s), 9.72(1H, s); MS m/z 342([M+H]+).

HPLC at 280 nm gave 99.6% purity.

Analysis of C ₂₂ H ₁₅ NO ₃ ·0.2H ₂ O:

Calculated: C: 76.60; N: 4.50; H: 4.06

Measured value: C: 76.46; N: 4.53; H: 3.80

Example 51

7-(4-Methoxy-3-pyridyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-methoxy-3- Pyridylboronic acid (168 mg, 1.1 mmol) was reacted to prepare the title compound to give a yellow solid (62.3 mg, 17%): mp > 220 °C;

¹ H NMR (DMSO-d ₆ ): δ3.94 (3H, s) 4.98 (2H, s), 6.64-6.67 (2H, m), 6.83 (1H, dd, J=8.5Hz, J=2.4Hz) , 6.93 (1H, dd, J=8.8Hz, J=2.3Hz), 6.98 (1H, d, J=8.5Hz), 7.69 (1H, dd, J=8.5Hz, J=2.4Hz), 7.78-7.85 (2H, m), 8.13 (1H, d, J=2.2Hz), 8.22 (1H, s), 9.60 (1H, s), 9.73 (1H, s); MS m/z 372 ([M+H] +).

HPLC at 280 nm gave 98.9% purity.

Analysis of C ₂₃ H ₁₇ NO ₄ ·0.2H ₂ O:

Calculated: C: 73.67; N: 4.68; H: 3.74

Measured value: C: 73.71; N: 4.40; H: 3.34

Example 52

  7-(pyrimidinyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with pyrimidinylboronic acid (370 mg, 3 mmol) to prepare the title compound, resulting in a brown solid (84.8 mg, 25%): melting point > 250 °C;

¹ H NMR (DMSO-d ₆ ): δ5.04 (2H, s), 6.62 (1H, d, J = 2.3Hz), 6.65 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =8.5Hz, J=2.6Hz), 6.97(1H, dd, J=11.8Hz, J=5.4Hz), 7.82-7.86(2H, m), 8.31(1H, s), 8.87(2H, s), 9.26(1H, s), 9.73(2H, br s); MS m/z 343([M+H]+).

HPLC at 300 nm gave 96.8% purity.

Analysis for C ₂₁ H ₁₄ N ₂ O ₃ ·0.3H ₂ O:

Calculated: C: 72.53; N: 4.23; H: 8.06

Measured value: C: 72.48; N: 4.17; H: 7.68

Example 53

 7-(5-methoxy-3-pyridyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (515 mg, 1.5 mmol) was mixed with 5-methoxy-3- Pyridylboronic acid (306 mg, 3 mmol) was reacted to prepare the title compound to give a tan solid (160.4 mg, 29%): mp > 250 °C;

¹ H NMR (DMSO-d ₆ ): δ3.88 (3H, s), 5.00 (2H, s), 6.64 (2H, dd, J=6.2Hz, J=2.4Hz), 6.85 (1H, dd, J =8.5Hz, J=2.6Hz), 6.94(1H, dd, J=8.8Hz, J=2.3Hz), 7.38(1H,dd, J=2.9Hz, J=1.7Hz), 7.81-7.85(2H, m), 8.14(1H, s), 8.25(1H, s), 8.36(1H, s), 9.61(1H, br s), 9.72(1H, br s); MS m/z 372([M+H ]+).

HPLC at 300 nm gave 99.0% purity.

Analysis of C ₂₃ H ₁₇ NO ₄ :

Calculated: C: 74.38; N: 4.61; H: 3.77

Measured value: C: 74.27; N: 4.49; H: 3.31

Example 54

  7-(2-pyridyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (686 mg, 2 mmol) was mixed with 2-pyridyltributyltin (306 mg, 3 mmol) to prepare the title compound, resulting in a brown solid (46.9 mg, 7%): melting point > 220 °C;

¹ H NMR (DMSO-d ₆ ): δ4.96 (2H, s), 6.57 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 2.4Hz), 6.85 (1H, dd, J =8.5Hz, J=2.4Hz), 6.92(1H, dd, J=8.8Hz, J=2.4Hz), 7.40-7.46(1H, m), 7.47(1H,dd, J=6.9Hz, J=1.0 Hz), 7.78(1H, d, J=8.8Hz), 7.84(1H, d, J=8.7Hz), 7.90-7.94(1H, m), 8.23(1H, s), 8.75(1H, d, J =2.4Hz), 9.52(1H, br s), 9.71(1H, br s); MS m/z 342([M+H]+).

HPLC at 210 nm gave 99.0% purity.

Analysis of C ₂₂ H ₁₅ NO ₃ ·0.5H ₂ O:

Calculated: C: 75.42; N: 4.60; H: 4.00

Measured value: C: 75.08; N: 4.20; H: 3.80

Example 55

  7-(3,4-Dichlorophenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 3,4-dichlorophenyl Boronic acid (572 mg, 3 mmol) was reacted to prepare the title compound to give a white solid (220 mg, 54%): mp 143-145°C;

¹ H NMR (DMSO-d ₆ ): δ4.99 (2H, s), 6.57 (1H, d, J = 2.4Hz), 6.65 (1H, d, J = 2.3Hz), 6.84 (1H, dd, J =8.5Hz, J=2.6Hz), 6.93(1H, dd, J=8.8Hz, J=2.4Hz), 7.35(1H,dd, J=8.2Hz, J=2.1Hz), 7.62(1H,d, J=1.9Hz), 7.77-7.84(3H,m), 8.24(1H,s), 9.59(1H,s), 9.72(1H,s); MS m/z 407/409([MH]-).

HPLC at 280 nm gave 97.7% purity.

Analysis of C ₂₂ H ₁₅ NO ₃ ·0.3H ₂ O:

Calculated: C: 66.62; H: 3.55

Measured value: C: 66.40; H: 3.39

Example 56

  7-(4-methylphenylthio)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-methylphenylthioboronic acid as described above in Example 23 (504 mg, 3 mmol) were reacted to prepare the title compound, resulting in a brown solid (250 mg, 65%): mp 195-198°C;

¹ H NMR (DMSO-d ₆ ): δ2.56 (3H, s), 4.95 (2H, s), 6.65 (2H, dd, J=10.6, J=2.4Hz), 6.84 (1H, dd, J= 8.5, Hz J = 2.6Hz), 6.91 (1H, dd, J = 8.8Hz, J = 2.4Hz), 7.28 (2H, d, J = 8.5Hz), 7.39 (2H, d, J = 8.5Hz), 7.77(1H,d,J=8.7Hz), 7.82(1H,d,J=8.6Hz), 8.18(1H,s), 9.52(1H,s), 9.69(1H,s); MS m/z 387 ([M+H]+).

HPLC at 280 nm gave 99.4% purity.

Analysis for C ₂₄ H ₁₈ SO ₃ ·0.3H ₂ O:

Calculated: C: 73.56; H: 4.78

Measured value: C: 73.66; H: 4.43

Example 57

  7-(4-cyanomethylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-cyanomethylphenyl as described above in Example 23 Boronic acid (483 mg, 3 mmol) was reacted to prepare the title compound to give a white solid (210 mg, 55%): mp 240-242°C;

¹ H NMR (DMSO-d ₆ ): δ4.14 (2H, s), 4.95 (2H, s), 6.63 (2H, d, J=2.3Hz), 6.85 (1H, dd, J=8.5, Hz J = 2.4Hz), 6.92 (1H, dd, J = 8.8Hz, J = 2.4Hz), 7.36 (2H, d, J = 8.2Hz), 7.49 (2H, d, J = 8.3Hz), 7.78 (1H, d,J=8.9Hz), 7.83(1H,d,J=8.6Hz), 8.20(1H,s), 9.55(1H,s), 9.70(1H,s); MS m/z 380([M+ H]+).

HPLC at 280 nm gave 98.6% purity.

Analysis of C ₂₅ H ₁₇ NO ₃ ·0.2H ₂ O:

Calculated: C: 78.40; N: 4.58; H: 3.66

Measured value: C: 78.45; N: 4.30; H: 3.56

Example 58

 7-(3-Trifluoromethoxyphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 3-trifluoromethoxybenzene as described above in Example 23 Boronic acid (618 mg, 3 mmol) was reacted to prepare the title compound, resulting in a white solid (140 mg, 33%): mp 110-112°C;

¹ H NMR (DMSO-d ₆ ): δ4.98 (2H, s), 6.61 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 1.9Hz), 6.85 (1H, dd, J =8.5, Hz J=2.4Hz), 6.93(1H, dd, J=8.8Hz, J=2.3Hz), 7.32(1H, s), 7.38-7.44(2H, m,), 7.64-7.68(1H, m), 7.80(1H, d, J=8.8Hz), 7.83(1H, d, J=8.6Hz), 8.23(1H, s), 9.61(1H, s), 9.72(1H, s); MS m /z 425([M+H]+).

HPLC at 280 nm gave 98.7% purity.

Analysis for C ₂₄ H ₁₅ F ₃ O ₄ 0.5H ₂ O:

Calculated: C: 66.51; H: 3.72

Measured value: C: 66.57; H: 3.29

Example 59

7-(4-Trifluoromethoxyphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-trifluoromethoxybenzene as described above in Example 23. Boronic acid (618 mg, 3 mmol) was reacted to prepare the title compound, resulting in a white solid (219 mg, 52%): mp 245-248°C;

¹ H NMR (DMSO-d ₆ ): δ4.97 (2H, s), 6.60 (1H, d, J = 2.3Hz), 6.64 (1H, d, J = 2.5Hz), 6.85 (1H, dd, J =8.5, Hz J=2.5Hz), 6.93(1H, dd, J=8.8Hz, J=2.3Hz), 7.32(1H, s), 7.38-7.44(2H, m,), 7.64-7.68(1H, m), 7.80(1H, d, J=8.8Hz), 7.83(1H, d, J=8.6Hz), 8.22(1H, s), 9.60(1H, s), 9.71(1H, s); MS m /z 425([M+H]+).

HPLC at 280 nm gave 99.9% purity.

Analysis of C ₂₄ H ₁₅ F ₃ O ₄ :

Calculated: C: 67.93; H: 3.56

Measured value: C: 67.76; H: 3.50

Example 60

  7-(4-tert-butylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-tert-butylphenylboronic acid as described above in Example 23 (534 mg, 3 mmol) were reacted to prepare the title compound, resulting in a white solid (144 mg, 36%): mp 162-165°C;

¹ H NMR (DMSO-d ₆ ): δ1.38 (9H, s), 4.94 (2H, s), 6.63 (1H, d, J=2.4Hz), 6.68 (1H, d, J=2.2Hz), 6.84 (1H, dd, J=8.5, Hz J=2.4Hz), 6.90 (1H, dd, J=8.8Hz, J=2.3Hz), 7.27 (2H, d, J=8.2Hz), 7.53 (2H, d, J = 8.3Hz), 7.79 (1H, d, J = 7.6Hz), 7.82 (1H, d, J = 8.6Hz), 8.18 (1H, s), 9.54 (1H, s), 9.69 (1H, s); MS m/z 397 ([M+H]+).

HPLC at 280 nm gave 99.2% purity.

Analysis for C ₂₇ H ₂₄ O ₃ ·0.5H ₂ O:

Calculated: C: 79.98; H: 6.21

Measured value: C: 79.24; H: 6.29

Example 61

  7-(Naphthyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 1-naphthylboronic acid (516 mg , 3 mmol) to prepare the title compound, resulting in a white solid (110 mg, 28%): melting point 160-162°C;

¹ H NMR (DMSO-d ₆ ): δ4.85 (2H, s), 6.24 (1H, d, J = 2.4Hz), 6.57 (1H, d, J = 2.3Hz), 6.84-6.92 (3H, m ), 7.26-7.51 (4H, m), 7.63-7.68 (1H, m), 7.83 (1H, d, J = 8.1Hz), 7.88 (1H, d, J = 8.5Hz), 8.02 (1H, d, J=8.2Hz), 8.30(1H,s), 9.39(1H,s), 9.70(1H,s); MS m/z 389([M+H]+).

HPLC at 280 nm gave 95.4% purity.

Analysis for C ₂₇ H ₁₈ O ₃ ·0.4H ₂ O:

Calculated: C: 81.55; H: 4.76

Measured value: C: 81.39; H: 4.87

Example 62

  7-(4-Ethylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 4-ethylphenylboronic acid ( 450 mg, 3 mmol) were reacted to prepare the title compound, resulting in a white solid (292 mg, 79%): mp 203-205°C;

¹ H NMR (DMSO-d ₆ ): δ1.29 (3H, t, J = 7.6Hz), 2.71 (2H, q, J = 2.2Hz), 4.94 (2H, s), 6.65 (2H, dd, J =13.7Hz, J=2.4Hz), 6.85(1H,dd, J=8.5Hz, J=2.6Hz), 6.90(1H,dd, J=8.8Hz, J=2.4Hz), 7.25(2H,d, J=7.9Hz), 7.34(2H, d, J=8.2Hz), 7.76(1H, d, J=8.8Hz), 7.82(1H, d, J=8.6Hz), 8.17(1H, s), 9.51 (1H, s), 9.69 (1H, s); MSm/z 369 ([M+H]+).

HPLC at 280 nm gave 99.8% purity.

Analysis of C ₂₅ H ₂₀ O ₃ ·0.5H ₂ O:

Calculated: C: 79.56; H: 5.61

Measured value: C: 79.86; H: 5.37

Example 63

7-(3,5-Dimethylphenyl)-5H-dibenzo[c,g]chromene-3,9-diol

7-Bromo-H-dibenzo[c,g]chromene-3,9-diol (343 mg, 1 mmol) was mixed with 3,5-dimethylbenzene as described above in Example 23 Boronic acid (450 mg, 3 mmol) was reacted to prepare the title compound, resulting in a white solid (270 mg, 73%): mp 220-223°C;

¹ H NMR (DMSO-d ₆ ): δ2.34 (6H, s), 4.93 (2H,), 6.62 (2H, dd, J=9.7Hz, J=2.4Hz), 6.84 (1H, dd, J= 8.5Hz, J=2.4Hz), 6.88-6.91(3H, m), 7.04(1H, s), 7.76(lH, d, J=8.9Hz), 7.82(1H, d, J=8.6Hz), 8.16 (1H,s), 9.49(1H,s), 9.69(1H,s); MS m/z 369([M+H]+).

HPLC at 280 nm gave 97.2% purity.

Analysis for C ₂₅ H ₂₀ O ₃ ·0.25H ₂ O:

Calculated: C: 80.52; H: 5.54

Measured value: C: 80.64; H: 5.32

Example 64

Ability to compete with 17β-estradiol for ERα and ERβ

Representative examples of the invention were evaluated for their ability to compete with 17[beta]-estradiol for ER[alpha] and ER[beta]. This assay provides a means of determining whether a particular compound binds to the estrogen receptor (and is therefore "estrogenic") and is selective for ERα or ERβ. These values are presented in the table below and expressed as _IC50 . 17β-estradiol was included as a standard reference for comparison. The method used is briefly described below. Crude lysates of Escherichia coli (E. coli) expressing the estrogen receptor ligand-binding domains (D, E, & F) of human ERα or ERβ were prepared. Both receptors and compounds were diluted in 1X Dulbecoo's PBS (DPBS) supplemented with 1 mM EDTA. Using high-binding masked microtiter plates, 100 microliters of receptors (luG/well) were bound to 2 mM [ ^3H ]-17[beta]-estradiol and various concentrations of compound. After 5 to 15 hours at room temperature, the plates were washed with DPBS/1 mM EDTA and bound radioactivity was measured using a liquid scintillation counter. _IC50 is defined as the concentration of compound that reduces total 17[beta]-estradiol binding by 50%. The results obtained are described in the table below.

Table 1.5H-dibenzo[c,g]chromene derivatives

Results obtained in standard pharmacological test procedures indicate that the compounds of the invention are estrogenic compounds, some with a strong preferential affinity for the ER[beta] receptor. The compounds of the invention range from high preferential affinity for ER[beta] over ER[alpha] to nearly equal affinity for both receptors. Thus, the compounds of the present invention span a range of activities based at least in part on their selective profile of receptor affinities. Furthermore, because each novel receptor-ligand complex is unique and thus unique in its interaction with various co-regulatory proteins, the compounds of the invention will exhibit differential regulation depending on their cellular environment. performance. For example, a compound may act as an estrogen agonist in some cell types and may act as an estrogen antagonist in other tissues. Compounds with this activity are sometimes referred to as SERMs (selective hormone receptor modulators). However, unlike many estrogens, many SERMs do not cause an increase in uterine wet weight. These compounds are antiestrogenic in the uterus and are able to completely antagonize the trophic effects of estrogen agonists in uterine tissue. However, these compounds act as estrogen agonists in the bone, cardiovascular and central nervous systems. Because of this tissue selectivity of these compounds, they are useful in the treatment or prevention of estrogen deficiency (in certain tissues such as bone or cardiovascular) or estrogen excess (in the uterus or breast) caused by or associated with Mammalian condition or syndrome.

Even in addition to this cell-specific modulation, the compounds of the invention have the potential to act as agonists at one receptor type and antagonists at another receptor type. For example, it has been shown that these compounds can be antagonists of ERβ and at the same time agonists of ERα (Meyers, Marvin J.; Sun, Jun; Carlson, Kathryn E.; Katzenellenbogen, Benita S.; Katzenellenbogen, John A.. J. Med . Chem. (1999), 42(13), 2456-2468). This ERSAA (estrogen receptor selective agonist antagonist) activity provides a pharmacologically distinct estrogenic activity to this series of compounds.

Standard pharmacological laboratory procedures for determining the activity profile of a given test compound are readily available. The following examples briefly summarize several typical experimental procedures. Standard pharmacological assays for SERMs are also provided by US Patents 4,418,068 and 5,998,402.

Example 65

    Pro-utero/anti-utero test method for rats

The estrogenic and antiestrogenic properties of compounds can be determined in a uterotropic test (4 days) in sexually immature rats (as previously described by LJ Black and RL Goode, Life Sciences, 26, 1453 (1980)). Sexually immature Sprague-Dawley rats (female, 18 days old) were tested in 6 groups. 50% DMSO/50% saline as injection vehicle, 10uG compound, 100uG compound, (100uG compound + 1uG 17β-estradiol) for detection of antiestrogenicity and 1uG 17β-estradiol for daily intraperitoneal injection for treatment animal. On day 4, animals were sacrificed by _CO2 asphyxiation, their uteri were removed, excess lipid stripped, any fluid removed and wet weight determined. Histological studies were performed on sections of one uterine horn, and the remainder was used to isolate total RNA to evaluate complement component 3 gene expression.

Example 66

    Experimental method for 6-week ovariectomized rats - bone and heart protection

Sprague Dawley CD rats were obtained from Taconic Farms (weight range 240-275 g) 1 day after ovariectomy or sham surgery. They were housed 3 or 4 rats/cage in a 12/12 (light/dark) cycle room with food (Purina 5K96C rat chow) and water provided ad libitum. All study treatments started 1 day after the animals' arrival and were dosed 7 days a week for 6 days as indicated. A group of age-corresponding sham-operated rats received no treatment and served as the complete estrogen-supplemented control group in each study.

All treatments were prepared in 1% tween 80 in normal saline at a given concentration such that the treatment volume was 0.1 ml/100 g body weight. 17β-estradiol was dissolved in corn oil (20 μg/ml) and delivered subcutaneously, 0.1 ml/rat. All doses were adjusted at three-week intervals according to group mean body weight measurements.

Bone mineral density (BMD) of each rat was assessed 5 weeks after initiation of treatment and 1 week prior to study termination. Total and trabecular densities of the proximal tibia were measured in anesthetized rats using the XCT-960M (pQCT; Stratec Medizintechnik, Pforzheim, Germany). Assays were performed as follows: 15 min prior to scanning, each rat was anesthetized with an intraperitoneal injection of 45 mg/kg ketamine, 8.5 mg/kg xylazine, and 1.5 mg/kg acepromazine.

The right hindlimb was strapped to an acrylic support frame at a 90° angle at the ankle joint and 180° at the knee joint by a 25 mm diameter polycarbonate tube. Polycarbonate tubes are fixed to the slide platform so that they are perpendicular to the aperture of the pQCT. The platform is adjusted so that the distal end of the femur and the proximal end of the tibia are within the scan range. The 2D scan length is 10 mm and the line resolution is 0.2 mm. After the scan image is displayed on a monitor, it is positioned at the proximal end of the tibia. Start the PQCT scan at a distance of 3.4 mm distal to this point. The thickness of the pQCT scan was 1 mm, the voxel (three-dimensional pixel value) was 0.140 mm, and the slice consisted of 145 projections.

After the pQCT scan is completed, the image is displayed on a monitor. Outline the region of interest including the tibia but excluding the fibula. Automatic soft tissue removal using an iterative algorithm. The density of the remaining bone (total density) is expressed in mg/cm3. Strip the outer 55% of the bone in a concentric spiral. The density of the remaining bone (trabecular density) is expressed in mg/cm3. One week after BMD assessment, rats were euthanized by carbon dioxide asphyxiation and blood was collected for cholesterol determination. The uterus is removed and weighed. Total cholesterol was determined using a Boehringer-Mannheim Hitachi 911 clinical analyzer, cholesterol/HP kit. Statistics were compared using one-way analysis of variance with Dunnet's test.

Example 67

       MCF-7/ERE anti-proliferation test method

Stock solutions of test compounds (typically 0.1 M) were prepared in DMSO and then diluted 10 to 100-fold in DMSO to prepare 1 or 10 mM working solutions. DMSO stock solutions were stored at 4°C (0.1M) or -20°C (<0.1M). Growth medium for MCF-7 cells [D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetal calf serum, 1% (v/v) penicillin-streptomycin and 2mMglutaMax-1 ] Passaging, twice a week. The cells were placed in ventilated bottles and cultured at 37°C in a 5% CO ₂ /95% humidity incubator. The day before treatment, cells were placed in 96-well plates at 25,000/well and growth medium, and cultured overnight at 37°C.

Cells were diluted with 50 μl/well 1:10 of adenovirus 5-ERE-tk-luciferase in the experimental medium [D-MEM/F-12 medium without phenol red, containing 10% (v/v) Heat-inactivated charcoal-adsorbed fetal bovine serum, 1% (v/v) penicillin-streptomycin, 2 mM glutaMax-1, 1 mM sodium pyruvate] for infection at 37°C. The wells were then washed once with 150 μλ assay medium. Finally, in replicate experiments using 8 wells per treatment, cells were treated for 24 hours at 37°C with 150 μλ/well of vehicle (≤0.1% v/v DMSO) or compound diluted ≥1000 times into the assay medium .

Primary screening of test compounds was performed with a single dose of 1 μM, either alone (agonist mode) or in combination with 0.1 nM 17[beta]-estradiol (EC ₈₀ , antagonist mode). Each 96-well plate also contained a vehicle control (0.1% v/v DMSO) and an agonist control (0.1 or 1 nM 17[beta]-estradiol). Dose-response experiments were performed on the active compounds in agonist and/or antagonist mode with log rises from 10 ⁻¹⁴ to 10 ⁵ M. From these dose response curves _EC50 and _IC50 values can be obtained respectively. In each treatment group, the last well contained 5 microliters of 3 x 10 ^-5 M ICI-182,780 (final concentration 10 ^-6 M) as an ER antagonist control.

After treatment, cells were lysed with 25 μl/well of 1× Cell Culture Lysis Reagent (Promega Corporation) for 15 minutes on a shaker. Cell lysates (20 microliters) were transferred to 96-well luminometer plates and luciferase was detected using 100 microliters/well of luciferase substrate (Promega Corporation) in a MicroLumat LB 96P luminometer (EG & G Berthold) active. A 1 second background detection was performed on each well prior to the addition of substrate. Substrate was added and luciferase activity was detected for 10 seconds after a 1 second delay. Data were transferred from the luminometer to a Macintosh personal computer and analyzed using JMP software (SAS Institute); this program removed background readings from the luciferase assay in each well and then calculated the mean and standard deviation for each treatment.

Luciferase values were logarithmic and extreme transformation values were downweighted using Huber Mestimator. Transformed and weighted data were analyzed using JMP software, performing one-way ANOVA (Dunnett's test). Compound treatments were compared to vehicle control results in agonist mode, or positive agonist control (0.1 nM 17[beta]-estradiol) results in antagonist mode. For initial single-dose experiments, if compound-treated results were significantly different (p<0.05) from the appropriate control, the results were expressed as a percentage relative to the 17β-estradiol control [i.e., ((compound-vehicle control)/( 17β-estradiol control-vehicle control))×100]. JMP software was also used to determine _EC50 and/or _IC50 from non-linear dose response curves.

Example 68

Inhibition of LDL Oxidation - Antioxidant Activity

Porcine aortas were obtained from slaughterhouses, washed, transferred to iced PBS, and aortic endothelial cells were collected. To collect cells, the intercostal vessels of the aorta were ligated and one end of the aorta was clamped. Fresh sterile-filtered 0.2% collagenase (Sigma type I) was placed in the vessel, and the other end of the vessel was clamped to form a closed system. The aorta was incubated at 37°C for 15-20 minutes, then the collagenase solution was collected and centrifuged at 2000xg for 5 minutes. The pellet was resuspended in 7 ml of endothelial cell culture medium consisting of charcoal-adsorbed FBS (5%), NuSerum (5%), L-glutamine (4 mM), penicillin-streptomycin (1000 U/ml, 100 μg/ml) and gentimicin (75 μg/ml) supplemented with phenol red-free DMEM/Ham's F12 medium, inoculated in 100 mm Petri dishes, and cultured at 37°C in 5% CO ₂ . After 20 minutes, the cells were rinsed with PBS, then fresh medium was added, and the operation was repeated after 24 hours. Cells were confluent after approximately 1 week. Endothelial cells were routinely fed twice a week, trypsinized when confluent, and inoculated at a ratio of 1:7. Cell-mediated oxidation of 12.5 [mu]g/ml LDL was carried out at 37[deg.] C. for 4 hours in the presence of test compound (5 [mu]M). Results are expressed as percent inhibition of the oxidation process, as measured by TBARS (thiobarbituric acid reactants) for the analysis of free aldehydes (Yagi K., Biochem Med 15:212-216 (1976)).

Example 69

D12 Hypothalamic Cell Experimental Method

D12 rat hypothalamic cells were subcloned from the RCF17 parental cell line and stored frozen. They were routinely grown in DMEM: F12 (1:1), glutaMAX-1 (2Mm), penicillin (100 U/ml)-streptomycin (100 μg/ml) and 10% fetal bovine serum (FBS). Cells were placed in phenol red-free medium (DMEM: F12, glutaMAX, penicillin-streptavidin) containing 2-10% charcoal-adsorbed FBS at a subconfluent density (1-4×10<6> wells/150 mm dish). prime). After 24 hours, medium containing 2% of the adsorbed serum was added to the cells again. To test for agonist activity, cells were treated with 10 nM 17β-estradiol or various doses of test compounds (1 mM, or 1 pM to 1 mM). To examine antagonist activity, cells were treated with 0.1 nM 17β-estradiol in the absence or presence of various doses (100 pM to 1 mM) of the test compound. Control dishes were also treated with DMSO as a negative control. Forty-eight hours after hormone addition, cells were lysed and binding assays performed.

For each binding assay, 100-150 mg of protein was incubated with 10 nM ³ H-R5020 in a volume of 150 microliters and a 100-fold excess of R5020. Reactions were prepared in triplicate (three with R5020, three without R5020) in 96-well plates. Add protein extract first, then add ³ H-R5020 or ³ H-R5020 and 100×unlabeled R5020. The reaction was carried out at room temperature for 1-2 hours. The reaction was terminated by adding 5% charcoal (Norit SX-4), 0.5% dextran 69K (Pharmacia) (pH 7.4) in 100 ml cold TE. After 5 minutes at room temperature, centrifugation (5 minutes, 1000 RCF, 4°C) separated bound and unbound ligand. The supernatant (approximately 150 mL) was removed and transferred to scintillation vials. After addition of scintillation fluid (Beckman Ready Protein+), samples were counted in a scintillation counter for 1 min.

Example 70

Progesterone receptors in the preoptic area of the CNS

Ovaryectomy was performed on 60-day-old female Sprague-Dawley rats. Animals were housed in an animal care facility with a photoperiod of 12 hours of light and 12 hours of darkness, with free access to tap water and rodent chow.

Ovariectomized animals were randomized into groups injected with vehicle (50% DMSO, 40% PBS, 10% ethanol vehicle), 17[beta]-estradiol (200 ng/kg) or the side compound. The antagonistic properties of the test compound were evaluated by injecting other animals with the test compound 1 hour before the injection of 17[beta]-estradiol. Six hours after the subcutaneous injection, the animals were euthanized with a lethal dose of _CO2 , and their brains were harvested and frozen.

Tissues collected from animals were sectioned at -16°C in a cryostat and pooled on silane-coated microscope slides. Slides with sections were then dried on a slide warmer maintained at 42°C and stored in a dry slide box at -80°C. Before processing, warm the dry slide box slowly to room temperature (-20°C for 12-16 hours; 4°C for 2 hours; room temperature for 1 hour) to eliminate the formation of condensation on the slide and thereby reduce tissue and RNA degradation. to minimum. Dried slides were placed on metal supports, postfixed in 4% paraformaldehyde (pH 9.0) for 5 minutes and processed as described above.

A plasmid containing an 815 bp fragment of rat PR cDNA 9 (ligand binding region) was linearized and used to generate an S35-UTP labeled probe that was partially complementary to rat PR mRNA. Processed slides with sections were hybridized with 20 ml of hybridization mix containing riboprobes (4-6 x 106 DPM/slide) and 50% formamide and incubated overnight at 55°C in a humidified chamber. In the morning slides were placed on metal supports immersed in 2×SSC (0.5M NaCl, 0.015M sodium citrate; pH 7.0)/10 mM DTT. The scaffolds were transferred whole to a large container and washed with 2×SSC/10 mM DTT for 15 minutes at room temperature with gentle agitation. Slides were then washed with RNase buffer at 37°C for 30 minutes, rinsed with RNase A (2 mg/ml) at 37°C for 30 minutes, and rinsed with 1×SSC at room temperature for 15 minutes. Subsequently, slides were rinsed (2×30 min) in 0.1×SSC at 65°C to remove non-specific markers, rinsed in 0.1×SSC for 15 minutes at room temperature and treated with a graded series of alcohol:ethyl acetate (70%, 95% and 100%) dehydrogenation. The air-dried slides were placed opposite the x-ray film for 3 days before processing for imaging. Slides from all animals were hybridized, washed, exposed, and photographed together to eliminate differences due to variation in conditions between experiments.

Example 71

Hot flashes in rats - CNS effects

Ovariectomized female 60-day-old Sprague-Dawley rats were obtained after surgery. Surgery was performed at least 8 days before the first treatment. The animals were housed individually, with a 12-hour light/dark cycle, and were given standard rat chow and water.

Two control groups were included in each study. Doses are based on mg/kg group mean body weight prepared in 10% DMSO in sesame oil (s.c. studies) or 1.0% tween 80 in saline (oral studies). Animals are administered test compounds at doses ranging from 0.01 to 10 mg/kg group mean body weight. Vehicle and ethinyl estradiol (EE) control (0.1 mg/kg sc, or 0.3 mg/kg po) control groups were included in each experiment. When compounds were tested for antagonist activity, EE was co-administered at 0.1 or 0.3 mg/kg for sc or oral studies, respectively. The test compound was applied until the daytime temperature of the tail skin was measured.

After an acclimatization period of 4 days, the animals are treated once daily with the compound of interest. There were 10 animals per treatment group. The compound was administered either by subcutaneous injection of 0.1 ml in the nape of the neck, or by oral administration in an amount of 0.5 ml. On the third day of treatment, a morphine pellet (75 mg morphine sulfate) was implanted subcutaneously. On the fifth day of treatment, one or two additional morphine pills were implanted. On day eight, approximately half of the animals were injected with ketamine (80 mg/kg, intramuscularly) and a thermocouple connected to the MacLab DataAcquisition System (API Instruments, Milford, MA) was strapped to the tail approximately 1 km from the base of the tail. inch. The system continuously measures tail skin temperature. Baseline temperature was measured for 15 minutes, followed by subcutaneous injection (0.2 mL) of naloxone (1.0 mg/kg) to block the effect of morphine and tail skin temperature was measured 1 hour thereafter. On day nine, the remaining animals were pooled and analyzed similarly.

Example 72

Vasomotor function in isolated rat aortic rings

Sprage-Dawley rats (240-260 g) were divided into 4 groups:

1. Normal unresectable (intact) ovaries

2. Ovariectomized (ovex), vehicle-treated

3. Removal of the ovaries. 17β-estradiol-treated (1 mg/kg/day)

4. Ovariectomized, Experimental Compound (i.e., 1 mg/kg/day) Treated Animals

Animals were ovariectomized approximately 3 weeks prior to treatment. Each animal was administered 17[beta]-estradiol sulfate or test compound (1 mg/kg/day) suspended in distilled deionized water containing 1% tween-80 by gastrointestinal gavage. Vehicle-treated animals were given the appropriate amount of vehicle used in the drug-treated groups.

After _CO inhalation and exsanguination, animals were euthanized. Their thoracic aortas were quickly taken out and placed in a physiological solution with the following components (mM) at 37°C: NaCl (54.7), KCl (5.0), NaHCO ₃ (25.0), MgCl ₂ 2H ₂ O (2.5), D - Glucose (11.8) and _CaCl2 (0.2), these solutions were gassed with _CO2 - _O2 95%/5% to reach a final pH value of 7.4. The adventitia was removed from the outer surface, and the vessels were cut into 2-3 mm wide rings. The ring was suspended in a 10 ml tissue bath with one end connected to the bottom of the water bath and the other end connected to a load cell. A static tension of 1 gram was applied to the rings. The ring was equilibrated for 1 hour, and the signal was acquired for analysis.

After equilibration, the rings were exposed to increasing concentrations of phenylephrine (10 ^-8 to 10 ^-4 M) and the tension was recorded. The water bath was then rinsed 3 times with fresh buffer. After rinsing, add 200mM L-NAME in tissue water bath and equilibrate for 30 minutes. The phenylephrine concentration-response curves were then repeated.

Example 73

  Eight Arm Radial Arm Maze - Cognitive Improvement

Male Sprague-Dawley, CD rats (Charles River, Kingston, NY) weighing 200-250 grams on arrival were used. Six rats per cage were given standard laboratory chow and water ad libitum for one week. Housing was performed in a colony room maintained at 22°C with a 12-hour light/dark cycle, with light on at 6:00 am. After acclimatization to the apparatus, animals were housed individually and maintained at 85% free-feeding weight. Once a stable weight was achieved, rats were placed in an 8-arm radial maze.

The structure of the maze was adapted from that of Peele and Baron (Pharmacology, Biochemistry, and Behavior, 29:143-150, 1988). The maze height was raised to 75.5 cm and included a circular area surrounded by 8 arms radiating equidistantly from each other from the center, each arm being 58 cm long by 13 cm high. Equipped with a transparent plexiglass cylinder, animals were trapped in the central part of the maze before the start of each experiment. Each arm of the maze is equipped with 3 sets of photocells connected to a data acquisition device, which in turn is connected to a computer. The movement of rats in the maze was tracked using photocells. A pill feeder located above a food cup at the end of each arm dispensed two 45 mg chocolate pellets when the arms' external photocells were first activated at a given time. The maze was located in a testing room with black and white geometric posters on each wall as visual cues. During all training and testing, white noise (approximately 70db) was audible.

The training method consists of five sessions, each lasting 5 or 10 minutes per day. There was a 10 second delay between placing the rat in the center portion of the maze and placing it on the raised cylinder to start timing. In Phase I, food-restricted rat pairs were placed in a maze for 10 minutes with 45 mg chocolate food pellets spread over the 8 arms of the maze. In Phase II, each rat was placed individually in the maze for 10 min with the bolus spread between the middle photocell in each arm to the food cup. In Phase III, each rat was placed in a maze for 10 min with food pellets located only in and near the food cups in each arm. In Phase IV, each rat was given 10 minutes to obtain two boluses on each arm. Re-entering an arm is considered an error. Rats were trained in this manner daily until they reached a performance standard of less than or equal to 2 total misses in three consecutive days of training. The total acclimatization and training time is approximately 3 weeks.

Test compounds were prepared in phosphate buffered saline and administered at 1 mg/kg. Scopolamine HBr (0.3 mg/kg, s.c.) acts as an impairing agent, which leads to increased error rates (impaired memory). On any given experimental day, the test compound was administered ip simultaneously with scopolamine 30 minutes before the first maze test.

To evaluate test compounds, an 8×8 balanced Latin square for repeated measures was designed to achieve high experimental efficiency with a minimum number of animals: 8 treatments (vehicle, scopolamine, 3 doses) were randomly assigned in each experiment Test compound in combination with scopolamine) 8 experiments were performed twice a week. Each treatment was used the same number of times as each other treatment. Therefore, the residual effect of each treatment can be estimated and removed from the direct treatment effect. After ANOVA, Dunnett's two sides test was performed for multiple comparisons on adjusted means.

Animals that did not make 4 correct choices within 5 minutes during the first trial, or that had not made a total of 8 choices by the end of the second trial, were considered timed out for that time period. Any animal that "timed out" after more than one dose of test compound was excluded from the analysis.

Example 74

Neuroprotection

Inhibition of time-dependent cell death in cultures of primary cortical neurons

Primary cortical neurons were obtained from 0-1 year old rat brains using a variation of the method described by Monyer et al., 1989, Brain Research 483:347-354. Dispersed brain tissues were grown in DMEM/10% PDHS (pregnant donor horse serum) for 3 days, and then treated with cytarabine (ARC) for 2 days to remove contaminating glial cells. On the fifth day, the ARC medium was removed and replaced with DMEM/10% PDHS. Neuronal cells were cultured for an additional 4-7 days before use.

Control primary neuron cultures exhibited progressive cell death between days 12 and 18 in culture. On day 9, the test compound was added to 6 cultures maintained in DMEM and 10% PDHS and the rest of the cultures were used as controls, and then 12 cultures were evaluated for lactate dehydrogenase (LDH) on days 12 and 16. )level. LD was detected using a variation of the method of Wroblewski et al. 1955, Proc. Soc. Exp. Biol. Med. 90:210-213. LD is a microenzyme commonly used in clinical and basic research to measure tissue viability. An increase in medium LD is directly related to cell death.

Neuroprotection against cytotoxicity induced by hypoglycemia

C6 glioma cells obtained from ATCC were placed in RPMI medium (in FALCON 25 cm2 tissue culture flasks) containing FBS at a concentration of 1 × 10<6> cells/ml. Four hours before the onset of hypoglycemia, the maintenance medium was discarded, the monolayers were rinsed twice in the appropriate medium, and then incubated for four hours at 37°C in serum-free or serum-free plus test compound. Monolayers were rinsed twice with Kreb’s Ringer phosphate buffer, followed by the addition of the appropriate glucose treatment. RPMI medium contained 2 mg glucose/ml; flasks were divided into 6 groups, each containing 100% glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2 mg/ml) or 0 % glucose (buffer) or supplemented with test compound. All flasks were incubated for 20 hours and then assessed for total viable and dead cell numbers using trypan blue.

Neuroprotection against excitotoxic amino acids

Five dishes containing SK-N-SH neuroblastoma cells were treated with test compound and five dishes were treated with RPMI medium. After 4 hours, all cells were treated with NMDA (500 mu M) for 5 minutes. Total viable and dead cells were then determined.

Neuroprotection against oxygen-glucose removal

Analysis of pyknotic nuclei to measure apoptosis: Cortical neurons were prepared from E18 rat fetuses and plated at a density of 100,000 cells/well precoated with poly-D-lysine (10 ng/ml) and serum Cloth on an 8-well chamber slide. Cells were placed in high glucose DMEM containing 10% FCS and placed in a 37 °C incubator containing 10% _CO2 /90% air. The next day, the medium was changed to high glucose DMEM with B27 supplement, thereby removing serum, and the cells were placed in the incubator without further medium changes until the day of the experiment. On day 6, slides were divided into two groups; control group and OGD group. Cells in the control group received DMEM with glucose and customB27 (no antioxidant). Cells in the OGD group received glucose-free DMEM containing custom B27, which had been degassed under vacuum for 15 minutes. Cells were purged with 90% _N2 /10% _CO2 for 10 min in an airtight chamber and incubated at 37 °C for 6 h. After 6 hours, the medium containing the vehicle (DMSO) or the test compound of the cells in the control group and the OGD group was replaced with DMEM containing glucose and custom B27. Return the cells to the normoxic incubator at 37 °C. After 24 hours, cells were fixed in 4% PFA for 10 minutes at 4°C and stained with Topro (a fluorescent nuclear binding dye). Apoptosis was assessed by measuring pyknotic nuclei using a laser scanning hemocytometer.

Measurement of LDH release as an indication of cell death: Cortical neurons were prepared from E18 rat fetuses and plated at a density of 150,000 cells/well precoated with poly-D-lysine (10 ng/ml) and serum 48-well culture plate. Cells were placed in high glucose DMEM containing 10% FCS and placed in a 37 °C incubator containing 10% _CO2 /90% air. The next day, the medium was changed to high glucose DMEM with B27 supplement to remove serum. On day 6, cells were divided into two groups; control group and OGD group. Cells in the control group received DMEM containing glucose and custom B27 (no antioxidant). Cells in the OGD group received glucose-free DMEM containing custom B27, which had been degassed under vacuum for 15 minutes. Cells were purged with 90% _N2 /10% _CO2 for 10 min in an airtight chamber and incubated at 37 °C for 6 h. After 6 hours, the medium containing the vehicle (DMSO) or the test compound of the cells in the control group and the OGD group was replaced with DMEM containing glucose and custom B27. Return the cells to the normoxic incubator at 37 °C. After 24 hours, cell death was assessed by measuring the release of LDH (lactate dehydrogenase) by the cells into the medium. In the LDH assay, aliquots of 50 microliters of medium were transferred to 96-well plates. After adding 140 microliters of 0.1 M potassium phosphate buffer (pH 7.5) and 100 microliters of 0.2 mg/ml NADH, the plate was left to stand in the dark at room temperature for 20 minutes. Add 10 μl of sodium pyruvate to initiate the reaction. Plates were read immediately at 340 nM in a Thermomax plate reader (Molecular Devices). Absorbance (an index of NADH concentration) was recorded every 6 seconds for 5 minutes and LDH activity was calculated using the slope representing the rate of NADH disappearance.

LDH activity (U/ml)=(ΔA/min)(TCF)(20)(0.0833)/(.78)

where 0.0833 = constant of proportionality

0.78 = instrument light path length (cm)

Example 75

  Experimental method for HLA rats - Crohn's disease and inflammatory bowel disease

Male HLA-B27 rats were obtained from Taconic and provided an unrestricted food source (PMI Lab Chow 5001) and water. Rats were 22-26 weeks old at the start of the study.

Rats were subcutaneously injected with one of the following formulations once a day for 7 days. There were 5 rats in each group and the last dose was administered 2 hours before euthanasia.

●Vehicle (50% DMSO/50% Dulbecco's PBS)

17α-ethynyl-17β-estradiol (10 μg/kg)

●Experimental compound

Stool quality was observed daily and graded according to the following criteria: diarrhea=3; soft stool=2; normal stool=1. At the end of the experimental procedure, serum was collected and stored at -70°C. Colon sections were prepared for histological analysis and another fragment was analyzed for myeloperoxidase activity.

Myeloperoxidase activity was measured using the following method. Colon tissue was harvested and snap frozen in liquid nitrogen. Use a representative sample of the entire colon to ensure consistency between samples. Tissues were stored at -80°C until use. Next, the tissue was weighed (approximately 500 mg) and homogenized in 1:15 w/v of ₅ mM _H2KPO4 (pH 6) wash buffer. The tissue was centrifuged at 20,000 xg for 45 minutes at 2-8°C in a Sorvall RC 5B centrifuge. Then discard the supernatant. Tissue was resuspended and homogenized in 2.5 ml of 50 _{mM H2KPO4} containing 10 mM EDTA and 0.5% Hex Ammonium Bromide (1:5 w/v) and homogenized to aid in solubilization of intracellular MPO. Tissues were frozen in liquid nitrogen, thawed in a 37°C-water bath and sonicated for 15 seconds to ensure cell membrane lysis. This process was repeated 3 times. Samples were then placed on ice for 20 min and centrifuged at 12,000 x g for 15 min at 2–8 °C. Supernatants were analyzed after these steps.

Add 2.9 mL of 50 mM _{H2KPO4 containing} 0.167 o-dianisidine and 0.0005% _H2O2 to the reaction tube to prepare the experimental _mixture . When degraded by hydrogen peroxide, o-dianisidine oxidizes and absorbs at 460 nm in a concentration-dependent manner. The mixture was heated to 25°C. Add 100 μl of tissue supernatant to the reaction tube, incubate at 25°C for 1 min, then transfer 1 ml to a disposable plastic test tube. The OD was measured at 460 nm every two minutes of reaction time against a blank containing 2.9 mL of reaction mixture and 100 μL of 0.5% ammonium bromide solution.

Units of enzyme activity were quantified by comparing the absorbance at 460 to a standard curve prepared with 31.1 units/vial of purified human MPO. MPO was reconstituted and _serially diluted to four known concentrations with 50 mM _H2KPO4 containing 10 mM EDTA and 0.5% Hex Ammonium Bromide. Sample absorbance is compared to this curve to determine activity.

Histological analysis was performed as follows. Colonic tissues were soaked in 10% neutral buffered formalin. Each colon sample was divided into four samples for evaluation. Formalin-fixed tissues were processed in a vacuum infiltration processor for paraffin embedding. Samples were sectioned at 5 microns and then stained with hematoxylin and eosin (H&E) for double-blind histological evaluation using criteria modified after Boughton-Smith. After scoring was complete, samples were identified, data were tabulated and analyzed by ANOVA linear modeling with multiple comparisons of means.

All patents, publications, and other documents cited herein are hereby incorporated by reference in their entirety.

Claims (19)

1. following formula: compound or its pharmacologically acceptable salt

Wherein

R ¹, R ², R ³, R ⁵, R ⁶, R ⁷And R ⁸Be selected from hydrogen, hydroxyl, C independently of one another ₁-C ₆Alkyl, C ₁-C ₆Alkoxy or halogen;

R ⁴Be hydrogen, C ₁-C ₆Alkyl, halogen, C ₁-C ₆Alkoxyl group ,-CN ,-C ₂-C ₈Alkenyl ,-CHO, aryl, furyl, thienyl, pyrimidyl or pyridyl; Condition is R ¹-R ⁸In at least one be not H;

Described alkyl, alkoxyl group, alkenyl, aryl, furyl, thienyl, pyrimidyl or pyridyl are all optional to be substituted.

2. compound according to claim 1, wherein R ⁴Be selected from hydrogen, C ₁-C ₆Alkyl, halogen, C ₁-C ₆Alkoxyl group ,-CN ,-C ₂-C ₇Alkenyl ,-CHO, phenyl, furyl, thienyl, pyrimidyl and pyridyl.

3. compound according to claim 1, wherein R ⁴Be selected from hydrogen, C ₁-C ₆Alkyl, halogen, C ₁-C ₆Alkoxyl group ,-CN, C ₂-C ₇Alkenyl, furyl, thienyl and pyridyl.

4. according to each described compound, wherein R in the claim 1 to 3 ⁵, R ⁷And R ⁸Be hydrogen or halogen independently of one another.

5. according to each described compound, wherein R in the claim 1 to 4 ¹, R ², R ³And R ⁶Be hydrogen, halogen or hydroxyl independently of one another.

6. according to each described compound, wherein R in the claim 1 to 5 ¹, R ², R ³And R ⁶In at least one be hydroxyl.

7. according to each described compound, wherein R in the claim 1 to 6 ²And R ⁶All are hydroxyls.

8. compound according to claim 1, it is one of following:

(a) 5H-dibenzo [c, g] chromene-3, the 9-glycol;

(b) 8-chloro-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(c) 7-chloro-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(d) 7-bromo-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(e) 3,9-dihydroxyl-5H-dibenzo [c, g] chromene-7-nitrile;

(f) 7-methoxyl group-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(g) 7-vinyl-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(h) 12-bromo-7-methoxyl group-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(i) 12-chloro-7-methoxyl group-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(j) 7-methyl-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(k) 7-[2-(methylol) phenyl]-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(l) 7-phenyl-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(m) 7-(2-tolyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(n) 7-(3-tolyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(o) 7-(4-tolyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(p) 7-(4-p-methoxy-phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(q) 7-(4-chloro-phenyl-)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(r) 7-(4-fluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(s) 7-thiophene-2-base-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(t) 7-thiene-3-yl--5H-dibenzo [c, g] chromene-3, the 9-glycol;

(u) 7-(3-fluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(v) 7-(3-chloro-phenyl-)-5H-dibenzo [c, g] chromene-3,9-glycol;

(w) 7-(3-p-methoxy-phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(x) 7-(2-chloro-phenyl-)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(y) 7-(3, the 4-difluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(z) 7-(4-pyridyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(aa) 7-(2-fluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(bb) 7-(3, the 4-3,5-dimethylphenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(cc) 7-(4-cyano-phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(dd) 7-(3-fluoro-4-aminomethyl phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ee) 7-(3, the 4-Dimethoxyphenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ff) 7-(3-trifluoromethyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(gg) 7-(3, the 5-difluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(hh) 7-(3, the 5-dichlorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ii) 7-(3-methyl-4-fluorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(jj) 7-(3-furyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(kk) 7-(2-furyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ll) 7-butyl-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(mm) 7-(3-pyridyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(nn) 7-(4-methoxyl group-3-pyridyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(oo) 7-(pyrimidyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(pp) 7-(5-methoxyl group-3-pyridyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(qq) 7-(2-pyridyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(rr) 7-(3, the 4-dichlorophenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ss) 7-(4-methylbenzene sulfenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(tt) 7-(4-cyano methyl phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(uu) 7-(3-Trifluoromethoxyphen-l)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(vv) 7-(4-Trifluoromethoxyphen-l)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(ww) 7-(4-tert-butyl-phenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(xx) 7-(naphthyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol;

(yy) 7-(4-ethylphenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol; Or

(zz) 7-(3, the 5-3,5-dimethylphenyl)-5H-dibenzo [c, g] chromene-3, the 9-glycol.

9. pharmaceutical composition, it comprises according to each described compound and pharmaceutical carrier in the claim 1 to 8.

10. one kind is suppressed osteoporotic method in the Mammals of needs, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

11. one kind is suppressed osteoarthritis, hypocalcemia, hypercalcinemia, Paget's disease, osteomalacia, osteohalsiteresis, multiple myeloma or osseous tissue is had other form method for cancer of detrimental action in the Mammals of needs, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

12. a method that in the Mammals of needs, suppresses the growth of optimum or pernicious abnormal structure, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

13. method according to claim 12, the growth of wherein said abnormal structure is prostatomegaly, leiomyoma of uterus, mammary cancer, endometriosis, carcinoma of endometrium, polycystic ovarian syndrome, endometrial polyp, optimum mammary cancer, endometriosis, ovarian cancer, melanoma, prostate cancer, colorectal carcinoma or CNS cancer.

14. a reducing cholesterol in the Mammals of needs, triglyceride level, Lp (a) or LDL level; Or inhibition hypercholesterolemia; Hyperlipidaemia; Cardiovascular disorder; Atherosclerosis; Peripheral vascular disease; Restenosis or vasospasm; Or the immune-mediated blood vessel injury that causes of the vascular damaged that suppresses to cause by the cell incident, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

15. a method that in the Mammals of needs, suppresses the illness that free radical causes, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

16. one kind provides cognitive and improves or neuroprotective in the Mammals of needs; Treatment or suppress senile dementia, Alzheimer's disease, cognitive ability reduces or the method for nerve degenerative diseases, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

17. one kind is suppressed inflammatory bowel disease, proctitis ulcerosa, crohn, colitis, hot flush, vagina or vaginal orifice atrophy, atrophic vaginitis, vaginal dryness, itch, sexual anhedonia, misnicturition, frequent micturition, the urinary incontinence, urinary tract infection, vasomotion syndrome in the Mammals of needs; Male pattern alopecia; Skin atrophy; Acne; Type ii diabetes; Functional disorder type uterine hemorrhage or infertile method, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

18. one kind is suppressed leukemia, endometrial ablation, chronic renal or hepatopathy in the Mammals of needs, or blood coagulation disease or disorderly method, it comprise give described Mammals significant quantity according to each described compound in the claim 1 to 8.

19. according to each described compound in the claim 1 to 8 as the purposes of medicine.