HK1018771B - Synthesis and use of retinoid compounds having negative hormone and/or antagonist - Google Patents

Description

Synthesis and use of retinoid having negative hormone and/or antagonist activity

Scope of the invention

The present invention relates to novel compounds having retinoid negative hormones (retinoids) and/or retinoid antagonist-like biological activities. More specifically, the present invention relates to 4-aryl substituted benzopyran, 4-aryl substituted benzothiopyran, 4-aryl substituted 1, 2-dihydroquinoline, and 8-aryl substituted 5, 6-dihydronaphthalene derivatives, which may also be substituted with substituted 3-oxo-1-propenyl groups. These novel compounds have retinoid antagonist-like activity for treating or preventing toxicity induced by retinoids and vitamin A and precursors of vitamin A in mammals, and as an adjuvant for treating mammals with retinoids in order to prevent or ameliorate undesirable side effects. The invention also relates to the use of retinoid negative hormones to increase the biological activity of other retinoids and steroid hormones and to inhibit the basal activity of non-ligand bound retinoic acid receptors.

Background

Compounds having retinoid-like activity are well known in the art and have been described in numerous U.S. and other national patent and scientific publications. Retinoid-like activity is well known and generally accepted in the art for the treatment of mammals, including humans, to cure or alleviate symptoms associated with many diseases.

Retinoids (vitamin a and its derivatives) are known to have a wide range of activities, including effects on cell proliferation and differentiation in different biological systems. This activity has led to the use of retinoids in the treatment of various diseases, including skin disorders and cancer. The prior art has developed a number of compounds with retinoid-like biological activity and a number of patents and chemical literature describe such compounds. Relevant patent documents include U.S. patent nos. 4980369, 5006550, 5015658, 5045551, 5089509, 5134159, 5162546, 5234926, 5248777, 5264578, 5272156, 5278318, 5324744, 5346895, 5346915, 5348972, 5348975, 5380877, 5399561, 5407937 (assigned to the same assignee as the present application), and patents and publications cited therein which describe or relate, inter alia, chroman, dihydrobenzothiopyran and 1, 2, 3, 4-tetrahydroquinoline derivatives having retinoid-like biological activity. In addition, several applications are pending, assigned to the assignee of the present application, which relate to other compounds having retinoid-like activity.

Us patent nos. 4740519 (short et al.), 4826969(Maignan et al.), 4326055(Loeliger et al.), 5130335 (chandartana et al.), 5037825(Klaus et al.), 5231113 (chandartana et al.), 5324840 (chandarrata), 5344959 (chandartana), 5130335 (chandartana et al.), published european patent application nos. 0176034a (Wuest et al.), 0350846A (Klaus et al.), 0176032A (Frickel et al.), 0176033A (Frickel et al.), 0253302A (Klaus et al.), 0303915a (breeet al.), uk patent application No. 2190378A (Klaus et al.), german patent application No. 356A (Klaus et al.), german patent application No. 3602473 (german patent No.: 3602473 et al): j.amer.acad.derm.15: 756-764(1986) (Sporn et al), chem.pharm.ball.33: 404-407(1985) (Shudo et al.), J.Med chem.31: 2182-,Chemistry and Biology of Synthetic RetinoidsCRCPress Inc.1990 pp.334-335, 354(Dawson et al) describes or relates to compounds comprising a tetrahydronaphthyl moiety and having retinoid-like or related biological activity. Tetralin derivatives for use in liquid crystal compositions are described in U.S. Pat. No. 4391731(Boller et al).

Kagechika et al, j.med.chem 32: 834(1989) describes certain 6- (3-oxo-1-propenyl) -1, 2, 3, 4-tetramethyl-1, 2, 3, 4-tetrahydronaphthalene derivatives and related flavone compounds having retinoid-like activity. Shudo et al in chem.pharm.Bull.33: 404(1985) and Jetten et al in Cancer Research 47: 3523(1987) describe or relate to other 3-oxo-1-propenyl derivatives (chalcone compounds) and their retinoid-like or related biological activities.

Unfortunately, compounds having retinoid-like activity (retinoids) also cause several undesirable side effects at therapeutic dose levels, including headache, teratogenesis, mucosal to skin toxicity, musculoskeletal toxicity, dyslipidemias (dyslipidemias), skin irritation, headache, and toxicity to the liver. These side effects limit the acceptability and utility of retinoids for treatment of disease.

It is now generally recognized in the art that: there are two major types of retinoid receptors in mammals (and other organisms). These two major types or families (family) of receptors are referred to as RARs and RXRs, respectively. Within each class there are subtypes: within the RAR family, the subtypes are referred to as RAR- α, RAR- β and RAR- γ, and in RXR, the subtypes are: RXR-alpha, RXR-beta and RXR-gamma. These two families of receptors are transcription factors that can be distinguished from each other by their ligand binding specificity. All trans-RA (ATRA) binds to and activates a class of retinoid receptors (RARs) including RAR- α, RAR- β and RAR- γ. Different ligands, 9-cis-RA (9C-RA), bind to and activate RARs and members of the Retinoid X Receptor (RXR) family.

It has also been established in the art that the distribution of the two major retinoid receptor types and several subtypes is heterogeneous in various tissues and organs of mammals. Furthermore, it has been generally recognized in the art that many of the unwanted side effects of retinoids are mediated by one or more RAR receptor subtypes. Thus, among compounds having agonist-like activity at retinoid receptors, specificity or selectivity for one of the major classes or families and even for one or more subtypes in one receptor family is considered to be a desirable pharmacological property.

More recently, compounds have been developed in the art that bind to RAR receptors without eliciting such a response, i.e., a response elicited by agonists of the same receptor. Thus, a compound or drug that binds to RAR receptors without eliciting a "retinoid" response is one that is capable of (to some extent, more or less) blocking the activity of RAR agonists in biological assays and systems. More specifically, in the scientific and patent literature in this field, published PCT application WO 94/14777 describes certain heterocyclic carboxylic acid derivatives that bind to RAR retinoid receptors and is said to be useful in the treatment of certain diseases such as acne, psoriasis, rheumatoid arthritis and viral infections. In the article by Yoshimura et al [ J med. chem.38: 3163 and 3173 (1995). Kaneko et al, med. chem res.1: 220-225 (1991); apfel et al, proc.natl.acad.sci.usa 89: 7129-; eckhardt et al, Toxicology Letters 70: 299-308 (1994); keidel et al Molecular and Cellular Biology 14: 287-298 (1994); and Eyrolles et al, J.Med.chem.37: 1508-1517(1994) describes compounds with antagonist-like activity against one or more RAR retinoid subtypes.

In addition to the undesirable side effects of treatment with retinoids, occasionally severe disease caused by an excess of vitamin a or vitamin a precursors occurs, either by taking an excess of vitamin supplement or by ingesting the liver of certain fish and animals that contain high levels of the vitamin. Chronic or acute toxicity with vitamin a overabundance syndrome includes headache, desquamation, bone toxicity, dyslipidemia, and the like. In recent years, toxicity caused by the use of vitamin a analogs, i.e., retinoids, has become apparent, essentially summarizing the toxicity of the vitamin a overabundance syndrome, suggesting a common biological cause, RAR activation. These toxicities are currently mainly treated by supportive approaches and avoid further exposure to causative factors, whether it be liver, vitamin supplements or retinoids. While some toxicity may be removed over time, others (e.g., premature epiphyseal closure) are permanent.

In general, a particular antidote is the best treatment for poisoning by a pharmaceutical agent, however, only about 24 compounds or compound types among thousands of compounds are known to be specific antidotes. Clearly, specific antidotes are valuable in treating vitamin a hyperactivity and retinoid toxicity. Indeed, with the increasing availability of effective retinoids for clinical use, specific antidotes for retinoid intoxication can save many lives.

Summary of the invention

The present invention includes compounds of formula I:formula 1

Wherein X is S, O, NR ', wherein R' is H or alkyl of 1 to 6 carbon atoms, or

X is [ C (R)₁)₂]_nWherein R is₁Independently is H or alkyl of 1-6 carbon atoms, and n is an integer between 0-2;

R₂is hydrogen, lower alkyl of 1-6 carbon atoms, F, Cl, Br, I, CF₃Fluorine substituted alkyl with 1-6 carbon atoms, OH, SH, alkoxy with 1-6 carbon atoms or alkylthio with 1-6 carbon atoms;

R₃is hydrogen, lower alkyl of 1 to 6 carbon atoms or F;

m is an integer of 0 to 3;

o is an integer of 0 to 3;

z is-C ≡ C-,

-N＝N-，

-N＝CR₁-，

-CR₁＝N，

-(CR₁＝CR₁)_n’-wherein n' is an integer from 0 to 5,

-CO-NR₁-，

-CS-NR₁-，

-NR₁-CO，

-NR₁-CS，

-COO-，

-OCO-，

-CSO-，

-OCS-；

y is phenyl or naphthyl or heteroaryl selected from pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrazolyl, said phenyl and heteroaryl optionally substituted by one or two R₂Is substituted by radicals, or

When Z is- (CR)₁＝CR₁)_n’And n' is 3, 4 or 5, then Y is represented at (CR)₁＝CR₁)_n’A direct bond between the group and B;

a is (CH)₂)_qWherein q is 0 to 5, a lower branched alkyl group having 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and 1 or 2 double bonds, an alkynyl group having 2 to 6 carbon atoms and 1 or 2 triple bonds;

B is hydrogen, COOH or a pharmaceutically acceptable salt thereof, COOR₈，CONR₉R₁₀，-CH₂OH，CH₂OR₁₁，CH₂OCOR₁₁，CHO，CH(OR₁₂)₂，CHOR₁₃O，-COR₇，CR₇(OR₁₂)₂，CR₇OR₁₃O or tri-lower alkylsilyl, wherein R₇Is alkyl, cycloalkyl or alkenyl having 1 to 5 carbon atoms, R₈Is an alkyl group of 1 to 10 carbon atoms or a trimethylsilylalkyl group wherein the alkyl group has 1 to 10 carbon atoms, or a cycloalkyl group of 5 to 10 carbon atoms, or R₈Is phenyl or lower alkylphenyl, R₉And R₁₀Independently hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, or phenyl or lower alkylphenyl, R₁₁Is lower alkyl, phenyl or lower alkylphenyl, R₁₂Is lower alkyl, and R₁₃Is a divalent alkyl radical of 2 to 5 carbon atoms, and

R₁₄is (R)₁₅)_r-phenyl, (R)₁₅)_r-naphthyl or (R)₁₅)_r-heteroaryl, wherein said heteroaryl has 1-3 heteroatoms selected from O, S and N, r is an integer from 0 to 5, and

R₁₅independently H, F, Cl, Br, I, NO₂，N(R₈)₂，N(R₈)COR₈，NR₈CON(R₈)₂，OH，OCOR₈，OR₈CN, an alkyl group having 1 to 10 carbon atoms, a fluoroalkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms and 1 to 3 double bonds, an alkynyl group having 1 to 10 carbon atoms and 1 to 3 triple bonds, or a trialkylsilyl or trialkylsiloxy group wherein the alkyl groups independently have 1 to 6 carbon atoms.

The present invention also includes compounds of formula 101: Formula 101

Wherein X is S, O, NR ', wherein R' is H or alkyl of 1 to 6 carbon atoms, or

X is [ C (R)₁)₂]_nWherein R is₁Independently is H or alkyl of 1-6 carbon atoms, and n is an integer between 0-2;

R₂is hydrogen, 1-6 carbon atomsLower alkyl of a subgroup, F, Cl, Br, I, CF₃Fluorine substituted alkyl with 1-6 carbon atoms, OH, SH, alkoxy with 1-6 carbon atoms or alkylthio with 1-6 carbon atoms;

R₃is hydrogen, lower alkyl of 1 to 6 carbon atoms or F;

m is an integer of 0 to 3;

o is an integer of 0 to 3;

y is phenyl or naphthyl or heteroaryl selected from pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrazolyl, said phenyl and heteroaryl optionally substituted by one or two R₂Substituted by groups;

a is (CH)₂)_qWherein q is 0 to 5, a lower branched alkyl group having 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and 1 or 2 double bonds, an alkynyl group having 2 to 6 carbon atoms and 1 or 2 triple bonds;

b is hydrogen, COOH or a pharmaceutically acceptable salt thereof, COOR₈，CONR₉R₁₀，-CH₂OH，CH₂OR₁₁，CH₂OCOR₁₁，CHO，CH(OR₁₂)₂，CHOR₁₃O，-COR₇，CR₇(OR₁₂)₂，CR₇OR₁₃O or tri-lower alkylsilyl, wherein R₇Is alkyl, cycloalkyl or alkenyl having 1 to 5 carbon atoms, R₈Is an alkyl group of 1 to 10 carbon atoms or a trimethylsilylalkyl group wherein the alkyl group has 1 to 10 carbon atoms, or a cycloalkyl group of 5 to 10 carbon atoms, or R ₈Is phenyl or lower alkylphenyl, R₉And R₁₀Independently hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, or phenyl or lower alkylphenyl, R₁₁Is lower alkyl, phenyl or lower alkylphenyl, R₁₂Is lower alkyl, and R₁₃Is a divalent alkyl radical of 2 to 5 carbon atoms, and

R₁₄is (R)₁₅)_r-phenyl, (R)₁₅)_r-naphthyl or (R)₁₅)_r-heteroaryl, wherein said heteroaryl has 1-3 heteroatoms selected from O, S and N, r is an integer from 0 to 5, and

R₁₅independently H, F, Cl, Br, I, NO₂，N(R₈)₂，N(R₈)COR₈，NR₈CON(R₈)₂，OH，OCOR₈，OR₈CN, an alkyl group having 1 to 10 carbon atoms, a fluoroalkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms and 1 to 3 double bonds, an alkynyl group having 1 to 10 carbon atoms and 1 to 3 triple bonds, or a trialkylsilyl or trialkylsiloxy group wherein the alkyl groups independently have 1 to 6 carbon atoms;

R₁₆is H, lower alkyl of 1 to 6 carbon atoms;

R₁₇is H, lower alkyl of 1-6 carbon atoms, OH or OCOR₁₁And are and

p is 0 or 1, provided that when p is 1, then no R is present₁₇And m is an integer of 0-2.

The compounds of the present invention are useful for the prophylactic administration of retinoids in order to treat or prevent certain undesirable side effects of retinoids in certain diseases. For this purpose, the compounds of the present invention may be co-administered with a retinoid. The compounds of the present invention are also useful in the treatment of acute or chronic toxicity resulting from overdose with retinoid drugs or vitamin a or poisoning by such drugs.

The invention further relates to the use of an RAR antagonist in a biological system (including a mammal) for blocking all or part of RAR receptor sites so as to prevent or reduce the effect of an RAR agonist at such receptor sites. More particularly, the invention relates to the use of RAR antagonists for (a) prophylaxis and (b) treatment of chronic or acute toxicity of retinoids (including vitamin a or vitamin a precursors) and side effects in retinoid therapy.

In one particular aspect of the invention, a method of treating a pathological condition in a mammal is provided. The disease treated is associated with retinoic acid receptor activity. The method comprises administering to the mammal an effective amount of a compound that binds to the following retinoic acid receptor subtypes: RAR_α、RAR_βAnd RAR_γA retinoid antagonist or a negative hormone. The antagonist or negative hormone is administered in a pharmaceutically effective amount to provide a beneficial therapeutic effect against the pathological condition of the mammal.

As antidotes for acute or chronic retinal derivatives or vitamin a poisoning, the compounds can be administered via the intestinal tract: gastric intubation or food/water mixtures, or parenteral administration, e.g., intraperitoneal, intramuscular, subcutaneous, or topical to the skin, etc. The only requirement for the route of administration is that it must be able to deliver the antagonist to the target tissue. The RAR antagonists can be formulated alone or in admixture with excipients. In the case of enteral administration, the RAR antagonist need not be formulated as a solution.

As an adjunct to treatment with a retinoid, and to prevent one or more side effects of administration of a retinoid drug, RAR antagonists may similarly be administered enterally or parenterally. The RAR antagonist and RAR agonist need not be administered by the same route of administration. The key is that a sufficient amount of the RAR antagonist is continuously present in the tissue of interest upon exposure to the RAR agonist. To prevent toxicity of the retinoid, it is desirable to administer the RAR antagonist simultaneously with or prior to treatment with the RAR agonist. In many cases, the RAR antagonist will be administered by a route other than an agonist. For example, topical administration of RAR antagonists to the skin can prevent or ameliorate the undesirable skin effects of enteral administration of retinoids.

Another aspect of the invention is a method for identifying a retinoid negative hormone. The method comprises the following steps: obtaining transfected cells containing a reporter gene responsive to transcription of binding to a recombinant retinoid receptor, said recombinant retinoid receptor having at least a protein domain located C-terminal to the DNA binding domain of the intact retinoid receptor, determining the basal level of reporter gene expression in untreated transfected cells, propagating said untreated transfected cells in the absence of added retinoid, treating the transfected cells with a retinoid to be tested for negative hormone activity, determining the level of reporter gene expression in treated cells, comparing the levels of reporter gene expression determined in treated cells and untreated cells, and identifying as retinoid negative hormones those retinoids which produce reduced levels of reporter gene expression in the treated cells compared to the basal levels of reporter gene expression measured in untreated cells. In some preferred embodiments of the present method, the intact receptors are RAR- α, RAR- β and RAR- γ subtypes. In other embodiments, the intact receptors are the RXR- α, RXR- β and RXR- γ subtypes. The recombinant receptor may also be a recombinant RAR or RXR receptor. In certain embodiments, the recombinant receptor is a retinoid receptor with constitutive transcriptional activator domain chimera (chimera). The constitutive transcriptional activator domain can include a number of amino acids having a net negative charge or an amino acid sequence having a viral transcriptional activator domain such as the herpes simplex virus VP-16 transcriptional activator domain. In embodiments in which the constitutive transcriptional activator domain has a net negative charge, the retinoid receptor may be recombinant and have a DNA binding domain deleted therefrom, e.g., a DNA binding domain specific for a cis-regulatory element other than a retinoic acid response element. These elements include estrogen responsive elements. Preferably, the transfected cells are propagated in a growth medium substantially lacking endogenous retinoids, such as a growth medium comprising charcoal-extracted serum. In this method, the reporter gene may be a luciferase gene, in which case the determining step may include luminescence (luminometry). The reporter gene may also be a beta-galactosidase gene, in which case the assay step will include a beta-galactosidase assay. The transfected cell may be a transfected mammalian cell, such as a green monkey cell or a human cell.

Another aspect of the invention is a method of potentiating the pharmacological activity of a steroid superfamily receptor agonist administered to a mammal. The method comprises co-administering to the mammal a retinoid negative hormone in a pharmaceutically effective amount in combination with a steroid superfamily receptor agonist to potentiate the pharmacological activity of the steroid superfamily receptor agonist. This pharmacological activity is measurable in an in vitro reporter gene transactivation assay, e.g., by testing anti-AP-1 activity. The pharmacological activity to be potentiated may be an antiproliferative activity, such as the type of activity measurable in retinal pigment epithelium (retinal pigment epithelium). The steroid superfamily receptor agonist may be any of the following agonists: a retinoid receptor agonist, a vitamin D receptor agonist, a glucocorticoid receptor agonist, a thyroid hormone receptor agonist, a peroxisome-activated proliferator an estrogen receptor agonist. The retinoid receptor agonist may be a RAR agonist such as all-trans-retinoic acid or 13-cis-retinoic acid. The retinoid receptor agonist may also be an RXR agonist. A preferred vitamin D receptor agonist is 1, 25-dihydroxyvitamin D3. A preferred glucocorticoid receptor agonist is dexamethasone. A preferred thyroid hormone receptor agonist is 3, 3', 5-triiodothyronine. The retinoid negative hormone is a RAR-specific retinoid negative hormone, preferably having a dissociation constant less than or about equal to 30 nM. Examples of such RAR-specific retinoid negative hormones include AGN 193109, AGN 193385, AGN 193389 and AGN 193871. Compositions containing a pharmaceutically effective amount of a retinoid negative hormone can be co-administered simultaneously as a steroid superfamily agonist and mixed prior to co-administration. They may also be administered together as separate components.

Brief description of the drawings

Fig. 1 shows the chemical structure of AGN 193109.

FIGS. 2A-2F are series of line graphs showing that AGN 139109 inhibits ATRA-dependent transactivation on RARs. FIGS. 2A and 2B represent activity at RAR- α receptors; FIGS. 2C and 2D represent activity at RAR- β receptors; FIGS. 2E and 2F represent activity at RAR-gamma receptors. In fig. 2A, 2C, and 2E, open squares represent retinoic acid treatment and filled circles represent AGN 193109 treatment. In FIGS. 2B, 2D and 2F, the individual lines are represented by 10^-8Luciferase activity measured after M ATRA and varying concentrations of AGN 193109 treatment.

FIGS. 3A and 3B represent line graphs of luciferase activity measured in CV-1 cells transfected with the reporter plasmid ERE-tk-Luc and the expression plasmid ER-RAR- α and stimulated with different concentrations of ATRA (FIG. 3A) or AGN 193109 (FIG. 3B). The value points represent the mean ± SEM of three independent luciferase assays. Results of transfection using different amounts of co-transfected ER-RAR- α (0.05, 0.1 and 0.2 μ g/well) are shown in each graph.

FIGS. 4A and 4B represent line graphs of luciferase activity in CV-1 cells transfected with the reporter plasmid ERE-tk-Luc and the expression plasmid ER-RAR- β and stimulated with different concentrations of ATRA (FIG. 4A) or AGN 193109 (FIG. 4B). The value points represent the mean ± SEM of three independent luciferase assays. Results of transfection using different amounts of co-transfected ER-RAR- β (0.05, 0.1 and 0.2. mu.g/well) are shown in each graph.

FIGS. 5A and 5B represent line graphs of luciferase activity measured in CV-1 cells transfected with the reporter plasmid ERE-tk-Luc and the expression plasmid ER-RAR-gamma and stimulated with different concentrations of ATRA (FIG. 5A) or AGN 193109 (FIG. 5B). The value points represent the mean ± SEM of three independent luciferase assays. Results of transfection using different amounts of co-transfected ER-RAR- γ (0.05, 0.1 and 0.2 μ g/well) are shown in each graph.

FIG. 6 shows the ATRA and AGN 193109 dose response of CV-1 cells co-transfected with the ERE-tk-Luc reporter plasmid and the ER-RXR-alpha chimeric receptor expression plasmid alone or together with the RAR- γ -VP-16 expression plasmid. ER-RXR- α co-transfected cells were treated with ATRA (squares) and AGN 193109 (diamonds). Cells co-transfected with ER-RXR- α and RAR- γ -VP-16 were treated with ATRA (circles) or AGN 193109 (triangles).

FIG. 7 shows a representation of a vector transfected with the ERE-tk-Luc reporter plasmid and ER-RAR-gamma expression construct, followed by 10^-8ATRA of M and a line graph showing the measured value of luciferase activity recorded in the lysate of CV-1 cells treated with test compound at different concentrations on the horizontal axis. The test compounds were AGN 193109 (squares), AGN 193357 (open diamonds), AGN 193385 (circles), AGN 193389 (triangles), AGN 193840 (solid squares) and AGN 192870 (solid diamonds).

FIG. 8 shows a line graph representing luciferase activity measurements recorded in lysates of CV-1 cells transfected with the ERE-tk-Luc reporter plasmid and RAR- γ -VP-16 and ER-RXR- α expression constructs, and then treated with test compounds at different concentrations on the horizontal axis. The test compounds were ATRA (open square), AGN 193109 (open circle), AGN 193174 (open triangle), AGN 193199 (solid square), AGN 193385 (solid circle), AGN 193389 (inverted triangle), AGN 193840 (oblique solid square) and AGN 193871 (semi-solid diamond).

FIGS. 9A, 9B and 9C diagrammatically show the mechanism by which AGN 193109 can modulate the interaction between RAR (shaded) and the negative coactivator protein (-) in the context of the transactivation test. Figure 9A shows the binding equilibrium of negative and positive coactivator proteins (+) with RAR. In the absence of ligand, basal levels of transcription of the reporter gene are generated. As shown in FIG. 9B, the addition of RAR agonists promoted the binding of positive coactivator protein to RAR and resulted in up-regulated (upregulated) reporter gene transcription. As depicted in figure 9C, the addition of AGN 193109 promoted the binding of negative coactivator protein to RAR and prevented transcription of the reporter gene.

FIG. 10 shows that the concentration is maintained at 10^-8AGN 193109 inhibition by M was determined as AGN191183 concentration (10)^-10-10^-12M) of the TPA-induced Str-AP1-CAT expression.The results obtained for the test performed with AGN191183 alone are shown as shaded bars, while the striped bars represent the results of the combined AGN 193109 and AGN191183 treatments.

Figure 11 diagrammatically shows the mechanism by which AGN 193109 can potentiate the activity of RARs and other nuclear receptor family members. As depicted in this diagram, induced RARs (with AB-C-DEF domain open rectangles) have increased sensitivity to RAR ligands in the anti-AP 1 assay, as negative coactivator proteins (ncps) present in a limited supply are separated into (sequence) RARs, resulting in two populations: RAR + ncp and RAR-ncp. RAR-ncp has increased sensitivity to ligands. non-RAR nuclear factors (shaded rectangles with AB-C-DEF domains) have increased sensitivity to cognate ligands, as ncp has been separated into RARs by the activity of AGN 193109. The mode domains of the nuclear receptor are designated using standard names such as "AB" (ligand independent transactivation domain), "C" (DNA binding domain) and "DEF" (ligand regulated transactivation and dimerization domains).

FIG. 12 is a graph showing AGN193109 versus 1, 25-dihydroxy vitamin D in CV-1 cells transfected with MTV-DR3-Luc reporter plasmid₃Line graph of dose response effect. With 1, 25-dihydroxyvitamin D₃(solid Square), 1, 25-dihydroxyvitamin D₃And 10^-8M AGN193109 (filled triangle), 1, 25-dihydroxy vitamin D₃And 10^-7Transfectants were treated with M AGN193109 (filled circles).

FIG. 13 is a graph showing AGN193109 (10nM) co-administration versus 1, 25-dihydroxy vitamin D₃Bar graph of mediated inhibition of TPA-induced Str-AP1-CAT activity. Solid bars represent for 1, 25-dihydroxyvitamin D alone₃Inhibition of CAT activity in treated transfected cells. Open bars represent for mixed 1, 25-dihydroxyvitamin D₃And inhibition of CAT activity in AGN 193109-treated transfected cells.

E.g. 14 is AGN193109 alone and in admixture with AGN191183Graph of the effect of the form on co-transfection of HeLa cells with RAR- γ and RAR-responsive MTV-TREP-Luc reporter constructs. The drug treatments introduced in this figure are: AGN193109 (Square) alone, and 10^-10AGN193109 (diamond) in combination with M AGN191183 and 10^-9AGN193109 for use with M AGN 191183.

Figure 15 is a line graph showing proliferating ECE16-1 cells as a response to EGF (filled squares), but not as a response to defined medium alone (open circles). Cells treated with AGN 193109 alone are represented by filled triangles. The filled circles represent results obtained by treating cells with 10nMAGN 191183 and 0-1000nM AGN 193109.

Figure 16 is a bar graph showing the effect of AGN 193109 on CaSki cell proliferation in the presence or absence of AGN 191183 retinoid agonist. All sample groups received 20ng/ml Epidermal Growth Factor (EGF) except for the samples propagated in Defined Medium (DM) alone (open bars). Striped bars represent samples propagated without AGN 193109. The filled bars represent samples propagated in the presence of 1000nM AGN 193109. The concentration of AGN 191183 used in the process is shown on the horizontal axis.

FIG. 17 is a dose response curve showing AGN 193109 potentiates ATRA antiproliferative activity on Retinal Pigment Epithelium (RPE) cells. Samples treated with ATRA alone were represented by solid squares. The use of ATRA and AGN 193109 (10) in combination is represented by the filled circles^-7M) treated samples. The concentration of ATRA used to treat the different samples is given on the horizontal axis.

FIG. 18 is a dose response curve showing that 13-cis-RA and ATRA inhibit RPE cell growth and AGN193109 potentiates the antiproliferative activity of 13-cis-RA. The different sample treatments shown in the dose response included 13-cis-RA alone (filled squares), and AGN193109 (10)^-6M) mixed 13-cis-RA (closed circle), with AGN193109 (10)^-8M) mixed 13-cis-RA (filled triangles) and ATRA (filled diamonds). The concentrations of 13-cis-RA and ATRA used in the sample treatment are indicated inOn the horizontal axis.

FIG. 19 is a dose response curve showing AGN193109 enhances the antiproliferative activity of dexamethasone in primary RPE cell culture. Different sample treatments shown in the dose response include ATRA (filled squares), dexamethasone alone (filled circles), and AGN193109 (10)^-8M) dexamethasone in combination (filled triangles) and AGN193109 (10)^-6M) dexamethasone in combination (filled diamonds). Dexamethasone and ATRA concentrations used in the sample treatment are shown on the horizontal axis.

FIG. 20 is a dose response curve showing AGN193109 enhances the antiproliferative activity of thyroid hormone (T3) in primary RPE cell culture. Different sample treatments shown in the dose response include ATRA (filled squares), T3 alone (filled circles), and AGN193109 (10) ^-8M) T3 (filled triangles) in combination with AGN 193109 (10)^-6M) combined T3 (filled diamonds). The T3 and ATRA concentrations used in the sample treatment are shown on the horizontal axis.

Detailed description of the invention Definition of

For the purposes of this invention, a RAR antagonist is defined as binding to one or more compounds having K_dLess than 1 micromole (K)_d< 1 μ M), however, it does not cause significant transcriptional activation of RAR subtype regulatory genes in the receptor co-transfection assay. Antagonists are generally chemical agents that inhibit the activity of agonists. Thus, the activity of a receptor antagonist is generally determined by its ability to inhibit the activity of an agonist.

RAR agonists are defined as binding to one or more compounds having K_dLess than 1 micromole (K)_d< 1 μ M) and causing transcriptional activation of RAR subtype regulatory genes in a receptor co-transfection assay. The term "RAR agonists" includes compounds that can bind to and/or activate receptors other than RARs, such as the RXR receptor.

Herein, a negative hormone or anti-stimulant is a receptor ligand that causes the receptor to adopt an inactive state relative to the basal state in the absence of any receptor. Thus, while antagonists may inhibit the activity of agonists, negative hormones are ligands that may alter the conformation of the receptor in the absence of agonists. Bond et al [ Nature 374: 272(1995) ]The concept of negative hormones or anti-irritants has been investigated. More precisely, Bond et al have proposed that there is a non-ligand bound β in equilibrium between the inactive conformation and the spontaneous active conformation₂-adrenergic receptors. Agonists are proposed to stabilize the receptor in the active conformation. In contrast, the anti-excitant is believed to stabilize the inactive receptor conformation. Thus, while antagonists exhibit their activity by inhibiting agonists, negative hormones may additionally exhibit their activity by inhibiting the spontaneous conversion of non-ligand bound receptors to an active conformation in the absence of agonists. Only one class of antagonists will function as negative hormones. AGN 193109 is an antagonist and a negative hormone, as described herein. To date, no other retinoid has been shown to have negative hormonal activity.

Herein, co-administration of two pharmacologically active compounds refers to either in vitro or in vivo administration of two separate chemical entities. Co-administration refers to simultaneous administration of separate formulations; simultaneously administering a mixture of the formulations; and administering one formulation followed by administration of a second formulation. In all cases, the co-administered formulations will act in conjunction with each other.

The term alkyl refers to (includes) any (all) of the known general alkyl, branched alkyl and cycloalkyl groups. The term alkenyl refers to (includes) general alkenyl, branched alkenyl and cyclic alkenyl groups having one or more sites of unsaturation. Similarly, the term alkynyl refers to (includes) both general alkynyl and branched alkynyl groups having one or more triple bonds.

Lower alkyl means in the case of general lower alkyl the above defined broad definition of alkyl having 1 to 6 carbon atoms, and lower branched and cyclic alkyl groups applicable to 3 to 6 carbon atoms. Lower alkenyl is similarly defined as general lower alkenyl groups having 2 to 6 carbon atoms, and branched and cyclic lower alkenyl groups of 3 to 6 carbon atoms. Similarly, lower alkynyl is also defined as general lower alkynyl having 2 to 6 carbon atoms and branched lower alkynyl having 4 to 6 carbon atoms.

The term "ester" as used herein refers to (includes) any compound within the scope of the definition of the term as generally used in organic chemistry. It includes organic and inorganic esters. When B (in formula 1 or formula 101) is-COOH, the term includes treatment of the functionality-derived product with an alcohol or thiol (preferably an aliphatic alcohol having 1 to 6 carbon atoms is used). When the ester is derived from a compound wherein B is-CH ₂OH, the term includes compounds derived from organic acids capable of forming esters including phosphorus-based acids and sulfur-based acids or compounds having the formula-CH₂OCOR₁₁Wherein R is₁₁Is any substituted or unsubstituted aliphatic, aromatic, heteroaromatic or aliphatic aryl group (preferably having 1-6 carbon atoms in the aliphatic moiety).

Unless otherwise indicated herein, preferred esters are derived from saturated aliphatic alcohols or acids of 10 or less than 10 carbon atoms or cyclic or saturated aliphatic cyclic alcohols and acids of 5 to 10 carbon atoms. Particularly preferred aliphatic esters are esters derived from lower alkanoic acids and alcohols. Also preferred are phenyl or lower alkyl phenyl esters.

Amides have the definition of terms in general organic chemistry. It includes both unsubstituted amides and all aliphatic and aromatic mono-and disubstituted amides. Unless otherwise indicated herein, preferred amides are mono-and di-substituted amides derived from saturated aliphatic groups of 10 or less than 10 carbon atoms or mono-or di-substituted amides derived from cyclic or saturated aliphatic cyclic groups of 5 to 10 carbon atoms. Particularly preferred amides are those derived from substituted and unsubstituted lower alkylamines. Also preferred are substituted and unsubstituted phenyl or lower alkyl phenyl amine derived mono or disubstituted amides. Unsubstituted amides are also preferred.

Acetals and ketals include groups of the formula-CK, where K is (-OR)₂. Herein, R is a lower alkyl group. K may also be-OR₇O-, wherein R₇Is lower alkyl of 2-5 carbon atoms, straight chain or branched chain.

Pharmaceutically acceptable salts of any of the compounds of the present invention having a functionality capable of forming a salt, such as an acid functionality, may be prepared. Pharmaceutically acceptable salts are salts that retain the activity of the parent compound and do not produce any deleterious or undue effects on the subject to whom they are administered.

Pharmaceutically acceptable salts may be derived from organic or inorganic bases. The salt may be a monovalent or multivalent ion. Of particular interest are the inorganic ions, sodium, potassium, calcium and magnesium. Organic salts, in particular ammonium salts, such as mono-, di-and tri-alkylamines or ethanolamines can be prepared using amines. Salts may also be formed using caffeine, trimethylaminomethane, and similar molecules. Wherein there is a nitrogen atom sufficiently basic to enable the formation of acid addition salts, may be formed using any inorganic or organic acid or alkylating agent, such as methyl iodide. Preferred salts are those formed using inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid. Any simple organic acid, such as any of the mono-, di-, or tri-acids, may also be used.

Some compounds of the invention may have both trans and cis (E and Z) isomers. Furthermore, the compounds of the present invention may include one or more chiral neutrals and, thus, may exist in enantiomeric or diastereomeric forms. The scope of the invention is intended to include all such isomers per se as well as mixtures of cis and trans isomers, as well as diastereomeric mixtures and racemic mixtures of enantiomers (optical isomers). In this application, when no particular reference is made to the configuration (cis, trans or R or S) of a compound (or asymmetric carbon atom), then a mixture of such isomers or any one of them is meant.

Aryl substituted benzopyrans, benzenes with retinoid antagonist-like biological activity And a thiopyran, 1, in a pharmaceutically acceptable carrier,derivatives of 2-dihydroquinoline and 5, 6-dihydronaphthalene

Preferred compounds of the invention for the symbol Y in formula 1 are those wherein Y is phenyl, pyridyl, thienyl or furyl. More preferred compounds are those wherein Y is phenyl or pyridyl, most preferably Y is phenyl. Preferred compounds for the substituents on the Y (phenyl) and Y (pyridyl) groups are compounds wherein the phenyl group is substituted with Z and A-B groups 1, 4 (para) and compounds wherein the pyridine ring is substituted with Z and A-B groups 2, 5. (substitution at the 2, 5 position in the "pyridine" nomenclature corresponds to substitution at the 6 position in the "nicotinic acid" nomenclature.) in preferred compounds of the invention, or alternatively, there is no optional R on the Y group ₂Substituent or said alternative R₂The substituent is fluorine (F).

Preferred compounds have the A-B group (CH)₂)_n-COOH or (CH)₂)_n-COOR₈Wherein n and R₈As defined above. Preferably n is zero and R₈Is lower alkyl, or n is zero and B is COOH or a pharmaceutically acceptable salt thereof.

In most preferred embodiments of the compounds of the present invention, X is [ C (R)₁)₂]_nWherein n is 1. Furthermore, compounds in which n is zero (indene derivatives) and in which X is S or O (benzothiopyrans and benzopyran derivatives) are also preferred. When X is [ C (R)₁)₂]_nAnd when n is 1, then R₁Preferably an alkyl group of 1 to 6 carbon atoms, more preferably a methyl group.

R preferably attached to the aromatic moiety of the tetrahydronaphthalene, benzopyran, benzothiopyran or dihydroquinoline moiety of the compound of formula 1₂The radical being H, F or CF₃。R₃Preferably hydrogen or methyl, more preferably hydrogen.

With regard to the group Z in the compounds of the invention and represented in formula 1, in the most preferred embodiments Z represents an acetylenic bond (Z ═ C ≡ C-). However, the "linking group" Z is also preferably a diazo group (Z ═ N —), preferablyAn alkene or polyene group (Z ═ CR)₁＝CR₁)_n’-) preferably an ester group (Z ═ COO-), amide (Z ═ CO-NR)₂-) or thioamides (Z ═ CS-NR₂-) bond.

Involving R₁₄Group, preferably wherein R ₁₄Compounds that are phenyl, 2-pyridyl, 3-pyridyl, 2-thienyl and 2-thiazolyl. Preferably R₁₅Group (R)₁₄Substituent(s) on) is hydrogen, lower alkyl, trifluoromethyl, chlorine, lower alkoxy or hydroxy.

The most preferred compounds of the invention relating to formula 2, formula 3, formula 4, formula 5 and formula 5a are shown in table 1.Formula 2 is a compound represented by formula 3,formula 4 formula 5Formula 5a

TABLE 1A compound of formula R₁₄ ^* Z R₂ ^* R₈ ^*# 124-methylphenyl-C.ident.C-H Et1a 2 phenyl-C.ident.C-H Et 223-methylphenyl-C.ident.C-H Et 322-methylphenyl-C.ident.C-H Et 423, 5-dimethylphenyl-C.ident.C-H Et 524-ethylphenyl-C.ident.C-H Et 624-butylphenyl-C.ident.C- H Et 724-chlorophenyl-C.ident.C-H Et 824-methoxyphenyl-C.ident.C-H Et 924-trifluoromethylphenyl-C.ident.C-H Et 1022-pyridyl-C.ident.C-H Et 1123-pyridyl-C.ident.C-H Et 1222-methyl-5-pyridyl-C.ident.C-H Et 1323-hydroxyphenyl-C.ident.C-H Et 1424-hydroxyphenyl-C.ident.C-H Et 1525-methyl-2-thiazolyl-C.ident.C-H Et15a 22-thiazolyl-C.ident.C-H Et 1624-methyl-2-thiazolyl-C.ident.C-H Et 1822-methyl-5-pyridyl-C.ident.C C-H Et 1822-C-C.ident.C -H H1922-pyridyl-C.ident.C-H H2023-methylphenyl-C.ident.C-H H2124-ethylphenyl-C.ident.C-H H2224-methoxyphenyl-C.ident.C-H H2324-trifluoromethylphenyl-C.ident.C-H H2423, 5-dimethylphenyl-C.ident.C-H H2524-chlorophenyl-C.ident.C-H H2623-pyridyl-C.ident.C-H H2722-methylphenyl-C.ident.C-H H282 3-hydroxyphenyl-C.ident.C-H H2924-hydroxyphenyl-C.ident.C-H H3025-methyl-2-thiazolyl-C.ident.C-H H30a 22-thiazolyl-C.ident.C-H H3124-methyl-2-thiazolyl-C.ident.C-H H3224, 5-dimethyl-2-thiazolyl-C.ident.C-H H3325-methyl-2-thienyl-C.ident.C-H Et33a 22-thienyl-C.ident.C-H34Et 25-methyl-2-thienyl-C.ident.C-H H34a 22-thienyl-C.ident.C-H H3524-methylphenyl-CONH-Et 3624-methylphenyl-CONH-H H3724-363724-methylphenyl-COO-H Et 3824-CONH-Et 3824-3 -Methylbenzenesuccinate-COO-H (CH) ₂)₂Si(CH₃) 3924-methylphenyl-COO-H H4024-methylphenyl-CONH-F Et 4124-methylphenyl-CONH F4224-methylphenyl-CSNH-H Et 4324-methylphenyl-CSNH-H H4424-methylphenyl-CH ═ CH-H Et 4524-methylphenyl-CH ═ CH-H H46a 24-methylphenyl-N ═ N-H Et46b 24-methylphenyl-N ═ N-H Et-46 bH4734-methylphenyl-C.ident.C-H Et 4834-methylphenyl-C.ident.C-H H4944-methylphenyl-C.ident.C-H Et 5044-methylphenyl-C.ident.C-H H5154-methylphenyl-Et 5254-methylphenyl-H6024-methylphenyl-C.ident.C-H H60a 2 phenyl-C.ident.C-H H6124-pinylphenyl-C.ident.C-H H6224-methylphenyl-CSNH F Et 6324-methylphenyl-CSNH F H645 a 4-methylphenyl- - - - - - -Et 655 a 4-methylphenyl- - - - - -H6622-furyl-C.ident.C-H Et 6722-furyl-C.ident.C-H H.

Aryl and (3-oxo-1-propenyl) compounds having retinoid antagonist-like biological activity Substituted benzopyran, benzothiopyran, 1, 2-dihydroquinoline and 5, 6-dihydronaphthalene derivatives

Referring to symbol Y in formula 101, preferred compounds of the present invention are those wherein Y is phenyl, pyridyl, thienyl or furyl. More preferred compounds are those wherein Y is phenyl or pyridyl, and most preferred compounds are those wherein Y is phenyl. As to the substituents on said Y (phenyl) and Y (pyridyl), preferred compounds are those wherein said phenyl group is substituted by-CR₁₆＝CR₁₇Compounds in which the pyridine ring is substituted by-CR₁₆＝CR₁₇And A-B groups 2, 5 substituted. (substitution at the 2, 5 position in the "pyridine" nomenclature corresponds to substitution at the 6 position in the "nicotinic acid" nomenclature.) the invention is particularly advantageousIn selected compounds, there is no optional R on the Y group₂And (4) a substituent.

Preferred compounds have the A-B group (CH)₂)_n-COOH or (CH)₂)_n-COOR₈Wherein n and R₈As defined above. Preferably n is zero and R₈Is lower alkyl, or n is zero and B is COOH or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the compounds of the invention, X is [ C (R)₁)₂]_nWherein n is 1. Furthermore, compounds in which X is S or O (benzothiopyran and benzopyran derivatives) are also preferred. When X is [ C (R) ₁)₂]_nAnd when n is 1, then R₁Preferably an alkyl group of 1 to 6 carbon atoms, more preferably a methyl group.

R preferably attached to the aromatic moiety of the tetrahydronaphthalene, benzopyran, benzothiopyran or dihydroquinoline moiety of a compound of formula 101₂The radical being H, F or CF₃。R₃Preferably hydrogen or methyl, more preferably hydrogen.

Involving R₁₄Group, preferably wherein R₁₄Compounds that are phenyl, 2-pyridyl, 3-pyridyl, 2-thienyl and 2-thiazolyl. Preferably R₁₅Group (R)₁₄Substituents of the groups) are hydrogen, lower alkyl, trifluoromethyl, chlorine, lower alkoxy or hydroxy.

Preferred compounds of the invention relating to formula 102 are shown in table 2.

Formula 102

TABLE 2Compound R₁₅ ^* R₈ ^*101 CH₃ H102 CH₃ Et103 H H104 H EtBiologically active, administration forms

As noted above, the compounds of the present invention are antagonists of one or more RAR receptor subtypes. This means that the compounds of the invention bind to one or more subtypes of RAR receptors, but do not trigger a response triggered by agonists of the same receptor. Some of the compounds of the invention are antagonists of all three RAR receptor subtypes (RAR- α, RAR- β, and RAR- γ), and are referred to as "RAR full antagonists". Other compounds are only antagonists of one or two RAR receptor subtypes. Some of the compounds within the scope of this invention are partial agonists of one or both of the RAR receptor subtypes and antagonists of the remaining subtypes. The compounds of the invention do not bind to RXR receptors and thus they are neither agonists nor antagonists of RXR.

Depending on the site and the nature of unwanted side effects which it is desired to inhibit or ameliorate, the compounds used according to the invention may be antagonists only of one or two RAR receptor subtypes. Part of the compounds used according to the invention may be partial agonists of one or two RAR receptor subtypes and antagonists of the remaining subtypes. In general, such compounds are useful according to the invention if the antagonist is acting on one or more subtypes of RAR receptors (which are or are the main cause of overeating toxicity or unwanted side effects). It is noted in this connection that, in general, a compound is considered to be an antagonist of a given receptor subtype if it does not cause significant transcriptional activation of the receptor-regulated reporter gene in the co-transfection assay described below, but nevertheless binds to a receptor with a Kd value below about 1. mu.M.

Whether a compound is a RAR antagonist and thus can be used according to the invention, can be tested in the following tests.

A detailed description of the chimeric receptor transactivation assay, which tests agonist-like activity in RAR- α, RAR- β and RAR- γ, RXR- α receptor subtypes, is described in published PCT application No. WO94/17796 (published at 8/18, 1994), which is based on work by Feigner P.L. and Holm. Focus Vol 11, No.2 (1989). The disclosure is the corresponding part of PCT in U.S. application serial No. 08/016404 (filed on 11/2/1993, granted by us patent 5455265). In this test, the compounds should not cause significant activation by specific receptor subtype (RAR- α, RAR- β and RAR- γ) reporter genes in order to be acceptable antagonists of RAR for use in the present invention.

A whole receptor transactivation assay and a ligand binding assay for determining the antagonist/agonist-like activity of the compounds of the present invention or their ability to bind to several retinoid receptor subtypes are described in published PCT application No. WO93/11755 (especially pages 30-33 and 37-41, published 24.6.1993), respectively. The following also provides a description of the all-receptor transactivation assay.Total receptor transactivation assay

CV1 cells (5000 cells/well) were not transfected with one of the RAR expression vectors (10ng) in an automated 96-well format (format) using the RAR reporter plasmid MTV-TREP-LUC (50ng), and by the calcium phosphate method of Heyman et al (Cell 68: 397-406). For the determination of the transactivation of RXR- α and RXR- γ, the RXR-response reporter plasmid CRBPII-tk-LUC (50ng) was used together with the appropriate RXR expression vector (10ng) essentially as described by Heyman et al (supra) and Allegretto et al (J.biol.chem.268: 26625-26633). For the RXR- β transactivation assay, the RXR-response reporter plasmid, CPRE-tk-LUC (50mg), and the RXR- β expression vehicle (10mg) were used as described above. These reporter plasmids contain the DRI element from human CRBPII and some DRI element from the promoter, respectively (see Magelsdorf et al. The Retinoids：Biology，Chemistry and Medicine，pp.319-349, Raven Press ltd., New York and Heyman et al, supra). Beta-galactosidase (50ng) expression vehicle was used as an internal control in transfection in order to normalize for variations in transfection efficiency. The cells were transfected in triplicate for 6 hours and then incubated with retinoids36 hours, and the extracts were assayed for luciferase and beta-galactosidase activity. Detailed experimental procedures for transactivation of the pan-receptors have been described by Heyman et al (supra) and Allegretto et al (supra). The results obtained in this assay are reported as EC₅₀Values are expressed as EC in the chimeric receptor transactivation assay₅₀And (4) numerical representation. Results of the ligand binding assay are given as K_dAnd (4) numerical representation. See Cheng et al biochemical 22: 3099-3108.

The compounds should not cause significant activation of the receptor gene by a certain receptor subtype (RAR- α, RAR- β or RAR- γ) in a whole receptor transactivation assay to qualify as RAR antagonists for use in the present invention. Last but not least, the compound should be at K_dLess than about 1 micromolar (K)_d< 1 μ M) to be able to function as an antagonist of said bound receptor subtype, provided that the same receptor subtype is not significantly activated by said compound.

For certain exemplary compounds of the present invention, the results of the full receptor transactivation assay are shown in table 3 below, and the efficiency (in percent) of the test compound assay in relation to all trans retinoic acid is presented in table 4. Table 5 shows the results of a ligand binding assay for certain exemplary compounds of the invention.

TABLE 3

Total receptor transactivation assay

Compound # EC₅₀ (nM)

RARα RARB RARγ RXRα RXRB RXRγ

18 0.00 0.00 0.00 0.00 0.00 0.00

19 0.00 0.00 0.00 0.00 0.00 0.00

20 0.00 0.00 0.00 0.00 0.00 0.00

21 0.00 0.00 0.00 0.00 0.00 0.00

22 0.00 0.00 0.00 0.00 0.00 0.00

23 0.00 0.00 0.00 0.00 0.00 0.00

24 0.00 0.00 0.00 0.00 0.00 0.00

25 0.00 0.00 0.00 0.00 0.00 0.00

26 0.00 0.00 0.00 0.00 0.00 0.00

27 0.00 0.00 0.00 0.00 0.00 0.00

28 0.00 0.00 0.00 0.00 0.00 0.00

29 0.00 0.00 0.00 0.00 0.00 0.00

30 0.00 0.00 0.00 0.00 0.00 0.00

31 0.00 0.00 0.00 0.00 0.00 0.00

32 0.00 0.00 0.00 0.00 0.00 0.00

34 0.00 0.00 0.00 0.00 0.00 0.00

36 0.00 0.00 0.00 0.00 0.00 0.00

39 0.00 0.00 0.00 0.00 0.00 0.00

41 0.00 0.00 0.00 0.00 0.00 0.00

45 0.00 0.00 0.00 0.00 0.00 0.00

46b 0.00 0.00 0.00 0.00 0.00 0.00

52 0.00 0.00 0.00 0.00 0.00 0.00

60 0.00 0.00 0.00 0.00 0.00 0.00

61 0.00 0.00 0.00 0.00 0.00 0.00

63 0.00 0.00 0.00 0.00 0.00 0.00

101 0.00 0.00 0.00 0.00 0.00 0.00

1030.000.000.000.000.000.00 in Table 3, 0.0 indicates that the compound has 20% less activity (efficacy) in this assay than all trans retinoic acid activity.

TABLE 4

All-trans activation assay efficiency (% equivalent to RA activity)Compound #

RARα RARB RARγ RXRα RXRB RXRγ18 4.00 1.00 0.00 2.00 10.00 1.019 0.00 5.0 3.0 0.0 9.0 4.020 3.0 4.0 0.00 4.00 0.00 3.021 2.00 2.00 2.00 3.00 0.00 3.0022 0.00 0.00 2.00 1.00 0.00 2.0023 0.00 6.00 3.00 1.00 0.00 4.0024 3.00 7.00 4.00 1.00 0.00 3.0025 2.00 3.00 3.00 5.00 0.00 3.0026 1.00 6.00 0.00 2.00 0.00 3.0027 9.00 14.00 6.00 2.00 0.00 4.0028 2.00 10.00 2.00 2.00 0.00 3.0029 0.00 6.00 11.00 0.00 6.00 2.0030 3.00 5.00 1.00 0.00 9.00 3.0031 4.00 14.00 2.00 1.00 8.00 6.0032 0.00 2.00 2.00 1.00 0.00 2.0034 3.00 5.00 2.00 1.00 0.00 3.0036 1.00 5.00 0.00 1.00 7.00 2.0039 1.00 7.00 9.00 2.00 0.00 1.0041 3.00 5.00 6.00 1.00 0.00 3.0045 2.00 0.00 7.00 3.00 8.00 0.0046b 4.00 5.00 3.00 2.00 0.00 4.0052 0.00 15.00 3.00 0.00 0.00 10.0060 0.00 1.00 4.00 3.00 0.00 3.0061 2.00 2.00 0.00 1.00 0.00 3.0063 2.00 2.00 7.00 1.00 0.00 1.00101 0.00 4.00 2.00 1.00 0.00 3.0103 4.00 12.0 7.0 0.00 0.0 2.0

TABLE 5

Ligand binding assays

Compound # K_d (nM)

RARα RARB RARγ RXRα RXRB RXRγ

18 24.00 11.00 24.00 0.00 0.00 0.00

19 565 210 659 0.00 0.00 0.00

20 130.00 22.0 34.00 0.00 0.00 0.00

21 16 9 13 0.00 0.00 0.00

22 24.0 17.0 27.0 0.00 0.00 0.00

23 32.00 25.00 31.00 0.00 0.00 0.00

24 699 235 286 0.00 0.00 0.00

25 50 17 20 0.00 0.00 0.00

26 40.00 31.00 36.00 0.00 0.00 0.00

27 69.00 14.00 26.00 0.00 0.00 0.00

28 669 77 236 0.00 0.00 0.00

29 234 48 80 0.00 0.00 0.00

30 683 141 219 0.00 0.00 0.00

31 370 52.00 100.00 0.00 0.00 0.00

32 0.00 89.00 169.00 0.00 0.00 0.00

34 52.00 30.00 17.00 0.00 0.00 0.00

36 13.00 550.00 0.00 0.00 0.00 0.00

39 67.00 38.00 113.00 0.00 0.00 0.00

41 5.1 491 725 0.00 0.00 0.00

45 12.0 2.80 17.0 0.00 0.00 0.00

46b 250 3.70 5.80 0.00 0.00 0.00

52 60.00 63.00 56.00 0.00 0.00 0.00

60 1.5 1.9 3.3 0.00 0.00 0.00

61 96 15 16 0.00 0.00 0.00

63 133 3219 0.00 0.00 0.00 0.00

101 750 143 637 0.00 0.00 0.00

1033012732610.000.000.00 Table 5 shows that 0.0 represents values greater than 1000 nM.

As can be seen from the test results attributed to tables 3, 4 and 5, the exemplary compounds of the invention shown therein are antagonists of the RAR receptor subtype, however do not have affinity for the RXR receptor subtype. (other compounds of the invention may be antagonists of some, but not all, of the RAR receptor subtypes and agonists of the remaining RAR subtypes.) due to this property, the compounds of the invention may be used to block the activity of RAR agonists in bioassays. In mammals, including humans, the compounds of the invention may be co-administered with RAR agonists by pharmacologically selective or site-specific delivery, preferably to prevent unwanted side effects of RAR agonists. The compounds of the present invention may also be used to treat chronic or acute vitamin a overages resulting from overdosing with vitamin a supplements or ingestion of the liver in certain fish and animals that contain high levels of vitamin a. In addition, the compounds of the present invention can also be used for acute or chronic toxicity caused by retinoid drugs. Known in the art are: the toxicity observed for the vitamin a hypersyndrome (headache, desquamation, bone toxicity, dyslipidemia) was similar or identical to that observed with other retinoids, suggesting a common biological factor, RAR activation. Because the compounds of the present invention block RAR activation, they are useful in treating the above-mentioned toxicities.

When the compounds of the present invention are co-administered topically to the skin, the compounds of the present invention are substantially capable of preventing skin irritation induced by RAR agonist retinoids. Similarly, the compounds of the invention can be administered topically to the skin to block skin irritation in patients or animals to which RAR agonist compounds are administered systemically. The compounds of the present invention can accelerate recovery from pre-existing retinoid toxicity, can block hypertriglyceridemia caused by co-administration of retinoids and can block bone toxicity caused by RAR agonists (retinoids).

In general, as a therapeutic use in mammals according to the present invention, the antagonist compound may be administered enterally or topically to the skin as an antidote to vitamin A, vitamin A precursors after administration of the causative agent (vitamin A precursor or other retinoid) has ceased or toxicity to the retinoid due to overdosing or prolonged exposure. The antagonist compound can optionally be co-administered with a retinoid drug according to the invention, in which case the retinoid provides a beneficial therapeutic effect and the co-administered antagonist ameliorates or eliminates one or more undesirable side effects of the retinoid. For this type of use, the antagonist may be administered at a specific site, for example as a cream or lotion applied to the skin surface, while the co-administered retinoid may be administered enterally.

For therapeutic use according to the present invention, the antagonist compounds are incorporated into pharmaceutical compositions such as tablets, pills, capsules, solutions, suspensions, creams, ointments, gels, salves, lotions, and the like, using pharmaceutically acceptable excipients and carriers well known in the art per se. The preparation of skin surface preparations is described in detail, for example, in Remington's Pharmaceutical Science (Edition 17, MackPublishing Company, Easton, Pennsylvania). For topical application to the skin, the antagonist compounds may also be administered as a powder or spray, especially as an aerosol. If the drug is administered systemically, it may be formulated as a powder, pill, tablet, etc., or as a syrup or elixir suitable for oral administration. For intravenous or intraperitoneal injection, the antagonist compounds are prepared as solutions or suspensions that can be administered by injection. In some cases, it is useful to formulate antagonist compounds for injection. In some cases, it may be useful to formulate the antagonist compounds as suppositories or as sustained release formulations for subcutaneous or intramuscular injection.

The antagonist compound is administered at a therapeutically effective dose according to the present invention. The therapeutic concentration is that concentration which alleviates or prevents the expansion of a particular condition (e.g., toxicity due to exposure to retinoids or vitamin A or side effects of retinoids drugs). It will be appreciated that when the antagonist compounds are co-administered in order to block retinoid-induced toxicity or side effects according to the invention, the antagonist compounds are used in a prophylactic manner in order to prevent the occurrence of specific conditions such as skin irritation.

Useful therapeutic or prophylactic concentrations vary from condition to condition, and in some cases, the concentration employed may vary with the severity of the disease being treated and the patient's sensitivity to the treatment. Thus, none of the concentrations can be used unchanged, but can be altered depending on the specificity of chronic or acute retinoid toxicity or the symptoms associated with treatment. The desired concentration can be obtained by routine experimentation. However, formulations containing 0.01-1.0 mg of antagonist compound per ml of formulation are expected to be effective therapeutic concentrations for topical application to the skin. If systemic administration is desired, it is expected that a therapeutic effect will be achieved at a daily dose of 0.01-5 mg per kg body weight.

The basis for the use of RAR antagonists to prevent or treat RAR agonist-induced toxicity is the competitive inhibition of the activation of RAR receptors by RAR agonists. The main difference between these two uses of RAR antagonists is the presence or absence of preexisting retinoid toxicity. Most of the examples described below relate to the use of retinoids to prevent retinoid toxicity, however, the general methods described herein are also useful for treating preexisting retinoid toxicity.

Test introduction showing RAR use for preventing or treating retinoid toxicity and/or side effects of retinoid drugsExample 1: topical antagonist treatment induced by topical dermal application of stimulants Skin irritation

The compound 4- [ (E) -2- (5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethylnaphthalen-2-yl) propen-1-yl ] benzoic acid (known as AGN 191183) is known in the art as a potent RAR agonist (see, e.g., the description section of U.S. patent 5324840 and figure 2 b). ("AGN" number is any designated reference number used by the cooperative assignee of the present invention for the purpose of identifying a compound)

4- [ (5, 6-dihydro-5, 5-dimethyl-8- (phenyl) -2-naphthyl) ethynyl ] benzoic acid (AGN192869 also known as compound 60a) is a compound in the preparation described below. The compounds are RAR antagonists.

Skin irritation induced by topical administration of the RAR agonist AGN 191183 was also blocked by topical administration of the RAR antagonist AGN192869 to the skin surface of nude mice.

More specifically, skin irritation was measured on a semi-quantitative scale by daily subjective evaluation of desquamation and abrasion. A single figure (skin surface irritation index) summarizes the skin irritation that occurs on animals during the course of the test. The skin irritation index was calculated as follows. The skin surface irritation index is the algebraic sum of the comprehensive peeling index and the comprehensive abrasion index. The combined indices for scaling and bruising ranged from 0-9 and 0-8, respectively, and the maximum severity, time of onset and average severity of the observed scaling and bruising were taken into account.

The severity of scaling was scored on a 5 scale and the severity of bruising on a 4 scale, with higher indices reflecting greater severity. The highest severity score for the composite index is the highest daily severity index given to an animal during the observation.

For the onset score of the composite index, the indices specified in the 0-4 range are as follows:

TABLE 6

Number of apparent peels or abrasions of severity 2 or more

(sky) Number of episodes

8 0

6-7 1

5 2

3-4 3

1-2 4

The average severity score for the composite index is the sum of the daily peeling or abrasion indices divided by the number of days observed. The first day of treatment is not counted because the drug has not yet worked at the time of the first treatment.

To calculate the composite peeling and abrasion index, the average severity and number of episodes index were summed and divided by 2. The results are added to the highest severity index. The combined peeling and abrasion indices are then summed to give a total skin surface irritation index. Each animal had its skin surface irritation index, the value of which is expressed as the mean of the individual indices of each group of animals ± SD. This value is approximated as an integer.

Female nude mice were surface treated with acetone, AGN191183, AGN 192869, or some mixture of AGN 192869 and AGN191183 for 5 consecutive days [ Crl: SKH1-hrBR ] (8-12 weeks old, n ═ 6). The dosages of the individual compounds are shown in table 7. The therapeutic agent was applied to the dorsal skin of the animals in a total volume of 4ml/kg (about 0.1 ml). Mice were observed daily and scored for desquamation and bruising until 3 days after the last treatment, i.e. day 8.

TABLE 7

Experimental design and results of example 1

Dose to dose molar ratio

AGN 191183 AGN 192869 (192869: skin surface)

Group of (mg/kg/d) (mg/kg/d) (191183 Stimulating fingerNumber of

A 0 0 -- 0±0

B 0.025 0 -- 8±2

C 0.025 0.06 2∶1 5±2

D 0.025 0.30 10∶1 2±1

E 0.025 1.5 50∶1 1±0

F 0 1.5 -- 0±0

The skin surface irritation scores of example 1 are given in table 7. At a dose of 1.5mg/kg/d (group F), acetone (vehicle) or AGN 192869 (antagonist) did not cause observable skin surface irritation. AGN 191183(RAR agonist) caused moderate skin surface irritation at the dose of 0.025 mg/kg/d. However, the use of AGN 192869 inhibited AGN 191183-induced skin surface irritation in a dose-dependent manner, with a 50-fold molar excess of AGN 192869, almost completely abrogating the irritation effect. This indicates that the skin surface RAR antagonist blocks skin irritation caused by the skin surface RAR agonist. When the RAR antagonist is more effective, such as the compound 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoic acid (AGN 193109 also referred to herein as compound 60), complete blockade of RAR agonist-induced skin irritation can be achieved using lower molar ratios of antagonist to agonist.

Example 2: blocking induction by oral stimulants by topical application of antagonists Skin irritation of

In this example, the potent RAR agonist agnet, agnet 191183(4- [ (E) -2- (5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethylnaphthalen-2-yl) propen-1-yl ] benzoic acid) and the potent RAR antagonist, 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoic acid (AGN 193109, compound 60) were used and the body weight of the test animal (mouse) was used as an indicator of systemic RAR agonist exposure.

Female nude mice in each group (8-12 weeks old, n ═ 6) were administered by intragastric intubation with corn oil or AGN 191183(0.26mg/kg) suspended in corn oil (5 ml/kg). Vehicle (97.6% acetone/2.4% dimethyl sulfoxide) or a solution of AGN193109 in vehicle (6ml/kg) was also topically administered to the dorsal skin of the mice. The specific doses for the different groups are shown in table 8. Daily administration was continued for 4 consecutive days. Mice were weighed and scored for daily skin surface irritation as described in example 1 until 1 day after the last trial. The percent change in body weight was calculated by subtracting the final body weight (day five) from the initial body weight (day one), dividing by the initial body weight and multiplying by 100%. The skin surface irritation index was calculated as described in example 1.

The surface irritation index and lost body weight for the different groups are shown in table 8. Combination treatments with topical and oral administration of vehicle, i.e., acetone and corn oil, respectively, did not cause skin surface irritation or loss of body weight. Similarly, combination therapy with the oral vehicle and the topical administration of the antagonist AGN193109 did not produce skin surface irritation or loss of body weight. Oral administration of AGN191183 alone resulted in significant loss of body weight and skin irritation. When used with a lower dose of AGN193109, the skin irritation induced by AGN191183 was significantly reduced, while at higher doses AGN193109 it was completely blocked. AGN193109, administered via the skin surface, also blocked AGN 191183-induced loss of body weight in a dose-related manner, however, the blockage was not complete. Thus, topical administration of AGN193109 to the skin preferably blocks the skin toxicity of AGN 191183. It was postulated that low doses of AGN193109 were absorbed systemically, partially blocking the loss of body weight induced by AGN 191183. However, such absorption may be lower in animals with lower permeability skin, such as humans. In addition, the inhibitory effect of AGN193109 on loss of body weight may be due to improved AGN 191183-induced skin irritation.

TABLE 8

Experimental design and results of example 2

Increased or lost in the surface of the skin by oral administration of AGN191183

Surface of skin

Dosage of AGN193109 by weight%

Group of (mg/kg/d) (mg/kg/d) Stimulating fingerNumber of

A 0 0 1±2 0±0

B 0 0.26 (21±6) 8±1

C 0.12 0.26 (9±5) 1±1

D 0.47 0.26 (3±5) 0±1

E 0.47 0 3±3 0±0

Thus, example 2 shows that topical administration of RAR antagonists can be used to preferably block skin irritation induced by oral administration of RAR agonists.

Example 3: acceleration of topical application of antagonists from pre-existing retinoids Recovery of toxicity

In this example, the test animals were treated by topical administration of the RAR agonist AGN191183 to induce weight loss followed by topical administration of vehicle or RAR antagonist AGN 193109.

Female nude mice (8-12 weeks old, n ═ 5) were surface treated with AGN191183 (0.13mg/kg/d) in vehicle (97.6% acetone/2.4% DMSO, 4ml/kg) transdermally daily for 2 consecutive days. Then, starting on the third day, the same mice in each group were surface treated with vehicle or AGN193109(4mg/kg) in vehicle daily through the skin for 3 consecutive days (n ═ 5). Mice were weighed on days 1-5 and 8. Body weight is expressed as mean ± SD. Means were statistically compared using unpaired, two-tailed t-test (two-tailed t-test). When P < 0.05, the difference was considered significant.

TABLE 9

Results of example 3

Treatment of body weight (g)

(3-5 days)

Day 1 Day 2 Day 3 Day 4 Day 5 Day 8

Solvent 24.6 + -1.523.9 + -1.221.4 + -1.220.3 + -1.721.0 + -1.424.7 + -1.0

AGN 193109 23.9±1.0 23.5±1.2 21.4±0.6 22.2±0.7 22.8±0.8 25.0±1.1

The process of change of the body weight with time in example 3 is shown in Table 9. The body weights of both groups of mice decreased in parallel on the second and third days as a result of the AGN 191183 treatment on the first and second days. However, AGN 193109 treatment significantly increased body weight on days four and five compared to vehicle treatment. These data show that recovery from AGN 191183-induced weight loss is accelerated by subsequent treatment with AGN 193109. On day eight, there was no significant difference in body weight between the two groups of mice, indicating that complete recovery could be achieved within a given sufficient time. Thus, the RAR antagonist may be effective to reduce RAR agonist-induced toxicity even if the RAR agonist-induced toxicity is prior to RAR antagonist treatment, i.e., in the RAR agonist toxicity regimen.

Example 4: oral administration of antagonists to block co-administration of retinal derivatives by oral administration Hypertriglyceride induced by excitant

5- [ (E) -2- (5, 6, 7, 8-tetrahydro-3, 5, 5, 8, 8-pentamethylnaphthalen-2-yl) propen-1-yl ] -2-thiophenecarboxylic acid is a known RAR/RXR all-agonist (see U.S. Pat. No. 5324840, Table 32) and is known as AGN 191659. The compound is orally administered for inducing acute hypertriglyceridemia in rats, and AGN193109 (compound 60) is co-administered orally in order to block AGN 191659-induced hypertriglyceridemia.

Male Fescher rats (6-7 weeks old, n-5) were administered via gastric tube using corn oil (vehicle), AGN 191659, AGN193109 or a mixture of AGN 191659 and AGN 193109. AGN 191659 and AGN193109 were administered as a fine suspension in corn oil. The experimental design, including the dosages used, is shown in table 10.

Blood samples were drawn from the inferior venous lumen under carbon dioxide anesthesia. Serum was separated from the blood by low speed centrifugation. Total serum triglycerides (triglycerides plus glycerol) were determined using a standard spectrophotometric endpoint assay commercially available in a kit and suitable for 96-well inoculation plates. Serum triglyceride levels are expressed as mean ± SD. The means were compared statistically by one-way analysis of variance and if significant differences were found, then Dunnett's test was performed. Differences were considered significant when P < 0.05.

As shown in table 10, AGN 191659 itself caused a significant increase in serum triglycerides versus vehicle treatment. AGN193109 by itself did not significantly increase serum triglycerides. Mixture of AGN193109 and AGN 191659 at 1: 1 and 5: 1 molar ratios significantly reduced serum glycemia

The oil triester to a level that did not significantly differ from the control.

Watch 10

Experimental design and results for example 4

Group of Treatment (dosage) Serum glycerol III (mg/dl)

Solvent A55.0 + -3.1

B AGN 193109(19.6mg/kg) 52.4±6.3

C AGN 191659(3.7mg/kg) 122.5±27.6

D AGN 193109(3.9mg/kg) 55.7±14.7

+AGN 191659(3.7mg/kg)

E AGN 193109(19.6mg/kg) 72.7±8.9

+AGN 191659(3.7mg/kg)

Example 4 shows that RAR antagonists can be used to block hypertriglyceridemia induced by co-administered retinal derivatives.Example 5: parenteral use of antagonists to block parenteral co-administration of retinoids Excitant-induced bone toxicity

Example 5 shows that RAR antagonists can block the osteotoxicity induced by RAR agonists. In this example, AGN193109 was used in order to block premature epiphyseal closure in guinea pigs caused by co-administration of the RAR agonist AGN 191183.

Osmotic pumps containing vehicle (20% dimethylsulfoxide/80% polyethylene glycol-300), AGN 191183(0.06mg/ml), or AGN 191183(0.06mg/ml) in combination with AGN193109 (0.34mg/ml) were implanted intraperitoneally into groups of male Hartley guinea pigs (approximately 3 weeks old, n-4). Osmotic pumps designed by the manufacturer deliver approximately 5 μ l of solution per hour for 14 consecutive days.

Animals were sacrificed by carbon dioxide asphyxiation 14 days after transplantation. The left tibia was removed and placed in 10% buffered formalin. The tibia was decalcified by contact with formic acid/formalin solution for 3-4 days, and paraffin sections were prepared. Sections were stained with hematoxylin and eosin by standard methods. The proximal tibial epiphyseal plate was examined and scored for closure or patency. To this end, epiphyseal closure is defined as the illustrated epiphyseal growth plate cartilage continuity break, i.e., is replaced by bone and/or fibroblast tissue.

None of the four weekly vehicle-treated guinea pigs showed epiphyseal closure at the end of the indicated trial. This is expected because the proximal tibial epiphyseal plate of guinea pigs generally does not close until the animal is at least 10 months of age. All four AGN 191183-treated guinea pigs showed partial or complete epiphyseal closure. However, treatment of either guinea pig with a mixture of AGN 191183 and AGN 193109 did not show epiphyseal closure. Thus, when these compounds were co-administered parenterally, AGN 193109 in a 5-fold molar excess completely blocked AGN 191183-induced bone toxicity.RAR antagonist compounds

The compound 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ]Benzoic acid (AGN 193109, Compound 60) and 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (phenyl) -2-naphthyl) ethynyl]Benzoic acid (AGN 192869, compound 60a) is an example of an RAR antagonist and is used in the above animal tests to block the RAR receptor according to the present invention. The compounds of the following formula (formula 1) are additional and general examples of other RAR antagonist compounds for use according to the present invention.

Formula 1

In formula 1, X is S, O, NR ', wherein R' is H or alkyl of 1-6 carbon atoms, or

X is [ C (R)₁)₂]_nWherein R is₁Is H or alkyl of 1 to 6 carbon atoms, and n is an integer of 0 or 1;

R₂is hydrogen, lower alkyl of 1-6 carbon atoms, F, CF₃Fluorine substituted alkyl with 1-6 carbon atoms, OH, SH, alkoxy with 1-6 carbon atoms or alkylthio with 1-6 carbon atoms;

R₃is hydrogen, lower alkyl of 1 to 6 carbon atoms or F;

m is an integer of 0 to 3;

o is an integer of 0 to 3;

z is-C ≡ C-,

-N＝N-，

-N＝CR₁-，

-CR₁＝N，

-(CR₁＝CR₁)_n’-wherein n' is an integer from 0 to 5,

-CO-NR₁-，

-CS-NR₁-，

-NR₁-CO，

-NR₁-CS，

-COO-，

-OCO-，

-CSO-，

-OCS-；

y is phenyl or naphthyl or is selected from pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazylHeteroaryl of oxazolyl, imidazolyl and pyrazolyl, said phenyl and heteroaryl being optionally substituted by one or two R ₂Is substituted by radicals, or

When Z is- (CR)₁＝CR₁)_nAnd n' is 3, 4 or 5, then Y is represented at (CR)₁＝CR₁)_nA direct bond between the group and B;

a is (CH)₂)_qWherein q is 0 to 5, a lower branched alkyl group having 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and 1 or 2 double bonds, an alkynyl group having 2 to 6 carbon atoms and 1 or 2 triple bonds;

b is hydrogen, COOH or a pharmaceutically acceptable salt thereof, COOR₈，CONR₉R₁₀，-CH₂OH，CH₂OR₁₁，CH₂OCOR₁₁，CHO，CH(OR₁₂)₂，CHOR₁₃O，-COR₇，CR₇(OR₁₂)₂，CR₇OR₁₃O or tri-lower alkylsilyl, wherein R₇Is an alkyl, cycloalkyl or alkenyl radical having 1 to 5 carbon atoms, R₈Is an alkyl group of 1 to 10 carbon atoms or a trimethylsilylalkyl group wherein the alkyl group has 1 to 10 carbon atoms, or a cycloalkyl group of 5 to 10 carbon atoms, or R₈Is phenyl or lower alkylphenyl, R₉And R₁₀Independently hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, or phenyl or lower alkylphenyl, R₁₁Is lower alkyl, phenyl or lower alkylphenyl, R₁₂Is lower alkyl, and R₁₃Is a divalent alkyl radical of 2 to 5 carbon atoms, and

R₁₄is (R)₁₅)_r-phenyl, (R)₁₅)_r-naphthyl or (R)₁₅)_r-heteroaryl, wherein said heteroaryl has 1-3 heteroatoms selected from O, S and N, r is an integer from 0 to 5, and

R₁₅independently H, F, Cl, Br, I, NO ₂，N(R₈)₂，N(R₈)COR₈，NR₈CON(R₈)₂，OH，OCOR₈，OR₈CN, an alkyl group having 1 to 10 carbon atoms, a fluoroalkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms and 1 to 3 double bonds, an alkynyl group having 1 to 10 carbon atoms and 1 to 3 triple bonds, or a trialkylsilyl or trialkylsiloxy group wherein the alkyl groups independently have 1 to 6 carbon atoms.Synthetic method of aryl substituted compound

Exemplary RAR antagonist compounds of formula 1 can be prepared by synthetic chemical routes described herein. Synthetic chemists will readily appreciate that the reaction conditions set forth herein are specific embodiments that can be generalized to any and all compounds represented by formula 1. Reaction scheme 1Reaction scheme 1 (continue)

Reaction scheme 1 shows the synthesis of a compound of formula 1, wherein the Z group is ethynyl (-C ≡ C-) and X is [ C (R ≡ C-)₁)₂]n, wherein n is 1. In other words, reaction scheme 1 shows the synthesis of the ethynyl substituted dihydronaphthalene derivatives of the present invention. According to the scheme, a tetrahydronaphthalen-1-one compound of formula 6 is brominated to provide a bromo derivative of formula 7. Said compound of formula 6 already carrying the desired R₁、R₂And R₃Substituents (which are defined according to formula 1 above). A preferred example of a compound of formula 6 is 3, 4-dihydro-4, 4-dimethyl-1 (2H) -naphthalene, which is described in the chemical literature [ Arnold et al.j.am.chem.soc.69: 2322-2325(1947) ]. The preferred route for the synthesis of this compound from 1-bromo-3-phenylpropane is also presented in the experimental part of the present application.

The compound of formula 7 is then reacted with (trimethylsilyl) acetylene to provide a (trimethylsilyl) ethynyl substituted-3, 4-dihydro-naphthalen-1 (2H) -one of formula 8. The reaction with (trimethylsilyl) acetylene is generally carried out in the presence of a ketone iodide, a suitable catalyst [ generally of the formula Pd (PPh)₃)₂Cl₂]Heating (about 100 ℃) in the presence of an acid acceptor (e.g., triethylamine) in an inert gas (argon). The reaction time is generally about 24 hours. Then, the (trimethylsilyl) ethynyl substituted-3, 4-dihydro-naphthalen-1 (2H) -one of formula 8 is reacted with a base (potassium hydroxide or potassium carbonate) in an alcohol solvent such as methanol to provide the ethynyl substituted-3, 4-dihydro-1-naphthalen-1 (2H) -one of formula 9. Then, the compound of formula 9 is reacted with an aromatic or heteroaromatic reagent X₁-Y(R₂) A-B' (formula 10) in the presence of a ketone iodide, a suitable catalyst (typically Pd (PPh)₃)₂Cl₂) In the presence of an acid acceptor (e.g. triethylamine) in an inert gas (argon). The zinc salt of the compound of formula 9 (or other suitable metal salt) can optionally be reacted with a reagent of formula 10 in Pd (PPh)₃)₄Or similar complexes. General and reagent X ₁-Y(R₂) The coupling reaction of-A-B' (formula 10) is carried out at room temperature or at moderately elevated temperature. Generally, the coupling process between an ethynylaryl derivative or zinc salt thereof and a halogen substituted aryl or heteroaryl compound (e.g., reagent of formula 10) is described in U.S. patent 5264456. The compounds of formula 11 are precursors to exemplary compounds of the invention or derivatives thereof that are protected in the B' group (which can be readily removed by reactions well known in the art). The compounds of formula 11 may be converted to other precursors of the exemplary compounds by such reactions and transformations well known in the art. Such reactions by conversion to "homologues and derivatives" are illustrated in scheme 1. One such transformation for the synthesis of several exemplary compounds is the saponification of the ester group (when B or B' is an ester group) to provide the free carboxylic acid or salt thereof.

In general, halogen substituted aryl or heteroaryl compounds of formula 10 can be obtained by reactions well known in the art. An example of such a compound is ethyl 4-iodobenzoate, which may be obtained, for example, by esterification of 4-iodobenzoic acid. Another example is ethyl 6-iodonicotinate, which can be obtained by halogen exchange reaction on 6-chloronicotinic acid followed by esterification. In general, the following general principles and procedures, which are well known and disclosed, can be utilized with respect to the derivatization of compounds of formula 11 and/or the synthesis of aryl and heteroaryl compounds of formula 10, which can then be reacted with compounds of formula 9.

The carboxylic acid is generally esterified by refluxing the acid in a solution of a suitable alcohol in the presence of an acidic catalyst such as hydrogen chloride or thionyl chloride. The carboxylic acid may optionally be condensed with a suitable alcohol in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. The ester is recovered and purified by conventional means. Acetals and ketals can be readily prepared by the method described by March (Advanced Organic Chemistry, 2nd edition, McGraw-Hill Book Company, p.810). Alcohols, aldehydes and ketones can all be protected by the formation of ethers and esters, acetals or ketals, respectively, by known methods such as those described in McOmie (Plenum Publishing Press, 1973) and Protecting Groups (Ed. Greene, John Wiley & Sons, 1981).

In order to increase the value of n in the compound of formula 10 before carrying out the coupling reaction of reaction scheme 1, in which the compound corresponding to formula 10 is not commercially available, aromatic or heteroaromatic carboxylic acids are made satisfactory (homologation) by continuous treatment under the Arndt-eisert conditions or other permissible course of the reaction. Derivatives other than carboxylic acids may also optionally be made desirable by suitable methods. The desired acid is then esterified by the general method described above.

Compounds of formula 10 (or other intermediate or example compounds) wherein a is alkenyl with one or more double bonds may be prepared, for example, by synthetic schemes well known to organic chemists having practical experience; the double bond is introduced, for example, by a Wittig reaction or the like or by removing halogen from an alpha-haloarylalkylcarboxylic acid, ester or similar carboxy aldehyde (carboxaldehyde). The compound of formula 10 (or other intermediate or example compound) wherein the a group has a triple bond (ethynyl) can be prepared by reacting the corresponding arylmethyl ketone with a strong base such as lithium diisopropylamide (with diethyl chlorophosphate followed by addition of lithium diisopropylamide).

Acids and salts derived from compounds of formula 11 (or other intermediate or example compounds) can be readily obtained from the corresponding esters. Alkaline saponification using an alkali metal base will provide the acid. For example, the ester of formula 11 (or other intermediate or example compound) may be dissolved in a polar solvent such as an alkanol, preferably under an inert gas at room temperature, using about 3 molar excess of a base such as lithium hydroxide or potassium hydroxide. The solution is stirred for a period of time, 15-20 hours, cooled, acidified and the hydrolysate recovered by conventional methods.

Amides may be formed from the corresponding esters or carboxylic acids by any suitable amidation method known in the art. One method of preparing such compounds is to convert the carboxylic acid to an acid chloride and then treat the compound with ammonium hydroxide or a suitable amine.

By using thionyl chloride or other methods (j. march,Advanced Organic Chemistry，2nd Edition, McGram-Hill Book Company) to convert the corresponding acid to the acid chloride, which is then reduced with sodium borohydride (March, lbid, p.1124) to produce the corresponding alcohol. Lithium aluminum hydride can be optionally used to reduce the ester at low temperatures. These alcohols are alkylated under Williamson reaction conditions (March, Ibid, p.357) using the appropriate alkyl halides to yield the corresponding ethers. The alcohol may be converted to an ester by reacting the alcohol with a suitable acid in the presence of an acidic catalyst or dicyclohexylcarbodiimide and dimethylaminopyridine.

Aldehydes can be prepared from the corresponding primary alcohols using mild oxidants such as pyridinium dichromate in dichloromethane (Corey, e.j., Schmidt, g., tet.lett.399, 1979) or dimethyl sulfoxide/oxalyl chloride in dichloromethane (Omura, k., Swern, d., Tetrahedron 34: 1651 (1978)).

Ketones can be prepared from the appropriate aldehyde by treating the aldehyde with an alkyl Grignard reagent or similar reagent followed by oxidation.

Acetals or ketals are prepared from the corresponding aldehydes or ketones by the method described in March, Ibid, p.810.

The compounds of formula 10 (or other intermediates or exemplary compounds) wherein B is hydrogen can be prepared from the corresponding halogenated aromatic or heteroaromatic compounds, preferably wherein the halogen is iodine.

Referring again to reaction scheme 1, a compound of formula 11 is reacted with sodium bis (trimethylsilyl) amide and 2- [ N, N-bis (trifluoromethylsulfonyl) amino]-5-chloropyridine is reacted in an inert ether solvent such as tetrahydrofuran at low temperatures (-78 ℃ C. and 0 ℃ C.). The disclosure is shown in reaction scheme 1, wherein the sodium salt intermediate, which is not isolated, is generally shown in parentheses, as in formula 12. The reaction produces a trifluoromethylsulfonyloxy derivative represented by formula 13. (Tf ═ SO)₂CF₃). Then, by reaction with said aryl or heteroaryl compound R₁₄H to convert the compound of formula 13 to the exemplary compound of the present invention represented by formula 14, wherein the organometallic derivative has the formula R₁₄Met (Met represents a monovalent metal), preferably R ₁₄Li(R₁₄As defined in formula 1). Said and organometallic derivatives (preferably of formula R)₁₄Lithium derivative of Li) in an inert ethereal solvent (e.g. tetrahydrofuran), in zinc chloride (ZnCl)₂) And tetrakis (triphenylphosphine) -palladium (0) (Pd (PPh)₃)₄) In the presence of (a). If the organolithium reagent R is₁₄Li is not commercially available and can be obtained from said compound R₁₄H (or halogen derivatives R thereof)₁₄-X₁Wherein X₁As halogen) in ethereal solvents known in the art. The reagent R₁₄The temperature range of the reaction between Li and the compound of formula 13 is generally within about-78 ℃ to 50 ℃. The compounds of formula 14 can be converted to other homologs and derivatives according to the reactions discussed above.

The intermediate 7-bromo-tetrahydronaphthalen-1-one compounds of formula 7 shown in scheme 1 can also be reacted with a compound having the formula R₁₄MgBr(R₁₄As defined in formula 1) to yield a tertiary alcohol of formula 15. By treatment with acidsDehydration of the tertiary alcohol provides the 3, 4-dihydro-7-bromonaphthalene derivative of formula 16 as an intermediate in the synthesis of other compounds of the present invention (see reaction schemes 6, 7 and 8). Reaction scheme 2

With reference to scheme 2, the synthetic route to the above compounds is illustrated, wherein for formula 1, X is S, O or NR' and Z is ethynyl (-C ≡ C-). The raw material in the reaction sequence is bromophenol, bromothiophenol or bromoaniline having a structure shown in formula 17. In order to simplify the present description, in the following description, it is considered that X is mainly sulfur and is used for the preparation of a benzothiopyran derivative. However, it should be remembered that: the schemes described herein, including modifications that would be apparent to those skilled in the art, are also suitable for use in the preparation of the benzopyran (X ═ O) and dihydroquinoline (X ═ NR') compounds of the present invention. Thus, a compound of formula 17, preferably p-bromothiophenol, p-bromophenol or p-bromoaniline, is reacted under basic conditions with a 3-bromocarboxylic acid of formula 18. In this reaction scheme, the symbols have the definitions set forth in formula 1. Examples of reactants of formula 18 (wherein R is ₃Is hydrogen) is 3-bromopropionic acid. Reaction with 3-bromocarboxylic acids of formula 18 yields compounds of formula 19. The latter is cyclized by treatment with an acid to produce a 6-bromothiochroman-4-one derivative (when X is S) or a 6-bromobenzodihydropyran derivative (when X is O) having formula 20. The bromo compound of formula 20 is then subjected to substantially the same reaction sequence under similar conditions as described in scheme 1 for converting the bromo compound of formula 7 to the compound of the present invention. Thus, the following is briefly summarized: reacting the bromo compound of formula 20 with (trimethylsilyl) acetylene provides a 6- (trimethylsilyl) ethynyl substituted thiochroman-4-one or thiochroman-4-one compound of formula 21. Then, the 6- (trimethylsilyl) ethynyl substituted thiochroman-4-one compound of formula 21 is reacted with a base (potassium hydroxide or potassium carbonate) to provide an ethynyl substituted 6-ethynyl substituted thiochroman-4-one compound of formula 22. Then, the compound of formula 22 is reacted with an aromatic or heteroaromatic reagent X₁-Y(R₂) -A-B' (formula 10) in reaction scheme 1Coupling under similar conditions as described in the analogous reaction gives compounds of formula 23.

The compound of formula 23 is then reacted with sodium bis (trimethylsilyl) amide and 2- [ N, N-bis (trifluoromethylsulfonyl) amino under similar conditions in a similar reaction as described in scheme 1 ]-5-chloropyridine reacting to produce 4-trifluoromethylsulfonyloxybenzothiopyran or a benzopyran derivative represented by formula 24. Then by reaction with an aryl or heteroaryl compound R as depicted in scheme 1₁₄An organometallic derivative of H, to convert the compound of formula 24 to the compound of formula 25.

Similar to the use of the intermediate 7-bromo-tetrahydronaphthalen-1-one compound of formula 7 of reaction scheme 1, the intermediate 6-bromothiochroman-4-one compound of formula 20 can also be used in the preparation of other compounds within the scope of the present invention (as described in reaction schemes 6, 7 and 8 below). The compounds of formula 25 can also be converted to other homologs and derivatives in a reaction similar to that described in scheme 1. Reaction scheme 3

Reaction scheme 3 illustrates wherein for formula 1, X is [ C (R)₁)₂]_nN is 0 and Z is acetylene functional group (-C ≡ C-). According to this reaction scheme, 6-bromo-2, 3-dihydro-1H-inden-1-one derivatives of formula 26 are reacted according to a reaction sequence similar to the reaction described in schemes 1 and 2 above (starting with trimethylsilylacetylene), providing indene derivatives of formula 31 via intermediates of formulae 27-30. In a preferred embodiment within the scope of scheme 3, the starting material is 6-bromo-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one, which is available according to the chemical literature (see Smith al. org. Prep. proced. Int.1978, 10, 123-. As described below, compounds of formula 26, such as 6-bromo-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one, can also be used in the synthesis of other exemplary compounds used in the present invention. Reaction scheme 4

With reference to reaction scheme 4, a synthetic route to an exemplary compound is presented wherein for formula 1, Z is- (CR)₁＝CR₁)_n-, n' is 3, 4 or 5, and Y represents in (CR)₁＝CR₁)_nDirect bond between group and B. For example where the X group is [ C (R)₁)₂]_nAnd n is 1 (dihydronaphthalene derivative). Furthermore, it will be appreciated that the reactions and synthetic processes described in scheme 4 and other schemes thereafter (including modifications which will be apparent to those skilled in the art) are also applicable to those in which X is S, O or NR' (a benzothiopyran, benzopyran or dihydroquinoline derivative) or [ C (R)₁)₂]_nAnd n is 0 (indene derivative).

According to reaction scheme 4, a 1, 2, 3, 4-tetrahydronaphthalene derivative of formula 32 is reacted with an acid chloride (R)₁COCl) under Friedel Crafts conditions and oxidizing the resulting acetylated product in, for example, a Jones oxidation reaction to produce a mixture of isomeric 6-and 7-acetyl-1 (2H) -naphthalenone derivatives of formula 33. In a particularly preferred embodiment of this reaction, the starting material of formula 32 is 1, 2, 3, 4-tetrahydro-1, 1-dimethylnaphthalene (a known compound), which can be prepared according to the methods described in the experimental section of this application. Reacting a 7-acetyl-1 (2H) -naphthalenone derivative of formula 33 with ethylene glycol in the presence of an acid to protect the oxo functionality of the exocyclic ketone moiety in the form of a ketal derivative of formula 34. Then, the ketal of formula 34 is reacted with R ₁₄Reaction of a Grignard reagent of MgBr (symbols as defined in formula 1) produces a tertiary alcohol of formula 35. Then, the dioxolane protecting group is removed and the 3, 4-dihydro-7-acetylnaphthalene derivative of formula 36 is provided by dehydration by treating the tertiary alcohol with an acid. The ketone functionality of the compound of formula 36 is subjected to reaction with Horner Emmons (or similar) using a phosphonate reagent of formula 37 under strongly basic conditions to yield, upon reduction, an aldehyde compound of formula 38. Another Horner Emmons (or similar) reaction using a reagent of formula 39 under strongly basic conditions provides a compound of formula 40. The latter can be converted into other homologues and derivatives according to the above reaction. Formula 37H for preparing preferred compoundsSpecific examples of the organ Emmons reagent are diethyl cyanomethylphosphonate; an example of Horner Emmons reagent of formula 39 is diethyl (E) -3-ethoxycarbonyl-2-methylallylphosphonate. Reaction scheme 5

Reaction scheme 5 illustrates a synthetic method for preparing compounds in which the Z group is azo (-N ═ N-). As with reaction scheme 4, for example, where the X group is [ C (R)₁)₂]_nAnd n is 1 (dihydronaphthalene derivative). Furthermore, it will be appreciated that the synthetic procedures described, including modifications which will be obvious to those skilled in the art, are also applicable to all azo compounds used in the present invention, i.e. where X is S, O or NR' (a benzothiopyran, benzopyran or dihydroquinoline derivative) or [ C (R) ₁)₂]_nAnd n is 0 (indene derivative). Thus, introduction of the nitro group into the starting compound of formula 6 under essentially standard nitration conditions produces the 3, 4-dihydro-7-nitro-1 (2H) -naphthalenone derivative of formula 41. Reducing the latter to a 3, 4-dihydro-7-amino-1 (2H) -naphthalenone derivative of formula 42, which is then reacted with a compound of formula ON-Y (R)₂) The nitroso compound of-A-B (formula 43) is reacted under the conditions (glacial acetic acid) generally used for the preparation of azo compounds. The nitroso compound of formula 43 can be obtained according to reactions known in the art. A specific example of such a compound for use in the synthesis of the preferred compound is ethyl 4-nitrosobenzoate. Then, the azo compound of formula 44 is reacted with sodium bis (trimethylsilyl) amide and 2- [ N, N-bis (trifluoromethylsulfonyl) amino]-5-chloropyridine to give a 4-trifluoromethylsulfonyloxy derivative represented by formula 45. Then by reaction with a compound derived from an aryl or heteroaryl compound R₁₄The organometallic derivative of H reacts to convert the compound of formula 45 to the azo compound shown in formula 46. These latter two reactions, the conversion of the 4-trifluoromethylsulfonyloxy derivative and the subsequent reaction with the organometallic derivative, have been described in the above reaction schemes 1, 2 and 3 and are used in several preferred synthetic methods to produce exemplary RAR antagonist compounds. Reaction scheme 6

Reaction scheme 6 illustrates a method for the preparation of compounds wherein for formula 1, the Z group is COO-or CONR₁(R₁Preferably H) is preferred. These ester and amide derivatives are prepared from 3, 4-dihydro-7-bromonaphthalene derivatives of formula 16 (which can be obtained as described in scheme 1). Thus, the compound of formula 16 is reacted with a strong base such as t-butyllithium in an inert ether solvent such as tetrahydrofuran at low temperature and carbon dioxide (CO) is added₂) 5, 6-dihydro-2-naphthalenecarboxylic acid derivatives of formula 47 are provided. Then, reacting the compound of formula 47 with formula X₂-Y(R₂) -A-B (formula 48) wherein X₂Represents OH or NR₁Group, R₁Hydrogen is preferred. Those skilled in the art will understand that: the compounds of formula 48 are aryl or heteroaryl hydroxy or amino derivatives which are obtainable according to the prior art. The reaction between the compounds of formula 47 and formula 48 can be carried out under a variety of known ester or amide forming conditions, for example, coupling of the two in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine. Optionally, the compound of formula 47 is converted to the corresponding acid chloride for coupling with the compound of formula 48 in the presence of a base. As described above, the amide or ester compound of formula 49 may be converted to other homologs and derivatives. Although as wherein the X group of formula 1 is [ C (R) ₁)₂]_nAnd n is 1 (dihydronaphthalene derivative) reaction scheme 6 is presented and shown, the process described herein is applicable to the preparation of benzopyran, benzothiopyran, dihydroquinoline, and indene derivatives.

According to the teachings of U.S. Pat. No. 5324744, wherein for formula 1, Z is-OCO-, NR₁The compounds of the invention of CO, as well as the corresponding thioester and thioamide homologs, may be prepared from intermediates derived from compounds of formula 16 wherein the bromo functionality is substituted with an amino or hydroxyl group. Reaction scheme 7

Reaction scheme 7 illustrates a scheme for the preparation wherein for formula 1, Z is- (CR)₁＝CR₁)_nPreferred synthesis of the compounds in which-and n' are 0. The compound of formula 50 can be obtained in a coupling reaction between a compound of formula 16 and a Grignard reagent derived from a halogenated compound of formula 10. The coupling reaction is generally carried out in the presence of a zinc salt and a nickel (Ni (0)) catalyst in an inert ethereal solvent such as tetrahydrofuran. As described above, the compounds of formula 50 can be converted to other homologs and derivatives. Reaction scheme 8

The method for preparing compounds wherein Z is- (CR) is described with reference to reaction scheme 8₁＝CR₁)_nPreferred synthesis of compounds where-and n' are 1. More precisely, reaction scheme 8 introduces a preferred method for preparing those compounds which are dihydronaphthalene derivatives and in which the Z group represents a vinyl (-CH ═ CH-) functional group. However, the general processes described herein (including modifications that would be obvious to one skilled in the art) are applicable to analogous benzopyran, benzothiopyran, dihydroquinoline compounds, and to compounds in which the vinyl group is substituted. Thus, according to reaction scheme 8, in a suitable catalyst [ generally of the formula Pd (PPh) ₃)]Reacting a 7-bromo-1 (2H) -naphthalenone derivative of formula 7 with a compound having the structure-CH in the presence of an acid acceptor (e.g., triethylamine) in an inert gas (argon gas)₂＝CH-Y(R₂) Vinyl derivatives of-A-B (formula 51). The conditions of this reaction are similar to those of the coupling process of the ethynyl derivative of formula 9 with the reagent of formula 10 (see, e.g., scheme 1), and such reactions are generally referred to in the art as Heck reactions. Vinyl derivatives of formula 51 are available according to the prior art, and an example of such a reagent for use in the synthesis of preferred compounds of the invention is ethyl 4-vinylbenzoate.

The product of the Heck coupling reaction is a vinyl derivative of formula 52, then prepared by reactingSodium bis (trimethylsilyl) amide and 2- [ N, N-bis (trifluoromethylsulfonyl) amino]Treatment with-5-chloropyridine yields 4-trifluoromethylsulfonyloxy derivatives of formula 53, which are thereafter, as described above, reacted with a compound R derived from an aryl or heteroaryl group₁₄The organometallic derivative of H reacts to convert it to the compound used in the present invention. The resulting compound of formula 54 may be converted to other homologs and derivatives.

Compounds of formula 54 can also be obtained by using the synthetic scheme of the Wittig or Horner Emmons reaction. For example, an intermediate of formula 33 (see scheme 4) can be reacted with triphenylphosphonium bromide (Wittig) reagent or more preferably with a compound of formula (EtO) ₂PO-CH₂-Y(R₂) -diethyl phosphonate (Horner Emmons) reagent reaction of a-B (e.g. Horner Emmons reaction similar to that described in us patent 5324840). The Horner Emmons reaction provides an intermediate compound similar in structure to formula 52 and converts this intermediate to the compound of formula 54 by the reaction sequence described for the compound of formula 52 in reaction scheme 8.Synthesis method of aryl and (3-oxo-1-propenyl) -substituted compound

Exemplary RAR antagonist compounds having formula 101 can be prepared by synthetic chemical routes described herein. Synthetic chemists readily understand that: the conditions set forth herein are specific embodiments that may be generalized to any and all compounds represented by formula 101. Reaction scheme 101Reaction scheme 101 (continuation)

Reaction scheme 101 illustrates the synthesis of compounds of formula 101 wherein X is [ C (R)₁)₂]_nN is 1, p is zero and R₁₇Is H or lower alkyl. In other words, scheme 101 illustrates the synthesis of compounds of the present invention, which are derivatives of 3, 4-dihydronaphthalene. According to the scheme, by R₃And R₂A tetralin compound of formula 103, suitably substituted with a group (as defined in formula 101) as starting material. Formula (II)A preferred example of a 103 compound is 1, 3, 3, 4-tetrahydro-1, 1-dimethyl-naphthalene, which is described in the chemical literature (Mathur et al tetrahedron, 1985, 41: 1509). The preferred route for the synthesis of this compound from 1-bromo-3-phenylpropane is also described in the experimental part of the present application.

In a Friedel Crafts type reaction, a compound of formula 103 is reacted with a compound having the structure R₁₆CH₂COCl(R₁₆As defined in formula 101) and then oxidized with chromium trioxide in acetic acid to provide the isomeric 6 and 7 acetyl-3, 4-dihydro-1 (2H) -naphthalenone derivatives. Only the structural formula of the 6-acyl compound (formula 104) which is important from the viewpoint of the present invention is shown in reaction scheme 101. In the preparation of preferred compounds of the invention, R₁The radicals representing methyl radicals R₂、R₃And R₁₆Is H, and thus, a preferred intermediate corresponding to formula 104 is 3, 4-dihydro-4, 4-dimethyl-6-acetyl-1 (2H) -naphthalenone.

The exo-cyclic ketone functionality of the compound of formula 104 is then protected as a ketal, for example by treatment with ethylene glycol in an acid, to provide the 1, 3-dioxolanyl derivative of formula 105. Then, reacting the compound of formula 105 with formula R₁₄MgBr(R₁₄As defined in formula 101) to yield a 1, 2, 3, 4-tetrahydro-1-hydroxy-naphthalene derivative of formula 106. The exocyclic ketone functionality of the compound of formula 106 is then deprotected by treatment with acid and dehydration to give the compound of formula 107. Another method for obtaining a compound of formula 107 from a compound of formula 105 is to combine a compound of formula 105 with sodium bis (trimethylsilyl) amide and 2- [ N, N-bis (trifluoromethylsulfonyl) amino ]-5-chloropyridine (Tf ═ SO)₂CF₃) In an inert ether solvent such as tetrahydrofuran at low temperature (-78 deg.C-0 deg.C). The reaction proceeds via a sodium salt intermediate, which is generally not isolated and is not shown in reaction scheme 101. The overall reaction produces a trifluoromethylsulfonyloxy derivative which is then reacted with a compound R derived from an aryl or heteroaryl group₁₄An organometallic derivative of H, such that said organometallic derivative has the formula R₁₄Met (Met represents a monovalent metal), preferably R₁₄Li(R₁₄As defined in equation 101). Said organometallic derivative is preferably of the formula R₁₄The reaction of lithium derivatives of Li is generally carried out in an inert ethereal solvent (for example tetrahydrofuran), in zinc chloride (ZnCl)₂) And tetrakis (triphenylphosphine) -palladium (0) (Pd (PPh)₃)₄) In the presence of oxygen. If not commercially available, compounds R can be prepared from the compounds according to methods known in the art₁₄H (or halogen derivatives R thereof)₁₄-X₁Wherein X is₁Halogen) in an ethereal solvent₁₄And Li. In general, the reagent R₁₄The temperature range of the reaction between Li and the trifluoromethylsulfonyloxy derivative is within about-78 ℃ to 50 ℃.

The condensation between the ketone compound of formula 107 and the aldehyde or ketone of formula 108 results in the formation of the compounds of the present invention. In preparing preferred exemplary compounds of the present invention, the reagent of formula 108 is 4-carboxybenzaldehyde (R) ₁₇-H). Examples of other reagents within formula 108 and suitable for condensation reactions and for the synthesis of compounds within the scope of the present invention (formula 101) are: 5-carboxy-pyridine-2-aldehyde, 4-carboxy-thiophene-2-aldehyde, 5-carboxy-thiophene-2-aldehyde, 4-carboxy-furan-2-aldehyde, 5-carboxy-furan-2-aldehyde, 4-carboxyacetophenone, 2-acetyl-pyridine-5-carboxylic acid, 2-acetyl-pyridine-4-carboxylic acid, 2-acetyl-thiophene-5-carboxylic acid, 2-acetyl-furan-4-carboxylic acid, and 2-acetyl-furan-5-carboxylic acid. The latter compounds are available from the chemical literature; see, for example, decrroix et al, j.chem.res. (S), 4: 134 (1978); dawson et al, j.med.chem.29: 1282 (1983); and Queguiner et al, Bull Soc Chimique de France No.10, pp.3678-3683 (1969). The condensation reaction between the compounds of formula 107 and 108 is carried out in an alcoholic solvent in the presence of a base. Preferably, the reaction is carried out in ethanol in the presence of sodium hydroxide. This condensation reaction is known to those skilled in the art as an aldol condensation and in the preferred embodiment described herein (condensation of a ketone of formula 107 with an aldehyde of formula 108) is known as the Claisen-Schmidt reaction. [ see March: Advanced Organic Chemistry：Reactions，Mechanisms，and Structure，pp.694-695，McGraw Hill(1968)]. The compounds of formula 109 are within the scope of the invention and may be further converted to yield other compounds of the invention. In addition, the A-B group of formula 108 may be a group as defined in formula 101 within the scope of the present invention only after one or more synthetic transformations whose nature is well known and within the skill of an experienced chemist. For example, the reaction carried out on the A-B group may be a deprotection, tolerance (homologation), esterification, saponification, amide formation, or the like reaction.

In general, for derivatization of compounds of formula 109 and/or synthesis of aryl and heteroaryl compounds of formula 108 (which may then be reacted with compounds of formula 107), the following well-known and disclosed general principles and synthetic procedures may be used.

As mentioned above, the carboxylic acid is generally esterified by refluxing the acid in a suitable alcohol solution in the presence of an acidic catalyst such as hydrochloric acid or thionyl chloride. In addition, the carboxylic acid may be condensed with a suitable alcohol in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. The ester can be recovered and purified by conventional methods. By means of the method in March,Advanced Organic Chemistryacetals and ketals can be easily prepared by the method described in (2nd Edition, McGraw-Hill Book Company, p.810). By known methods (e.g. in McOmie, Plenum Press, 1973 and Protecting Groups, Ed. Greene, John Wiley) &Sons, 1981) can protect all alcohols, aldehydes and ketones by forming the corresponding ethers and esters, acetals or ketals, respectively.

To increase the value of n in the compound of formula 108 prior to conducting the condensation reaction of reaction scheme 101, wherein the compound corresponding to formula 108 is not commercially available, an aromatic or heteroaromatic carboxylic acid is made satisfactory (while the aldehyde group is protected) by continuous processing under the Arndt-eisert conditions or other permissible reaction processes. Derivatives other than carboxylic acids may also optionally be made desirable by suitable methods. The desired acid is then esterified by the general method described above.

Compounds of formula 108 wherein A is alkenyl having one or more double bonds (or other intermediates of the invention, where applicable) may be prepared, for example, by synthetic schemes well known to organic chemists having practical experience, such as by Wittig et al reactions or by introducing double bonds by removing halogen from α -haloarylalkylcarboxylic acids, esters, or similar carboxy aldehydes. Compounds of formula 108 (or other intermediates of the invention where applicable) in which the a group has a triple bond (ethynyl) can be prepared by reacting the corresponding arylmethyl ketone with a strong base such as lithium diisopropylamide (with diethyl chlorophosphate followed by addition of lithium diisopropylamide).

The acids and salts derived from the compounds of formula 109 (or other intermediates or compounds of the invention, where applicable) can be readily obtained directly as a result of the condensation reaction or from the corresponding esters. Alkaline saponification using an alkali metal base will provide the acid. For example, the ester of formula 109 (or other intermediate or compound of the invention, where applicable) may be dissolved in a polar solvent such as an alkanol, preferably under an inert gas at room temperature, using about 3 molar excess of a base such as lithium hydroxide or potassium hydroxide. The solution is stirred for a period of time, 15-20 hours, cooled, acidified and the hydrolysate recovered by conventional methods.

Amides may be formed from the corresponding esters or carboxylic acids by any suitable amidation method known in the art. One method of preparing such compounds is to convert the acid to an acid chloride and then treat the compound with ammonium hydroxide or a suitable amine.

By using thionyl chloride or other methods (j. march,Advanced Organic Chemistry2nd Edition, McGram-Hill Book Company) to convert the corresponding acid to the acid chloride, which is then reduced with sodium borohydride (March, lbid, p.1124) to produce the corresponding alcohol. Lithium aluminum hydride can be optionally used to reduce the ester at low temperatures. Use under Williamson reaction conditions (March, Ibid, p.357) Suitable alkyl halides alkylate these alcohols to yield the corresponding ethers. The alcohol may be converted to an ester by reacting the alcohol with a suitable acid in the presence of an acidic catalyst or dicyclohexylcarbodiimide and dimethylaminopyridine.

Aldehydes can be prepared from the corresponding primary alcohols using mild oxidants such as pyridinium dichromate in dichloromethane (Corey, e.j., Schmidt, g., tet.lett.399, 1979) or dimethyl sulfoxide/oxalyl chloride in dichloromethane (Omura, k., Swern, d., Tetrahedron 34: 1651 (1978)).

Ketones can be prepared from the appropriate aldehyde by treating the aldehyde with an alkyl Grignard reagent or similar reagent followed by oxidation.

Acetals or ketals can be prepared from the corresponding aldehydes or ketones by the method described in March, Ibid, p.810. Reaction scheme 102Reaction scheme 102 (continue)

Referring to reaction scheme 102, where p is zero for formula 101, R is in the aryl portion of the condensed ring structure₂Is OH and R₁₇Those compounds of the invention which are OH. Those skilled in the art will readily understand these compounds as beta-diketones in their enol form. Reaction scheme 102 also illustrates the synthetic route to those compounds of the present invention wherein p is 1. The latter compounds are readily understood by those skilled in the art as flavonoids. Thus, according to this reaction scheme, a 1, 2, 3, 4-tetrahydro-6-methoxynaphthalen-1-one derivative of formula 110 is reacted with dihydrocarbylzinc (R) ₁Zn) in the presence of titanium tetrachloride in a suitable solvent such as dichloromethane with geminal dihydrocarbyl radicals R₁R₁In place of the oxo function, to give compounds in which R₁A compound of formula 111 which is lower alkyl. In a preferred example of a compound of the invention prepared according to reaction scheme 102, R₃The radicals are hydrogen and R1 is methyl. Thus, the dihydrocarbyl zinc is dimethylZinc, and the preferred starting material of formula 110 is 1, 2, 3, 4-tetrahydro-6-methoxynaphthalen-1-one. The latter compound is commercially available, for example, from Aldrich Chemical Company. The 1, 2, 3, 4-tetrahydro-1, 2-dialkyl-6-methoxynaphthalene derivative of formula 111 is then oxidized with chromium trioxide in acetic acid and acetic anhydride to produce the 1, 2, 3, 4-tetrahydro-3, 4-dialkyl-7-methoxynaphthalene-1-one derivative of formula 112. Contacting a ketone compound of formula 112 with a Grignard reagent (R)₁₄MgBr，R₁₄As defined in formula 101) to produce a 1-hydroxy-1-aryl-3, 4-dihydro-3, 4-dialkyl-7-methoxynaphthalene derivative of formula 113. The hydroxy compound of formula 113 is subjected to an elimination process by heating, preferably in an acid, to produce a dihydronaphthalene compound of formula 114. Removal of the methyl group from the phenomethyl ether functional group of the compound of formula 114 by treatment with boron tribromide in a suitable solvent such as dichloromethane, followed by introduction of R ₁₆CH₂The acylating agent of the CO group acylating the phenolic hydroxyl group to produce the compound of formula 115. In a preferred embodiment, R₁₆Is H, whereby the acylating agent is acetyl chloride or acetic anhydride. The acylation reaction is carried out in a basic solvent such as pyridine. The acylated phenol compound of formula 115 is reacted with aluminum trichloride at an elevated temperature, which undergoes Fries rearrangement and produces the 1-aryl-3, 4-dialkyl-3, 4-dihydro-6-acetyl-7-hydroxy-naphthalene compound of formula 116. By introducing CO-Y (R)₂) The acylating agent (e.g., acid chloride) of the-A-B group acylating the phenolic hydroxyl group of the compound of formula 116 to produce the compound of formula 117. In the acid chloride reagent Cl-CO-Y (R)₂) in-A-B (or a similar acylating agent), symbol Y, R₂And A-B have the definition in formula 101. In the preparation of preferred compounds of the invention according to this reaction scheme, the reagent is ClCOC₆H₄COOEt (half ethyl ester half acid chloride of terephthalic acid).

Reacting a compound of formula 117 with a strong base, such as potassium hydroxide in pyridine, produces a compound of formula 118 as a result of an intramolecular Claisen condensation reaction. Compounds of formula 118 are within the scope of this invention and formula 101 wherein the aromatic moiety of the condensation ring moiety has a hydroxyl group as R₂A substituent, and R ₁₇Is a hydroxyl group. According to the foregoingThe reactions (intramolecular Claisen condensation) and Fries rearrangement previously mentioned, it is noted that possible reaction mechanisms are mentioned in this specification in order to fully explain the reactions described herein and to assist one skilled in the art in carrying out the work described herein to react and prepare the compounds of the invention. Furthermore, the inventors do not wish to be bound by theory or mechanism of reaction and thus the invention presented herein should not be limited.

Reacting a compound of formula 118 with a strong acid, such as sulfuric acid, in a suitable protic solvent, such as acetic acid, produces a flavone compound of formula 119. Compounds of formula 119 are also compounds of the present invention within the scope of formula 101 (where p is 1). The compounds of formula 118 and formula 119 can be subjected to further reactions and transformations to provide other homologs and derivatives as described in scheme 101 above. The process of conversion to homologues and derivatives is shown in reaction scheme 102. Reaction scheme 103

Referring to reaction scheme 103, shown is a scheme wherein for formula 101, X is S, O or NR', p is zero and R₁₇Synthetic routes to those compounds of the invention which are H or lower alkyl. However, using the general synthetic principles shown in scheme 102, one skilled in the art can modify the synthetic methods shown and can also obtain compounds in which X is S, O or NR 'and p is 1 or in which X is S, O or NR' and p is zero, and in which R is in the aromatic portion of the condensed ring portion ₂The radicals being OH and R₁₇A compound of the present invention which is OH.

The starting material of reaction scheme 103 is a phenol, thiophenol, or aniline derivative of formula 120. In preferred compounds of the invention, R₂And R₁₆The radicals are all hydrogen, the preferred starting materials of formula 120 are 3-vinylthiophenol or 3-vinyl-phenol, which are compounds known from the chemical literature [ Nuyken, et al, polym. bull (Berlin) 11: 165(1984)]. For simplicity of description, in the description that follows, it will be considered that X is mainly sulfur and is used for the preparation of the benzothiopyran derivatives of the present invention. However, it should be remembered that: therein, theThe depicted scheme, including modifications that would be apparent to one skilled in the art, is also applicable to the preparation of benzopyran (X ═ O) and dihydroquinoline (X ═ NR') compounds within the scope of the present invention. Thus, the compound of formula 120 is reacted with a 3-bromocarboxylic acid of formula 121 under basic conditions. In the reaction scheme, the symbols have the definitions set forth in formula 101. Examples of reagents of formula 121 (wherein R is₃Is hydrogen) is 3-bromopropionic acid. Reaction with a 3-bromocarboxylic acid of formula 121 yields a compound of formula 122. The latter is cyclized by treatment with an acid to produce a 7-vinyl-thiochroman-4-one derivative (when X is S) or a 7-vinyl-chroman derivative (when X is O) having the formula 123. Then reacting the 7-vinyl-thiochroman-4-one derivative or 7-vinyl-chroman-4-one derivative of formula 123 in Pd (II) Cl ₂And CuCl₂Oxidation in the presence of a catalyst provides the corresponding 7-acyl (ketone) compound of formula 124. The latter reaction is known to those skilled in the art as Wacker oxidation. The exocyclic keto group of the compound of formula 124 is protected as a ketal, for example, by treatment with ethylene glycol in an acid, providing a 1, 3-dioxolanyl derivative of formula 125. The compound of formula 125 is then subjected to a reaction sequence analogous to that of the compound of formula 105 in reaction scheme 101. Thus, reacting a 1, 3-dioxolanyl derivative of formula 125 with a compound of formula R₁₄Reaction of a Grignard reagent of MgBr to produce a tertiary alcohol of formula 126, which is then dehydrated in acid to provide a benzothiopyran (X is S), benzopyran (X is O) or dihydroquinoline derivative of formula 127 (X is NR'). The ketone compound of formula 127 is then reacted with a reagent of formula 108 in an aldol condensation (Claisen Schmidt) reaction in the presence of a base to provide the compound of the invention of formula 128. The compound of formula 128 can be further converted to homologues and derivatives as described in reaction schemes 101 and 102 above.

Specific embodiments 2-hydroxy-2-methyl-5-phenylpentane

100.0g (0.492mol) of 1-bromo-3-phenylpropane in 100ml of diethyl ether are added to a mixture of 13.16g (0.541mol) of magnesium turnings in 200ml of dry diethyl ether, and after addition of 5 to 10ml of the solution, the addition is stopped until the formation of the Grignard reagent has begun. The remaining bromide was then added over 1 hour. The Grignard reagent was stirred for 20 min at 35 ℃ and then 31.64g (0.541mol) of acetone were added over 45 min. The reaction was stirred at room temperature overnight before cooling to 0 ℃ and acidified carefully with 20% hydrochloric acid. The aqueous layer was extracted with ether (3X 200ml), and the combined organic layers were washed with water and saturated aqueous sodium chloride solution before drying over magnesium sulfate. The solvent was removed under reduced pressure and the residue was distilled to give 63.0g (72%) of the product as a pale yellow oil:

bp99-102℃/0.5mm Hg.1H NMR(CDCl₃)：δ7.28-7.18(5H，m)，2.63(2H，t，J＝7.5Hz)，1.68 (2H，m)，1.52 (2H，m)，1.20 (6H，s).1, 2, 3, 4-tetrahydro-1, 1-dimethylnaphthalene

A mixture of phosphorus pentoxide (55.3g, 0.390mol) in 400ml of methanesulfonic acid was heated at 105 ℃ under argon until the solid dissolved. The resulting solution was cooled to room temperature and 2-hydroxy-2-methyl-5-phenylpentane (63.0g, 0.354mol) was added slowly with stirring. After 4 hours, the reaction was quenched by carefully pouring the solution into 1 liter of ice. The resulting mixture was extracted with ether (4X 125ml), and the combined organic layers were washed with water, saturated aqueous sodium bicarbonate, water and saturated aqueous sodium chloride before drying over magnesium sulfate. The solution was concentrated under reduced pressure and then distilled to yield 51.0g (90%) of a clear colorless oil:

bp.65.67℃/1.1mmHg.1H NMR(CDCl₃)：δ7.32(1H，d，J＝7.4Hz)，7.16-7.05(3H，m)，2.77(2H，t，J＝6.3Hz)，1.80 (2H，m)，1.66 (2H，m)，1.28(6H，s).3, 4-dihydro-4, 4-dimethyl-1 (2H) -naphthalenone (Compound A)

A solution of 350ml of glacial acetic acid and 170ml of acetic anhydride is cooled to 0 ℃ and 25.0g (0.25mol) of chromium trioxide are carefully added in small portions. The resulting mixture was stirred for 30 minutes before 120ml of benzene was added. 1, 2, 3, 4-tetrahydro-1, 1-dimethylnaphthalene in 30ml of benzene solution was slowly added. After the addition was complete, the reaction was stirred at 0 ℃ for 4 hours. The solution was diluted with water (200ml) and extracted with diethyl ether (5X 50 ml). The combined organic layers were washed with water, saturated aqueous sodium carbonate solution and saturated aqueous sodium chloride solution before drying over magnesium sulfate. The solvent was removed under reduced pressure and distilled to yield 16.0g (74%) of the product as a pale yellow oil:

bp 93-96℃/0.3mm Hg 1H NMR(CDCl₃)：δ8.02(1H，dd，J＝1.3，7.8Hz)，7.53(1H，m)，7.42(1H，d，J＝7.9Hz)，7.29(1H，m)，2.74(2H，t，J＝6.8Hz)，2.02(2H，t，J＝6.8Hz)，1.40(6H，s).3, 4-dihydro-4, 4-dimethyl-7-bromo-1 (2H) -naphthalenone (Compound B)

A mixture of 9.5g (71.4mmol) of aluminum trichloride and 3ml of methylene chloride was charged into a 100ml three-necked flask equipped with a high-efficiency reflux condenser, a drying tube and an addition funnel. 3, 4-dihydro-4, 4-dimethyl-1 (2H) -naphthalenone (5.0g, 28.7mmol) (attention: exothermic reaction!) was added dropwise to the mixture at room temperature with stirring. Then, 5.5g (34.5mmol) of bromine was added very slowly, and the resulting mixture was stirred at room temperature for 2 hours. (note: if stirring was stopped, the mixture temperature rose to 70 ℃ until stirring was started again.) then the reaction was quenched by slow addition of ice-cooled 6M hydrochloric acid. The mixture was extracted with ether, and the combined organic layers were washed with water, saturated aqueous sodium bicarbonate solution and saturated sodium chloride before being dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was distilled to afford 5.8g (80%) of the product as a pale yellow oil (solidified upon standing): bp: 140 deg.C/0.4 mm Hg.1H NMR (CDCl)₃)：δ8.11(1H，d，J＝3.0Hz)，7.61(1H，dd，J＝3.0，9.0Hz)，7.31(1H，d，J＝9.0Hz)，2.72(2H，t，J＝6.0Hz)，2.01(2H，t，J＝6.0Hz)，1.28(6H，s).1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-7-bromonaphthalene (Compound C)

A solution of 4-bromotoluene (5.40g, 31.8mmol) in 10ml THF was added in two portions to a mixture of magnesium turnings (648.0mg, 27.0mmol) in 25ml THF. The reaction was started by adding 2ml of the solution and then the rest of the solution was slowly added through the addition funnel. The mixture was stirred at room temperature for 1 hour, and then the solution was transferred to a second flask with a cannula. A solution of 4.0g (15.9mmol) of 3, 4-dihydro-4, 4-dimethyl-7-bromo-1 (2H) -naphthalenone (compound B) in 15ml of THF is added to the Grignard reagent formed. The resulting solution was heated to reflux overnight, cooled to room temperature, and the reaction quenched by the careful addition of ice-cooled 10% hydrochloric acid. The combined organic layers were extracted with ether and then washed with water and saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. Removal of the solvent under reduced pressure afforded the product as an oil which was purified by column chromatography (hexane/ethyl acetate, 96: 4) to afford the colorless product:

1H NMR(CDCl₃)：δ7.36 (1H，dd，J ＝ 2.1，7.6Hz)，7.26(3H，m)，7.12(3H，s)，2.34(3H，s)，2.24-2.04(2H，m)，1.81(1H，m)，1.55(1H，m)，1.35(3H，s)，1.30(3H，s).3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-7-bromonaphthalene (Compound D)

3.4g (9.85mmol) of 1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-7-bromonaphthalene (compound C) and 40ml of benzene were introduced into a flask with a Dean-Stark trap. Catalytic amounts of p-toluenesulfonic acid monohydrate were added and the resulting solution was heated to reflux for 2 hours. After cooling to room temperature, diethyl ether was added and the solution was washed with water, saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. The solvent was removed under reduced pressure and column chromatography purification (100% hexane/silica gel) afforded the title compound as a colorless solid:

1H NMR(CDCl₃)：δ7.32(1H，dd，J＝2.1，8.2Hz)，7.21(5H，m)，7.15(1H，d，J＝2.1Hz)，5.98(1H，t，J＝4.7Hz)，2.40(3H，s)，2.32(2H，d，J＝4.7Hz)，1.30(6H，s).7-ethynyl-3, 4-dihydro-4, 4-dimethylnaphthalen-1 (2H) -one (Compound E)

0.97g (1.3mmol) of bis (triphenylphosphine) palladium (II) chloride and 0.26g (1.3mmol) of cuprous iodide were added to a solution of 7g (27.6mmol) of 3, 4-dihydro-4, 4-dimethyl-7-bromo-1 (2H) -naphthalenone (compound B) in 150ml of triethylamine (15 min. with argon flow). Argon was bubbled through the reaction mixture for 5 minutes, then 39ml (36.6mmol) of (trimethylsilyl) acetylene was added. The reaction mixture was sealed in a pressure tube and placed in a preheated oil bath (100 ℃) for 24 hours. The reaction mixture was then filtered through Celite (Celite), washed with ether and the filtrate was concentrated in vacuo to give crude 7- (trimethylsilyl) ethynyl-3, 4-dihydro-4, 4-dimethylnaphthalen-1 (2H) -one. 0.6g (4.3mmol) of potassium carbonate was added to a solution of the crude TMS-ethynyl compound in 50ml of methanol. The mixture was stirred at room temperature for 8 hours and then filtered. The filtrate was concentrated in vacuo, diluted with ether, washed with water, 10% hydrochloric acid and brine, dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica gel, 10% ethyl acetate-hexanes) gave the desired compound as a white solid:

PMR(CDCl₃)：δ1.39(6H，s)，2.02(2H，t，J＝7.0Hz)，2.73(2H，t，J＝7.0Hz)，3.08(1H，s)，7.39(1H，d，J＝8.2Hz)，7.61(1H，dd，J＝1.8，8.2Hz)，8.14(1H，d，J＝91.8Hz).4-Iodobenzoic acid ethyl ester

2ml of thionyl chloride was added to a suspension of 10g (40.32mmol) of 4-iodobenzoic acid in 100ml of anhydrous ethanol, and the mixture was then heated under reflux for 3 hours. The solvent was removed in vacuo and the residue was dissolved in 100ml of diethyl ether. The ether solution was washed with saturated sodium bicarbonate and saturated sodium chloride solution and dried over magnesium sulfate. The solvent was then removed in vacuo and the residue was subjected to Kugelrohr distillation (100 ℃, 0.55mm) to yield the desired compound as a colorless oil: PMR (CDCl)₃)：δ1.42(3H，t，J～7Hz)，4.4(2H，q，J～7Hz)，7.8(4H).6-iodonicotinic acid

Mixing sodium iodide (20.59g, 137.40 m)mol) was cooled to-78 ℃ under argon and hydroiodic acid (97.13g, 759.34mmol) was added. The cooling bath was removed and the suspension was stirred for 5 minutes. 6-Chloronicotinic acid (22.09g, 140.20mmol) was added to the mixture and the resulting mixture was slowly warmed to room temperature and stirred. The mixture was heated to reflux at 125 ℃ for 24 h, cooled to room temperature and poured into 0 ℃ acetone (500 ml). The yellow solid was collected by filtration and washed with 200ml of 1N aqueous sodium bisulfate solution. Recrystallization from methanol (washing the crystals with diethyl ether) affords the title compound as white crystals: mp177-179 ℃ lith.mp 187-: nicotinic Acid Derivatives j. org. chem.51: 953-954(1986).1H NMR (DMSO-d 6): δ 8.81(1H, dd, J ═ 0.8, 2.4Hz), 8.01(1H, dd, J ═ 0.8, 8.2Hz), 7.91(1H, dd, J ═ 2.4, 8.2Hz). 6-Iodonicotinic acid ethyl ester

A solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (19.86g, 103.6mmol) in dichloromethane (250ml) was added to a suspension of 6-iodonicotinic acid (23.38g, 94.20mmol) in dichloromethane (100 ml). Ethanol (12.40g, 269.27mmol) followed by dimethylaminopyridine (1.15g, 9.41mmol) was added to the mixture. The mixture was heated at 50 ℃ for 24.5 h, concentrated in vacuo and diluted with water (200ml) and then extracted with diethyl ether (550 ml). The combined organic layers were washed with saturated aqueous sodium chloride, dried (magnesium sulfate) and concentrated to a yellow solid. Purification by flash chromatography (silica gel, 10% ethyl acetate-hexanes) afforded the title compound as white needles: as white threads: mp 48-49 ℃; 1H NMR (CDCl)₃)：δ8.94(1H，d，J＝2.1Hz)，7.91(1H，dd，J＝2.1，8.2Hz)，7.85(1H，d，J＝8.2Hz)，4.41(2H，q，J＝7.1Hz)，1.41(3H，t，J＝7.1Hz).4- [ (5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) ethynyl group]Benzoic acid ethyl ester (Compound) F)

5g (7.2mmol) of bis (triphenylphosphine) palladium (II) chloride and 1.4g (7.2mmol) of cuprous iodide were added to a solution of 4g (21.7mmol) of 7-ethynyl-3, 4-dihydro-4, 4-dimethylnaphthalen-1 (2H) -one (compound E) (15 min. with argon flow) and 6g (21.7mmol) of ethyl 4-iodobenzoate in 100ml of triethylamine. Argon gas was introduced into the mixture for 5 minutes, followed by stirring at room temperature for 18 hours. The reaction mixture was filtered through celite and the filtrate was concentrated in vacuo. Purification by flash chromatography (silica gel, 10% ethyl acetate-hexanes) gave the title compound as a white solid:

PMR(CDCl₃)：δ1.41(3H，t，J＝7.2Hz)，1.41(6H，s)，2.04(2H，t，J＝6.5Hz)，2.76(2H，t，J＝6.5Hz)，4.40(2H，q，J＝7.2Hz)，7.44(1H，d，J＝8.2Hz)，7.59 (2H，d，J＝8.4Hz)，7.68(1H，dd，J＝1.8，8.2Hz)，8.04(2H，d，J＝8.4Hz)，8.15(1H，d，J＝1.8Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl]Benzyl benzene Ethyl acid ester (Compound G)

A solution of 500.0mg (1.44mmol) of ethyl 4- (5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) ethynyl ] benzoate (compound F) in 4.0ml of THF is added to a cold solution (-78 ℃ C.) of 291.6mg (1.59mmol) of sodium bis (trimethylsilyl) amide in 5.6ml of THF. The reaction mixture was stirred at-78 ℃ for 35 minutes, then a solution of 601.2mg (1.59mmol) of 5-chloro (2-bis-trifluoromethylsulfonyl) imide in 4.0ml THF was added. After stirring at-78 ℃ for 1 hour, the solution was warmed to 0 ℃ and stirred for 2 hours. The reaction was quenched by addition of saturated aqueous ammonium chloride solution. The mixture was extracted with ethyl acetate (50ml) and the combined organic layers were washed with 5% aqueous sodium hydroxide solution, water and brine. The organic phase was dried over sodium sulfate and concentrated in vacuo to a yellow oil. Purification by column chromatography (silica gel, 7% ethyl acetate-hexanes) gave the title compound as a colorless solid:

1H NMR(CDCl₃)：δ8.04(2H，dd，J＝1.8，8.4Hz)，7.60(2H，dd，J＝1.8，8.4Hz)，7.51(2H，m)，7.32(1H，d，J＝8.0Hz)，4.40(2H，q，J＝7.1Hz)，6.02(1H，t，J＝5.0Hz)，2.44(2H，d，J＝5.0Hz)，1.43(3H，t，J＝7.1Hz)，1.33(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl)) Ethynyl group]Benzoic acid ethyl ester Compound 1)

A solution of 4-tolyllithium (lithiotomelene) was prepared by adding 189.9mg (1.74ml, 2.96mmol) of t-butyllithium (1.7M in hexane) to 253.6mg (1.482mmol) of 4-bromotoluene in 2.0ml of THF as a cold solution (-78 ℃). After stirring for 30 minutes, a solution of 269.4mg (1.977mmol) of zinc chloride in 3.0ml of THF was added. The resulting solution was warmed to room temperature, stirred for 30 minutes and added via cannula to 472.9mg (0.988mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ]Ethyl benzoate (Compound G) and 50mg (0.04mmol) of tetrakis (triphenylphosphine) palladium (0) in 4.0ml of THF. The resulting solution was heated at 50 ℃ for 45 minutes, cooled to room temperature, and diluted with saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate (40ml) and the combined organic layers were washed with water and brine. The organic phase was dried over sodium sulfate and concentrated in vacuo to a yellow oil. Purification by column chromatography (silica gel, 5% ethyl acetate-hexanes) gave the title compound as a colorless solid.¹H NMR(d₆-acetone):

δt.35(6H，s)，1.40(3H，t，J＝7.1Hz)，2.36(2H，d，J＝4.7Hz)，2.42(3H，s)，4.38(2H，q，J＝7.1Hz)，5.99(1H，t，J＝4.7Hz)，7.25(5H，m)，7.35(2H，m)，7.52(2H，d，J＝8.5Hz)，7.98(2H，d，J ＝8.5Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8-phenyl-2-naphthyl) ethynyl]Ethyl benzoate (Compound 1a)

Using the same general procedure as for the preparation of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoate (compound 1), 203.8mg (0.43mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ] benzoate (compound G) was converted using 58.2mg (0.36ml, 0.69mmol) phenyllithium (1.8M solution in cyclohexane/diethyl ether), 116.1mg (0.85mmol) zinc chloride and 13.8mg (0.01mmol) tetrakis (triphenylphosphine) palladium (0) into the desired compound (colorless solid):

PMR(CDCl₃)：δ1.36(6H，s)，1.40(3H，t，J＝7.1Hz)，2.37(2H，d，J＝4.7Hz)，4.38(2H，q，J＝7.1Hz)，6.02(1H，t，J＝4.7Hz)，7.20(1H，d，J＝1.5Hz)，7.27 (1H，m)，7.39(6H，m)，7.52(2H，d，J＝8.2Hz)，7.98(2H，d，J＝8.2Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-methylphenyl) -2-naphthyl) ethynyl ]Benzoic acid ethyl ester Compound 2)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was used, using 284.8mg (2.090mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 3-tolyllithium [ prepared by adding 201.2mg (1.86ml, 3.14mmol) of tert-butyllithium (1.7M solution in pentane) to 274.0mg (1.568mmol) of 3-methylbromobenzene in 2.0ml of THF (-78 ℃ C.) in 2.0ml of THF]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.99(2H，d，J＝8.4Hz)，7.51(2H，d，J＝8.4Hz)，7.39-7.14(7H，m)，5.99(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.60(3H，s)，2.35(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methylphenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Compound 3)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was used, using 199.4mg (1.463mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 2-tolyllithium [ prepared by adding 133.9mg (1.23ml, 2.09mmol) of tert-butyllithium (1.7M solution in pentane) to 178.7mg (1.045mmol) of a cold solution (-78 ℃ C.) of 2-methylbromobenzene in 2.0ml of THF in 4.0ml of THF ]200.0mg (0.418mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Benzoic acid ethyl esterEster (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.97(2H，d，J＝8.4Hz)，7.50(2H，d，J＝8.4Hz)，7.49-7.19(6H，m)，6.81(1H，d，J＝1.6Hz)，5.89(1H，t，J＝4.5Hz)，4.36(2H，q，J＝7.1Hz)，2.43-2.14(2H，dq，J＝3.7，5.4Hz)，2.15(3H，s)，1.39-1.34(9H，m).

4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3, 5-dimethylphenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Ester (Compound 4)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed using 249.0mg (1.827mmol) zinc chloride, 24mg (0.02mmol) tetrakis (triphenylphosphine) palladium (0) and 3, 5-dimethylphenyllithium in 2.0ml THF [ prepared by adding 167.7mg (1.54ml, 2.62mmol) tert-butyllithium (1.7M solution in pentane) to 249.0mg (1.305mmol) of a cold (-78 ℃ C.) solution of 3, 5-dimethylbromobenzene in 2.0ml THF]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl) ₃)：δ7.98 (2H，d，J＝8.4Hz)，7.52(2H，d，J＝8.4Hz)，7.40-7.33(2H，m)，7.20(1H，d，J＝1.6Hz)，7.00(1H，s)，6.97 (2H，s)，5.97(1H，t，J＝4.8Hz)，4.37(2H，q，J＝7.1Hz)，2.36-(6H，s)，2.34(2H，d，J＝4.8Hz)，1.39(3H，t，J＝7.1Hz)，1.37(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-ethylphenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Compound 5)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed using 249.0mg (1.827mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 4-ethylphenyl in 2.0ml of THFLithium [ prepared by adding 167.7mg (1.54ml, 2.62mmol) of tert-butyllithium (1.7M solution in pentane) to 244.0mg (1.305mmol) of 4-ethylbromobenzene in 2.0ml of THF (-78 ℃ C.)]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.99(2H，d，J＝8.4Hz)，7.51(2H，d，J＝8.4Hz)，7.42-7.24(7H，m)，5.99(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.71(2H，q，J＝7.6Hz)，2.35(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- (1, 1-dimethylethyl) phenyl) -2-naphthyl) ethynyl group]Benzene and its derivatives Ethyl formate (Compound 6)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl]The same general procedure for ethyl benzoate (Compound 1) was followed using 142.4mg (1.045mmol) of zinc chloride and 4-tert-butylphenyl-lithium [ prepared by adding 100.6mg (0.97ml, 1.57mmol) of tert-butyllithium (1.5M solution in pentane) to 167.0mg (0.78mmol) of a cold solution (-78 ℃ C.) of 4-tert-butylbromobenzene in 1.0ml of THF ]250.0mg (0.52mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.99(2H，d，J＝8.4Hz)，7.55(2H，d，J＝8.4Hz)，7.28-7.45(7H，m)，6.02(1H，t，J＝4.9Hz)，4.38(2H，q，J＝7.2Hz)，2.36(2H，d，J＝4.9Hz)，1.59(3H，s)，1.40(3H，t，J＝7.2Hz)，1.39(9H，s)，1.35(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-chlorophenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester (Compound No.) Thing 7)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed using 249.0mg (1.827mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 4-chlorophenyllithium in 2.0ml of THF[ prepared by adding 167.7mg (1.54ml, 2.62mmol) of tert-butyllithium (1.7M solution in pentane) to a cold solution (-78 ℃ C.) of 252.4mg (1.305mmol) of 4-chloro-1-bromobenzene in 2.0ml THF]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.98(2H，d，J＝8.4Hz)，7.53(2H，d，J＝8.4Hz)，7.40-7.27(6H，m)，7.12(1H，d，J＝1.6Hz)，6.00(1H，t，J＝4.8Hz)，4.37(2H，q，J＝7.1Hz)，2.35(2H，d，J＝4.8Hz)，1.40(2H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methoxyphenyl) -2-naphthyl) ethynyl group ]Benzoic acid ethyl ester (Compound 8)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was used, using 249.0mg (1.827mmol) zinc chloride, 24mg (0.02mmol) tetrakis (triphenylphosphine) palladium (0) and 4-methoxyphenyllithium [ prepared by adding 167.7mg (1.54ml, 2.62mmol) of tert-butyllithium (1.7M solution in pentane) to 244.1mg (1.305mmol) of a cold (-78 ℃ C.) solution of 4-methoxy-1-bromobenzene in 2.0ml THF]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.98(2H，d，J＝8.5Hz)，7.52(2H，d，J＝8.6Hz)，7.40-7.21(5H，m)，6.95(2H，d，J＝8.7Hz)，5.97(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，4.34(3H，s)，2.34(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-trifluoromethylphenyl) -2-naphthyl) ethynyl group]Benzoic acid ethyl ester Ester (Compound 9)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methyl) amino-4-carbonylPhenyl) -2-naphthyl) ethynyl]Ethyl benzoate (Compound 1) the same general procedure was used, using 249.0mg (1.827mmol) zinc chloride, 24mg (0.02mmol) tetrakis (triphenylphosphine) palladium (0) and 4-trifluoromethylphenyl lithium in 2.0ml THF [ prepared by adding 167.7mg (1.54ml, 2.62mmol) tert-butyllithium (1.7M solution in pentane) to 296.6mg (1.305mmol) of a cold solution of 4-trifluoromethylbromobenzene in 2.0ml THF (-78 ℃ C.) ]250.0mg (0.522mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.98(2H，d，J＝8.5Hz)，7.67(2H，d，J＝8.3 Hz)，7.54-7.36(6H，m)，7.10(1H，d，J＝1.6Hz)，6.06(1H，t，J＝4.8Hz)，4.37(2H，q，J＝7.1Hz)，2.38(2H，d，J＝4.8Hz)，1.39(3H，t，J＝7.1Hz)，1.35(6H，s)。4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-pyridinyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester (Compound No.) Thing 10)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]The same general procedure for Ethyl benzoate (Compound 1) was followed using 142.4mg (1.045mmol) of zinc chloride and lithium 2-pyridinide [ prepared by adding 100.6mg (0.97ml, 1.57mmol) of tert-butyllithium (1.5M solution in pentane) to 123.8mg (0.784mmol) of 2-bromopyridine in 1.0ml of THF (-78 ℃ C.) ]]250.0mg (0.52mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (d6. acetone): Δ 8.64(1H, m), 7.99(2H, d, J ═ 8.5Hz), 7.85(1H, ddd, J ═ 1.8, 7.7, 9.5Hz), 7.58(2H, d, J ═ 8.4Hz), 7.50(1H, d, J ═ 7.7Hz), 7.47 (2H, d, J ═ 1.1Hz), 7.35(2H, m), 6.32(1H, t, J ═ 4.8Hz), 4.34(2H, q, J ═ 7.2Hz), 2.42(2H, d, J ═ 7.4Hz), 1.35(3H, t, J ═ 7.0Hz), 1.35(6H, s). 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-pyridinyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester (Compound No.) Thing 11)

Use and preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8-)(4-methylphenyl) -2-naphthyl) ethynyl]The same general procedure for ethyl benzoate (Compound 1) was followed using 142.4mg (1.045mmol) of zinc chloride and 3-pyridyllithium [ prepared by adding 100.2mg (0.92ml, 1.56mmol) of tert-butyllithium (1.5M solution in pentane) to 123.8mg (0.784mmol) of 3-bromopyridine in 1.0ml of THF (-78 ℃ C.)]170.0mg (0.35mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ8.63-8.61(2H，dd，J＝1.7Hz)，7.992H，d，J＝8.4Hz)，7.67(1H，dt，J＝7.9Hz)，7.52(2H，d，J＝8.4Hz)，7.43-7.34(3H，m)，7.10(1H，d，J＝1.6Hz)，6.07(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.40(2H，d，J＝4.7Hz)，1.390(3H，t，J＝7.1Hz)，1.36(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methyl-5-pyridyl) -2-naphthyl) ethynyl group]Benzoic acid ethyl ester Ester (Compound 12)

Using the same general procedure as for the preparation of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoate (Compound 1), using 142.4mg (1.045mmol) of zinc chloride and lithium 2-methyl-5-pyridinium [ prepared by adding 100.5mg (0.92ml, 1.57mmol) of tert-butyllithium (1.7M solution in pentane) to 134.8mg (0.784mmol) of ethyl 2-methyl-5-bromopyridine in 1.0ml of THF (-78 ℃ C.) ] 250.0mg (0.522mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ] benzoate (Compound G) are converted into the desired compound (colorless solid):

1H NMR(CDCl₃)：δ8.50(1H，d，J＝2.2Hz)，7.99(2H，d，J＝8.3Hz)，7.56(1H，dd，J＝2.3，8.0Hz)，7.53(2H，d，J＝8.4Hz)，7.43(1H，dd，J＝2.3，8.0Hz)，7.37(2Hd，J＝8.0Hz)，7.21(1H，d，J＝8.1Hz)，7.11(1H，d，J＝1.5Hz)，6.04(1H，t，J＝4.7Hz)，4.38(2H，q，J＝7.2Hz)，2.63(3H，s)，2.38(2H，d，J＝4.6Hz)，1.40(3H，t，J＝7.1Hz)，1.35(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3)- ((2, 2-dimethylethyl) -dimethylsilyloxy) phenyl) -2- Naphthyl) ethynyl]Benzoic acid ethyl ester (Compound H)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound G) the same general procedure was followed using 150.0mg (1.10mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 3- (2, 2-dimethylethyl) -dimethylsiloxy) phenyllithium in 2.0ml of THF [ prepared by adding 100.2mg (0.92ml, 1.564mmol) of tert-butyllithium (1.7M solution in pentane) to 226.0mg (0.787mmol) of a cold (-78 ℃ C.) solution of 3- ((2, 2-dimethylethyl) -dimethylsiloxy) bromobenzene in 2.0ml of THF]150.0mg (0.314mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)δ7.98(2H，d，J＝8.4Hz)，7.51(2H，d，J＝8.4Hz)，7.40-7.22(4H，m)，6.95(1H，d，J＝7.6Hz)，6.84-6.82(2H，m)，6.00(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.35(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.34(3H，s)，0.99(9H，s)，0.23 (6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- ((2, 2-dimethylethyl) -dimethylsilyloxy) phenyl) -2- Naphthyl) ethynyl]Benzoic acid ethyl ester (Compound I)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group ]Ethyl benzoate (Compound 1) the same general procedure was followed using 209.0mg (1.53mmol) of zinc chloride, 24mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 4- ((2, 2-dimethylethyl) -dimethylsiloxy) phenyllithium in 2.0ml THF [ prepared by adding 140.3mg (1.30ml, 2.19mmol) of tert-butyllithium (1.7M solution in pentane) to 315.0mg (1.09mmol) of a cold (-78 ℃ C.) solution of 4- ((2, 2-dimethylethyl) -dimethylsiloxy) bromobenzene in 2.0ml THF]210.0mg (0.439mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Conversion of Ethyl benzoate (Compound G) to the desired CompoundCompound (colorless solid): 1H NMR (CDCl)₃)：δ7.98 (2H，d，J＝8.4Hz)，7.51(2H，d，J＝8.4Hz)，7.39-7.25(3H，m)，7.21(2H，d，J＝8.5Hz)，5.87(2H，d，J＝8.5Hz)，5.96(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.33(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.33(6H，s)，1.01(9H，s)，0.25(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-hydroxyphenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Compound 13)

91.5mg (0.35ml, 0.35mmol) of tetrabutylammonium fluoride (1M solution in THF) are added at room temperature to 60.0mg (0.114mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3- ((2, 2-dimethylethyl) -dimethylsilyloxy) -phenyl) -2-naphthyl) ethynyl]Ethyl benzoate (Compound H) in a solution of 1.0ml THF. After stirring overnight, the solution was diluted with ethyl acetate and washed with water and saturated aqueous sodium chloride solution before drying over magnesium sulfate. The solvent was removed under reduced pressure, and then purified by column chromatography (4: 1, hexane: ethyl acetate) to give the objective compound as a colorless solid: 1H NMR (CDCl) ₃)：δ7.98(2H，d，J＝7.8Hz)，7.52(2H，d，J＝8.3Hz)，7.39-7.21(4H，m)，6.93(1H，d，J＝7.5Hz)，6.84(1H，d，7.1Hz)，6.83(1H，s)，6.01 1H，t，J＝4.7Hz)，4.91(1H，s)，4.39(2H，q，J＝7.1Hz)，2.35(2H，d，J＝4.7Hz)，1.39(3H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-hydroxyphenyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Compound 14)

73.2mg (0.29ml, 0.29mmol) of tetrabutylammonium fluoride (1M solution in THF) are added at room temperature to 50.0mg (0.095mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- ((2, 2-dimethylethyl) -dimethylsiloxy) phenyl) -2-naphthyl) ethynyl]Ethyl benzoate (Compound I) in a solution of 1.0ml THF. After stirring overnight, the solution was diluted with ethyl acetate, and before drying over magnesium sulfate, water and saturated chlorine were addedAnd washing with an aqueous sodium chloride solution. The solvent was removed under reduced pressure, and then purified by column chromatography (4: 1, hexane: ethyl acetate) to give the objective compound as a colorless solid: 1H NMR (CDCl)₃)：δ7.98(2H，d，J＝8.2Hz)，7.52(2H，d，J＝8.3Hz)，7.41-7.20(5H，m)，6.88(2H， d，J＝8.4Hz)5.96(1H，t，J＝4.5Hz)，4.37(2H，q，J＝7.1Hz)，2.34(2H，d，J＝4.5Hz)，1.39(3H，t，J＝7.1Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methylthiazol-2-yl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Ester (Compound 15)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was used, 150.0mg (1.10mmol) of zinc chloride, 14mg (0.012mmol) of tetrakis (triphenylphosphine) palladium (0) and 5-methylthiazol-2-yl lithium [ prepared by adding 53.2mg (0.53ml, 0.83mmol) of n-butyllithium (1.55M solution in pentane) to 82.0mg (0.83mmol) of a cold solution (-78 ℃ C.) of 5-methylthiazole in 5.0ml of THF in 4.0ml of THF ]264.0mg (0.552mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃)：δ7.99(2H，d，J＝7.8Hz)，7.88(1H，d，J＝1.5Hz)，7.55(2H，d，J＝7.8Hz)，7.54(1H，s)，7.45(1H，dd，J＝1.5，8.0Hz)，7.35(1H，d，J＝7.9Hz)，6.48(1H，t，J＝4.8Hz)，4.38(2H，q，J＝7.1Hz)，2.51(3H，s)，2.38(2H，d，J＝4.8Hz)，1.40(3H，s)，1.32(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester (Compound No.) Thing 15a)

A solution of lithium 2-thiazolate (2-lithiothiazole) was prepared by adding 41.2mg (0.42ml, 0.63mmol) of n-butyllithium (1.5M solution in hexane) to a cooled solution of 53.4mg (0.63mmol) of thiazole in 1.0ml of THF (-78 ℃). The solution was stirred for 30 minutes, then a solution of 113.9mg (0.84mmol) zinc chloride in 1.5ml THF was added. The resulting solution was warmed to room temperature, stirred for 30 minutes, and then the organozinc was added via cannula to a solution of 200.0mg (0.42mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ] benzoate (compound G) and 12.4mg (0.01mmol) of tetrakis (triphenylphosphine) palladium (0) in 1.5ml of THF. The resulting solution was heated at 50 ℃ for 45 minutes, cooled to room temperature and diluted with saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate (40ml), and the combined organic layers were washed with water and brine. The organic phase was dried over sodium sulfate and concentrated in vacuo to a yellow oil. Purification by column chromatography (silica gel, 20% ethyl acetate-hexanes) gave the title compound as a colorless oil:

PMR(CDCl₃)：δ1.35(6H，s)，1.40(3H，t，J＝7.1Hz)，2.42(2H，d，J＝4.8Hz)，4.38(2H，q，J＝7.1Hz)，6.57(1H，t，J＝4.8Hz)，7.33(1H，d，J＝3.3Hz)，7.36(1H，d，J＝8.0Hz)，7.46(1H，dd，J＝1.7，8.1Hz)，7.55(2H，d，J＝8.4Hz)，7.87(1H，d，J＝1.7Hz)，7.92(1H，d，J＝3.3Hz)，8.00(2H，d，J＝8.4Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylthiazol-2-yl) -2-naphthyl) ethynyl group]Benzoic acid ethyl ester Ester (Compound 16)

Using the same general procedure as for the preparation of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoate (Compound 1), 295.0mg (0.617mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ] benzoate (Compound G) was converted to the desired compound (Compound G) using 168.0mg (1.23mmol) of zinc chloride, 16mg (0.014mmol) of tetrakis (triphenylphosphine) palladium (0) and 4-methylthiazol-2-yl lithium in 6.0ml of THF [ prepared by adding 59.6mg (0.60ml, 0.93mmol) of n-butyllithium (1.55M solution in hexane) to 92.0mg (0.93mmol) of 4-methylthiazole in 6.0ml of cold (-78 ℃ C.) ] of THF Color solid):

1H NMR(CDCl₃)：δ8.00(2H，d，J＝8.4Hz)，7.80(1H，d，J＝1.7Hz)，7.55(2H，d，J＝8.4Hz)，7.45(1H，dd，J＝1.7，8.0Hz)，7.35(1H，d，J＝8.0Hz)，6.87(1H，s)，6.52(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.2Hz)，2.54(3H，s)，2.39(2H，d，J＝4.7Hz)，1.40(3H，t，J＝7.2Hz)，1.33(3H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4, 5-dimethylthiazol-2-yl) -2-naphthyl) ethynyl group]Benzyl benzene Ethyl acid ester (Compound 17)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed using 110.0mg (0.84mmol) of zinc chloride, 12mg (0.011mmol) of tetrakis (triphenylphosphine) palladium (0) and 4, 5-dimethylthiazol-2-yl lithium in 2.0ml of THF [ prepared by adding 40.2mg (0.39ml, 0.63mmol) of n-butyllithium (1.55M solution in hexane) to 71.0mg (0.63mmol) of a cold (-78 ℃ C.) solution of 4, 5-dimethylthiazole in 2.0ml of THF ]200.0mg (0.418mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): 1H NMR (CDCl)₃):δ8.00(2H，d，J＝8.4Hz)，7.82(1H，d，J＝1.7Hz)，7.54(2H，d，J＝8.4Hz)，7.43(1H，dd，J＝1.7，8.0Hz)，7.33 91H，d，J＝8.0Hz)，6.45(1H，t，J＝4.9Hz)，4.38(2H，q，J＝7.1Hz)，2.41(3H，s)，2.40(3H，s)，2.37(2H，d，J＝4.9Hz)，1.40(3H，t，J＝7.1Hz)，1.32(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methyl-5-pyridyl) -2-naphthyl) ethynyl group]Benzoic acid (chemical) Compound 18)

81.7mg (0.194mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methyl-5-pyridyl) -2-naphthyl) ethynyl group are stirred at room temperature]Ethyl benzoate (Compound 12) and 40.7mg (0.969mmol) of LiOH-H₂A solution of O in 3ml THF/water (3: 1, v/v) was left overnight. The reaction was inhibited by addition of saturated aqueous ammonium chloride solution and ethyl acetateAnd (4) ester extraction. The combined organic layers were washed with water and brine, dried over sodium sulfate and concentrated in vacuo to yield the desired compound as a colorless solid: 1H NMR (d 6-DMSO): δ 8.41(1H, d, J ═ 1.9Hz), 7.90(2H, d, J ═ 8.3Hz), 7.63(1H, dd, J ═ 2.3, 7.9Hz), 7.59(2H, d, J ═ 8.3Hz), 7.49(2H, m), 7.33(1H, d, J ═ 7.8Hz), 6.95(1H, s), 6.11(1H, t, J ═ 4.5Hz), 2.52 (3H, s), 2.37(2H, d, J ═ 4.6Hz), 1.31(6H, s). 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-pyridinyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 19)

80.0mg (0.196mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-pyridyl) -2-naphthyl) ethynyl group are stirred at room temperature]Ethyl benzoate (Compound 10) and 20.6mg (0.491mmol) of LiOH-H₂A solution of O in 3ml THF/water (3: 1, v/v) was left overnight. The reaction was quenched by addition of saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over sodium sulfate and concentrated in vacuo to yield the desired compound as a colorless solid: 1H NMR (d 6-DMSO): δ 8.64(1H, m), 7.94(2H, d, J ═ 8.3Hz), 7.87(1H, dt, J ═ 1.7, 7.8Hz), 7.58(2H, d, J ═ 8.3Hz), 7.50(1H, d, J ═ 8.2Hz), 7.47(2H, s), 7.37(1H, m), 7.25(1H, s), 6.30(1H, t, J ═ 4.6Hz), 2.39 (2H, d, J ═ 4.6Hz), 1.31(6H, s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-methylphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 20)

28.0mg (0.70mmol, 0.7ml) of sodium hydroxide (1M in water) was added to 30.0mg (0.071mmol) of 4- ((5, 6-dihydro-5, 5-dimethyl-8- (3-methylphenyl) -2-naphthyl) ethynyl]Ethyl benzoate (Compound 2) in a solution of 3ml ethanol and 2ml THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1H NMR (DMSO): δ 7.90(2H, d, J ═ 8.5Hz), 7.59(2H, d, J ═ 8.5Hz), 7.46(2H, s), 7.32-7.13(4H, m), 7.10(1H, s), 6.98(1H, t, J ═ 4.5Hz), 2.34(3H, s), 2.31(2H, d, J ═ 4.5H, s) z)，1.30(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-ethylphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 21)

28.0mg (0.70mmol, 0.7ml) of sodium hydroxide (1M aqueous solution) was added to 47.0mg (0.108mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-ethylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 5) in a solution of 3ml ethanol and 2ml THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1H NMR (DMSO): δ 7.90(2H, d, J ═ 8.3Hz), 7.59(2H, d, J ═ 8.3Hz), 7.46(2H, s), 7.29-7.21(4H, m), 7.02(1H, s), 6.01(1H, t, J ═ 4.5Hz), 2.64(2H, q, J ═ 7.5Hz), 2.33(2H, d, J ═ 4.5Hz), 1.29(6H, s), 1.22(3H, t, J ═ 7.5Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methoxyphenyl) -2-naphthyl) ethynyl group]Benzoic acid (chemical combination) Thing 22)

40.0mg (1.00mmol, 1.0ml) of sodium hydroxide (1M aqueous solution) was added to 80.0mg (0.183mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methoxyphenyl) -2-naphthyl) ethynyl group ]Ethyl benzoate (Compound 8) in a solution of 3ml ethanol and 2ml THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1H NMR (DMSO): δ 7.90(2H, d, J ═ 8.3Hz), 7.60(2H, d, J ═ 8.3Hz), 7.45(2H, s), 7.24(2H, d, J ═ 8.6Hz), 7.02-5.89(3H, m), 5.98(1H, t, J ═ 4.4Hz), 3.79(3H, s), 2.31(2H, d, J ═ 4.7Hz), 1.29(6H, s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-trifluoromethylphenyl) -2-naphthyl) ethynyl group]Benzoic acid (chemical) Compound 23)

60.0mg (1.50mmol, 1.50ml) of sodium hydroxide (1.0M in water) are added to a solution of 70.0mg (0.148mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-trifluoromethylphenyl) -2-naphthyl) ethynyl ] benzoate (compound 9) in 3ml of ethanol and 2ml of THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid:

1H NMR(DMSO)：δ7.90(2H，d，J＝8.3Hz)，7.80(2H，d，J＝8.1Hz)，7.61-7.47(6H，m)，6.97(2H，s)，6.16(1H，t，J＝4.5Hz)，2.37(2H，d，J＝4.6Hz)，1.30(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3, 5-dimethylphenyl) -2-naphthyl) ethynyl ]Benzoic acid (chemical) Compound 24)

48.0mg (1.20mmol, 1.20ml) of sodium hydroxide (1.0M in water) are added to 90.0mg (0.207mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3, 5-dimethylphenyl) -2-naphthyl) ethynyl]Ethyl benzoate (Compound 4) in a solution of 3ml ethanol and 2ml THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1hnmr (dmso): δ 7.90(2H, d, J ═ 8.2Hz), 7.59(2H, d, J ═ 8.2Hz), 7.45(2H, s), 7.00(1H, s), 6.97(1H, s), 5.97(1H, t, J ═ 4.5Hz), 2.31(2H, d, J ═ 4.5Hz), 2.30(6H, s), 1.29(6H, s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-chlorophenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 25)

48.0mg (1.20mmol, 1.20ml) of sodium hydroxide (1.0M in water) are added to a solution of 80.0mg (0.181mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-chlorophenyl) -2-naphthyl) ethynyl ] benzoate (compound 7) in 3ml of ethanol and 2ml of THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid:

1H NMR(DMSO)：δ7.90(2H，d，J＝8.3Hz)，7.60(2H，d，J＝8.3Hz)，7.51-7.48(4H，m)，7.34(2H，d，J＝8.4Hz)，6.97(1H，s)，6.07(1H，t，J＝4.5Hz)，2.34(2H，d，J＝4.6Hz)，1.29(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-pyridinyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 26)

48.0mg (1.20mmol, 1.20ml) of sodium hydroxide (1.0M aqueous solution) was added to 45.0mg (0.110mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-pyridyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 11) is dissolved in 3ml of ethanol and 2ml of THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1H NMR (DMSO): δ 8.60(1H, d, J ═ 4.6Hz), 8.55(1H, s), 7.90(2H, d, J ═ 8.3Hz), 7.76(1H, d, J ═ 7.5Hz), 7.60(2H, d, J ═ 8.3Hz), 7.51-7.46(3H, m), 6.94(1H, s), 6.14(1H, t, J ═ 4.5Hz), 2.37(2H, d, J ═ 4.5Hz), 1.31(6H, s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methylphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 27)

60.0mg (1.50mmol, 1.50ml) of sodium hydroxide (1.0M in water) was added to 80.0mg (0.190mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-methylphenyl) -2-naphthyl) ethynyl group ]Ethyl benzoate (Compound 3) in a solution of 3ml ethanol and 2ml THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid: 1HNMR(DMSO)：δ7.89(2H，d，J＝8.4Hz)，7.57(2H，d，J＝8.4Hz)，7.46(2H，s)，7.29-7.14(4H，m)，6.59(1H，s)，5.90(1H，t，J＝4.7Hz)，2.39(2H，m)，2.60(3H，s)，1.39(3H，s)，1.29(3H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3-hydroxyphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 28)

40.0mg (1.00mmol, 1.00ml) of sodium hydroxide (1.0M in water) are added to a solution of 40.0mg (0.076mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (3- ((2, 2-dimethylethyl) -dimethylsiloxy) phenyl) -2-naphthyl) ethynyl ] benzoate (compound H) in 3ml of ethanol and 2ml of THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid:

1H NMR (d 6-acetone): δ 7.90(2H, d, J ═ 8.3Hz), 7.49(2H, d, J ═ 8.4 z), 7.35(2H, s), 7.15-7.07(2H, m), 6.77-6.69(3H, m), 5.92(1H, t, J ═ 4.7Hz), 2.25(2H, d, J ═ 4.7Hz), 1.23(6H, s). 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-hydroxyphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 29)

60.0mg (1.50mmol, 1.50ml) of sodium hydroxide (1.0M in water) are added to a solution of 75.0mg (0.143mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- ((2, 2-dimethylethyl) -dimethylsiloxy) phenyl) -2-naphthyl) ethynyl ] benzoate (compound I) in 3ml of ethanol and 2ml of THF. The solution was heated at 50 ℃ for 2 hours, cooled to room temperature and acidified with 10% hydrochloric acid. Extraction with ethyl acetate, followed by drying over sodium sulfate and removal of the solvent under reduced pressure afforded the title compound as a colorless solid:

1H NMR (d 6-acetone): delta 8.01(2H, d, J-8.3 Hz), 7.59(2H, d, J-8.4 Hz), 7.45(2H, s), 7.20-7.17(3H, m), 6.92-6.89(2H, m), 5.97(1H, t, J)＝4.7Hz)，2.35(2H，d，J＝4.7Hz)，1.34(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methylthiazol-2-yl) -2-naphthyl) ethynyl]Benzoic acid (chemical) Compound 30)

Aqueous sodium hydroxide (1ml, 1M, 1mmol) was added to 100mg (0.23mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methylthiazol-2-yl) -2-naphthyl) at room temperature]Acetylenic ethyl benzoate (compound 15) and 4ml ethanol. The resulting solution was heated at 50 ℃ for 1 hour and concentrated in vacuo. The residue was suspended in a solution of dichloromethane and ether (5: 1) and acidified to pH 5 with 1M aqueous hydrochloric acid. The layers were separated and the organic layer was washed with brine, dried (sodium sulfate), filtered and the solvent was removed under reduced pressure to give the desired compound as a white solid: 1H NMR (d 6-DMSO): δ 7.96(1H, d, J ═ 1.7Hz), 7.95(2H, d, J ═ 8.0Hz), 7.65(2H, d, J ═ 8.0Hz), 7.64(1H, s), 7.53(1H, dd, J ═ 1.7, 8.0Hz), 7.46(1H, d, J ═ 8.0Hz), 6.59(1H, t, J ═ 4.5Hz), 2.50(3H, s), 2.39(2H, d, J ═ 4.5Hz), 1.27(6H, s). 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 30a)

33.9mg (0.08mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl group are stirred at room temperature]Ethyl benzoate (Compound 15a) and 8.5mg (0.20mmol) of LiOH-H₂A solution of O in 3ml THF/water (3: 1, v/v) was left overnight. The reaction was quenched by addition of saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over sodium sulfate and concentrated in vacuo to yield the desired compound as a colorless solid:

PMR(d₆-DMSO)：δ1.29(6H，s)，2.42(2H，d，J＝4.6Hz)，6.68(1H，t，J＝4.6Hz)，7.51(2H，m)，7.62(2H，d，J＝8.2Hz)，7.77(1H，d，J＝3.3Hz)，7.93(2H，d，J＝8.2Hz)，7.98(1H，d，J＝3.3Hz).4- [ (5, 6-dihydro-5, 5-)Dimethyl-8- (4-methylthiazol-2-yl) -2-naphthyl) ethynyl]Benzoic acid (chemical) Compound 31)

Aqueous sodium hydroxide (1ml, 1M, 1mmol) was added to a solution of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylthiazol-2-yl) -2-naphthyl ] ethynylbenzoate (compound 16) (145.0mg, 0.34mmol) and 4ml ethanol at room temperature the resulting solution was heated at 50 ℃ for 1 hour and concentrated in vacuo the residue was suspended in a solution of dichloromethane and ether (5: 1), acidified to pH 5 with aqueous 1M hydrochloric acid, allowed to separate layers, and its organic layer was washed with brine, dried (sodium sulfate), filtered and the solvent removed under reduced pressure to yield the desired compound as a white solid:

1H NMR(d6-DMSO)：δ7.94(2H，d，J＝8.1Hz)，7.87(1H，d，J＝1.6Hz)，7.63(2H，d，J＝8.3Hz)，7.50(1H，dd，J＝1.6，8.1Hz)，7.45(1H，d，J＝8.1Hz)，7.27(1H，s)，6.58(1H，t，J＝4.8Hz)，2.43(3H，s)，2.37(2H，d，J＝4.8Hz)，1.26(6H，s).4- [ (5, 6-dioxo-5, 5-dimethyl-8- (4, 5-dimethylthiazol-2-yl) -2-naphthyl) ethynyl group]Benzyl benzene Acid (Compound 32)

Aqueous sodium hydroxide (1ml, 1M, 1mmol) was added to a solution of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4, 5-dimethylthiazol-2-yl) -2-naphthyl ] ethynylbenzoate (compound 17) (58.0mg, 0.13mmol) and 4ml ethanol at room temperature the resulting solution was heated at 50 ℃ for 1 hour and concentrated in vacuo the residue was suspended in a solution of dichloromethane and ether (5: 1), acidified to pH 5 with 1M aqueous hydrochloric acid, partitioned, and its organic layer was washed with brine, dried (sodium sulfate), filtered and the solvent removed under reduced pressure to give the desired compound as a white solid:

1H NMR(d6-DMSO)：δ7.94(2H，d，J＝8.4Hz)，7.86(1H，d，J＝1.6Hz)，7.61(2H，d，J＝8.3Hz)，7.50(1H，dd，J＝1.6，8.0Hz)，7.45(1H，d，J＝8.0Hz)，6.51(1H，t，J＝4.9Hz)，2.37(3H，s)，2.36(2H，d，J＝4.6Hz)，2.32(3H，s)，1.26(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methyl-2-thienyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester Ester (Compound 33)

Using the same general procedure as for the preparation of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl ] benzoate (Compound 1), 170.0mg (0.366mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl ] benzoate (Compound G) was converted into the desired compound (colorless solid) using 202.0mg (1.48mmol) of zinc chloride, 24mg (0.022mmol) of tetrakis (triphenylphosphine) palladium (0) and 5-methyl-2-thienyllithium [ prepared by adding 58.6mg (0.36ml, 0.915mmol) of n-butyllithium (2.5M solution in hexane) to 89.8mg (0.915mmol) of 2-methylthiophene in 2.0ml of cold (-78 ℃ C.) of THF ] in 2.0ml of THF Body):

1H NMR(CDCl₃)：δ8.00(2H，d，J＝8.3Hz)，7.59(1H，d，J＝1.7Hz)，7.55(2H，d，J＝8.2Hz)，7.43(1H，dd，J＝1.7，8.0Hz)，7.35(1H，d，J＝8.0Hz)，6.87(1H，d，J＝3.5Hz)，6.74(1H，d，J＝2.8Hz)，6.15(1H，t，J＝4.8Hz)，4.38(2H，q，J＝7.1Hz)，2.52(3H，s)，2.32(2H，d，J＝4.8Hz).1.40(3H，t，7.1Hz)，1.32(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thienyl) -2-naphthyl) ethynyl]Benzoic acid ethyl ester (Compound No.) Object 33a)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed, using 186.8mg (1.37mmol) of zinc chloride, 37.1mg (0.03mmol) of tetrakis (triphenylphosphine) palladium (0) and 2-thienyllithium [ prepared by adding 65.9mg (0.69ml, 1.03mmol) of n-butyllithium (1.5M solution in hexane) to 86.5mg (1.03mmol) of thiophene in 1.0ml of cold solution of THF (-78 ℃ C.)]250.0mg (0.52mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): PMR(CDCl₃)：δ1.33(6H，s)，1.36(3H，t，J＝7.1Hz)，2.38(2H，d，J＝4.7Hz)，4.34(2H，q，J＝7.2Hz)，6.25(1H，t，J＝4.7Hz)，7.13(2H，m)，7.47(4H，m)，7.62(2H，d，J＝8.5Hz)，8.00(2H，d，J＝8.5Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methyl-2-thienyl) -2-naphthyl) ethynyl]Benzoic acid (chemical) Compound 34)

Aqueous sodium hydroxide (1ml, 1M, 1mmol) was added to a solution of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (5-methyl-2-thienyl) -2-naphthyl ] ethynylbenzoate (compound 33) (35.0mg, 0.082mmol) in 2ml ethanol and 1ml THF at room temperature the resulting solution was stirred overnight at room temperature then acidified with 10% hydrochloric acid, extracted with ethyl acetate then dried over sodium sulfate and the solvent removed under reduced pressure to afford the title compound as a colorless solid:

1H NMR (d 6-acetone): δ 8.03(2H, d, J ═ 8.6Hz), 7.63(2H, d, J ═ 8.6Hz), 7.54-7.48(3H, m), 6.89(1H, m), 6.18(1H, t, J ═ 4.7Hz), 2.49(3H, s), 2.35(2H, d, J ═ 4.7Hz), 1.32(6H, s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thienyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 34a)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl]The same procedure as for benzoic acid (compound 30a) using 17.8mg (0.42mmol) of LiOH in water, 70.0mg (0.17mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thienyl) -2-naphthyl) ethynyl group]Ethyl benzoate (compound 33a) was converted to the desired compound (colorless solid): PMR (d)₆-DMSO)：δ1.27(6H，s)，2.33(2H，d，J＝4.9Hz)，6.23(1H，t，J＝4.9Hz)，7.14(2H，m)，7.38-7.56(4H，m)，7.61(2H，d，J＝8.3Hz)，7.92(2H，d，J＝8.3Hz).5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthoic acid (Compound K)

A solution of 3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-7-bromonaphthalene (compound D) (250.0mg, 0.764mmol) in 2.0ml THF was cooled to-78 deg.C and 1.0ml tert-butyllithium (1.68mmol, 1.7M pentane solution) was added slowly. After stirring at-78 ℃ for 1 hour, gaseous carbon dioxide (generated by evaporation of dry ice, passing through a drying tube) was bubbled into the reaction for 1 hour. Then, the reaction solution was warmed to room temperature, and the reaction was inhibited by adding 10% hydrochloric acid. The extract was extracted with ethyl acetate, and then the combined organic layers were washed with water and saturated aqueous sodium chloride solution and dried over magnesium sulfate. The solvent was removed under reduced pressure and the solid was washed with hexane to afford the desired compound as a colorless solid: 1H NMR (CDCl) ₃)δ7.94(1H，dd，J＝1.8，8.1Hz)，7.76(1H，d，J＝1.8Hz)，7.45(1H，d，J＝8.1Hz)，7.24(4H，m)，6.01(1H，t，J＝4.7Hz)，2.40(3H，s)，2.36(2H，d，J＝4.7Hz)，1.35(6H，s).4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Amino group]Benzoic acid ethyl ester (Compound 35)

A solution of 170.0mg (0.58mmol) of 5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthoic acid (compound K), 115.0mg (0.70mmol) of ethyl 4-aminobenzoate, 145.0mg (0.76mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 92.4mg (0.76mmol) of 4-dimethylaminopyridine in 6.0ml of DMF is stirred at room temperature overnight. Ethyl acetate was added, and the resulting solution was washed with water, a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. After removal of the solvent under reduced pressure, the product was isolated by column chromatography (10-15% ethyl acetate/hexane) as a colorless solid:

1H NMR(CDCl₃)：δ8.02(2H，d，J＝8.7Hz)，7.72(2H，m)，7.65(2H，d，J＝8.7Hz)，7.52(1H，d，J＝1.8Hz)，7.48(1H，d，J＝8.0-Hz)，7.25(4H，m)，6.15(1H，t，J＝4.9Hz)，4.36(2H，q，J＝7.1Hz)，2.40(3H，s)，2.38(2H，d，J＝4.9Hz)，1.39(3H，t，J＝7.1Hz)，1.37(6H，s).4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Amino group]Benzoic acid (chemical) Compound 36)

240.1mg of sodium hydroxide (6.00mmol, 3.0ml of a 2M aqueous solution) are added to 26.5mg (0.06mmol) of 4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Amino group]Ethyl benzoate (Compound 35) in a solution of 3.0ml ethanol and 4.0ml THF. After stirring at room temperature for 72 hours, the reaction was inhibited by adding 10% hydrochloric acid. Extraction with ethyl acetate, drying of the organic layer over magnesium sulfate, removal of the solvent under reduced pressure afforded a solid. Crystallization from acetonitrile afforded the title compound as a colorless solid: 1H NMR (d 6-DMSO): δ 10.4(1H, s), 7.91-7.81(5H, m), 7.54(1H, d, J ═ 8.1Hz), 7.45(1H, d, J ═ 1.7Hz), 7.23(4H, s), 6.04(1H, t, J ═ 4.7Hz), 2.35(5H.s).1.33(6H, s). 4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Oxy radical]Benzoic acid ethyl ester (Compound 37)

A solution of 25.0mg (0.086mmol) of 5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthoic acid (compound K), 17.5mg (0.103mmol) of ethyl 4-hydroxybenzoate, 21.4mg (0.112mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 12.6mg (0.103mmol) of 4-dimethylaminopyridine in 2.0ml of DMF is stirred at room temperature overnight. Ethyl acetate was added, and the resulting solution was washed with water, a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution before being dried over magnesium sulfate. After removal of the solvent under reduced pressure, the product was isolated by column chromatography (10% ethyl acetate/hexane) as a light yellow solid:

1H NMR(CDCl₃)：δ8.08(2H，d，J＝8.1Hz)，8.05(1H，dd，J＝1.8，8.1Hz)，7.89(1H，d，J＝1.8Hz)，7.50(2H，d，J＝8.1Hz)，7.22(5H，m)，6.05(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.39(2H，d，J＝4.7Hz)，2.38(3H，s)，1.39(3H，t，J＝7.1Hz)，1.37(6H，s).4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Oxy radical]Benzoic acid 2-tris Silylethyl ester (Compound 38)

A solution of 93.5mg (0.320mmol) of 5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthoic acid (compound K), 76.0mg (0.319mmol) of 2-trimethylsilylethyl 4-hydroxybenzoate, 80.0mg (0.417mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 51.0mg (0.417mmol) of 4-dimethylaminopyridine in 4.0ml of DMF is stirred at room temperature overnight. Ethyl acetate was added, and the resulting solution was washed with water, a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution before being dried over magnesium sulfate. After removal of the solvent under reduced pressure, the product was isolated by column chromatography (5% ethyl acetate/hexane) as a colorless solid: 1H NMR (CDCl) ₃)：δ8.08(2H，d，J＝8.8Hz)，8.05(1H，dd，J＝1.8，8.1Hz)，7.50(1H，d，J＝8.1Hz)，7.26-7.18(6H，m)，6.05(1H，t，J＝4，7Hz)，4.42(2H，t，J＝8.4Hz)，2.40(2H，d，J＝4.7Hz)，2.39(3H，s)，1.38(6H，s)，0.09(9H，s).4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Oxy radical]Benzoic acid (chemical) Compound 39)

A solution of 110.0mg (0.213mmol) of 2-trimethylsilylethyl 4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl ] oxy ] benzoate (compound 38) and 167.3mg tetrabutylammonium fluoride (0.640mmol, 0.64ml of a 1M solution in THF) in 2.0ml THF is stirred at room temperature for 22 h. Ethyl acetate was added, and the resulting solution was washed with water and a saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the residual solid was washed with ethyl acetate and acetonitrile to afford the desired compound as a colorless solid:

1H NMR (d 6-acetone): δ 8.10(2H, d, J ═ 8.8Hz), 8.06(1H, dd, J ═ 2.0, 8.1Hz), 7.82(1H, d, J ═ 1.9Hz), 7.64(1H, d, J ═ 8.1Hz), 7.35(2H, d, J ═ 8.6Hz), 7.25(4H, m), 6.08(1H, t, J ═ 4.7Hz), 2.42(2H, d, J ═ 4.7Hz), 2.35(3H, s), 1.39(6H, s).2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl ]Amino group]Benzyl benzene Ethyl acid ester (Compound 40)

A solution of 115.0mg (0.41mmol) of 5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthoic acid (compound K), 89.0mg (0.49mmol) of ethyl 2-fluoro-4-aminobenzoate, 102.0mg (0.53mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 65.0mg (0.53mmol) of 4-dimethylaminopyridine in 5.0ml of DMF is stirred at 50 ℃ for 1 hour and then at room temperature overnight. Ethyl acetate was added, and the resulting solution was washed with water, a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution before being dried over magnesium sulfate. After removal of the solvent under reduced pressure, the product was isolated by column chromatography (20% ethyl acetate/hexane) as a colorless solid:

1H NMR(CDCl₃)：δ7.96(1H，s)，7.89(1H，t，J＝8.4Hz)，7.70(2H，m)，7.52(1H，d，J＝1.9Hz)，7.45(1H，d，J＝8.1Hz)，7.23(5H，m)，6.04(1H，t，J＝4.8Hz)，4.36(2H，q，J＝7.1Hz)，2.38(3H，s)，2.35(2H，d，J＝4.8Hz)，1.39(3H，t，J＝7.1Hz)，1.36(6H，s).2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Amino group]Benzyl benzene Acid (Compound 41)

40.0mg of sodium hydroxide (1.00mmol, 1.0ml of an aqueous solution of 1M) were added to 41.6mg (0.091mmol) of 2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl]Amino group]A solution of ethyl benzoate (Compound 40) in 2.0ml of ethanol and 2.0ml of THF. After stirring overnight at room temperature, the reaction was inhibited by the addition of 10% hydrochloric acid. With ethyl acetate Extraction, drying of the organic layer over magnesium sulfate, removal of the solvent under reduced pressure afforded a solid. Crystallization from acetonitrile afforded the title compound as a light yellow solid: 1H NMR (d 6-acetone): δ 9.84(1H, s), 7.94-7.83(3H, m), 7.64(1H, dd, J ═ 2.0Hz), 7.53(2H, d, J ═ 8.1Hz), 7.23(4H, s), 6.04(1H, t, J ═ 4.7Hz), 2.38(2H, d, J ═ 4.7Hz), 2.36(3H, s), 1.35(6H, s).4- [ [5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl]Amino group]Benzoic acid Ethyl ester (Compound 42)

A solution of 110.0mg (0.25mmol) of ethyl 4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl ] amino ] benzoate (compound 35) and 121.0mg (0.30mmol) of [2, 4-bis (4-methoxyphenyl) -1, 3-dithio-2, 4-diphosphetane-2, 4-disulfide ] (Lawesson's reagent) in 12.0ml of benzene was refluxed overnight. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated under reduced pressure. The title compound was isolated via column chromatography (10-25% ethyl acetate/hexanes) as a yellow solid:

1H NMR(CDCl₃)：δ8.92(1H，s)，8.06(2H，t，J＝8.5Hz)，7.88-7.70(3H，m)，7.42(2H，d，J＝8.1Hz)，7.18(4H，m)，6.03(1H，t，J＝4.7Hz)，4.37(2H，q，J＝7.1Hz)，2.38(3H，s)，2，36(2H，d，J＝4.7Hz)，1.56(3H，t，J＝7.1Hz)，1.35(6H，s).4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl ]Amino group]Benzoic acid (Compound 43)

60.0mg of sodium hydroxide (1.50mmol, 1.5ml of a 1M aqueous solution) are added to a solution of 84.0mg (0.184mmol) of ethyl 4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl ] amino ] benzoate (compound 42) in 2.0ml of ethanol and 2.0ml of THF. After stirring overnight at room temperature, the reaction was inhibited by the addition of 10% hydrochloric acid. Extraction with ethyl acetate, drying of the organic layer over magnesium sulfate, removal of the solvent under reduced pressure afforded a solid. Crystallization from acetonitrile afforded the title compound as a yellow solid:

1H NMR (d 6-acetone): δ 10.96(1H, s), 8.05(4H, m), 7.72(1H, dd, J ═ 2.0, 8.0Hz), 7.54(1H, s), 7.46(1H, d, J ═ 8.1Hz), 7.20(4H, m), 6.04(1H, t, J ═ 4.7Hz), 2.38(2H, d, J ═ 4.7Hz), 2.33(3H, s), 1.35(6H, s).2-acetyl-6-bromonaphthalene (Compound L)

21.0g (267mmol) of acetyl chloride are added to a cold (10 ℃ C.) mixture of 44.0g (0.212mol) of 2-bromonaphthalene and 34.0g (0.255mol) of aluminum trichloride in 400ml of nitrobenzene. The reaction mixture with mechanical stirring was warmed to room temperature and heated at 40 ℃ for 18 hours. After cooling to 0 ℃ in an ice bath, the reaction was inhibited by the addition of 12M hydrochloric acid (70 ml). The layers were separated and the organic phase was washed with water and dilute aqueous sodium bicarbonate. Kugelrohr distillation followed by recrystallization from 10% ethyl acetate-hexanes gave 23g of the title compound as a tan solid: 1H NMR (CDCl) ₃):δ8.44(1H，br s)，8.04-8.10(2H，m)，7.85(1H，d，J＝8.5Hz)，7.82(1H，d，J＝8.8Hz)，7.64(1H，d，J＝8.8Hz)，2.73(3H，s).6-bromo-2-naphthoic acid (Compound M)

A solution of 4g (16.06mmol) of 2-acetyl-6-bromonaphthalene (compound L) in 50ml of 1, 4-dioxane was added to a solution of sodium hypochlorite (62ml, 5.25% aqueous solution (w/w), 3.6g, 48.18mmol) and sodium hydroxide (6.4g, 160.6mmol) in 50ml of water. The yellow solution was heated in an oil bath at 70 ℃ for 2 hours, cooled to room temperature and extracted with diethyl ether (2X 50 ml). The aqueous layer was diluted with sodium bisulfite solution (until the potassium iodide indicator remained colorless) and then acidified with 1N sulfuric acid (pH < 2) to give a white precipitate. The mixture was extracted with ether and the combined organic phases were washed with saturated aqueous sodium chloride solution, dried (magnesium sulfate) and concentrated to yield 3.54g (88%) of the title compound as a solid: 1H NMR (DMSO-d 6): Δ 8.63(1H, br s), 8.32(1H，d，J＝2.0Hz)，8.10(1H，d，J＝8.8Hz)，8.00-8.05(2H，m)，7.74(1H，dd，J＝2.0，8.8Hz).6-bromo-2-naphthoic acid ethyl ester (Compound N)

18M sulfuric acid (2ml) was added to a solution of 3.1g (12.43mmol) of 6-bromo-2-naphthoic acid (compound M) in ethanol (30ml, 23.55g, 511.0 mmol). The solution was refluxed for 30 minutes, cooled to room temperature and the reaction mixture was partitioned between pentane (100ml) and water (100 ml). The aqueous phase was extracted with pentane (100ml), the combined organic layers were washed with saturated aqueous sodium chloride (100ml), dried (magnesium sulfate) and concentrated to give an off-white solid. Purification by flash chromatography (silica gel, 10% EtOAc: hexanes) afforded the title compound as a white solid:

1H NMR(CDCl₃)：δ8.58(1H，br s)，8.10(1H，dd，J＝1.7，9Hz)，8.06(1H，d，J＝2Hz)，7.83(1H，d，J＝9Hz)，7.80(1H，d，J＝9Hz)，7.62(1H，dd，J＝2，9Hz).(E) -4- [2-5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) vinyl 1-benzoic acid ethyl ester (Compound O)

124.0mg (0.40mmol) of tris (2-methylphenyl) phosphorus and then 44.0mg (0.20mmol) of palladium (II) acetate are added to a solution of 520.0mg (2.00mmol) of 3, 4-dihydro-4, 4-dimethyl-7-bromo-1 (2H) -naphthalenone (compound B) and 510.0mg (2.90mmol) of ethyl 4-vinylbenzoate in 4.0ml of triethylamine degassed by passing argon for 25 minutes. The resulting solution was heated at 95 ℃ for 2.5 hours, cooled to room temperature, and concentrated under reduced pressure. Purification by column chromatography (10% ethyl acetate/hexanes) afforded the title compound as a colorless solid: 1HNMR (CDCl)₃)：δ8.19(1H，d，J＝2.0Hz)，8.03(2H，d，J＝8.4Hz)，7.69(1H，dd，J＝2.0，8.2Hz)，7.57(2H，d，J＝8.4Hz)，7.45(1H，d，J＝8.2Hz)，7.20(2H，s)，4.39(2H，q，J＝7.1Hz)，2.76(2H，t，J＝6.5Hz)，2.04(2H，t，J＝6.5Hz)，1.41(3H，t，J＝7.1Hz，and 6H，s).(E) -4- [2- (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) acetylene Base of]-benzoic acid ethyl esterEster (Compound P)

700.0mg (2.00mmol) of ethyl (E) -4- [2- (5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) vinyl ] -benzoate (compound O) in 25.0ml of THF are added to a cold (-78 ℃ C.) solution of 440.0mg (2.40mmol) of sodium bis (trimethylsilyl) amide in 10.0ml of THF. After stirring for 1.5 h at-78 deg.C 960.0mg (2.40mmol) of 2[ N, N-bis (trifluoromethylsulfonyl) amino ] -5-chloropyridine were added in one portion and after 30 min the solution was warmed to 0 deg.C and stirred for 3 h. The reaction was quenched by addition of saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The combined extracts were washed with 5% aqueous sodium hydroxide, dried (sodium sulfate) and the solvent removed under reduced pressure. The title compound was isolated via column chromatography (7% ethyl acetate/hexanes) as a colorless solid:

1H NMR(CDCl₃)：δ8.04(1H，d，J＝8.4Hz)，7.57(2H，d，J＝8.4Hz)，7.52(1H，s)，7.49(1H，d，J＝8.0Hz)，7.33(1H，d，J＝8.0Hz)，7.20(1H，d，J＝16.4Hz)，7.10(1H，d，J＝16.4Hz)，6.00(1H，t，J＝4.9Hz)，4.39(2H，q，J＝7.1Hz)，2.43(2H，d，J＝4.9Hz)，1.41(3H，t，J＝7.1Hz)，1.32(6H，s).(E) -4- [2- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethenyl]-benzoic acid Ethyl ester (Compound 44)

A solution of 4-tolyllithium was prepared at-78 ℃ by adding 130.7mg of tert-butyllithium (2.04mmol, 1.20ml of a 1.7M solution in pentane) to a solution of 374.5mg (2.20mmol) of 4-bromotoluene in 2.5ml of THF. After 30 minutes, a solution of 313.4mg (2.30mmol) of zinc chloride in 2.0ml of THF is added. The resulting solution was warmed to room temperature, stirred for 1.25 h, then added to 285.0mg (0.590mmol) of (E) -4- [2- (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethenyl via cannula]Ethyl benzoate (compound P) and 29.0mg (0.025mmol) of tetrakis (triphenylphosphine) palladium (0) in 2.0ml THF. The resulting solution was stirred at room temperature for 1 hour, then at 55 ℃ for 2 hours. After cooling to room temperature, the reaction was quenched by addition of saturated aqueous ammonium chloride. Extracting the mixture with ethyl acetateThe combined extracts were washed with 5% aqueous sodium hydroxide solution and saturated aqueous sodium chloride solution, and dried over sodium sulfate before being concentrated under reduced pressure. The title compound was isolated via column chromatography (10% ethyl acetate/hexanes) as a colorless solid: 1H NMR (CDCl) ₃)：δ7.96(2H，d，J＝8.1Hz)，7.47(2H，d，J＝8.1Hz)，7.43-7.16(7H，m)，7.07(1H，d，J＝16.3Hz)，6.93(1H，d，J＝16.3Hz)，5.97(1H，t，J＝4.7Hz)，4.39(2H，q，J＝7.0Hz)，2.41(3H，s)，2.33(1H，d，J＝4.7Hz)，1.38(3H，t，J＝7.0Hz)，1.33(6H，s).(E) -4- [2- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethenyl]-benzoic acid (Compound 45)

30.0mg of lithium hydroxide (0.909mmol, 1.0ml of 1.1M solution) and 1.0ml of methanol were added to 65.0mg (0.190mmol) of (E) -4- [2- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethenyl]-ethyl benzoate (compound 44) in a solution of 4.0ml THF. The solution was heated at 55 ℃ for 3 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was dissolved in water and extracted with hexane. The aqueous layer was acidified to pH1 with 10% hydrochloric acid and extracted with ether. The combined organic layers were washed with saturated aqueous sodium chloride, diluted with ethyl acetate to give a clear solution, and dried over sodium sulfate. Removal of the solvent under reduced pressure gave the title compound as a colorless solid: 1H NMR (d 6-DMSO): δ 7.86(2H, d, J ═ 8.4Hz), 7.66(2H, d, J ═ 8.4Hz), 7.58(1H, dd, J ═ 1.7, 8.1Hz), 7.41(1H, d, J ═ 8.1Hz), 7.28(1H, d, J ═ 16.5Hz), 7.23(4H, s), 7.08(1H, d, J ═ 1.7Hz), 7.07(1H, d, J ═ 16.5Hz), 5.97(1H, t, J ═ 4.6Hz), 2.35(3H, s), 2.31(1H, d, J ═ 4.6Hz), 1.29(6H, s). 4- [2- (1, 1-dimethyl-3- (4-methylphenyl) -5-indenyl) ethynyl]-benzoic acid ethyl ester (compound) 47)

A solution of 32.0mg (0.187mmol) of 4-bromotoluene in 1.0ml of THF is cooled to-78 ℃ and 24.0mg of tert-butyllithium (0.375mmol, 0.22ml of a 1.7 molar solution in pentane) are added slowly. Mixing the yellow colorThe solution is stirred for 30 minutes, while 29.8mg (0.219mmol) of zinc chloride in 1ml of THF are added. The resulting solution was warmed to room temperature and after 30 minutes was added to 29.0mg (0.062mmol) of 4- [2- (1, 1-dimethyl-3- (trifluoromethylsulfonyl) oxy-5-indenyl) ethynyl containing 1ml THF]In a second flask ethyl benzoate (compound FF) and 2.9mg (0.003mmol) of tetrakis (triphenylphosphine) palladium (0). The resulting solution was heated at 50 ℃ for 1 hour and then stirred at room temperature for 4 hours. The reaction was quenched by addition of saturated aqueous ammonium chloride solution and then extracted with ether. The combined organic layers were washed with water, saturated aqueous sodium chloride solution and dried over magnesium sulfate before concentration under reduced pressure. The desired compound was isolated as a colorless oil by column chromatography (10% diethyl ether/hexane): 1HNMR (300MHz, CDCl) ₃)：δ8.03(2H，d，J＝8.5Hz)，7.66(1H，s)，7.58(2H，d，J＝8.5 Hz)，7.50(2H，d，J＝8.0Hz)，7.46(1H，d，J＝7.9Hz)，7.38(1H，d，J＝7.7Hz)，7.28(2H，d，J＝9Hz)，6.43(1H，s)，4.40(2H，q，J＝7.2Hz)，2.43(3H，s)，1.41(3H，t；+6H，s).4- [2- (1, 1-dimethyl-3- (4-methylphenyl) -5-indenyl) ethynyl]-benzoic acid (compound 48)

5.2mg (0.12mmol) of LiOH.H₂O10.0 mg (0.025mmol) of 4- [2- (1, 1-dimethyl-3- (4-methylphenyl) -5-indenyl) ethynyl group]-Ethyl benzoate (Compound 47) in 0.5ml THF/H₂O (3: 1 v/v). After stirring at room temperature for 48 hours, the solution was extracted with hexane and the aqueous layer thereof was acidified with a saturated aqueous ammonium chloride solution. Solid sodium chloride was added and the resulting mixture was extracted with ethyl acetate. The combined organic layers were dried (sodium sulfate) and concentrated under reduced pressure to give the desired compound as a colorless solid: 1H NMR (300MHz, d)₆-DMSO)：δ7.95(2H，d，J＝8.3Hz)，7.65(2H，d，J＝8.3Hz)，7.57(2H，m)，7.49(3H，m)，7.30(2H，d，J＝7.9Hz)，6.61(1H，s)，2.36(3H，s)，1.36(6H，s).3- (4-Bromophenylthio) propionic acid

6.79g (35.7mmol) of 4-bromothiophenol are added to a solution of 1.44g (35.7mmol) of sodium hydroxide in 20.0ml of degassed water (argon being passed). The resulting mixture was stirred at room temperature for 30 minutes. 2.26g (16.3mmol) of potassium carbonate and 15ml of degassed water are introduced into the second flask. 5.00g (32.7mmol) of 3-bromopropionic acid (in portions) were added to the solution. The potassium carboxylate solution formed was added to the sodium thiol solution and the resulting mixture was stirred at room temperature for 48 hours. The mixture was filtered and the filtrate was extracted with benzene, and the combined organic layers were discarded. The aqueous layer was acidified with 10% hydrochloric acid and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, and concentrated under reduced pressure. Recrystallization of the solid formed from ether-hexane gives the desired compound as off-white crystals:

1H NMR(CDCl₃)：δ7.43(2H，d，J＝8.4Hz)，7.25(2H，d，J＝8.4Hz)，3.15(2H，t，J＝7.3Hz)，2.68(2H，t，J＝7.3Hz).2, 3-dihydro-6-bromo- (4H) -1-benzothiopyran-4-one

A solution of 3.63g (13.9mmol) of 3- (4-bromophenylthiooxy) propionic acid in 60ml of methanesulfonic acid was heated at 75 ℃ for 1.5 hours. After cooling to room temperature, the solution was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with 2N aqueous sodium hydroxide solution, water and saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. The solvent was removed under reduced pressure to afford a yellow solid from which the product was isolated via column chromatography (3% ethyl acetate-hexanes) as a light yellow solid:

1H NMR(CDCl₃)：δ8.22(1H，d，J＝2.1Hz)，7.48 1H，dd，J＝2.1，8.3Hz)，7.17(1H，d，J＝8.5Hz)，3.24(2H，t，J＝6.4Hz)，2.98(2H，t，J＝6.7Hz).2, 3-dihydro-6- (2-trimethylsilylethynyl) - (4H) -1-thiochroman-4-one

1.00g (4.11mmol) of 2, 3-dihydro-6-bromo- (4H) -1-Benzothiopyran-4-one and 78.3mg (0.41mmol) of Cul in 15.0ml of THF and 6.0ml of Et₂The solution in NH was purged with argon for 5 minutes. 2.0ml (1.39g, 14.2mmol) of (trimethylsilyl) acetylene, then 288.5mg (0.41mmol) of bis (triphenylphosphonium) palladium (II) chloride were added to the solution. The resulting dark solution was stirred at room temperature for 3 days, then filtered through a pad of celite, washing with ethyl acetate. The filtrate was washed with water and saturated aqueous sodium chloride solution before being dried over magnesium sulfate. The target compound was isolated as an orange oil by column chromatography (4% ethyl acetate-hexanes):

1H NMR(CDCl₃)：δ8.13(1H，d，J＝1.9Hz)，7.36(1H，dd，J＝2.1，8.2Hz)，7.14(1H，d，J＝8.2Hz)，3.19(2H，d，J＝6.3Hz)，2.91(2H，d.J＝6.3Hz)，0.21(9H，s).2, 3-dihydro-6-ethynyl- (4H) -1-benzothiopyran-4-one

A solution containing 600.0mg (2.25mmol) of 2, 3-dihydro-6- (2-trimethylsilylethynyl) - (4H) -1-thiochroman-4-one and 100.0mg (0.72mmol) of potassium carbonate in 15ml of methanol was stirred at room temperature for 20 hours. The solution was diluted with water and extracted with ether. The combined organic layers were washed with water and saturated aqueous sodium chloride solution before drying over magnesium sulfate. Removal of the solvent under reduced pressure afforded the title compound as an orange solid: 1H NMR (CDCl)₃)：δ8.17(1H，d，J＝1.8Hz)，7.40(1H，dd，J＝1.8，8.2Hz)，7.19(1H，d，J＝8.2Hz)，3.22(2H，t，J＝6.3Hz)，3.08(1H，s)2.94(2H，t，J＝6.3Hz).4- [2- (6- (2, 3-dihydro- (4H) -1-benzothiopyran-4-onyl)) ethynyl group]Benzoic acid ethyl ester

Argon was bubbled through a solution of 405.0mg (2.15mmol)2, 3-dihydro-6-ethynyl- (4H) -1-benzothiopyran-4-one and 594.0mg (2.15mmol) ethyl 4-iodobenzoate in 15ml triethylamine and 3ml THF for 15 minutes. 503.0mg (0.72mmol) of bis (triphenylphosphonium) palladium (II) chloride and 137.0mg (0.72mmol) of Cul are added to the solution. The solution was stirred at room temperature for 20 hours, then filtered through a pad of celite, washing with ethyl acetate. Removal of the solvent under reduced pressure afforded a brown solid. Column chromatography purification (3% ethyl acetate: hexanes) afforded the title compound as an orange solid:

1H NMR(d₆-acetone): δ 8.15(1H, d, J ═ 2.0Hz), 8.02(2H, d, J ═ 8.5Hz), 7.69(2H, d, J ═ 8.5Hz), 7.61(1H, dd, J ═ 2.1, 8.3Hz), 7.40(1H, d, J ═ 8.2Hz), 4.35(2H, q, J ═ 7.1Hz), 3.40(2H, t, J ═ 6.3Hz), 2.96(2H, t, J ═ 6.3Hz), 1.37(3H, t, J ═ 7.1Hz).4- [2- (6- (4- (trifluoromethylsulfonyl) oxy- (2H) -1-benzothiopyranyl)) ethynyl group]Benzoic acid Ethyl ester

370.0mg (1.10mmol) of ethyl 4- [2- (6- (2, 3-dihydro- (4H) -1-benzothiopyran-4-onyl)) ethynyl ] benzoate in 4.0ml THF are added to a solution of 221.9mg (1.21mmol) of sodium bis (trimethylsilyl) amide in 3.0ml THF, cooled to-78 ℃. After 30 minutes, a solution of 2- [ N, N-bis (trifluoromethylsulfonyl) amino ] -5-chloropyridine in 4.0ml THF was slowly added. The reaction was slowly warmed to room temperature and after 5 hours the reaction was quenched by addition of saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate, and the combined organic layers were washed with 5% aqueous sodium hydroxide solution, water and saturated aqueous sodium chloride solution before being dried over magnesium sulfate. The solvent was removed under reduced pressure, followed by column chromatography (4% ethyl acetate-hexane) to give the objective compound as a light yellow solid:

1H NMR(d₆Acetone): δ 8.12(2H, d, J ═ 8.5Hz), 7.66(2H, d, J ═ 8.5Hz), 7.56(1H, d, J ═ 1.7Hz), 7.49(1H, dd, J ═ 1.7, 8, 1Hz), 7.40(1H, d, J ═ 8.1Hz), 6.33(1H, t, J ═ 5.7Hz), 4.35(2H, q, J ═ 7.1Hz), 3.82(2H, d, J ═ 5.7Hz), 1.37(3H, t, J ═ 7.1Hz).4- [2- (6- (4- (4-methylphenyl) - (2H) -1-benzothiopyranyl)) ethynyl group]Benzoic acid ethyl ester (Compound No.) Thing 49)

88.4mg (1.38mmol, 0.81ml of a 1.7M solution in pentane) of tert-butyllithium are added at-78 ℃ to a solution of 120.8mg (0.70mmol) of 4-bromotoluene in 2.0ml of THF. After 30 minutes, a solution of 131.6mg (0.97mmol) of zinc chloride in 2.0ml of THF was added and the resulting pale yellow solution was allowed to warm to room temperature. After stirring for 40 min, the solution was added to a second flask containing 129.2mg (0.28mmol) of ethyl 4- [2- (6- (4- (trifluoromethylsulfonyl) oxy- (2H) -1-thiochromanyl)) ethynyl ] benzoate, 14.0mg (0.012mmol) of tetrakis (triphenylphosphine) palladium (0) and 2.0ml of THF. The resulting solution was heated at 50 ℃ for 5 hours, cooled to room temperature and the reaction was quenched by the addition of saturated ammonium chloride solution. The mixture was extracted with ethyl acetate and the combined organic layers were washed with water and saturated aqueous sodium chloride solution, then dried (magnesium sulfate) and concentrated to an orange oil. The target compound was isolated via column chromatography (3-5% ethyl acetate-hexanes) as a colorless solid:

1H NMR(d₆-acetone): δ 7.98(2H, d, J ═ 8.3Hz), 7.58(2H, d, J ═ 8.2Hz), 7.44-7.38(2H, m), 7.26-7.15(5H, m), 6.14(1H, t, J ═ 5.8Hz), 4.34(2H, q, J ═ 7.1Hz), 3.53(2H, d, J ═ 5.8Hz), 2.37(2H, s), 1.35(3H, t, J ═ 7.1Hz).4- [2- (6- (4- (4-methylphenyl) - (2H) -1-benzothiopyranyl)) ethynyl group]Benzoic acid (Compound 50)

160.0mg (4.00mmol, 2.0ml of a 2M aqueous solution) were added to a solution of 29.0mg (0, 07mmol) of ethyl 4- [2- (6- (4- (4-methylphenyl) - (2H) -1-benzothiopyranyl)) ethynyl ] benzoate (compound 49) in 2.0ml of THF and 2.0ml of ethanol. The resulting solution was stirred at 35 ℃ for 2 hours, then cooled to room temperature and stirred for an additional 2 hours. The reaction was quenched by addition of 10% aqueous hydrochloric acid and extracted with ethyl acetate. The combined organic layers were washed with water and saturated aqueous sodium chloride solution and dried over sodium sulfate. The solvent was removed under reduced pressure to afford a solid, which was washed with acetonitrile and dried under high vacuum to yield the title compound as a light yellow solid:

¹H NMR(d₆-DMSO)：δ7.90(2H，d，J＝8.4Hz)，7.59(2H，d，J＝8.4Hz)，7.40(4H，m)，7.25-7.13(4H，m)，7.02(1H，d，J＝1.7Hz)，6.11(1H，t，J＝5.7Hz)，3.54(2H，d，J＝5.7Hz)，2.34(3H，s).3, 4-dihydro-4, 4-dimethyl-7-acetyl-1 (2H) -naphthalenone (compound R); 3, 4-dihydro-4, 4-di Methyl-6-acetyl-1 (2H) -naphthalenone (Compound S)

Acetyl chloride (15g, 192mmol) and 1, 2, 3, 4-tetrahydro-1, 1-dimethylnaphthalene (24.4g, 152mmol) in dichloromethane (20ml) were added over 20 minutes to a cooled (0 ℃ C.) mixture of aluminum trichloride (26.3g, 199.0mmol) in dichloromethane (55 ml). The reaction mixture was warmed to room temperature and stirred for 4 hours. Ice (200g) was added to the reaction flask and the mixture was diluted with ether (400 ml). The layers were separated and the organic phase was washed with 10% hydrochloric acid (50ml), water (50ml), 10% aqueous sodium bicarbonate and saturated aqueous sodium chloride (50ml) before drying over magnesium sulphate. The solvent was distilled off to give a yellow oil dissolved in benzene (50 ml).

Chromium trioxide (50g, 503mmol) was added in small portions to a cold (0 ℃) solution of acetic acid (240ml) and acetic anhydride (120ml) under argon over 20 minutes. The mixture was stirred at 0 ℃ for 30 minutes and diluted with benzene (120 ml). The benzene solution prepared above was added via addition funnel with stirring over 20 minutes. After 8 hours, the reaction was inhibited by the addition of isopropanol (50ml) followed by water (100ml) at 0 ℃. After 15 minutes, the reaction mixture was diluted with ether (1100ml) and water (200ml) and then neutralized with solid sodium bicarbonate (200 g). The ether layer was washed with water (100ml) and saturated aqueous sodium chloride (2X 100ml), and dried over magnesium sulfate. Removal of the solvent under reduced pressure provided a mixture of isomeric diketones, which can be separated by chromatography (5% ethyl acetate/hexane). (Compound R):

1H NMR(CDCl₃)：δ8.55(1H，d，J＝2.0Hz)，8.13(1H，dd，J＝2.0，8.3Hz)，7.53(1H，d，J＝8.3Hz)，2.77(2H，t，J＝6.6Hz)，2.62(3Hs), 2.05(2H, t, J ═ 6.6Hz), 1.41(6H, S), (compound S): 1H NMR (CDCl 3): δ 8.10(1H, d, J ═ 8.1Hz), 8.02(1H, d, J ═ 1.6Hz), 7.82(1H, dd, J ═ 1.6, 8.1Hz), 2.77(2H, t, J ═ 7.1Hz), 2.64 (3H, s), 2.05(2H, t, J ═ 7.1Hz), 1.44(6H, s).3, 4-dihydro-4, 4-dimethyl-7- (2- (2-methyl-1, 3-dioxolanyl)) -1(2H) -naphthalenone (compound T)

A mixture of 3, 4-dihydro-4, 4-dimethyl-7-acetyl-1 (2H) -naphthalenone (compound R) (140.0mg, 0.60mmol), ethylene glycol (55.0mg, 0.90mmol), p-toluenesulfonic acid monohydrate (4mg), and benzene (25ml) was refluxed for 12 hours using a Dean-Stark apparatus. The reaction was inhibited by the addition of 10% aqueous sodium bicarbonate and extracted with ether (2X 75 ml). The combined organic layers were washed with water (5ml) and saturated aqueous sodium chloride (5ml) and dried over magnesium sulfate. Removal of the solvent under reduced pressure afforded the desired compound as an oil: 1H NMR (CDCl)₃)：δ8.13(1H，d，J＝2.0Hz)，7.64(1H，dd，J＝2.0，8.2Hz)，7.40(1H，d，J＝8.2Hz)，3.97-4.10(2H，m)，3.70-3.83(2H，m)，2.73(2H，t，J＝6.5Hz)，2.01(2H，t，J＝6.5Hz)，1.64(3H，s)，1.39(6H，s).1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-7- (2- (2-methyl-1, 3-dioxy) benzene Pentylring group)) naphthalene (Compound U)

A solution of 3, 4-dihydro-4, 4-dimethyl-7- (2- (2-methyl-1, 3-dioxolanyl)) -1(2H) -naphthalenone (compound T) (135.0mg, 0.52mmol) in 5ml THF was added to a solution of 195.4mg (1.00mmol) of p-toluoylmagnesium bromide (1.0ml, 1M in ether) in 2ml THF. The solution was refluxed for 16 h, cooled to room temperature and diluted with ether (50 ml). The solution was washed with water (5ml) and saturated aqueous ammonium chloride (5ml) and dried over magnesium sulfate. The solvent was removed under reduced pressure and column chromatography (5% ethyl acetate/hexanes) afforded the title compound as a solid: 1H NMR (CDCl) ₃)：δ7.37(2H，d)，7.21(1H，s)，7.13(2H，d，J＝8.5Hz)，7.08(2H，d，J＝8.5Hz)，3.88-3.99(2H，m)，3.58-3.75(2H，m)，2.34(3H，s)，2.12-2.30(2H，m)，1.79-1.90(1H，m)，1.57(3H，s)，1.48-1.58(1H，m)，1.38(3H，s)，1.31(3H，s).3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-7-acetylnaphthalene (Compound V)

A mixture of 130.0mg (0.38mmol) of 1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-7- (2- (2-methyl-1, 3-dioxolanyl)) naphthalene (compound U), p-toluenesulfonic acid monohydrate (4mg) and benzene (5ml) was refluxed for 16 hours. After cooling to room temperature, the reaction mixture was diluted with ether (100ml) and washed with 10% aqueous sodium bicarbonate, water and saturated sodium chloride solution. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure to give the desired compound as a solid: 1H NMR (CDCl)₃)：δ7.83(1H，dd，J＝1.8，8.0Hz)，7.66(1H，d，J＝1.8Hz)，7.45(1H，d，J＝8.0Hz)，7.25(2H，d，J＝8.5Hz)，7.22(2H，d，J＝8.5Hz)，6.03(1H，t，J＝6.3Hz)，2.47(3H，s)，2.41(3H，s)，2.37(2H，d，J＝6.3Hz)，1.36(6H，s).(E) -3- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2-butenenitrile (compound) W)

Diethyl cyanomethylphosphonate (450.0mg, 2.50mmol) was added to a slurry of sodium hydride (48.0mg, 2.00mmol) in THF (6 ml). After 40 minutes, 95.0mg (0.33mmol) of 3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-7-acetylnaphthalene (compound V) in THF (4ml) were added. The mixture was stirred for 16 h, diluted with ether (100ml) and washed with water and saturated aqueous sodium chloride solution before drying over magnesium sulphate. The solvent was removed under reduced pressure and purified by column chromatography (3% ethyl acetate: hexane) to provide the desired compound as a solid: 1H NMR (CDCl) ₃)：δ7.39(1H，d，J＝1H)，7.32(1H，dd，J＝2.0，8.1Hz)，7.20-7.25(4H，brs)，7.15(1H，d，J＝2.0Hz)，6.03(1H，t，J＝6.0Hz)，5.44(1H，s)，2.42(3H，s)，2.36(2H，d，J＝6.0 Hz)，2.35(3H，s)，1.35(6H，s).(E) -3- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2-butenal (compound X)

0.50ml (0.50mmol) of diisobutylaluminum hydride (1M in dichloromethane) are added to a cold (-78 ℃ C.) solution of 84.0mg (0.29mmol) of (E) -3- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2-butenenitrile (compound W) in dichloromethane (4 ml). After stirring for 1 hour, the reaction was inhibited at-78 ℃ by adding 2-propanol (1ml) diluted with ether (100 ml). After warming to room temperature, the solution was washed with water, 10% hydrochloric acid and saturated aqueous sodium chloride solution. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure to give the desired compound as an oil: 1H NMR (CDCl)₃)：δ10.12(1H，d，J＝7.9Hz)，7.43(2H，s)，7.19-7.28(5H，m)，6.27(1H，d，J＝7.9Hz)，6.03(1H，t，J＝4.8Hz)，2.47(3H，s)，2.42(3H，s)，2.37(2H，d，J＝4.8Hz)，1.37(6H，s).(E, E, E) -3-methyl-7- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) - -2-naphthyl) -2, 4, 6-octan Trienoic acid ethyl ester (Compound 51)

26.0mg (0.41mmol, 0.65ml) of n-butyllithium (1.6M solution) in hexane was added to 264.0mg (1.00mmol) of diethyl (E) -3-ethoxycarbonyl-2-methylallyl phosphate [ according to J.Org.chem.39: 821(1974) preparation]To a cooled (-78 ℃ C.) solution of THF (2ml) was then immediately added 82.0mg (0.26mmol) of (E) -3- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2-butenal (Compound X) in THF (3 ml). After 1 hour, the reaction mixture was diluted with ether (60ml), washed with water (5ml), saturated aqueous sodium chloride (5ml) and dried over magnesium sulfate. After removal of the solvent under reduced pressure, the target compound was isolated as an oil by column chromatography (5% ethyl acetate/hexane followed by HPLC using 1% ethyl acetate/hexane): 1H NMR (acetone-d 6): δ 7.36-7.43(2H, m), 7.18-7.27(4H, m), 7.17(1H, d, J ═ 1.7Hz), 7.08(1H, dd, J ═ 11.2, 15.2Hz), 6.46(1H, d, J ═ 11.2Hz), 6.38(1H, d, J ═ 15.2Hz), 5.98(1H, t, J ═ 4.7Hz), 5.78 (1H, s), 4.10(2H, q, J ═ 7.1Hz), 2.35 (3H, s), 2.33(3H, s), 2.32(2H, d, J ═ 4.7Hz), 2.12(3H, s), 1.31(6H, s), 1.22(3H, t, J ═ 1.7.7 Hz). (E，E，E)-3-methyl-7- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2, 4, 6-octan Trienoic acid (Compound 52)

12.0mg (0.50mmol) of LiOH (0.5ml, 1M solution) are added to a solution of 85.0mg (0.20mmol) of ethyl (E, E, E) -3-methyl-7- (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) -2, 4, 6-octenoic acid (compound 51) in THF (1ml) and methanol (1 ml). The mixture was stirred for 6h, diluted with ether (60ml) and acidified with 10% hydrochloric acid (1 ml). The solution was washed with water and saturated aqueous sodium chloride solution before drying over magnesium sulfate. Removal of the solvent under reduced pressure afforded the target compound as a solid, which was purified by recrystallization from acetone:

1H NMR (acetone-d 6): δ 7.35-7.45(2H, m), 7.19-7.28(4H, m), 7.17(1H, d, J ═ 1.8Hz), 7.09(1H, dd, J ═ 11.5, 15.1Hz), 6.48(1H, d, J ═ 11.5Hz), 6.42(1H, d, J ═ 15.1Hz), 5.99(1H, t, J ═ 4.7Hz), 5.82(1H, s), 2.36(3H, s), 2.33(2H, d, J ═ 4.7Hz), 2.32(3H, s), 2.13(3H, s), 1.32(6H, s). 3, 4-dihydro-4, 4-dimethyl-7-nitro-1 (2H) -naphthalenone (Compound Y)

783.0mg (4.49mmol) of 3, 4-dihydro-4, 4-dimethyl-1 (2H) -naphthalenone were slowly added to 1.7ml (3.0g, 30.6mmol, 18M) of sulfuric acid in-5 deg.C (a bath of glacial sodium chloride). A solution of 426.7mg (6.88mmol, 0.43ml, 16M) of nitric acid and 1.31g (0.013mol, 0.74ml, 18M) of sulfuric acid was slowly added. After 20 minutes, ice was added and the resulting mixture was extracted with ethyl acetate. The combined extracts were concentrated under reduced pressure to give a residue from which the desired compound was isolated via column chromatography (10% ethyl acetate: hexanes) as a light yellow solid:

1H NMR(CDCl₃)：δ8.83(1H，d，J＝2.6Hz)，8.31(1H，dd，J＝2.8，8.9Hz)，7.62(1H，d，J＝8.7Hz)，2.81(2H，t，J＝6.5Hz)，2.08(2H，t，J＝6.5Hz)，1.45(6H，s).3, 4-dihydro-4, 4-dimethyl-7-amino-1 (2H) -naphthalenone (Compound Z)

A solution of 230.0mg (1.05mmol) of 3, 4-dihydro-4, 4-dimethyl-7-nitro-1 (2H) -naphthalenone (compound Y) in 5.0ml of ethyl acetate is stirred at room temperature under 1 atmosphere of hydrogen using a catalytic amount of 10% palladium on charcoal for 24 hours. The catalyst was removed by filtration through a pad of celite and the filtrate was concentrated under reduced pressure to give the desired compound as a dark green oil:

1H NMR(CDCl₃)：δ7.30(1H，d，J＝2.7Hz)，7.22(1H，d，J＝8.4Hz)，6.88(1H，dd，J＝2.7，8.5Hz)，2.70(2H，t，J＝6.6Hz)，1.97(2H，t，J＝6.6HZ)，1.34(6H，s).4- [ (5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) azo]Benzoic acid ethyl ester (Compound) AA)

180.0mg (1.00mmol) of ethyl 4-nitrosobenzoate are added to a solution of 198.7mg (1.05mmol) of 3, 4-dihydro-4, 4-dimethyl-7-amino-1 (2H) -naphthalenone (compound Z) in 5.0ml of glacial acetic acid. The resulting solution was stirred at room temperature overnight and then concentrated under reduced pressure. The product was isolated from its residual oil by column chromatography (15% ethyl acetate: hexanes) as a red solid: 1H NMR (CDCl) ₃)δ8.57(1H，d，J＝2.0Hz)，8.19(2H，d，J＝8.4Hz)，8.07(1H，d，J＝8.0Hz)，7.94(2H，d，J＝8.4Hz)，7.58(1H，d，J＝8.6Hz)，4.41(2H，q，J＝7.1Hz)，2.79(2H，t，J＝6.6Hz)，2.07(2H，t，J＝7.02Hz)，1.44(6H，s)，1.42(3H，t，J＝7.1Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) azo]-benzoic acid Ethyl ester (Compound BB)

153.0mg (0.437mmol) of ethyl 4- [ (5, 6, 7, 8-tetrahydro-5, 5-dimethyl-8-oxo-2-naphthyl) azo ] benzoate (compound AA) in 2ml of THF are added at-78 ℃ to a solution of 90.4mg of sodium bis (trimethylsilyl) amide (0.48mmol, 0.48ml of a 1.0M solution in THF) in 2.0ml of THF. The dark red solution was stirred at-78 ℃ for 30 minutes, then a solution of 204.0mg (0.520mmol) of 2- [ N, N-bis (trifluoromethylsulfonyl) amino ] -5-chloropyridine in 2.0ml THF was added. The reaction mixture was allowed to warm to room temperature and after 3 hours the reaction was quenched by the addition of water. The organic layer was concentrated under reduced pressure to a red oil and the product was isolated by column chromatography (25% ethyl acetate: hexanes) as a red oil:

1H NMR(CDCl₃)：δ8.21(2H，d，J＝8.6Hz)，7.96(2H，d，J＝8.6Hz)，7.94(2H，m)，7.49(1H，d，J＝8.2Hz)，6.08(1H，t，J＝2.5Hz)，4.42(2H，q，J＝7.1Hz)，2.49(2H，d，J＝4.8Hz)，1.44(3H，t，J＝7.1Hz)，1.38(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) azo]Benzoic acid ethyl ester (Compound No.) Thing 46a)

A solution of 4-tolyllithium was prepared by adding 62.9mg (0.58ml, 0.98mmol) of t-butyllithium (1.7M in pentane) to 84.0mg (0.491mmol) of 4-bromotoluene in 1.0ml of THF as a cold solution (-78 ℃ C.). After stirring for 30 minutes, a solution of 107.0mg (0.785mmol) of zinc chloride in 2.0ml of THF is added. The resulting solution was warmed to room temperature, stirred for 30 minutes and added via cannula to 94.7mg (0.196mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) azo ]Ethyl benzoate (compound BB) and a solution of 25mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) in 2.0ml THF. The resulting solution was heated at 50 ℃ for 1.5 hours, cooled to room temperature, and diluted with saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate (40ml), and the combined organic layers were washed with water and brine. The organic phase is dried over sodium sulfate, concentrated in vacuo, and the desired compound is isolated via column chromatography (25% ethyl acetate: hexanes) as a red solid: 1H NMR (CDCl)₃)：δ8.21(2H，d，J＝8.6Hz)，7.96(2H，d，J＝8.6Hz)，7.94(2H，m)，7.49(1H，d，J＝8.2Hz)，6.08(1H，t，J＝2.5Hz)，4.42(2H，q，J＝7.1Hz)，2.49(2H，d，J＝4.8Hz)，1.44(3H，t，J＝7.1Hz)，1.38(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) azo]Benzoic acid (Compound) 46b)

80.0mg (2.00mmol) of sodium hydroxide (2.0ml, 1M in water) are added to 16.5mg (0.042mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) azo]Ethyl benzoate (Compound 46a) was dissolved in a solution of THF (2ml) and ethanol (1 ml). The mixture was stirred at room temperature for 12 hours, acidified with 10% hydrochloric acid and extracted with ethyl acetate. The combined organic layers were washed with water and saturated aqueous sodium chloride solution, and then dried over magnesium sulfate. After removal of the solvent under reduced pressure, the residue was recrystallized from ethyl acetate/hexane to provide the desired compound as a red solid: 1H NMR (acetone-d 6): δ 8.19(2H, d, J ═ 8.4Hz), 7.92(2H, d, J ═ 8.5Hz), 7.88(2H, dd, J ═ 2.1, 6.1Hz), 7.66(1H, s), 7.64(2H, d, J ═ 2.3Hz), 7.28(4H, d, J ═ 3.0Hz), 6.09(1H, t, J ═ 2.5Hz), 2.42(2H, d, J ═ 4.8Hz), 2.39(3H, s), 1.40(6H, s). 6- (2-trimethylsilyl) ethynyl-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one (Compound) CC)

259.6mg (1.363mmol) of iodoidene ketone (I), 956.9mg (1.363mmol) of bis (triphenylphosphonium) palladium (II) chloride and 3.14g (34.08mmol) of (trimethylsilyl) acetylene are added to a solution of 815.0mg (3.41mmol) of 6-bromo-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one (see Smith et al. org. Prep. proced. int.197810123-131) in 100ml of degassed triethylamine (20 min. argon gas feed). The mixture was heated at 70 ℃ for 42 hours, cooled to room temperature, filtered through a pad of silica gel, and washed with ether. Before drying over magnesium sulfate, the filtrate was washed with water, 1M hydrochloric acid, water and finally with saturated aqueous sodium chloride solution. The solution was concentrated under reduced pressure and then purified by column chromatography (silica gel, 10% diethyl ether-hexanes) to afford the title compound as a brown oil:

1H NMR(300MHz，CDCl₃)：δ7.79(1H，d，J＝1.4Hz)，7.69(1H，dd，J＝1.6，8.3Hz)，7.42(1H，d，J＝8.5Hz)，2.60(2H，s)，1.41(6H，s)，0.26(9H，s).6-ethynyl-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one (Compound DD)

197.3mg (1.43mmol) of potassium carbonate are added in one portion to a solution of 875.0mg (3.41mmol) of 6- (2-trimethylsilyl) ethynyl-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one (compound CC) in 28ml of methanol. After stirring at room temperature for 6 hours, the mixture was passed through a celite pad and the filtrate was concentrated under reduced pressure. The residual oil was placed on a silica gel column and eluted with 5% ethyl acetate-hexanes to give the desired product as a colorless oil:

1H NMR(300MHz，CDCl3)：δ7.82(1H，s)，7.72(1H，dd，J＝1.6，7.8Hz)，7.47(1H，d，J＝8.4Hz)，3.11(1H，s)，2.61(2H，s)，1.43(6H，s).4- [2- (5, 6-dihydro-5, 5-dimethyl-7-oxo-2-indenyl) ethynyl]Benzoic acid ethyl ester (Compound) EE)

A solution of 280.0mg (1.520mmol) of 6-ethynyl-2, 3-dihydro-3, 3-dimethyl-1H-inden-1-one (compound DD) and 419.6mg (1.520mmol) of ethyl 4-iodobenzoate in 5ml of triethylamine is purged with argon for 40 minutes. 271.0mg (1.033mmol) of triphenylphosphine, 53.5mg (0.281mmol) of iodoidene (I) and 53.5mg (0.076mmol) of bis (triphenylphosphine) palladium (II) chloride were added to the solution. The resulting mixture was heated at reflux for 2.5 hours, cooled to room temperature, and diluted with ether. After filtration through a celite pad, the filtrate was washed with water, 1M hydrochloric acid and saturated aqueous sodium chloride solution, then dried over magnesium sulfate and concentrated under reduced pressure. The target compound was isolated by column chromatography (15% ethyl acetate-hexanes) as a light yellow solid: 1H NMR (300MHz, d 6-acetone): δ 8.05(2H, d, J ═ 8.6Hz), 7.87(1H, dd, J ═ 1.4,8.1Hz)，7.75(2H，m)，7.70(2H，d，J＝8.5Hz)，4.36(2H，q，J＝7.1Hz)，2.60(2H，s)，1.45(6H，s)，1.37(3H，t，J＝7.1Hz).4- [2- (1, 1-dimethyl-3- (trifluoromethyl-sulfonyl) oxy-5-indenyl) ethynyl]Benzoic acid ethyl ester (Compound FF)

A solution of 88.0mg (0.48mmol) of sodium bis (trimethylsilyl) amide in 0.5ml of THF is cooled to-78 ℃ and a solution of 145.0mg (0.436mmol) of ethyl 4- [2- (5, 6-dihydro-5, 5-dimethyl-7-oxo-2-indenyl) ethynyl ] benzoate (compound EE) in 1.0ml of THF is added. After 30 minutes, a solution of 181.7mg (0.480mmol) of 2- [ N, N-bis (trifluoromethanesulfonyl) amino ] -5-chloropyridine in 1.0ml of THF was added. The reaction was allowed to warm slowly to room temperature and after 5 hours the reaction was quenched by addition of saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate, and the combined organic layers were washed with 5% aqueous sodium hydroxide solution, water and saturated aqueous sodium chloride solution, then dried (magnesium sulfate) and concentrated under reduced pressure. The product was isolated by column chromatography (10% ether-hexanes) as a colorless solid:

1H NMR (300MHz, d 6-; acetone): δ 8.05(2H, d, J ═ 8.3Hz), 7.69(2H, d, J ═ 8.4Hz), 7.63(2H, s), 7.55(1H, s), 4.36(2H, q, J ═ 7.1Hz), 1.44(6H, s), 1.37(3H, t, J ═ 7.1Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 60)

142.6mg (0.339mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group are stirred at room temperature ]Ethyl benzoate (Compound 1) and 35.6mg (0.848mmol) of LiOH-H₂A solution of O in 12ml THF/water (4: 1, v/v) was left overnight. The reaction mixture was extracted with hexane, and the hexane fraction was extracted with 5% aqueous sodium hydroxide solution. The aqueous layers were combined and acidified with 1M hydrochloric acid and then extracted with ethyl acetate and ether. The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the desired compound as a colorless solid: 1H NMR (d)₆-DMSO)：δ7.91(2H，d，J＝8.4Hz)，7.60(2H，d，J＝8.4Hz)，7.47(2H，s)，7.23(4H，q，J＝8.1Hz)，7.01(1H，s)，6.01(1H，t，J＝4.6Hz)，2.35(3H，s)，2.33(2H，d，J＝4.8Hz)，1.30(6H，s).4- [ (5, 6-dihydro-5, 5-dimethyl-8-phenyl-2-naphthyl) ethynyl]Benzoic acid (Compound 60a)

Using the same general procedure as for the preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl ] benzoic acid (compound 30a), 27.0mg (0.07mmol) of ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8-phenyl-2-naphthyl) ethynyl ] benzoate (compound 1a) was converted to the desired compound (colorless solid) using 5.9mg (0.14mmol) of LiOH in water:

PMR(d₆-DMSO)：δ1.31(6H，s)，2.35(2H，d，J＝4.5Hz)，6.05(1H，t，J＝J＝J＝4.5Hz)，7.00(1H，s)，7.33(2H，d，J＝6.2Hz)，7.44(4H，m)，7.59(2H，d，J＝8.1Hz)，7.90(2H，d，J＝8.1Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- (1, 1-dimethylethyl) phenyl) -2-naphthyl) ethynyl group]Benzene and its derivatives Formic acid (Compound 61)

80.0mg (0.173mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4- (1, 1-dimethylethyl) phenyl) -2-naphthyl) ethynyl group are stirred at room temperature ]Ethyl benzoate (Compound 6) and 18.1mg (0.432mmol) of LiOH-H₂A solution of O in 6ml THF/water (3: 1, v/v) was left overnight. The reaction mixture was extracted with hexane, and the remaining aqueous layer was acidified with 1M hydrochloric acid, and then extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the desired compound as a colorless solid: 1H NMR (d)₆-DMSO)：δ7.82(2H，d，J＝8.2Hz)，7.44(6H，m)，7.25(2H，d，J＝8.3Hz)，7.02(1H，s)，6.01(1H，t，J＝4.6Hz)，2.32(2H，d，J＝4.7Hz)，1.32(9H，s)，1.29(6H，s).2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl]Amino group] Ethyl benzoate (Compound 62)

A solution of 54.4mg (0.119mmol) of ethyl 2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) carbonyl ] amino ] benzoate (compound 40) and 57.7mg (0.143mmol) of [2, 4-bis (4-methoxyphenyl) -1, 3-dithio-2, 4-diphosphetane-2, 4-disulfide ] (Lawesson's reagent) in 12.0ml of benzene was refluxed overnight. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated under reduced pressure. The title compound was isolated via column chromatography (10-25% ethyl acetate/hexanes) as a yellow solid:

1H NMR(CDCl₃)：δ9.08(1H，s)，7.92(1H，br s)，7.90(1H，t，J＝8.2Hz)，7.66(1H，dd，J＝2.0，6.0Hz)，7.38(3H，m)，7.18(4H，m)，6.01(1H，t，J＝4.7 Hz)，4.35(2H，q，J＝7.1Hz)，2.36(3H，s)，2.33(2H，d，J＝4.7Hz)，1.38(3H，t，J＝7.1Hz)，1.33(6H，s)·2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl]Amino group] Benzoic acid (Compound 63)

55mg of sodium hydroxide (1.4mmol) and 1.0ml of water are added to a solution of 46.5mg (0.098mmol) of ethyl 2-fluoro-4- [ [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) thiocarbonyl ] amino ] benzoate (compound 62) in 1.0ml of ethanol and 1.0ml of THF. After stirring overnight at room temperature, ethyl acetate was added and the reaction was inhibited by the addition of 10% hydrochloric acid. The combined organic layers were washed with water, saturated aqueous sodium chloride solution and dried over magnesium sulfate. Removal of the solvent under reduced pressure afforded a solid which was crystallized from acetonitrile to afford the title compound as a light yellow solid:

1H NMR(d₆-acetone): δ 11.05(1H, s), 8.02(1H, m), 7.99(1H, t, J ═ 8.3Hz), 7.75(1H, m), 7.69(1H, dd, J ═ 2.0, 6.1Hz), 7.52(1H, s), 7.46(1H, d, J ═ 8.1Hz), 7.21(4H, m), 6.04(1H, t, J ═ 4.8Hz), 2.37(2H, d, J ═ 4.8Hz), 2.33(3H, s), 1.36(6H, s).5 ', 6' -dihydro-5 ', 5' -dimethyl-8 '- (4-methylphenyl) - [2, 2' -binaphthyl]-6-Carboxylic acid ethyl ester (Compound No.) Thing 64)

Magnesium turnings (0.044g, 1.82mmol) were added to a solution of 3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-7-bromonaphthalene (compound D) (0.45g, 1.40mmol) and THF (2.1ml) at room temperature under argon. Two drops of dibromoethane were added and the solution (slowly cloudy and yellow) was heated at reflux for 1.5 hours. In a second flask, zinc chloride (0.210g, 1.54mmol) was added, which was melted under high vacuum, cooled to room temperature and dissolved in THF (3 mL). The Grignard reagent was added to the second flask and after 30 minutes at room temperature a solution of 0.293g (1.05mmol) of ethyl 6-bromo-2-naphthoate (compound N) and THF (2ml) was added. In a third flask, Ni (PPh) was prepared₃)₄And THF as follows: A1M solution of diisobutylaluminum hydride and hexane (2.5ml, 2.5mmol) were added to NiCl ₂(PPh₃)₂(0.82g, 1.25mmol) and PPh₃(0.66g, 2.5mmol) in THF (3.5ml) and the resulting solution diluted with THF to a total volume of 15ml and stirred at room temperature for 15 minutes. Three 0.60ml aliquots of Ni (PPh) were added at 15 minute intervals₃)₄The solution was added to a second flask. The resulting suspension was stirred at room temperature for 2 hours. The reaction was inhibited by adding 5ml of 1N aqueous hydrochloric acid and stirred for 1 hour before its product was extracted with ethyl acetate. The organic layers were combined, washed with brine, dried (magnesium sulfate), filtered and the solvent removed in vacuo. The residue was crystallized from hexane to yield 130mg of pure material. The mother liquor was concentrated under reduced pressure and the residue was purified by silica gel chromatography (95: 5 hexane: ethyl acetate) to yield an additional 170mg of the title compound as a colorless solid (total yield 300mg, 64%):

1H NMR(CDCl₃) δ 8.57(s, 1H), 8.05(dd, 1H, J ═ 1.7, 8.0Hz), 7.84-7.95 (overlap d's, 3H), 7.66(dd, 1H, J ═ 1.7, 8.5Hz), 7.58(dd, 1H, J ═ 2.0, 8.0Hz), 7.48(d, 1H, J ═ 8.0Hz), 7.43(d, 1H, J ═ 2.0Hz), 7.32(d, 2H, J ═ 8.0Hz), 7.21(d, 2H, J ═ 8.0Hz), 6.04(t, 1H, J ═ 2H ═ 8.0Hz)4.8Hz)，4.44(q，2H，J＝7.1Hz)，2.40(s，3H)，2.39(d，2H，J＝4.8Hz)，1.45(t，3H，J＝7.1Hz)，1.39(s，6H).5 ', 6' -dihydro-5 ', 5' -dimethyl-8 '- (4-methylphenyl) - [2, 2' -binaphthyl ]-6-carboxylic acid (Compound 65)

Mixing 5 ', 6' -dihydro-5 ', 5' -dimethyl-8 '- (4-methylphenyl) - [2, 2' -binaphthyl ]]A solution of ethyl-6-carboxylate (compound 64) (0.19g, 0.43mmol), ethanol (8ml) and 1N aqueous sodium hydroxide (2ml) was heated at 60 ℃ for 3 hours. The solution was cooled to 0 ℃ and acidified with 1N aqueous hydrochloric acid. The product was extracted with ethyl acetate, the organic layers were combined, washed with water, brine, dried (magnesium sulfate), filtered and the solvent was removed in vacuo. The residue was recrystallized from THF/ethyl acetate at 0 ℃ to yield 35mg of pure material. The mother liquor was concentrated under reduced pressure and the residue was purified by silica gel chromatography (100% ethyl acetate) to yield an additional 125mg of the title compound as a colorless solid (total yield 160mg, 90%): 1H NMR (DMSO-d)₆) δ 8.57(S, 1H), 8.11(d, 1H, J ═ 8.7Hz), 7.96-7.82 (overlap d' S, 3H), 7.65(d, 2H, J ═ 7.6Hz), 7.50(d, 1H, J ═ 7.9Hz), 7.28(S, 1H), 7.26(d, 2H, J ═ 8.3Hz), 7.21(d, 2H, J ═ 8.3Hz), 6.01(t, 1H, J ═ 4.5Hz), 3.34(br S, 1H), 2.31(S, 3H), 2.31(d, 2H, J ═ 4.5Hz), 1.31(S, 6H).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-furyl) -2-naphthyl) ethynyl ]Benzoic acid ethyl ester (Compound No.) Thing 66)

Preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (4-methylphenyl) -2-naphthyl) ethynyl group]Ethyl benzoate (Compound 1) the same general procedure was followed using 142.4mg (1.045mmol) of zinc chloride, 24.1mg (0.02mmol) of tetrakis (triphenylphosphine) palladium (0) and 2-furyllithium [ prepared by adding 53.4mg (0.52ml, 0.78mmol) of n-butyllithium (1.5M solution in hexane) to 53.4mg (0.784mmol) of furan in 1.0ml of THF (-78 ℃ C.) ]]250.0mg (0.52mmol) of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (trifluoromethylsulfonyl) oxy-2-naphthyl) ethynyl group]Ethyl benzoate (compound G) was converted to the desired compound (colorless solid): PMR(CDCl₃)：δ1.32(6H，s)，1.41(3H，t，J＝7.1Hz)，2.35(2H，d，J＝5.0Hz)，4.39(2H，q，J＝7.1Hz)，6.41(1H，t，J＝5.0Hz)，6.50(2H，s)，7.36(1H，d，J＝8.0Hz)，7.45(1H，dd，J＝1.7，8.0Hz)，7.49(1H，s)，7.57(2H，d，J＝8.2Hz)，7.63(1H，d，J＝1.7Hz)，8.02(2H，d，J＝8.2Hz).4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-furyl) -2-naphthyl) ethynyl]Benzoic acid (Compound) 67)

Using the same general procedure as for the preparation of 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-thiazolyl) -2-naphthyl) ethynyl ] benzoic acid (compound 30a), ethyl 4- [ (5, 6-dihydro-5, 5-dimethyl-8- (2-furyl) -2-naphthyl) ethynyl ] benzoate (compound 66) was converted to the desired compound (colorless solid) using 16.0mg (0.38mmol) of LiOH in water:

PMR(d₆-DMSO)：δ1.26(6H，s)，2.33(2H，d，J＝4.9Hz)，6.41(1H，t，J＝4.9Hz)，6.60(2H，m)，7.45-7.53(3H，m)，7.64(2H，d，J＝8.3Hz)，7.75(1H，d，J＝1.6Hz)，7.93(2H，d，J＝8.3Hz).3, 4-dihydro-4, 4-dimethyl-7-acetyl-1 (2H) -naphthalenone (Compound 100C) and 3, 4-dihydro- 4, 4-dimethyl-6-acetyl-1 (2H) -naphthalenone (Compound 100D)

Acetyl chloride (15g, 192mmol) and 1, 2, 3, 4-tetrahydro-1, 1-dimethylnaphthalene (24.4g, 152mmol) in dichloromethane (20ml) were added to a cold (0 ℃) mixture of aluminum trichloride (26.3g, 199.0mmol) in dichloromethane (55ml) over 20 minutes. The reaction mixture was warmed to room temperature and stirred for 4 hours. Ice (200g) was added to the reaction flask and the mixture was diluted with ether (400 ml). The aqueous layer and the organic layer were separated, and the organic phase was washed with 10% hydrochloric acid (50ml), water (50ml), 10% aqueous sodium hydrogencarbonate solution and saturated aqueous sodium chloride solution (50ml), followed by drying (magnesium sulfate). The solvent was distilled off to give a yellow oil dissolved in benzene (50 ml).

Chromium trioxide (50g, 503mmol) was added in small portions to a cold solution (0 ℃) of acetic acid (240ml) and acetic anhydride (120ml) over 20 minutes under argon. The mixture was stirred at 0 ℃ for 30 minutes and diluted with benzene (120 ml). The benzene solution prepared above was added via an addition funnel over 20 minutes with stirring. After 8 hours, the reaction was inhibited by careful addition of isopropanol (50ml) followed by water (100ml) at 0 ℃. After 15 minutes, the reaction mixture was diluted with ether (1100ml) and water (200ml) and then neutralized with solid sodium bicarbonate (200 g). The ether layer was washed with water (100ml), then with saturated aqueous sodium chloride (2X 100ml) and dried over magnesium sulfate. The solvent was removed under reduced pressure to provide a mixture of isomeric diketones, which can be separated by chromatography (5% ethyl acetate/hexane). (Compound 100C):

1H NMR(CDCl₃): δ 8.55(1H, D, J ═ 2.0Hz), 8.13(1H, dd, J ═ 2.0, 8.3Hz), 7.53(1H, D, J ═ 8.3Hz), 2.77(2H, t, J ═ 6.6Hz), 2.62(3H, s), 2.05(2H, t, J ═ 6.6Hz), 1.41(6H, s) (compound 100D): 1H NMR (CDCl)₃)：δ8.10(1H，d，J＝8.1Hz)，8.02(1H，d，J＝1.6Hz)，7.82(1H，dd，J＝1.6，8.1Hz)，2.77(2H，t，J＝7.1Hz)，2.64(3H，s)，2.05(2H，t，J＝7.1Hz)，1.44(6H，s)，3, 4-dihydro-4, 4-dimethyl-6- (2- (2-methyl-1, 3-dioxolanyl)) -1(2H) -naphthalenone (compound 100E)

A solution of 1: 5 mixture of 1.80g (8.34mmol) of 3, 4-dihydro-4, 4-dimethyl-7-acetyl-1 (2H) -naphthalenone (compound 100C) and 3, 4-dihydro-4, 4-dimethyl-6-acetyl-1 (2H) -naphthalenone (compound 100D) in 50ml of benzene was combined with 517.7mg (8.34mmol) of ethylene glycol and 20.0mg (0.11mmol) of p-toluenesulfonic acid monohydrate. The resulting solution was heated under reflux for 18 hours, cooled to room temperature, and concentrated under reduced pressure. The objective compound was isolated as a colorless oil by column chromatography (10% ethyl acetate-hexane).

1H NMR(CDCl₃)：δ 8.01(1H，d，J＝8.2Hz)，7.51(1H，s)，7.43(1H，dd，J＝1.7，6.4Hz)，4.07(2H，m)，3.79(2H，m)，2.74(2H，t，J＝6.5Hz)，2.04(2H，t，J＝7.1Hz)，1.67(3H，s)，1.46(6H，s).1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-6- (2- (2-methyl-1, 3-dioxy) benzene Pentylring) naphthalene (Compound 100F)

A solution of 3, 4-dihydro-4, 4-dimethyl-6- (2- (2-methyl-1, 3-dioxolanyl)) -1(2H) -naphthalenone (compound 100E, 200.0mg, 0.769mmol) in THF (5ml) was added to a solution of 496.2mg (2.54mmol) of p-tolylmagnesium bromide in 20ml of THF (2.54ml, 1M ether solution). The solution was refluxed for 16 hours, cooled to room temperature, washed with water, saturated aqueous ammonium chloride solution, and dried over magnesium sulfate. The solvent was removed under reduced pressure and purified by column chromatography (10% ethyl acetate/hexane) to afford the title compound as a colorless solid:

1H NMR(CDCl₃)：δ7.49(1H)，d，J＝1.7Hz)，7.19(2H，m)，7.10(2H，d，J＝7.9Hz)，7.04(1H，d，J＝8.2Hz)，4.05(2H，m)，3.80(2H，m)，2.34(3H，s)，2.21(1H，m)，2.10(1H，m)，1.88(1H，m)，1.65(3H，s)，1.54(1H，m)，1.39(3H，s)，1.33(3H，s).3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-6-acetylnaphthalene (Compound 100G)

A solution of 1, 2, 3, 4-tetrahydro-1-hydroxy-1- (4-methylphenyl) -4, 4-dimethyl-6- (2- (2-methyl-1, 3-dioxolanyl)) naphthalene (compound 100F, 160.0mg, 0.52mmol), p-toluenesulfonic acid monohydrate (4mg) and 30ml of benzene was refluxed for 12 hours. After cooling to room temperature, the reaction mixture was diluted with ether (100ml) and washed with 10% aqueous sodium bicarbonate, water and saturated aqueous sodium chloride. The organic layer was dried over magnesium sulfate, the solvent was removed under reduced pressure to give the objective compound, which was separated by column chromatography (10% ethyl acetate-hexane) to give the objective compound as a yellow oil: 1H NMR (CDCl)₃)：δ7.97(1H，d，J＝1.8Hz)7.67(1H，dd，J＝1.7，6.4Hz)，7.22(4H，s)，7.13(1H，d，J＝8.1Hz)，6.10(1H，t，J＝4.5Hz)，2.59(3H，s)，2.40(3H，s)，2.38(2H，d，J＝4.7Hz)，1.38(6H，s).4- [ 3-oxo-3- (7, 8-dihydro-5- (4-methylphenyl) -8, 8-dimethyl-2-naphthyl) -1-propenyl]-benzene Formic acid (Compound 101)

53.1mg (0.354mmol) of 4-carboxybenzaldehyde and 80mg (2.00mmol, 2.0ml of 1M aqueous sodium hydroxide solution) were added to a solution of 78.7mg (0.272mmol) of 3, 4-dihydro-1- (4-methylphenyl) -4, 4-dimethyl-6-acetylnaphthalene (compound 100G) in 4.0ml of methanol. The resulting solution was stirred at room temperature for 12 hours, concentrated under reduced pressure, and the residual oil was dissolved in ethyl acetate. The solution was treated with 10% hydrochloric acid, and the organic layer thereof was washed with water and a saturated aqueous sodium chloride solution, and then dried over sodium sulfate. Removal of the solvent under reduced pressure gave the title compound as a colorless solid, which was purified by recrystallization from acetonitrile: 1H NMR (acetone d 6): δ 8.00(7H, m), 7.83(1H, d, J ═ 15.6Hz), 7.24(4H, s), 7.13(1H, d, J ═ 8.1Hz), 6.12(1H, t, J ═ 4.5Hz), 2.42(2H, d, J ═ 4.8Hz), 2.38(3H, s), 1.41(6H, s). 3, 4-dihydro-1-phenyl-4, 4-dimethyl-6-acetylnaphthalene (Compound 100H)

496.2mg of phenylmagnesium bromide (2.54mmol, 2.54ml of 1M in diethyl ether) are added to a solution of 508.0mg (1.95mmol) of 3, 4-dihydro-4, 4-dimethyl-6- (2- (2-methyl-1, 3-dioxolanyl)) -1(2H) -naphthalenone (compound 100E) in 10ml of THF. The resulting solution was heated under reflux for 8 hours, water was added, and heating was continued for 30 minutes. THF was removed under reduced pressure and the aqueous residue was extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, concentrated under reduced pressure, and the objective compound was isolated from the residue by column chromatography (10% ethyl acetate-hexane) as a colorless oil: 1H NMR (CDCl)₃)：δ7.97(1H，d，J＝1.8Hz)，7.67(1H，dd，J＝2.1，8.0Hz)，7.34 (5H，m)，7.10(1H，d，J＝8.1Hz)，6.12(1H，d，J＝4.6Hz)，2.59(3H，s)，2.39(2H，d，J＝4.8Hz)，1.38(6H，s).4- [ 3-oxo-3- (7, 8-dihydro-5-phenyl-8, 8-dimethyl-2-naphthyl)) -1-propenyl group]Benzoic acid (section) Compound 103)120.0mg of sodium hydroxide (3.00mmol, 3.0ml of 1M aqueous solution) are added to a solution of 115.0mg (0.42mmol) of 3, 4-dihydro-1-phenyl-4, 4-dimethyl-6-acetylnaphthalene (compound 100H) and 65.0mg (0.43mmol) of 4-formyl-benzoic acid in 5.0ml of ethanol and 1.0ml of THF. The resulting yellow solution was stirred at room temperature for 12 hours. The solution was acidified with 6% aqueous hydrochloric acid and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, concentrated under reduced pressure, and column chromatographed (50% ethyl acetate-hexanes) to isolate the desired compound as a light yellow solid: 1H NMR (CDCl) ₃)：δ8.13(2H，d，J＝7.7Hz)，8.04(1H，s)，7.81(1H，d，J＝15.5Hz)，7.75(3H，m)，7.60(1H，d，J＝15.5Hz)，7.35(5H，m)，7.14(1H，d，J＝8.1Hz)，6.15(1H，t，J.＝4.2Hz)，2.41(2H，d，J＝4.2Hz)，1.41(6H，s).

Method for enhancing nuclear receptor agonists Overview and introduction

It has been found that retinoid antagonists as a subset, which exhibit negative hormone activity, are useful for enhancing the biological activity of other retinoids and steroid receptor superfamily hormones. The other retinoids and steroid receptor superfamily hormones may be endogenous hormones or pharmaceutical agents. Thus, for example, certain activities of a pharmaceutical retinoid agonist are potentiated in eliciting specific biological effects when used in conjunction with a retinoid negative hormone. This method of co-administration is advantageous in minimizing the undesirable side effects of the pharmaceutical retinoid since lower doses of the pharmaceutical retinoid can be used to improve the therapeutic effect.

More specifically, AGN 193109 (a synthetic retinoid having the structure shown in FIG. 1) has been found to exhibit unique and unexpected pharmacological activity. AGN 193109 shows high affinity for RAR subtype nuclear receptors without activating these receptors or stimulating transcription of retinoid response genes. AGN 193109, on the other hand, inhibits RARs activated by retinoid agonists, and thus acts as a retinoid antagonist.

In addition, it has been discovered that retinoid negative hormones can be used without co-administration of retinoid agonists or steroid hormones to control certain disease symptoms. More specifically, the retinoid negative hormones disclosed herein can down-regulate high levels of basal transcription of responsive genes to RARs that are not bound to ligands. For example, if uncontrolled cell proliferation results from activity of a gene in response to RARs not bound to a ligand, gene activity can be reduced by administration of a retinoid negative hormone that inactivates RARs. As a result, cell proliferation dependent on the activity of RARs not bound to a ligand can be inhibited by the negative hormone. Inhibition of RARs that are not bound to the ligand cannot be achieved using conventional antagonists.

Importantly, AGN 193109 has been found to inhibit both the basal activity of RAR and sometimes to potentiate the activity of other retinoids and steroid receptor superfamily hormonal agonists. In the context of the present invention, a negative hormone such as AGN 193109 is considered to enhance a hormonal stimulant if a reduced concentration of stimulant in the presence of the negative hormone elicits substantially the same quantitative response as that obtainable by the stimulant alone. The quantitative response may be determined, for example, in an in vitro reporter gene assay. Thus, if used with AGN 193109, AGN 193109 can potentiate a therapeutic retinoid that elicits a desired response when used at a particular dose and concentration, and a lower dose or concentration of the therapeutic retinoid can be used to produce substantially the same effect as a higher dose or concentration of the therapeutic retinoid when used alone. Agonists that may be enhanced by co-administration of AGN 193109 include RAR agonists, vitamin D receptor agonists, glucocorticoid receptor agonists, and thyroid hormone receptor agonists. More specifically, specific agonists that can be enhanced by co-administration include: ATRA, 13-cis retinoic acid, synthetic RAR agonist AGN 191183, 1, 25-dihydroxy vitamin D3, dexamethasone, and thyroid hormone (3, 3', 5-triiodothyronine). Also disclosed herein are methods that can be used to identify other hormones that can be enhanced by co-administration with AGN 193109.

Thus, AGN 193109 is not expected in a sense to be a simple retinoid antagonist, but rather a negative hormone that can potentiate the activity of various members of the nuclear receptor family. We also disclose a possible mechanism that could explain the negative hormone activity and the ability of AGN 193109 to enhance the activity of other nuclear receptor ligands. This mechanism combines elements (elements) known to be involved in retinoid-dependent information transmission channels and additionally novel negative regulatory components.

One of ordinary skill in the art will appreciate that RARs, which are high affinity targets for AGN 193109 binding, are transcription factors that regulate the expression of various retinoid response genes. The binding site for cis-regulatory DNA as RARs has been identified as a proximal gene that is transcriptionally regulated in a retinoid-dependent manner. RARs, known as Retinoic Acid Response Elements (RAREs), which bind to the DNA site have been defined in detail. It is important that the RAREs bind to a heterodimer consisting of one RAR and one RXR. The RXR component of the heterodimer functions to promote high affinity interactions between RAR/RXR heterodimers and RARE's (Magelsdorf et al The Retinoids：Biology，Chemistry and Medicine，2nd edition，eds.Sporn et al.，Raven Press，Ltd.，New York 1994)。

As described in detail below, findings relating to the negative hormonal activity of AGN193109 are consistent with a mechanism involving the interaction of a putative Negative Coactivator Protein (NCP) with RAR. According to the proposed mechanism, this interaction is stabilized by AGN 193109.

Our results further show that AGN193109 can modulate the intracellular effectiveness of NCPs that interact with nuclear receptors other than the RARs occupied by AGN 193109. Second, AGN193109 may enhance transcriptional regulatory delivery channels including nuclear receptors sharing the ability to bind NCP with such RARs. In this regard, AGN193109 showed the ability to modulate different nuclear receptor transport pathways and to modulate activities not expected for conventional retinoid antagonists. Therefore, AGN193109 is useful as an agent that enhances the activity of nuclear receptor ligands, including endogenous hormones and prescription drugs. This example of specificity illustrates the more general principle that any nuclear receptor negative hormone will enhance the activity of other nuclear receptors that competitively bind NCP.

Although other materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. General references that can be used to accomplish the various nucleic acid processing and procedures described herein can be found in Molecular Cloning: the laboratory Manual (Sambrook et al eds. Cold Spring Harbor Lab publ.1989) and Current Protocols in Molecular Biology (Autobel et al eds., Greene Publishing associations and Wiley-Interscience 1987). The following is a description of the experiments and results leading to the formation of the present invention.

Example 6 describes a method for demonstrating that AGN 193109 binds with high affinity to each of three RARs without activating retinoid-dependent gene expression.

Example 6

AGN 193109 binds RARs with high affinity without transactivating retinal activity Derivative dependent gene expression

The composition was prepared using a method essentially according to Allegretto et al [ j.biol.chem.268: 26625(1993)]The method adopts a baculovirus expression system to respectively express human RAR-alpha, RAR-beta and RAR-gamma receptors as recombinant proteins. The recombinant receptor proteins were used separately to generate a protein using Heyman et al [ Cell 68: 397(1992)]The [ 2 ]³H]ATRA displacement assay to determine the binding affinity of AGN 193109.According to Cheng et al Biochemical Pharmacology 22: 3099(1973)]The method determines the dissociation constant (Kds).

AGN 193109 was also tested for its ability to transactivate RARs in CV-1 cells transiently co-transfected with RAR expressing vectors and retinoid response reporter constructs. The receptor expression vector pRShRAR- α [ Giguere et. Nature 330: 624(1987)]、pRShRAR-β[Benbrook et al.Nature333：669(1988)]And pRShRAR- γ [ Ishikawa et al. mol. Endocrinol.4: 837(1990) ]Co-transfection was performed separately. The delta MTV-TREP-Luc plasmid was compared with Umesono et al [ Nature 336: 262(1988)]The Δ MTV-TREP-CAT reporter construct is essentially identical except that the Chloramphenicol Acetyltransferase (CAT) reporter gene is replaced by a firefly luciferase encoded by the polynucleotide sequence. Molecular Cloning was used: a Laboratory Manual (Sambrook et al eds. Cold Spring Harbor Lab publ.1989) described calcium phosphate co-precipitation procedure to complete the transfection procedure of green monkey CV-1 cells. At a density of 4 × 10⁴Perwell, flat CV-1 cells were seeded onto 12-well multi-well flat-plated plates and transiently transfected with calcium phosphate pellets containing 0.7. mu.g reporter plasmid and 0.1. mu.g recipient plasmid according to standard protocols. After 18 hours, the cells were washed to transfer the pellet and added to Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10% charcoal-extracted bovine embryo serum (Gemini Bio-Products). Vehicle (ethanol) or AGN 193109 (10) alone^-9-10^-6M) cells were treated for 18 hours. Cell lysates were prepared in 0.1M KPO4(pH7.8), 1.0% TRION X-100, 1.0mM DTT, 2mM EDTA. According to de Wet et al [ mol.cell.biol.7: 725(1987)]The method uses firefly luciferin (antibiotic luminescence Laboratory) and EG &The luciferase activity was measured by a G Berthold 96-well planar seeded plate luminometer. The reported luciferase values are expressed as mean ± SEM of triplicate determinations.

The results in Table 11 show that AGN 193109 binds with high affinity to each of RAR- α, RAR- β and RAR- γ without activating retinoid-dependent gene expression. More precisely, AGN193109 binds to each of three receptors with Kd values in the range of 2-3 nM. Despite this tight binding, AGN 193109 did not activate gene expression when compared to induction stimulated by ATRA. Thus, half the maximum effective concentration of AGN 193109 (EC)₅₀) It could not be measured. Although not shown in the table, AGN was also found to have an undetectable affinity for RXRs.

TABLE 11

AGN 193109 binding and transactivation of RARs

	RAR-α	RAR-β	RAR-γ
				EC50(nM)	Is inactive	Is inactive	Is inactive
Kd(nM)	2	2	3

Example 7 describes a method for demonstrating that AGN 193109 is an antagonist of ATRA-dependent gene expression.

Example 7

AGN 193109-dependent inhibition of RAR transactivation by ATRA

The ability of AGN 193109 to antagonize ATRA-mediated RAR activation was studied in the co-transfection of CV-1 cells by the calcium phosphate co-precipitation method of Sambrook et al (Molecular Cloning: A Laboratory Manual Spring Harbor Lab Publ.1989). The cells were collected using a Hollenberg et al [ Cell 55: 899(1988) ]The Δ MTV-Luc reporter plasmid encodes a eukaryotic cell expression vector pRShRAR- α [ Giguere et al Nature 330: 624(1987)]、pRShRAR-β[Benbrook et al.Nature 333：669(1988)]And pRShRAR- γ [ Ishikawa et al. mol. Endocrinol.4: 837(1990)]And (4) carrying out cotransfection. The reporter plasmid contains two replicative TRE-recurrent response elements strikingly. The calcium phosphate transfection procedure was performed exactly as described in example 6. With the single solvent (ethanol), ATRA (10)^-9-10^-6M)、AGN 193109(10^-9-10^-6M) or with AGN 193109 (10)^-9-10^-6M) mixed 10^-8MATRA treated cells for 18 hours. The cell lysates and luciferase activity were also assayed as described in example 6.

The results of these procedures are shown in FIGS. 2A-2F, where luciferase values are expressed as mean. + -. SEM of triplicate determinations. More specifically, the results shown in FIGS. 2A, 2C and 2E indicate that stimulation of transfected cells with ATRA results in an increase in dose response in luciferase activity. This confirms that ATRA activates each of the three RARs in the experimental system and provides a basis for comparison as a measure of antagonist activity. The plot results shown in figures 2B, 2D and 2F show that co-treatment of transfected cells with 10nM ATRA and increasing the concentration of AGN 193109 results in inhibition of luciferase activity. Especially the same dose of AGN 193109 and ATRA resulted in greater than 50% inhibition of all three RAR subtypes compared to ATRA alone. Comparison of ATRA dose responses in the presence of different concentrations of AGN 193109 showed that ATRA was competitively inhibited by AGN 193109. It is apparent that the horizontal axis on all graphs shown in FIG. 2 represents the log of the concentration of retinoid. These results demonstrate that AGN 193109 is a potent RAR antagonist.

Next, we performed experiments to elucidate the mechanism of the activity of the AGN193109 antagonist below. One of ordinary skill in the art will understand that: nuclear receptor activation is thought to involve conformational changes in the receptor induced by ligand binding. The results of the protease protection assay have indeed demonstrated that nuclear hormone agonists and antagonists cause receptor proteins to adopt different conformational forms [ Keidel et al mol cell biol 14: 287 (1994); alan et al.j.biol.chem.267: 19513(1992)]. We used this test to determine if AGN193109 and ATRA caused RAR- α to adopt different conformations. AGN 193583 (RAR-. alpha.selective antagonist) was used as a positive control and was known to be an antagonist-specific model of protease sensitivity.

Example 8 describes a method for detecting a conformational change in RAR- α resulting from the binding of AGN 193109. As shown below, the results of this procedure unexpectedly show that AGN193109 leads to a trypsin sensitivity model that is essentially the same as that induced by ATRA (RAR agonist), but different from that induced by a model of a model RAR antagonist. This finding suggests that AGN193109 has properties that differ from those of other retinoid antagonists.

Example 8

Protease protection assay

Constructed in the vector pGEM3Z (Pharmacia) and containing RAR- α cDNA [ Giguereet al. Nature 330: 624(1987)]The plasmid of (a) is used in combination with a TNT-coupled reticulocyte lysate in vitro transcription-translation system (Promega) to prepare³⁵S]Methionine-labeling RAR-. alpha.s. Limited proteolytic digestion of the labeled protein RAR- α was performed according to Keidel et al [ mol.cell.biol.14: 287(1994)]The method is carried out. Is prepared by mixing equal parts of³⁵S]Mesocyte lysates of methionine-labelled RAR-. alpha.were incubated with ATRA, AGN 193583 or AGN193109 in a total volume of 9. mu.l for 45 min under freezing. For all experiments, retinitis yellowThe final concentration of aldehyde derivative was 100nM for ATRA and AGN193109 and 1000nM for AGN 193583. The difference between the final concentrations of the retinoid was about 10-fold greater based on the difference in relative affinities of ATRA and AGN193109 (with Kd at RAR-. alpha.of 2 and 10nM, respectively) and AGN 193583 (with Kd at RAR-. alpha.of 100 nM). After ligand binding, 1 μ l of trypsin at the appropriate concentration was added to the mixture to give a final concentration of 25, 50 or 100 μ g/ml. The samples were incubated at room temperature for 10 minutes and the tryptic digestion was stopped by adding SDS-sample buffer. Samples were subjected to polyacrylamide gel electrophoresis and autoradiography according to standard methods.

Both the agonist and antagonist produce different models of trypsin sensitivity that differ from the results obtained from digestion of receptors that are not bound to ligands. The results of the autoradiography show that the radiolabeled RAR- α was completely digested with trypsin at a concentration of 25, 50 or 100 μ g/ml at room temperature within 10 minutes without the addition of retinoids. Pre-association of ATRA produces the appearance of two major protease-resistant species. Pre-binding of the RAR- α selective antagonist AGN 193583 produces protease-resistant species with a molecular weight lower than that of the species produced by pre-binding of ATRA. The results show that retinoid agonists and antagonists cause conformational changes detectable by altered trypsin sensitivity. Surprisingly, AGN193109 prebonds to produce a protease protection pattern that is indistinguishable from that produced by ATRA prebonding.

The above results confirm that AGN193109 binds to RAR- α and changes its conformation. Interestingly, this conformational change is more similar in nature to that produced by agonist (ATRA) binding compared to that produced by antagonist (AGN 193583) binding. Clearly, the mechanism of AGN 193109-dependent antagonism is unique.

We considered possible mechanisms that could explain the activity of AGN193109 antagonists. In particular, we used standard gel shift assays to test whether AGN193109 interfered with RAR/RXR heterodimer formation or inhibited the interaction between RAR and its cognate DNA binding site.

Example 9 presents a gel electrophoresis mobility shift assay to demonstrate that AGN193109 neither inhibits RAR/RXR dimerization nor dimer binding to targeted DNA.

Example 9

Gel shift analysis

Except for omitting³⁵In addition to the S-labeled methionine, in vitro translated RAR- α was generated essentially as described in example 8. The samples were collected using a cell containing a Mangelsdorf et al [ Nature 345: 224-229(1990)]The pBluescript (II) (SK) -based vector of the RXR-alpha cDNA was used as a template for in vitro transcript generation to similarly generate in vitro translated RXR-alpha. (ii) reacting said labelled RAR-alpha and RXR-alpha (alone or in admixture) or with AGN193109 (10)^-6M) was previously combined (alone or in mixture) with a peptide having the sequence 5'-TCAGGTCACCAGGAGGTCAGA-3' (SEQ id no: 1) the end-labeled DR-5 RARE double-stranded probe of (1) interacts. The binding mixture was electrophoresed and autoradiographed on a native polyacrylamide gel according to standard laboratory procedures. The individual delayed species (species) present on the autoradiogram spectrum, common to all paths on the gel, represent undefined bound probe factors present in reticulocyte lysates. Only the RAR/RXR combination produces retinoid receptor-specific delayed species. Neither RAR alone nor RXR alone in combination with the probe produced such a shift. The presence of AGN193109 does not reduce this interaction.

These results show that AGN 193109 does not substantially alter the homo-or hetero-dimeric nature of RAR- α. In addition, AGN 193109 did not inhibit the interaction of receptor dimers with DNA fragments containing cognate binding sites.

In view of the unique properties that characterize AGN 193109, we continued to investigate whether this antagonist would otherwise inhibit the activity of RARs that are not bound to ligand. The receptor/reporter plasmid system used to perform this assay advantageously exhibits high levels of constitutive activity in the absence of added retinoid agonists. More specifically, these methods employ ER-RAR chimeric receptors and the ERE-tk-Luc reporter system. The ERE-tk-Luc plasmid includes the-397 to-87 regions of estrogen response in the 5' -flanking region of the Xenopus vitellogenin a2 gene upstream of the plasmid tk-Luc linking the HSV thymidine kinase promoter and the fluorescein reporter gene [ as described by Klein-Hitpass et al, Cell 46: 1053-1061(1986)]. The ER-RAR chimeric receptor consists of an estrogen receptor DNA binding domain fused to the "D-E-F" domain of the RARs. One of ordinary skill in the art will appreciate that this "D-E-F" domain functions to bind to retinoids, to provide retinoid-induced transactivation and to provide a contact site for heterodimerization with RXR. Thus, luciferase expression in the reporter system is dependent on activation of the transfected chimeric receptor construct.

Example 10 describes a method for demonstrating that AGN 193109 inhibits the basal gene activity resulting from RARs not bound to ligand. These processes are carried out without the addition of a retinoid agonist. The results presented below provide the first demonstration that AGN 193109 exhibits negative hormonal activity.

Example 10

Gene for retinoid-regulated reporter plasmids in transient cotransfected cell lines Repression of basal Gene Activity

CV-1 cells were co-transfected with the ERE-tk-Luc reporter plasmid and either the ER-RAR- α, ER-RAR- β or ER-RAR- γ expression plasmids. The ERE-tk-Luc plasmid contains the estrogen responsive promoter element of the Xenopuslaevis vitellogenin A2 gene and is compatible with the estrogen responsive promoter element of Klein-Hitpass et al [ Cell 46: 1053(1986)]Except that the CAT reporter gene is replaced by luciferase encoded by the polynucleotide sequence. Graupner et al [ biochem.biophysis.res.comm.179: 1554(1991)]Polynucleotide encoding ER-RAR-alpha, ER-RAR-beta or ER-RAR-gamma chimeric receptors for use in such co-transfection has been describedAnd (4) acid. These polynucleotides were compared to Ellis et al [ Cell 45: 721(1986)]The pECE expression vector is ligated and expressed under the transcriptional control of the SV-40 promoter. The calcium phosphate transfection procedure was performed exactly as described in example 6, using 0.5. mu.g/well of reporter plasmid and 0.05. mu.g, 0.10. mu.g or 0.2. mu.g/well of recipient plasmid. With the single solvent (ethanol), ATRA (10) ^-9-10^-6M) or AGN 193109 (10)^-9-10^-6M) cells were treated for 18 hours. The determination of cell lysates and luciferase activity was performed as described in example 6.

The results shown in FIGS. 3A, 4A and 5A confirm that ATRA strongly induces luciferase expression in all transfectants. The basal levels of luciferase, which are isoforms of these three transfected chimeric RAR, were expressed in the range of about 7000-40000 relative light units (light units) (rlu) and were somewhat dependent on the amount of recipient plasmid used in the transfection. Thus, as expected, these three chimeric receptors may be ATRA activated. More precisely, all three receptors bind to ATRA and activate transcription of the luciferase reporter gene that resides transiently on the ERE-tk-Luc plasmid.

Figures 3B, 4B and 5B show AGN 193109 dose response curves obtained in the absence of any exogenous retinoid agonist. Interestingly, ER-RAR- α (FIG. 3B) was essentially unaffected by AGN 193109, while ER-RAR- β and ER-RAR- γ chimeric receptors (FIGS. 4B and 5B, respectively) showed a decrease in luciferase reporter plasmid activity in response to AGN 193109 dose.

We additionally investigated negative hormone activity by examining the ability of AGN 193109 to inhibit gene expression mediated by chimeric RAR-gamma receptors designed to have a constitutive transcription activator domain. More specifically, we used a constitutively active RAR- γ chimeric receptor fused to the acidic activator domain of HSV VP-16 (termed RAR- γ -VP-16) in two types of luciferase reporter systems. The first consisted of an ERE-tk-Luc reporter plasmid co-transfected with ER-RARs and ER-RXR- α. The second method utilizes a delta MTV-TREP-Luc reporter plasmid to replace an ERE-tk-Luc reporter plasmid.

Example 11 presents a method for demonstrating that AGN 193109 can inhibit the transcriptional activator domain activity of RAR. The results shown below demonstrate that AGN 193109 can inhibit RAR-dependent gene expression in the absence of an agonist and confirm that AGN 193109 exhibits negative hormonal activity.

Example 11

Repression of RAR-VP-16 Activity in transiently transfected cells

CV-1 cells were transiently co-transfected with 0.5. mu.g/well of the ERE-tk-Luc luciferase reporter plasmid, 0.1. mu.g/well of the ER-RXR- α chimeric receptor expression plasmid, and 0. mu.g or 0.1. mu.g/well of the RAR- γ -VP-16 expression plasmid, according to the calcium phosphate co-precipitation technique described in example 6. The chimeric receptor ER-RXR- α consists of a fusion estrogen receptor DNA binding domain [ gracener et al. 1554(1991) ] RXR- α [ Magelsdorf, et al. Nature 345: 224-229(1990) hormone binding domain (amino acids 181-458) and consists of a polypeptide based on the sequence of Ellis et al [ Cell 45: 721(1986), and SV-40 expression of the expression vector pECE. RAR- γ -VP-16 was compared with Nagpal et al [ EMBO J.12: 2349(1993) the VP16RAR- γ 1 expression plasmid is identical and encodes a chimeric protein with the activation domain of the VP-16 protein of HSV fused to the amino-terminus of full-length RAR- γ. 18 hours after transfection, cells were rinsed with Phosphate Buffered Saline (PBS) and DMEM (Gibco-BRL) containing 10% FBS (Gemini Bio-Products) from which retinoids had been removed by charcoal extraction was added. Cells were treated with AGN 193109 or ATRA in ethanol vehicle or ethanol alone at appropriate dilutions for 18 hours, then rinsed with PBS and lysed with 0.1MKPO4(pH7.8), 1.0% TRITON X-100, 1.0nM DTT, 2nM EDTA. According to de Wet et al [ mol.cell.biol.7: 725(1987), luciferase activity was measured using firefly luciferin (Analytical luminence Laboratory) and an EG & G Berthold 96-well planar seeded luminometer. Luciferase values are expressed as mean + -SEM of triplicate determinations.

As shown in fig. 6, CV-1 cells were transfected with ERE-tk-Luc reporter constructs, and the ER-RAR- α chimeric expression plasmid showed weak activation of luciferase activity by ATRA, probably due to isomerization of ATRA to 9C-RA, a natural ligand for RXRs [ Heyman et al, cell 68: 397(1992)]. Cells transfected with the same mixture of reporter plasmid and chimeric receptor plasmid, but treated with AGN 193109 did not show any effect on luciferase activity. This latter result is expected because AGN 193109 does not bind to RXRs. CV-1 cells were similarly transfected with the ERE-tk-Luc reporter plasmid, however the ER-RAR chimeric receptor expression plasmid was substituted for ER-RXR- α which showed a very strong induction of luciferase activity after ATRA treatment.

In contrast, the inclusion of the RAR- γ -VP-16 expression plasmid with the ER-RXR- α and ERE-tk-Luc plasmids in the transfection mixture resulted in a significant increase in basal luciferase activity when assayed without any added retinoid. The observed increase in basal luciferase activity for ER-RXR- α/RAR- γ -VP-16 cotransfectants indicates that recombinant ER-RXR- α and RAR- γ -VP-16 proteins can heterodimerize when compared to results obtained for cells transfected with ER-RXR- α alone. The interaction of this heterodimer with the cis-regulatory estrogen response element results in targeting of the VP-16 activation domain to the promoter region of the ERE-tk-Luc reporter plasmid. Treatment of the triple transfected cells with ATRA resulted in a modest increase in luciferase activity above the high basal level. However, treatment of this triple transfectant with AGN 193109 resulted in a dose-dependent decrease in luciferase activity. Importantly, FIG. 6 shows that AGN 193109 treatment of cells co-transfected with ER-RXR- α and RAR- γ -VP-16 resulted in a cell size of about 10 ^-8The repression of luciferase activity with maximal inhibition occurred under M AGN 193109.

Our observation that AGN 193109 represses the constitutive transcriptional activation function of RAR- γ -VP-16 in the presence of RXR is explained by a model in which binding of AGN 193109 to RAR results in a conformational change in RAR that stabilizes the negative conformation that promotes binding of the trans-acting negative coactivator protein. When AGN 193109/RAR complex is bound by NCP, RAR can up-regulate transcription of genes that are normally responsive to activated RARs. Our model further suggests that the intracellular stores of NCP are in some way limited in concentration and can be depleted by the stimulation of complexation by AGN 193109 to RARs.

The results shown in fig. 6 additionally show even at 10^-6Under M AGN 193109, ER-RXR-alpha and RAR-gamma-VP-16 proteins can interact to form heterodimers that can activate the transfection of the reporter gene. More specifically, transfection with ER-RXR-alpha and RAR-gamma-VP-16 was performed at a concentration sufficient to provide maximal inhibition (10)^-8-10^-6M) gave a luciferase activity with an indication of about 16000 rlu. In contrast, transfection with ER-RXR-alpha alone was followed by concentrations as high as 10 ^-6M AGN193109 treated cells showed luciferase expression levels of only about 8000 rlu. Even at 10^-6The fact that higher levels of luciferase activity were obtained in cells expressing ER-RXR- α and RAR- γ -VP-16 in the presence of M AGN193109 indicates that there is a sustained interaction between these two recombinant receptors. Repression of RAR- γ -VP-16 activity by AGN193109 suggests that modulation of the (codon) NCP interaction can be co-controlled with VP-16 activation. We therefore recognized that it is possible to modulate the expression of genes that are not normally regulated by retinoids in an AGN 193109-dependent manner.

Candidates for the AGN193109 modulatory gene include those which are activated by transcription factor complexes and which are composed of RARs binding or heterodimeric non-RAR factors which do not require RAR agonist activation. Whereas stimulation with RAR agonists had essentially no effect on the expression of this gene, administration of AGN193109 promoted the formation of an inactive transcription complex containing AGN 193109/RAR/NCP. Second, the addition of AGN193109 retinoid negative hormone down regulates transcription of other retinoid-insensitive genes.

The same mechanism may explain that AGN 193109 can repress tissue transglutaminase (TGase) gene activity in HL-60 cells. The retinoid-responsive element, consisting of three typical retinoid half-sites separated by 5 and 7 base pairs, was always identical in the transcriptional control region of the gene. Although TGase can be induced by RXR selective agonists, it is not responsive to RAR selective agonists. TGase retinoid response elements were bound by RAR/RXR heterodimers (Davies et al. in Press). Interestingly, AGN 193109 was able to block TGase activity induced by RXR agonists. The ability to block the activity of the relevant RXR by separating NCPs from the RAR component of the heterodimer by this negative hormone could explain this AGN 193109 mediated repression.

We also obtained results supporting conclusions consistent with those described in example 11 by using RAR- γ -VP-16 and expression constructs and using the AMTV-TREP-Luc reporter plasmid in combination with the ERE-tk-Luc reporter plasmid instead of RAR- γ -VP-16 and ER-RXR- α expression constructs. In agreement with the above results, we found that AGN 193109 inhibited the activity of RAR- γ -VP-16 on the Δ MTV-TREP-Luc reporter plasmid. Thus, AGN 193109 represses the activity of RAR- γ -VP-16 when the chimeric receptor binds directly to the retinoic acid receptor response element instead of indirectly to the estrogen response element in the promoter region of the reporter plasmid. These findings demonstrate that assays for identifying agents having negative hormone activity need not be limited to the use of a particular reporter plasmid. Instead, an important feature included in the experimental system for the identification of retinoid negative hormones involves testing the ability of the compounds to suppress the activity of RARs designed to contain constitutive transcriptional activation domains.

In general, retinoid negative hormones can be identified as a subset of retinoids that repress substantial levels of expression of reporter genes in transfected cells as a transcriptional response to direct or indirect binding to a retinoid receptor or chimeric receptor comprising a retinoid receptor domain located at least C-terminal to the DNA binding domain of the receptor. This method has been adopted as a screening method for identifying a retinoid negative hormone. In various embodiments of the screening methods of the present invention, the receptor structure used to look for negative hormones is variable. More specifically, the retinoid receptor may be a subtype of RAR or RXR. The receptor may optionally be designed to include a constitutive transcriptional activator domain. Retinoid receptors used to screen for negative hormones may optionally contain a heterogeneous DNA binding domain as a surrogate for the DNA binding domain endogenous to the native receptor. However, when a second receptor is used in the screening method, wherein the second receptor can dimerize with a retinoid receptor used to look for negative hormones, then the retinoid receptor may not require a DNA binding domain because it can be linked to the transcriptional control region of the receptor gene indirectly through dimerization with the second receptor itself binding to the transcriptional control region.

In the practice of the screening method, the ability of a compound to repress basal expression of a reporter plasmid is typically determined in an in vitro assay. Basal expression represents the baseline level of reporter plasmid expression in transfected cells in the absence of exogenously added retinoid agonist. Steps can optionally be taken to remove endogenous retinoid ligands from the periphery of the transfected cells by methods such as extraction of serum from the cells for in vitro culture by activated charcoal.

Examples of reporter genes for use in the screening methods include those encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, or cell surface antigens that can be detected by immunochemical methods. Indeed, it was not expected that the nature of the reporter gene was critical to the operability of the method. However, the transcriptional regulatory region of the construct of the reporter gene must include one or more cis-regulatory elements that are targets for finding transcription factors for negative hormones. For example, if one wishes to identify a RAR negative hormone, the reporter construct transcriptional regulatory region may contain cis-regulatory elements that may be bound by a RAR-containing protein. In this example, there should be a corresponding relationship between the DNA binding domain of the RAR and the cis-regulatory element of the transcriptional regulatory region of the reporter construct. Thus, if a chimeric RAR with a constitutive transcription activator domain and a DNA binding domain (which can bind to a cis-regulatory estrogen response element) is used in the screening method, the transcriptional regulatory region of the reporter construct should contain an estrogen response element.

Mangelsdorf et al [ The Retinoid Receptors in The Retinoids: examples of cis-regulatory elements that directly bind to retinoid receptors (RAREs) for use in reporter gene assays are disclosed by Biology, chemistry and Medicine, 2nd edition, eds. Examples of cis-regulatory elements that indirectly bind to a chimeric receptor include a DNA binding site for any DNA binding protein, wherein the DNA binding domain of the protein can be added to a chimeric receptor consisting of the DNA binding domain linked to a retinoid receptor. Specific examples of heterogeneous DNA binding domains that can be designed into chimeric receptors and will recognize heterogeneous cis regulatory elements include those that recognize estrogen response elements. Thus, the retinoid receptor portion of the chimeric receptors used in the identification methods need not contain the DNA binding of the retinoid receptor, but must at least contain the ligand binding domain of the retinoid receptor.

Other examples of indirect retinoid receptors that bind the cis-regulatory element include the use of proteins that bind the cis-regulatory element and dimerize with the retinoid receptor. In this case, the retinoid receptor binds to the cis-regulatory element only by binding to the protein responsible for DNA binding. Examples of such systems would include the use of fusion proteins consisting of a heterogeneous DNA binding domain fused to RXR and containing at least the domain responsible for RXR dimerization with RARs. Co-induced RARs can dimerize with the fusion protein that binds cis-regulatory elements. We believe that any cis-regulatory element-binding protein that dimerizes RARs resulting in indirect binding of the RARs to the cis-regulatory element would also be suitable for use in negative hormone screening methods.

In a preferred embodiment of the screening method, a retinoid negative hormone is identified as one that suppresses basal expression of a designed RAR transcription factor that has increased basal activity. Although not necessary for the operability of the screening method, the design RARs used in the following examples include a constitutive transcriptional activation domain. The use of chimeric receptors in the absence of any retinoid provides a good means for increasing basal expression of reporter genes. Although transient transfection has been used in the methods detailed above, stably transfected cell lines constitutively expressing chimeric receptors are also used in the screening methods.

Chimeric retinoid receptors with constitutive transcriptional activator domains are heterodimeric for designing a second receptor with a DNA binding domain specific for an estrogen responsive cis-regulatory element, as disclosed in the following examples. In this case, a chimeric retinoid receptor with a constitutive transcription activator domain is indirectly associated with the cis-regulatory region controlling reporter gene expression via binding to a second receptor that binds to a DNA targeting sequence. More specifically, the second receptor is designed to contain a DNA binding domain that recognizes an estrogen responsive element. The reporter gene with an estrogen response element in the upstream promoter region is more favorable for retinoid agonist non-response in the absence of a transfected chimeric receptor with a constitutive transcriptional activator domain. Thus, all reporter gene activity is due to the transfected receptor. The use of the estrogen responsive element DNA binding domain in combination with the estrogen responsive element cis regulatory element is for illustrative purposes only. One of ordinary skill in the art will appreciate that other combinations of engineered (engineered) receptors specific for non-RARE cis-regulatory elements are also useful in the practice of the screening methods of the present invention.

The cells used in the screening method are eukaryotic cells that can be transfected. These cells may be animal cells such as human, primate or rodent cells. We have achieved very good results using CV-1 cells, however, it is well anticipated that other cultured cell lines may be successfully used. Any conventional transfection method known in the art can be used to induce expression constructs encoding chimeric retinoid receptors with constitutive transcriptional activator domains.

The constitutive transcriptional activator domain consists of a majority of amino acids, which may have an overall acidity represented by one negative charge at neutral pH. For example, the constitutive transcription activator domain can have an amino acid sequence that is also found in viral transcription factors. An example of a viral transcription factor with a constitutive transcription activator domain is herpes simplex virus 16. However, other viral or synthetic transcriptional activator domains may also be used in the construction of expression constructs encoding chimeric retinoid receptors with constitutive transcriptional activator domains.

We have developed a generalized screening method for identifying retinoid negative hormones, as described below. The screening method provides a means to distinguish simple antagonists from negative hormones. Table 12 lists several retinoid compounds that show effective affinity for RAR- γ, except ATRA, which do not transactivate the receptor in a transient co-transfection transactivation assay. Thus, we examined whether these compounds are RAR- γ antagonists, if any, of which exhibit negative hormonal activity.

Example 12

Testing for retinoid negative hormones

By the method described in Molecular Cloning: a Laboratory Manual (Sambrook et al eds. Cold Spring Harbor Lab Publ.1989) using 0.5 μ g of ERE-tk-Luc reporter plasmid and 0.1 μ gER-RAR- γ [ Grauper et al biochem. Biophys. Res. Comm.179: 1554(1991)]Chimeric expression plasmid 4X 10⁴CV-1 cell transfection. After 18 hours, the cells were rinsed with PBS and DMEM (Gibco-BRL) containing 10% charcoal-extracted FBS (Gemini Bio-Products) was added. 10 in ethanol^-8M ATRA or ethanol alone treated cells. In addition, use 10^-9、10^-8、10^-7Or 10^-6M compounds listed in table 12 treated ATRA-treated cells. After 18 hours, cells were rinsed in PBS and lysed at 0.1M KPO₄(pH7.8), 1.0% TRITON X-100, 1.0mM DTT, 2mM EDTA. According to de Wet et al [ mol.cell.biol.7: 725(1987)]The method measures luciferase activity.

TABLE 12Compound Kd (nM) @ RAR-gamma^a EC₅₀(nM)@RAR-γ^bATRA 1217 AGN 1931096 na (Compound 60) AGN 19317452 na (Compound 34a) AGN 19319930 naAGN 19338525 na (Compound 23) AGN 19338913 na (Compound 25) AGN 19384040 naAGN 19387130 na (Compound 50)

a is passing through³H-ATRA competes for binding to baculovirus-expressed RAR-gamma and relative affinity (Kd) as determined using the Cheng-Prussof equation.

b EC determined in CV-1 cells transiently co-transfected with. DELTA.MTV-TREP-Luc and RS-RAR-gamma₅₀. "na" represents no activity.

As shown in part by the results shown in figure 7 and table 12, with the exception of ATRA, all compounds listed in table 12 are retinoid antagonists at RAR- γ.

Second, RAR-gamma antagonists identified in Table 2 were screened to determine if any were retinoid negative hormones. According to the method in Molecular Cloning: a laboratory Manual (Sambrook et al eds. Cold Spring Harbor Lab Publ.1989) described calcium phosphate procedure with 0.5 μ g of ERE-tk-Luc reporter plasmid and 0.1 μ gER-RXR- α [ Grauper et al biochem. Biophys. Res. Comm.179: 1554(1991)]And 0.2 μ g RAR- γ -VP-16[ Nagpal et al EMBO J.12: 2349(1993)]Chimeric expression plasmid 4X 10⁴CV-1 cell transfection. After 18 hours, the cells were rinsed with PBS and DMEM (Gibco-BRL) containing 10% charcoal-extracted FBS (Gemini Bio-Products) was added. By 10^-9、10^-8、10^-7Or 10^-6M each compound listed in table 12 treated cells. Cells were treated with ethanol vehicle alone as a negative control. After 18 hours, cells were rinsed in PBS and lysed at 0.1M KPO ₄(pH7.8), 1.0% TRITON X-100, 1.0mM DTT, 2mM EDTA. According to de Wet et al [ mol.cell.biol.7: 725(1987)]The luciferase activity was measured by the method described above.

As shown in fig. 8, the retinoid antagonists in table 12 can be classified into two classes based on their effects on constitutive transfection-activating function of RAR- γ -VP-16 chimeric retinoid receptors. One class (including AGN 193174, AGN 193199, and AGN 193840) does not block RAR- γ -VP-16 activity despite their ATRA antagonists. In contrast, AGN193109, AGN 193385, AGN 193389 and AGN 193871 showed dose-dependent repression of RAR- γ -VP-16 constitutive activity. Thus, while both groups of compounds are RAR-gamma antagonists, only the second group of compounds exhibits negative hormonal activity. This test method can advantageously distinguish retinoid negative hormones from simple retinoid antagonists.

The results of the foregoing tests indicate that AGN193109 meets the criteria for defining a negative hormone. More specifically, the results shown in example 11 demonstrate that AGN193109 has the ability to exert inhibitory activity on RARs even in the absence of exogenously added retinoid ligands. Thus, the biologically active compounds are not dependent on blocking the interaction between RARs and agonists such as ATRA and AGN 191183. These findings lead to the conclusion that: AGN193109 stabilizes the interaction between RARs and NCPs. As shown in FIG. 9, the interaction of NCP/RAR/PCP exists in an equilibrium state. The effect of the stimulant is to increase PCP interaction and decrease NCP interaction. As previously described, our experimental results revealed that the intracellular effectiveness of NCP for other receptors can be modulated by administration of AGN 193109. More specifically, we found that AGN193109 could promote the complexing action of NCP with RARs, thereby reducing the intracellular stores of NCP available for interaction with transcription factors other than RARs.

Second, we examined the effect of AGN 193109 on agonist-mediated inhibition of AP-1-dependent gene expression. In endocr.rev.14: 651(1993), Pfhal discloses that retinoids can down-regulate gene expression by mechanisms including inhibition of AP-1 activity. We hypothesized that AGN 193109 may have one of two effects when used in combination with a retinoid agonist in a model system designated to measure AP-1 activity. First, it is believed that AGN 193109 has antagonized the effects of the agonist, thereby alleviating agonist-dependent inhibition of AP-1 activity. Furthermore, AGN 193109 may have enhanced the activity of the agonist, thereby exaggerating the agonist-dependent inhibition of AP-1 activity.

Example 13 describes a method for demonstrating that AGN 193109 enhances the anti-AP-1 activity of retinoid agonists. AGN 191183 retinoid agonists weakly inhibited AP-1 dependent gene expression as described below. AGN 193109 in combination with a retinoid agonist strongly inhibited AP-1 dependent gene expression. AGN 193109 itself has essentially no activity against AP-1.

Example 13

AGN 193109 enhances anti-AP-1 activity of retinoid agonists

Such as Giguere et al [ Nature 33: 624(1987) ] HeLa cells were transfected using LIOFECTAMINE (Life Technologies, Inc.), using 1. mu.g of Str-AP1-CAT reporter construct and 0.2. mu.g of plasmid pRS-hRAR α. The expression of the promoter was determined by cloning the HindIII-BamHI site corresponding to rat pBLCAT3 [ LuckoW et al, nucleic acids res.15: 5490(1987) the stromelysin-1 promoter [ matridian et al, mol.cell.biol.6: 1679(1986) ] Str-AP1-CAT was prepared from the DNA fragment at position-84 to + 1. The sequence of the stromelysin-1 promoter contains the AP1 domain unit as its sole enhancer element [ Nicholson et al, EMBO j.9: 4443(1990)]. The promoter sequence was prepared by annealing two synthetic oligonucleotides having the following sequence: 5'-AGAAGCTTATGGAAGCAATTATGAGTCAGTTTGCGGGTGACTCTGCAAATACTGCCACTCTATAAAAGTTGGGCTCAGAAAGGTGGACCTCGAGGATCCAG-3' (SEQ ID NO: 2) and 5'-CTGGATCCTCGAGGTCCACCTTTCTGAGCCCAACTTTTATAGAGTGGCAGTATTTGCAGAGTCACCCGCAAACTGACTCATAATTGCTTCCATAAGCTTCT-3' (SEQ ID NO: 3) as described in Nagpal et al [ J.biol.chem.270: 923(1995), a procedure comprising transfection, treatment with an appropriate compound and determination of CAT activity is accomplished.

The results of these procedures indicate that AGN193109 potentiates the anti-AP-1 activity of AGN 191183, the retinoid agonist. More precisely, at a concentration of 10^-12-10^-10In the range of M, AGN 191183 did not inhibit TPA-induced Str-AP1-CAT expression. Using AGN193109 at a concentration of 10^-10-10^-8Treatment in the M range does not substantially inhibit AP-1 mediated reporter activity. However, the results shown in FIG. 10 indicate that the concentration is 10^-12-10^-10In the range of M, AGN193109 (10) was used^-8M) and AGN 191183 in combination to stimulate the transfectant to substantially inhibit TPA-induced Str-AP1-CAT expression in an amount of 12% to 21%. Thus, AGN193109 potentiates AGN 191183 anti-AP-1 activity under conditions in which AGN 191183, a retinoid agonist, does not generally inhibit AP-1 activity.

We believe that AGN193109 potentiates the agonist-mediated repression of AP-1 activity by a mechanism that may involve AGN 193109-dependent receptor cohesion of NCPs to RARs. RARs belong to the superfamily of nuclear receptors, which also include receptors for 1, 25-dihydroxy vitamin D3, glucocorticoids, thyroid hormones, estrogens and progestins. It is reasonable to assume that the ability to bind NCPs can be shared by different members of the nuclear receptor superfamily. This led us to speculate that AGN193109 may potentiate anti-AP-1 activity of one or more ligands that interact with this superfamily of nuclear receptors.

The results described in the previous examples clearly show that AGN193109 potentiates the anti-AP-1 activity of retinoid agonists. More specifically, AGN193109 decreased the threshold dose at which anti-AP-1 activity of AGN 191183 could be detected. Since AGN193109 itself has essentially no anti-AP-1 activity, it acts synergistically with respect to the action of nuclear receptor agonists. We also found AGN193109 negative hormone potentiation as vitamin D₃1, 25-dihydroxyvitamin D as a natural ligand for receptors₃anti-AP-1 activity of (1).

The synergy observed in the previous examples between AGN193109 and AGN 191183 necessarily means that the anti-AP-1 activity of the retinoid agonist and the AGN 193109-mediated potentiation of this activity necessarily follow different mechanisms. If the mechanism of action of the two formulations is the same, the combined effect of AGN193109 and the stimulant is additive. However, the combined results showed more efficacy than either agent alone, and the effect was not expected before this finding.

The AGN 193109-mediated potentiation of the RAR agonist was effectively accomplished using an about 100-fold molar excess of AGN193109 over the retinoid agonist. Thus, most of the RARs should be bound by AGN193109, leaving very few RARs for agonist binding. Despite this fact, RARs that do not bind to AGN193109 are able to bind to retinoid agonists and strongly stimulate agonist-dependent responses measurable as inhibition of reporter gene expression. Thus, our data suggest a possible heterogeneity of RARs induced by AGN 193109.

The negative hormonal activity of AGN193109 is due to its ability to promote the interaction of RARs and NCPs, providing the basis for understanding the synergistic effect between AGN193109 and retinoid agonists. Our results are in complete agreement with the model in which AGN193109 treatment of cells promoted the binding of RARs and NCPs, thereby reducing the amount of extracellular free NCP and free RAR. This results in two RARs populations that differ in function. The first group is represented by RARs which bind to NCPs. The AGN 193109/RAR/NCP complex is not activated by retinoid agonists. The second group consists of RARs, which are not bound by NCP and retain interaction with agonists. The latter cell population is termed "RAR" and indicates free RARs in an environment substantially depleted of NCP.

RAR have reduced the likelihood of association with NCP by equilibrium binding and have increased sensitivity to measurable retinoid agonists, for example as anti-AP-1 activity. This is so because although the intracellular storage of NCP is depleted by administration of AGN193109, the storage of PCP is not yet depleted. Thus, free RAR^*s can bind to retinoid agonists and interact with PCP factors in an environment substantially depleted of NCP. The ability of AGN193109 to increase the sensitivity of other nuclear receptors to their respective agonists can be attributed to the ability of these different nuclear receptors to interact with the same NCPs (which interact with the AGN 193109/RAR complex). This model of AGN 193109-mediated modulation of NCP useful as a nuclear receptor family member is represented in graphical form in figure 11.

This graphical model enables us to predict that AGN 193109 may modulate the activity of nuclear receptor ligands other than retinoid agonists. As described in the examples below, we demonstrate that AGN 193109 potentiates the activity of 1, 25-dihydroxy vitamin D3 in an in vitro transactivation assay.

Example 14 describes a method for demonstrating that AGN 193109 increases the activity of 1, 25-dihydroxy vitamin D3 in a transactivation assay.

Example 14

AGN 193109 fortifying 1, 25-dihydroxy vitamin D₃Activity of (2)

Felgner et al [ proc.natl.acad.sci.usa 84: 7413(1987)]The cationic liposome-mediated transfection method transfects Hela cells. Will be 5X 10⁴Cell planes were seeded on 12-well multi-well plating and grown in DMEM supplemented with 10% FBS. LIPOFECTAMINE reagent (Life Technologies, Inc.) was used at 2 μ g/well, and the cells were transfected with a plasmid containing the nucleic acid sequence derived from the ligation reporter plasmid Δ MTV-Luc [ Heyman et al in Cell 68: 397(1992)]1, 25-dihydroxyvitamin D two copies of the mouse osteopontin gene₃Response element 5'-GTACAAGGTTCACGAGGTTCACGTCTTA-3' (SEQ ID NO: 4) [ Ferraraet al.J.biol.chem.269: 2971(1994) ]And 0.3. mu.g of plasmid pGEM3Z (Pharmacia, Inc.) as vector DNA so that the final concentration of DNA was 1.0. mu.g/well, and cells were co-transfected in serum-free medium. 6 hours after transfection, cells were supplied with growth medium containing activated charcoal-extracted FBS at a final concentration of 10%. 18 hours after transfection, with vehicle (ethanol) alone or at a final concentration of 10^-8Or 10^-7M AGN 193109 in ethanol. After 6 hours, 1, 25-dihydroxyvitamin D in ethanol was added₃To a final concentration of 10^-10-10^-7And M. With 1, 25-dihydroxyvitamin D₃Cells were lysed and harvested 18 hours after treatment. Luciferase activity was measured as described above. The experimental system can be used for monitoring and reacting 1, 25-dihydroxyvitamin D₃A convenient method for the quantification of dependent gene expression.

The results shown in FIG. 12 indicate that 1, 25-dihydroxy vitamin D, when used alone₃When compared with the results obtained with 1, 25-dihydroxyvitamin D₃Co-administration of AGN 193109 shifts its dose response curve to the left. This demonstrates that AGN 193109 potentiates 1, 25-dihydroxy vitamin D in an in vitro transactivation assay₃Effect ofForce. More specifically, FIG. 12 graphically depicts AGN 193109 at concentrations as low as 10-100 nM for 1, 25-dihydroxyvitamin D ₃Approximately 10-fold increase in activity. Need 10^-81, 25-dihydroxyvitamin D in M concentration₃So as to produce luciferase expression of about 2000 rlu, when the vitamin is added to a concentration of 10^-8-10^-7When M AGN193109 was co-administered, only 1, 25-dihydroxyvitamin D was required to produce the same amount of luciferase₃Amount of concentration 1/10. Although not shown in the graph in fig. 12, a concentration of 10 is used^-9-10^-8Substantially the same results can be obtained with M AGN 193109. Thus, co-administration of AGN193109 substantially reduces the 1, 25-dihydroxy vitamin D requirement for a similar effect in the absence of the negative hormone₃The amount of (c).

Interestingly, 1, 25-dihydroxy vitamin D was enhanced at AGN193109 when the above procedure was repeated with a co-transfected Vitamin D Receptor (VDR) expression plasmid₃There was a consistent decrease in the ability to activate. Our interpretation of this result is that over-expression of VDRs can affect AGN193109 to potentiate 1, 25-dihydroxyvitamin D₃The ability to be active. Thus, the intracellular concentration of a ligand receptor (which may vary in a tissue-specific manner) may affect the ability of AGN193109 to potentiate the activity of a ligand that binds to the receptor. This is again consistent with the model in which the tirtable NCPs help to regulate the response of vitamin D3 and support the model presented above.

As described in the examples below, we also demonstrated that AGN 193109 also potentiates the anti-AP-1 activity of 1, 25-dihydroxy vitamin D3. Our model for the action activity of AGN 193109 explains this observation by causing the urgent binding of NCPs to RARs in the presence of this drug. Endogenous vitamin D receptors present in Hela cells may be given more for 1, 25-dihydroxyvitamin D₃The sensitivity of the ligand, as a result of which the ligand inhibits the expression of reporter factor from Str-AP1-CAT, is exaggerated.

Example 15 describes a method for demonstrating that AGN 193109 potentiates the anti-AP-1 activity of 1, 25-dihydroxy vitamin D3.

Example 15

AGN 193109 fortifying 1, 25-dihydroxy vitamin D₃Against AP-1 activity

According to Nagpal et al [ j.biol.chem.270: 923(1995)]The method, using LIPOFECTAMINE, Lela cells were transfected with 1. mu.g of Str-AP 1-CAT. With a single AGN 193109 (10)^-9-10^-7M), 1, 25-dihydroxyvitamin D alone₃(10^-12-10^-7M) or at 10^-81, 25-dihydroxy vitamin D in the presence of M AGN 193109₃(10^-12-10^-7M) treating the transfected cells.

The results of these procedures indicate that AGN 193109 potentiates 1, 25-dihydroxy vitamin D ₃The ability to inhibit TPA-induced AP-1 activity. When it is 10^-9-10^-7When the concentration range of M was used alone, AGN193109 showed no detectable anti-AP-1 activity. The results shown in FIG. 13 show 1, 25-dihydroxyvitamin D₃Is only at 10^-8-10^-7M concentration ranges that repress TPA-stimulated activity. Analysis is at 10^-81, 25-dihydroxy vitamin D in the presence of M AGN193109₃Mediation of repression of CAT Activity on TPA stimulation indicates that anti-AP-1 Activity is at 10^-10-10^-9M1, 25-dihydroxyvitamin D₃Is detectable in combination with 1, 25-dihydroxyvitamin D alone₃Therapeutic contrast, 10^-8-10^-7Activity increased at dose M. The AGN 193109-dependent regulation of 1, 25-dihydroxyvitamin D₃The mediated anti-AP-1 activity is consistent with a model in which chelation of RARs by NCP renders the NCP unavailable for interaction with other nuclear receptor family members. Thus, the receptor was made specific for 1, 25-dihydroxyvitamin D₃The treatment is more sensitive.

The following mechanisms for RAR-mediated transactivation and anti-AP-1 activity may differ. The conclusion is based onOur observation is that high doses of AGN193109 completely inhibit transactivation without substantially inhibiting anti-AP-1 activity. Thus, additional evidence is desired to support RAR as mediated by AGN193109 therapy ^*And (4) forming a model. To this end, we investigated whether AGN193109 could potentiate the activity of the RAR-specific agonist AGN191183 in an in vitro transactivation assay.

Example 16 describes a method for demonstrating that 193109 potentiates the activity of the RAR-specific agonist AGN 191183. The results of this procedure show that in certain cases, AGN193109 increases the efficacy of RAR-specific retinoids and provides AGN193109 to promote RAR^*Strong evidence of formation.

Example 16

Potentiation of retinoid efficacy by co-administration of AGN193109

Felgner et al [ proc.natl.acad.sci.usa 84: 7413(1987)]The cationic liposome-mediated transfection method transfects Hela cells. Will be 5X 10⁴Cell planes were seeded on 12-well multi-well plating and grown in DMEM supplemented with 10% FBS. LIOFECTAMINE reagent (2 μ g/well, Life Technologies, Inc.) was used, with a reporter plasmid containing an insert Δ MTV-Luc [ Heyman et al in Cell 68: 397(1992)]0.7. mu.g of the reporter plasmid MTV-TREP-Luc replicating the TREPAl response element 5'-TCAGGTCATGACCTGA-3' (SEQ ID NO: 5) and 0.1. mu.g of the RAR-gamma expression plasmid pRShRAR-gamma (Ishikawa et al. mol. Endocrinol.4: 837(1990)) were co-transfected in serum-free medium. 6 hours after transfection, cells were supplied with growth medium containing activated charcoal-extracted FBS at a final concentration of 10%. 18 hours after transfection, with vehicle (ethanol) alone or at a final concentration of 10 ^-11-10^-8M AGN193109 in ethanol. After 6 hours, AGN191183 in ethanol was added to a final concentration of 0, 10^-10Or 10^-9And M. Cells were harvested 18 hours after treatment with AGN191183 and luciferase activity was measured as described above.

Preliminary experiments show that 10^-9AGN193109 of M inhibits binding to 10^-9The AGN191183 response of M in Hela cells was relatively ineffective. This is in accordance with 10^-9AGN 193109M inhibits 10 in CV-1 cells^-8M ATRA (FIG. 2) formed the control.

The results shown in figure 14 support that AGN193109 stimulates RAR^*A prediction is formed. Consistent with the identification of antagonists and negative hormone activities of AGN193109, treatment with AGN193109 resulted in a biphasic dose-response curve. Minimum dose of AGN193109 (10)^-11-10^-10M) resulted in stimulation of luciferase activity beyond AGN191183 alone. This effect suggests that RAR is produced by AGN 193109. Curiously, this result was also seen with AGN193109 alone, suggesting RAR^*' s may respond to endogenous ligands. AGN191183 is a synthetic retinoid agonist that, like ATRA, activates transcription through RARs. Substitution of AGN191183 for ATRA in example 7 would give quantitative similar results (i.e. AGN193109 would antagonize the effect of 10nM AGN 191183). Example 16 illustrates: although AGN193109 may act as an antagonist of RAR agonists, dosing conditions are readily identifiable where AGN193109 co-administration potentiates activation mediated by RAR agonists. It is important to note that the dose of the compound used in example 16 is substantially lower than the dose used in the method described in example 7. It is believed that AGN193109 treatment may lead to RAR-heterogeneous RARs for RAR ^*And s. Apparent heterogeneity (i.e., capacity for potentiation) appears to have a distinct window in transactivation for AP-1 repression. The reason for the biphasic profile is that as the amount of AGN 193109 increases, RAR available for binding to the agonist decreases proportionally. This does not appear to be the case for AP-1 repression, and we still speculate that this difference necessarily reflects two distinct mechanisms of transactivation and AP-1 repression by the same receptor species.

Clinical results have demonstrated that some retinoids are useful in inhibiting the growth of premalignant and malignant neck lesions. An exemplary study to support this conclusion has been conducted by Graham et al in West j.med.145: 192(1986), by Lippman et al, j. natl. cancer inst.84: 241(1992) and by Weiner et al in invest.new Drugs 4: 241 (1986).

Similar conclusions are supported by the results of in vitro studies for culturing cells to quantify the antiproliferative effect of different retinoids. More specifically, Agarwal et al, Cancer Res.51: 3982(1991) uses the ECE16-1 cell line in an early stage model of cervical dysplasia and demonstrates that retinoic acid inhibits Epidermal Growth Factor (EGF) -dependent cell proliferation.

Example 17 describes a method for demonstrating that AGN193109 antagonizes the activity of AGN 191183 retinoid agonists, which inhibits the proliferation of the ECE16-1 cell line.

Example 17

AGN193109 antagonizes the antiproliferative effects of retinoids in ECE16-1 cells

In a DMEM-containing atmosphere: f12 (3: 1), nonessential amino acids, 5% FBS, 5. mu.g/ml transferrin, 2nM of 3, 3', 5-triiodothyronine (thyroid hormone or "T₃"), cholera toxin 0.1nM, L-glutamine 2nM, 1.8X 10^-4M adenine and 10ng/ml EGF at a density of 1X 10⁴Cells/cm₂ECE16-1 cells were seeded. Cells were attached to the seeding plane overnight and then transferred to a culture medium containing DMEM: f12 (3: 1), 2mM L-glutamine, non-essential amino acids, 0.1% bovine serum albumin, 1.8X 10^-4Adenine of M, transferrin at 5. mu.g/ml, T at 2nM₃50. mu.g/ml ascorbic acid, 100. mu.g/ml streptomycin, 100 units/ml penicillin and 50. mu.g/ml gentamicin. EGF was supplemented at 10ng/ml in Defined Medium (DM). EGF treated cells received 10nM of AGN 191183 retinoid agonist in combination with 0, 0.1, 1.0, 10, 100, or 1000nM AGN193109 alone. After 3 days of treatment, as in Hembree et al [ Cancer Res.54: 3160(1994) ]The cells are harvested andcell numbers were measured using a COULTER counter.

The results shown in figure 15 demonstrate that ECE16-1 cells proliferated in response to EGF but not in defined medium alone. This confirmed the results obtained by Andreatta-van Leyen et al [ j.cell.physio.160: 265(1994), and by hembres et al Cancer res.54: 3160 (1994). Addition of 10nM AGN 191183 and 0nM AGN193109 completely inhibited EGF-mediated proliferation. Thus, AGN 191183 is a potent antiproliferative retinoid. Increasing the concentration of AGN193109 antagonizes AGN 191183 mediated growth inhibition by 0nM to 10nM by about 50%. A 10-fold molar excess of AGN193109 completely reversed the antiproliferative effect of AGN 191183. Treatment of cells with 1000nM 193109 alone had no effect on EGF-mediated increased proliferation. These results demonstrate that AGN193109 antagonizes the antiproliferative effect of retinoids, whereas when used to treat cells representing the cervical epithelium, which are sensitive to the growth inhibition of retinoids such as AGN 191183, it has essentially no antiproliferative effect on its own. Remarkably, there is no evidence that AGN193109 potentiates the antiproliferative effects of AGN 191183 agonists using the ECE16-1 model system.

In contrast to the model system represented by the ECE16-1 cell line, there are other examples in which cell proliferation associated with cervical dysplasia cannot be inhibited by retinoid agonists. For example, Agarwal et al [ Cancer Res.54: 2108(1994) describes the use of CaSki cells as a model of neck tumors, which are unresponsive to retinoid therapy. As described below, retinoid treatment had essentially no effect on the growth rate of CaSki cells, except for inhibiting cell proliferation. The following examples highlight the effect of the AGN 193109 negative hormone on the proliferation rate of this cell line. The results unexpectedly demonstrate that AGN 193109 inhibits the proliferation of neck tumor cells that are unresponsive to the antiproliferative effects of retinoid agonists.

Example 18 presents a method for demonstrating the inhibition of growth of a neck tumor cell line in which AGN 193109 is unresponsive to the antiproliferative effects of other retinoids, such as AGN 191183. AGN 193109 clearly showed antiproliferative activity in the absence of added retinoid.

Example 18

AGN 193109 inhibits the rate of proliferation from CaSki cervical cancer cell line

We examined EGF for 10^-6Effect of M concentration on CaSki cell proliferation alone or in combination with AGN191183 retinoid agonists and/or AGN 193109 negative hormones. Cell proliferation assays were performed as described above in relation to the study of ECE16-1 cells. EGF was added to the retinoid treated cultures to produce a final concentration of 20 ng/ml. In the presence or absence of 10^-6AGF 193109 for M AGN191183 (10)^-10-10^-6M) cells were treated for a total of 3 days. The medium was replaced daily with new medium and each of the two retinoids as appropriate. Cell numbers were determined as described above using a coulter counter.

The results shown in fig. 16 indicate that CaSki cells should not substantially exert an effect on retinoid agonists and AGN 193109 should exhibit antiproliferative activity in the absence of added retinoids. The presence of EGF in the medium stimulates the growth of CaSki cells. This conclusion is based on a comparison of the diagonal bars representing AGN-free 191183 and the open bars representing growth medium ("DM") defined alone. AGN191183 treatment had no antiproliferative effect on CaSki tumor cell lines. We disregarded any slight increase in cell proliferation rate associated with retinoid agonists, as a 1 ten thousand fold increase in the retinoid concentration correlates with only about a 20% increase in proliferation rate. Thus, the AGN191183 agonist had essentially no effect on the proliferation rate of CaSki cells.

The results shown in fig. 16 also indicate that AGN 193109 inhibits proliferation of CaSki cervical epidermal cell line. This conclusion is based on a comparison of the results of the measurements as a black bar of "0" AGN191183 and a diagonal bar of "0" AGN 191183. Thus, AGN 193109, when used to treat neck tumor cells that are inhibited from growing by retinoid agonists such as AGN191183, is capable of stimulating a biological response in the absence of added retinoid.

Our finding that AGN 193109 negative hormone is able to inhibit cell proliferation is consistent with a model in which RAR not bound to ligand mediates the expression of genes required for proliferation. Whereas RAR agonists such as AGN191183 have essentially no or slightly promoting effect on cell proliferation, AGN 193109 has an antiproliferative effect. The AGN 193109 negative hormone may bind to RARs, thereby promoting NCP binding and allowing RARs to adopt an inactive conformation. According to our model, it represses gene activity positively regulated by RARs not bound to ligand. The ability of AGN 193109 subtype to modulate the activity of RARs that are not bound to ligands may arise from its ability to promote the binding of RARs to NCPs.

One of ordinary skill in the art will appreciate the undesirable consequences of certain retinoid agonists for controlling cell growth after retinal detachment. Following retinal detachment, retinal pigment epithelial cells (RPEs) become inter-metaplastic, proliferate and migrate into the subretinal space. This procedure has an adverse effect on the success of the surgical procedure for retinal reattachment. Campochiaro et al [ invest. opthal & vis. sci.32: 65(1991) RAR agonists such as ATRA have been shown to exhibit antiproliferative effects on the growth of primary human RPE cultures. Retinoid agonists have also been shown to reduce the chance of retinal detachment after retinal reattachment surgery [ Fekrat et al, opthamology 102: 412(1994)]. As disclosed in the following examples, we analyzed the ability of AGN 193109 negative hormone to inhibit growth in primary human RPE cultures.

Example 19 describes a method for demonstrating that AGN 193109 potentiates the antiproliferative effect of retinoid antagonists in primary cultures of human retinal pigmented epithelial cells.

Example 19

AGN 193109 potentiates the antiproliferative activity of ATRA

According to Campochiaro et al [ invest&Vis.Sci.32：65(1991)]The method described in (1) establishes a primary culture of human retinal pigment epithelial cells (RPE). Will be 5X 10⁴The cell plane was seeded in 16-mm well DMEM (Gibco) containing 5% FBS in a 24-well multi-well seeding plate. Cell simulation with ethanol vehicle alone, ATRA in ethanol (10)^-10-10^-6M), AGN 193109 in ethanol (10)^-10-10^-6M) or ATRA (10)^-10-10^-6M) and 10^-6AGN 193109 treatment of M. The cells were supplied with new medium containing appropriate concentrations of these compounds every two days for a total of 5 days of treatment. Cells were transferred from the plates by gentle digestion with trypsin and cell numbers were recorded using an electronic cell counter.

The results shown in fig. 17 show that AGN 193109 dramatically potentiates the antiproliferative activity of ATRA on RPE cells. Treatment of primary RPE cells with ATRA resulted in a dose-dependent decrease in RPE cell proliferation at 10 compared to control cultures ^-6A decrease of about 40% in M ATRA. At any concentration detected during the process, AGN193109 treatment did not substantially alter the RPE cell growth rate. Unexpectedly, ATRA (10)^-11-10^-6M) and 10^-6The combination of M AGN193109 had a stronger antiproliferative activity than ATRA alone. Thus, AGN193109 co-treatment potentiates the anti-proliferative effects of ATRA. More precisely, the results shown in the figure indicate that only 10 is used^-10M ATRA binding 10^-7MAGN 193109 can obtain 10^-8Antiproliferative effects of M ATRA. Thus, AGN193109 negative hormone advantageously increases the antiproliferative activity of ATRA by about 100-fold.

In separate experiments, ATRA (10) was compared^-11-10^-6M) with ATRA and 10^-6The antiproliferative effect of AGN193109 by M again indicates a significant increase in sensitivity of primary RPE cells to ATRA in the presence of AGN 193109. In this system, AGN193109, when applied alone, is not considered to be retinoidsThe aldehyde derivative antagonists act and also do not exhibit an antiproliferative effect. However, co-administration of AGN193109 potentiates the antiproliferative effect of the retinoid agonist.

AGN193109 was tested for potentiating the antiproliferative effect of 13-cis retinoic acid (13-cis RA) in primary RPE cultures using the conditions and techniques described above for determining RPE cell proliferation. Remarkably, 13-cis RA is a clinically important compound. More specifically, 13-cis RA binds interferon 2 α [ Lippman et al.j. natl. cancer inst.84: 241 (1992); moore et al, search in Hematology 31: 31(1994) for the treatment of several diseases, including acne [ Peck et al.n.engl.j.med.300: 329 (1977); jones et al Br.J.Dermatol.108: 333(1980) and squamous cell carcinoma of the skin and cervix.

The results shown in FIG. 18 show 13-cis RA (10)^-12-10^-6M) and ATRA (10)^-12-10^-6M) can effectively inhibit the growth of RPE cells. Remarkably, the 13-cis isomer is approximately two orders of magnitude less potent in this assay when compared to ATRA. Similar to results obtained with the co-administration of AGN193109 and ATRA (supra), AGN193109 (10) was co-administered^-8Or 10^-6M) and 13-cis RA (10)^-12-10^-6M) significantly increases the efficacy of 13-cis RA in the regulated expression of RPE cell proliferation. Co-administration of AGN193109 increased the efficacy of 13-cis RA compared to treatment with 13-cis RA alone. Thus, AGN193109 potentiates the antiproliferative activity of 13-cis RA.

Second, we examined the ability of AGN193109 to potentiate the activity of other nuclear receptor hormones in primary RPE cell cultures. Dexamethasone (a synthetic glucocorticoid receptor agonist) is one member of a class of compounds that has been used clinically for its potent anti-inflammatory and immunosuppressive properties. Thyroid hormone (T3; 3, 3 ', 5' -triiodothyronine) is a natural thyroid hormone receptor agonist used primarily in hormone replacement therapy for the treatment of hypothyroidism. The methods used in these experiments were consistent with those described above for the procedure using ATRA and 13-cis RA.

The results of these methods show that co-administration of AGN193109 and the nuclear receptor agonist potentiates the antiproliferative activity of the nuclear receptor agonist. More specifically, the results shown in FIG. 19 show dexamethasone (10)^-11-10^-6M) or ATRA (10)^-12-10^-6M) single drug treatment of RPE cells was essentially incapable of inhibiting RPE cell proliferation. However, with dexamethasone (10)^-11-10^-6M) and 10^-8Or 10^-6AGN193109 from M treated RPE cells repressed RPE cell proliferation to about the extent of inhibition induced by treatment with ATRA. Similarly, the results shown in fig. 20 show that AGN193109 potentiates the antiproliferative activity of thyroid hormone. Similar to the results obtained with dexamethasone, proliferation of RPE cells was relative to thyroid hormone (10)^-11-10^-6M) drug treatment alone is refractory. However, thyroid hormone (10) is used^-11-10^-6M) and AGN193109 (10)^-8Or 10^-6M) co-treating the RPE cells to inhibit proliferation of the RPE cells in a thyroid hormone-dependent manner. We conclude that: AGN193109 sensitizes primary RPE cultures to the antiproliferative effects of these nuclear receptor agonists. The mechanism by which AGN193109 mediates these effects may be involved in the modulation of NCP/RAR interactions.

In addition, we examined the effect of AGN193109 on marker gene expression in other experimental systems sensitive to retinoid agonists. MRP8 and stromelysin genes are known to be inhibited by retinoid agonists in a variety of biological systems. For example, Wilkinson et al [ j.cell sci.91: 221(1988) and Madsen et al [ j. invest. dermaltol.99: 299(1992) ] it has been disclosed that the expression of MRP8 gene is increased in psoriasis. In contrast, in human psoriatic skin (Nagpal et al, submitted 1995), in human keratinocyte cultures [ Chandraratna et al, j. invest. dermatol.102: 625(1994), and human neonatal foreskin keratinocytes in culture [ Thacher et al.j. invest.dermaltol.104: 594(1995), the retinoid agonist AGN 190168 represses the expression of MRP8 gene. Nagpal et al [ j.biol.chem.270: 923(1995) have disclosed that retinoid agonists such as AGN 190168 repress the level of stromelysin mRNA in cultured human neonatal foreskin keratinocytes. We analyzed the regulated expression of these genes after treatment of cultured human neonatal foreskin keratinocytes with AGN 191183 agonist or AGN 193109.

Example 20 is presented to show that AGN 193109 inhibits the expression of MRP-8 in keratinocytes in culture.

Example 20

AGN 193109 inhibits expression of MRP-8 in keratinocytes

According to Nagpal et al [ j.biol.chem.270: 923(1995)]The method isolates and cultures naive foreskin keratinocytes in Keratinocyte Growth Medium (KGM) purchased from Clonetics. With AGN 191183 (10)^-7M) or AGN 193109 (10)^-6M) 3 days after treatment, total cellular RNA was isolated from the treated and control keratinocytes according to standard methods. Its mRNA is reverse transcribed into cDNA using primers specific for the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) or MRP-8, which then serves as a template in the amplification record. The GAPDH primers have the following sequences: 5'-CCACCCATGGCAAATTCCATGGCA-3' (SEQ ID NO: 6) and 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (SEQ ID NO: 7). The MRP-8 primer has the sequence as follows: 5'-ACGCGTCCGGAAGACCTGGT-3' (SEQ ID NO: 8) and 5'-ATTCTGCAGGTACATGTCCA-3' (SEQ ID NO: 9). After each cycle of PCR amplification, starting at 12 cycles and ending at 21 cycles, aliquots (10. mu.l) from the MRP-8 amplification reaction were transferred. Similarly, aliquots of the GAPDH amplification reaction were transferred after each PCR cycle starting from 15 cycles to the end of 24 cycles. The samples were electrophoresed in 2% agarose gel and the separated amplification products were detected by staining with 3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide. The staining intensity of the amplification product is determined quantitatively as the starting mRNA specific for a given primer set.

The process isThe results of the procedure show that AGN 191183 and AGN 193109 independently inhibit the expression of MRP-8 in keratinocytes. The intensity of the stained GAPDH amplification product was essentially equal in the flow channel of the gel representing the starting material isolated from the control, AGN 191183 on AGN 193109 treated keratinocytes. The weak band representing the GAPDH amplification product was first detectable in the flow channel corresponding to the sample transferred after 18 cycles of PCR amplification. Equal staining intensity in different flow channels of the gel indicates that equal amounts of starting material were used in all samples. Thus, differences in the intensity of the stained bands representing the MRP-8 amplification product are indicative of differences in MRP-8 mRNA expression in the different starting samples. As expected, at AGN 191183 (10) compared to untreated controls^-7M) results in suppression of MRP-8 amplification signal in the treated medium. AGN 193109 (10) as judged by lower intensity of the stained amplification product^-6M) treatment of cultured keratinocytes also repressed the expression of MRP 8.

AGN 193109 also inhibited the expression of the second marker gene in keratinocytes, as described in the examples below. Nagpal et al [ j.biol.chem.270: 923(1995) disclose that stromelysin mRNA expression is down-regulated by RAR-specific agonists in cultured human neonatal foreskin keratinocytes. Nicholson et al [ EMBO j.9: 4443(1990) ] discloses that the AP-1 promoter element plays an important role in retinoid-dependent negative regulation of the stromelysin-1 gene. Thus, it is important to determine whether AGN 193109 can alter the expression of the gene.

Example 21 describes a protocol demonstrating that AGN 193109 inhibits the expression of the stromelysin-1 gene in the absence of an exogenously added retinoid agonist.

Example 21

AGN 193109 inhibits the expression of stromelysin-1 in keratinocytes in culture

AGN 191183 (10) as RAR agonist^-7M) or AGN 193109 (10)^-6M) cornification of the initial foreskinCell pattern treatment or treatment for 24 hours. Total RNA prepared from model-treated and retinoid-treated keratinocytes was trans-transcribed and fully expressed as per Nagpal et al [ j.biol.chem.270: 923(1995)]The method uses beta-actin or stromelysin-1 oligo primers to PCR amplify the formed cDNAfor. After every 3 cycles starting from 18 cycles of PCR amplification, samples (10 μ Ι) were transferred from the PCR amplification reaction. The sample was electrophoresed in 2% agarose gel and detected after 3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide staining.

The results of these processes indicate that AGN 193109 inhibits stromelysin gene expression in the absence of exogenously added retinoid agonists. More specifically, 3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide staining bands representing β -actin amplification products were readily detectable in agarose gels after 18 cycles of PCR. Although all band intensities increased with increasing cycles of amplification, there was a slight decrease in the intensity of the staining bands in the samples representing AGN 191183-treated cells. This indicates that a slightly reduced amount of RNA must already be present in the starting sample of cells treated with AGN 191183. The results also indicate that stromelysin-1 mRNA was detectable in pattern-treated keratinocytes beginning at 33 cycles of PCR amplification. As expected, AGN 191183 (10), as judged by the strength of the weaker band compared to the samples from the pattern-processed samples (agv) ^-7M) the expression of stromelysin-1 mRNA was inhibited. AGN 193109 of keratinocytes when normalized to the intensity of β -actin amplification product and consistent with the results obtained in the MRP-8 expression assay (10)^-6M) treatment results in down-regulation of the level of stromelysin-1 mRNA. Indeed, the downregulation of stimulation by AGN 193109 treatment was indistinguishable from downregulation by keratinocytes treated with the RAR agonist AGN 191183.

As disclosed herein, AGN 193109 may have any of three possible effects of modulating the activity of co-administered steroid superfamily agonists. First, AGN 193109 may have no effect. Second, AGN 193109 can antagonize the action of the stimulant, resulting in a decrease in the stimulant activity. Finally, AGN 193109 can potentiate the activity of the stimulant, resulting in stimulation of the measured effect produced by the stimulant.

Compounds having activity that can be modulated by AGN 193109 include retinoid receptor agonists and agonists that bind to other members of the steroid receptor superfamily. The latter class of agonists include vitamin D receptor agonists, glucocorticoid receptor agonists, and thyroid hormone receptor agonists. Peroxidase proliferator-activated receptors, estrogen receptors and orphan receptors with currently unknown ligands can also be augmented by AGN 193109. Where the steroid superfamily agonist is a RAR agonist, AGN 193109 may antagonize or potentiate the activity of the agonist. Where the agonist used in conjunction with AGN 193109 is a compound that binds to a nuclear receptor other than RAR, co-administration of AGN 193109 will have no effect or become susceptible to the agonist system such that the activity of the agonist is potentiated.

A generalized exemplary method for detecting which of three possible activities AGN 193109 would have in a particular system is as follows. The specification describes each possible outcome of co-administration of AGN 193109 with agonists of the steroid receptor superfamily. Biological systems for assessing the ability of AGN 193109 to modulate nuclear receptor agonist activity include (but are not limited to): tissue culture cell lines, virus transformed cell lines, in vitro-in vivo primary cultured cells, and in vivo studies using living bodies have been established. In such systems, the determination of the biological effect of AGN 193109 can include the determination of any of a variety of biological end points (endpoints). These endpoints include: cell proliferation assays, programmed cell death assays (apoptosis), analysis of the differential status of cells tested by gene expression, analysis of the ability of cells to form tumors in nude mice, and analysis of gene expression following transient and stable introduction of reporter constructs.

To illustrate, the mRNA species designated mRNA "X" is "Y" in a primary culture isolated from organ "Z"Expressed in the cell by the gene "X". Under standard culture conditions, in which several "Y" cytogenetic markers are retained, including expression of gene "X", addition of a retinoid agonist results in a decrease in the abundance of "X" mRNA. Analysis of the expression of gene X can be assessed by isolating cellular mRNA and determining the level of XmRNA redundancy by polymerase chain reaction, ribonuclease protection or Northern blot methods such as Northern analysis. After isolation of organ Z, primary Y cells are cultured in a suitable growth medium. This primary culture is then inoculated into a tissue culture inoculation plate for amplifying the cell population. This step helps to separate the cells into 4 sample groups so that various doses of retinoid agonist and AGN 193109 can be used. The first group is a control group and receives vehicle only. The second group is added in an amount sufficient to provide a final concentration of 10 ^-11-10^-6The amount of M received RAR agonist, retinoic acid (given in ethanol). The minimum dose can be determined empirically based on the sensitivity of the system. This determination is within the ordinary skill in the art of ordinary experimentation. The third group will receive the nuclear receptor agonist and constant AGN193109 at the same dose used to treat the second group of cells. The dosage of AGN193109 to treat the third group of cells will be determined empirically, but should be close to the affinity constant (Kd) of AGN193109 for RAR subtypes (i.e., at least 10)^-8M). The fourth group received the lowest dose of AGN193109, including for use in the co-administration of stimulant in the third group. As a variation of this dosage regimen, AGN193109 would be used in place of the retinoid agonist described in the above examples, particularly in the second group, and AGN193109 would be used in place of a constant retinoid agonist, particularly in the third and fourth groups. After an appropriate incubation period, the cells are harvested in a manner suitable for detecting biological endpoints determined as indicators of stimulant activity.

For example, analysis of the effect of AGN193109 on retinoic acid-dependent regulation of gene expression included comparing the abundance of mRNA species X in mRNA stocks harvested from cells treated according to each of the four protocols described above. RNA from the control cells will be used as a baseline for determining XmRNA expression and represent the conditions corresponding to no repression. The effect of the stimulant on gene expression can be determined by comparing the levels to levels determined in a store of cell-derived mRNA treated with retinoic acid. Quantitative levels of expression of specific mRNAs resulting from retinoic acid treatment can then be compared to mRNA abundance from cells treated in parallel with AGN193109 alone or AGN193109 bound to retinoic acid. While this generalized example describes the analysis of the effect of co-administration of AGN193109 on gene expression repressed by retinoid agonists, this example additionally describes the analysis of the effect of co-administration of AGN193109 on genes induced by retinoid agonists. Important features for determining whether AGN193109 will function as an agonist, as a negative hormone, or not in a particular system include quantitatively comparing the magnitude of the effect in the presence and absence of AGN 193109.

An example where AGN193109 potentiates the activity of a co-administered stimulant is where co-administration of AGN193109 with retinoic acid results in a level of further repressed xmmrna expression relative to that measured in cells treated with retinoic acid alone. More precisely, a dose response curve comparing the biological effect on the Y-axis (i.e. the repression of xmna redundancy) to the dose of stimulant on the X-axis (logarithmic scale) can compare the repression of xmna redundancy mediated by a stimulant in the presence and absence of AGN193109 co-administration. The ability of AGN193109 to sensitize the biological response to the stimulant, thereby potentiating the activity of the stimulant, is indicated by a leftward shift in the dose-response curve. More specifically, in the presence of AGN193109, less stimulant would be required in order to achieve the same biological effect as that achieved using the stimulant alone.

An example of an AGN 193109-mediated antagonism of a co-administered stimulant is where AGN193109 co-administration with retinoic acid results in a level of suppressed X mRNA expression compared to the level measured in cells treated with retinoic acid alone. Comparing the dose response curves for XmRNA repression versus log of agonist dose in the presence and absence of AGN193109 indicates a shift to the right in the dose response curve. More specifically, in the presence of AGN193109, more stimulant is needed in order to obtain the same biological effect as that obtained with the stimulant alone.

The above examples wherein AGN193109 mediates antagonism or potentiation describe the results of experiments in which AGN193109 is co-administered with a retinoid agonist. However, if the agonist co-administered with AGN193109 is capable of binding to and activating a member of the steroid receptor superfamily other than RAR, then instead of antagonizing the agonist, AGN193109 may have no effect on the activity of the agonist. If AGN193109 co-treatment with such agonists produces the same level of mRNA expression as measured by treatment of cells with the agonist alone, then AGN 193109: the ability of NCP binding to affect the effectiveness of NCPs is not shown in this system. This is an example where AGN193109 has no effect on co-administered stimulants.Examples of antagonism

The method described in the generalized example for determining the effect of co-administration of AGN193109 with a retinoid agonist is exemplified in the method described in example 7. Ethanol (control, group 1) was used at a final concentration of 10^-9-10^-6AGN193109 (group 2), and 10 for M^-8M retinoic acid co-administered to a final concentration of 10^-9-10^-6AGN193109 (group 3) or retinoic acid (10) of M^-8M, group 4) treatment of CV-1 cells cotransfected with one of the three retinoic acid receptors and the retinoid agonist-induced MTV-TREP-Luc receptor construct. Comparing the luciferase activity of group 1 to the luciferase activity of group 4 enables determination of retinoid agonist induced luciferase reporter expression in the absence of added AGN 193109. Comparison of luciferase reporter expression in group 3 cells with the results measured in group 4 cells indicates that AGN193109 functions as an inhibitor of retinoid agonists in this system. Examples of antagonism

The method described in the general example for determining the effect of co-administration of AGN193109 with a retinoid agonist was similarly used in example 17 to determine the function of AGN193109 as an antagonist of the retinoid agonist-mediated repression of EGF-stimulated cell proliferation in ECE-16-1 transformed cervical epithelial cells. In this process, the treatment of ECE-16-1 cells involved treating the control sample (group 1) with EGF alone, and the combination of EGF and AGN193109 at a final concentration of 10^-6M (group 2) treatment of the samples with a final concentration of 10^-10-10^-6Composition and concentration of M EGF and AGN193109 10^-8A single dose of M of the retinoid agonist AGN191183 was co-administered (group 3) to treat the samples and used at a concentration of 10^-8The samples were treated with the EGF and AGN191183 composition of M (group 4). Three days after treatment, the cell proliferation rate was determined. It is possible to determine cells that have stimulated proliferation by EGF because other control treatments are included, in which the cells are placed in defined medium without EGF. Comparison of the number of cells in group 1 with the number of cells in group 4 enables determination of the ability of the RAR agonist AGN191183 to suppress EGF-stimulated proliferation of ECE-16-1 cells. Comparing group 3 with group 4 shows that AGN193109 antagonizes the activity of RAR agonists in this system. Examples of the enhancing action

The method described in the generalized example for determining the effect of co-administration of AGN193109 with a retinoid agonist was also used in example 14 to determine that AGN193109 potentiates the activity of a nuclear receptor agonist in Hela cells transfected with the MTV-VDRE-Luc reporter gene induced by 1, 25-dihydroxyvitamin D3. Treatment of transfected cells included vehicle alone (control, group 1) at a final concentration of 10^-10-10^-7M1, 25-dihydroxyvitamin D₃(group 2) final concentration of 10^-10-10^-7M1, 25-dihydroxyvitamin D₃And final concentration of 10^-8Or 10^-7AGN 193109M was co-administered (group 3) and at a final concentration of 10^-8Or 10^-7Single drug treatment of AGN193109 for M (group 4). Comparison of the luciferase activity measured in group 1 (control) cells with the luciferase activity of group 2 cells enabled the determination of 1, 25-bisHydroxy vitamin D₃Stimulation of luciferase activity is dose-dependent. Comparing the luciferase activity measured in the group 4(AGN 193109 alone treatment) cells with the luciferase activity measured in the group 3 (AGN 193109 co-administered) cells, it is similarly possible to measure dose-dependent 1, 25-dihydroxyvitamin D in the presence of a given concentration of AGN193109₃Stimulated luciferase activity. In this case, a zero value represents luciferase activity in cells treated with AGN193109 alone (group 4). This dosing regimen considered a comparison of 1, 25-dihydroxyvitamin D ₃Three dose response curves. Comparison of 1, 25-dihydroxy vitamin D in the absence of AGN193109₃The dose response curve of (1) represents co-administration of AGN193109 (10)^-8Or 10^-7M) demonstrated enhanced activity of the agonist as evidenced by a shift to the left in half maximal response.Examples of the enhancing action

In a generalized example for determining the effect of co-administration of AGN193109 with a retinoid agonist the method was further used to determine in example 19 that AGN193109 potentiates the antiproliferative activity of RAR agonists in primary cultures of human retinal pigment epithelial cells. The treatment of the cells comprises: ethanol vehicle alone (group 1) at a final concentration of 10^-10-10^-6Retinoic acid of M (group 2) at a final concentration of 10^-10-10^-6Co-administration of M retinoic acid 10^-6M AGN193109 (group 3) and final concentration of 10^-10-10^-6M AGN193109 alone (group 4). Comparing the results of the assays obtained using group 1 and group 2 cells allows for a determination of dose-dependent inhibition of proliferation of these cells by retinoic acid. Similarly, comparison of the results obtained using the group 3 cells with those obtained in group 1 allows for the determination of dose-dependent inhibition of proliferation of these cells by retinoic acid in the case of co-administration of AGN 193109. Group 4 demonstrated that AGN193109, when administered as a single treatment drug, had substantially no ability to alter the rate of proliferation of these cells. Comparing the dose response curves for repression of retinoic acid-mediated cell proliferation generated in groups 2 and 3 to that of AGN193109 primary RPE cells to the RAR agonist The antiproliferative effect of (b) is sensitive, thus providing a basis for the conclusion of potentiating the activity of RAR agonists.

As described above, Agarwal et al [ Cancer res.54: 2108(1994) it is believed that unlike the growth of HPV immortalized ECE-16-1 cells, the growth of CaSki cells is not inhibited by treatment with retinoid agonists. As disclosed herein, we unexpectedly found that the growth of CaSki cells was inhibited by AGN193109 in the absence of a retinoid agonist. The following example illustrates how AGN193109 can be used to inhibit the growth of CaSki cell tumors in vivo.

Example 22

Inhibition of CaSki cell tumor growth in nude mice after administration of AGN193109

Will be 1 × 10⁶CaSki cells were injected into each of a group of nude mice. Tumor formation is assessed using techniques well known to those skilled in the art. After injection, mice were randomly divided into control and test groups. The control group received blank drug. The test group was administered AGN 193109. Animals given placebo received gastric intubation with corn oil. During the treatment period, the animals in the test group received 20 μmol/kg of AGN193109 in corn oil per day. Tumor volume in cubic milliliters was measured using a graduated caliper. Tumor volume was plotted as a function of time. Mice receiving AGN193109 showed significantly lower tumor growth rates compared to the tumors of control mice, as judged by the size and number of tumors during the study. This result provides in vivo evidence that AGN193109 inhibits the growth of advanced neck cancer that is resistant to treatment that includes administration of a retinoid agonist.

As described above, CaSki cells are a model of cervical tumors and they do not respond to retinoid agonist treatment. However, we disclose that AGN 193109 inhibits the growth of CaSki cells without treatment with retinoid agonists. The ability of AGN 193109 to inhibit CaSki cell proliferation suggests that AGN 193109 may be useful in the treatment of neck cancer that is not responsive to retinoid agonist treatment. The following example presents a method that can be used to evaluate the efficacy of AGN 193109 in the treatment of neck cancer.

Example 23

Evaluation of the therapeutic Effect of AGN 193109 in patients with cervical cancer

Patients with advanced neck cancer were first identified. The cervical tissue biopsy is performed according to methods well known to those of ordinary skill in the art. Cells from disseminated tumors were propagated in tissue culture medium according to standard techniques to provide a sufficient number of cells to be divided into three sample groups. For this purpose, Agarwal et al [ Cancer res.54: 2108(1994)]The culture conditions are described. The first group served as a control group and received vehicle (ethanol) alone. The concentration of the second group is 10^-10-10^-6RAR agonist retinoic acid treatment of M. The dosage range of the third group is 10 ^-10-10^-6AGN193109 treatment of M. The cells were supplied daily with new growth medium and the retinoid mentioned above as appropriate for each sample set. After three days, the cell number was recorded using an electronic cell counter. The results of comparing the number of cells in the control culture to the number of cells in the retinoic acid treated culture indicate that the RAR agonists do not substantially inhibit the growth rate of cultured cervical cancer cells. In contrast, cells treated with AGN193109 showed a dose-dependent decrease in cell number when compared to cell counts in the control group. The results in which AGN193109 treatment inhibited proliferation of cultured neck cancer cells showed that AGN193109 is a useful therapeutic agent for treating neck cancer patients with metastasis.

In a randomized clinical study aimed at demonstrating the beneficial therapeutic role of AGN193109 in the disease, neck cancer patients who had undergone surgical removal of the major tumor were enrolled. Patients were divided into two groups. The first group was a control group and the second group was treated with AGN 193109. AGN193109 is mixed with pharmaceutically acceptable excipients to produce a composition suitable for systemic administration, using techniques well known to those skilled in the art. The control group was administered with a blank formulation test group administered with the formulation containing AGN193109 negative hormone. The patient is administered every other day at the maximum tolerated dose for three months to one year. The results of the study were quantified by measuring disease-free survival after a certain time. Each patient receiving AGN193109 showed a significant increase in disease-free survival, including some disproportionate patients who showed complete remission of their metastatic disease. This result shows that AGN193109 has utility in the in vivo treatment of neck cancer that is not responsive to the antiproliferative effects of retinoid agonists such as retinoic acid.

As described above, AGN193109 potentiates the antiproliferative activity of RAR agonists in primary cultures of human retinal pigment epithelial cells. Thus, it is reasonable to expect that co-administration of AGN193109 and a RAR agonist in vivo would increase the therapeutic index of the agonist, since a smaller amount of RAR agonist would be required to achieve the same therapeutic endpoint. Furthermore, AGN193109 has been shown to sensitize primary cultures of human retinal pigment epithelial cells to the antiproliferative effects of glucocorticoids and thyroid hormone receptor agonists. The following PVR rabbit model will be used in two separate studies to demonstrate an increase in therapeutic index by co-administration of AGN193109 and a RAR agonist (13-cis retinoic acid) or separate administrations of thyroid hormone receptor agonists. Of note is the expression of protein by Sen et al [ arch.opthalmol.106: 1291(1988) ] a model for rabbit retinal detachment that has been proposed to demonstrate that retinoid agonists that inhibit primary RPE cell proliferation in vitro also reduce the number of retinal detachments in vivo [ Araia et al invest. opthalmol.34: 522(1993)]. Thus, for their use as agents for preventing retinal detachment, a link between the activity of agonists of retinoids in vitro and in vivo has been established. The following example illustrates how AGN193109 may be used in therapeutic applications directed to the prevention of retinal detachment.

Example 24

AGN193109 in the treatment ofIncreasing steroid hyperandrogenism in Proliferative Vitreoretinopathy (PVR) Therapeutic use of family receptor agonists

In the first study, the response was determined according to Sen et al [ arch.opthalmol.106: 1291(1988) ] the method described human RPE cells were injected into the vitreous chamber of rabbit eyes. After intravitreal injection, rabbits were divided into five groups: the first group (control) received vehicle alone by intravitreal injection. The second group received retinoic acid (100 μ g) as a single drug treatment by intravitreal injection. The third group received AGN193109(100 μ g) as a single drug treatment by intravitreal injection. The fourth group received a dose of 1/10 of RAR agonist (retinoic acid) administered in group 2 by intravitreal injection (10 μ g). The fifth group received a composition of AGN193109(100 μ g) and retinoic acid (10 μ g) by intravitreal injection. One day after intravitreal injection of human RPE cells animals receive a separate intravitreal injection of the appropriate therapeutic agent. Rabbits were examined by indirect ophthalmoscopy on days 7, 14 and 28 and scored for the number and severity of traction retinal detachments. Rabbits in the 100 μ g retinoic acid injection group showed a significant reduction in the number and severity of retinal detachments compared to control rabbits or rabbits receiving AGN193109 or retinoic acid (10 μ g) alone. Rabbits in the group co-administered with AGN193109 and retinoic acid (10 μ g) showed significantly reduced number and severity of retinal detachments compared to rabbits in the control, AGN193109, or retinoic acid (10 μ g) groups. These results demonstrate that AGN193109 improves the therapeutic index of RAR agonist retinoic acid in the PVR in vivo model.

In a second study, human RPE cells were first provided to rabbits injected into the vitreous chamber of rabbit eyes and then divided into four groups. The first group (control) received vehicle alone by intravitreal injection. The second group received thyroid hormone (100 μ g) as a single drug treatment by intravitreal injection. The third group was administered AGN193109(100 μ g) as a single drug treatment by intravitreal injection. The fourth group was given a combination of AGN193109(100 μ g) and thyroid hormone (100 μ g). Rabbits were examined by indirect ophthalmoscopy on days 7, 14 and 28 and scored for the number and severity of traction retinal detachments. The results comparing the number and severity of retinal detachments in the four groups showed that treatment with AGN193109 or thyroid hormone alone did not inhibit retinal detachments when compared to control rabbits. In contrast, rabbits of the group to which AGN193109 and thyroid hormone composition were administered showed a significant reduction in the number and severity of retinal detachments. This result demonstrates that AGN193109 improves the therapeutic index of thyroid hormone in the PVR in vivo model.

The following example describes how AGN193109 can be used to increase the therapeutic index of RAR agonists for treatment of patients after retinal reattachment surgery.

Example 25

Increasing the therapeutic index of RAR agonist 13-cis retinoic acid

Some adult volunteers with retinal detachment caused by PVR were first identified. Each individual underwent surgical repair of retinal detachment using techniques standard in the art. Then, the patients were divided into five groups. The control group consisted of patients who underwent surgical repair of retinal detachment and did not receive any retinoid. The second group received 40mg of 13-cis retinoic acid orally twice daily for four weeks after surgery. The third group received 40mg AGN 103109 orally twice daily for four weeks after surgery. The fourth group received 4mg 13-cis retinoic acid orally twice daily for four weeks after surgery. Orally receive 40mg of AGN 103109 in combination with 4mg of 13-cis retinoic acid twice daily for four weeks after surgery. Substantially in accordance with Fekrat et al [ Ophthalmology 102: 412(1995) protocol and evaluation of drug efficacy.

The number and severity of postoperative patient retinal detachments in all five groups over a 9 month period was monitored using ophthalmoscopy techniques well known to those of ordinary skill in the art. Patients receiving 40mg oral 13-cis retinoic acid when compared to control patients, patients receiving 4mg 13-cis retinoic acid twice daily, or patients receiving 4mg of 13-cis retinoic acid twice daily Patients with 0mg AGN 193109 showed a significant reduction in the number of retinal detachments when compared. Examination of the results of the patient group receiving 40mg AGN 103109 orally in combination with 4mg 13-cis retinoic acid orally for four weeks post-surgery twice daily showed that the treatment results in this patient group were equal to or better than the treatment results in patients receiving 40mg 13-cis retinoic acid orally for four weeks post-surgery twice daily. This result demonstrates that the AGN 193109 negative hormone improves the therapeutic index of RAR agonists by reducing the number and severity of retinal detachments in PVR patients.Generalized assay for the identification of nuclear receptor negative hormones

We have demonstrated above that AGN 193109 can act as a negative hormone capable of repressing the basal transcriptional activity of RAR nuclear receptors. Next, we have described an assay using CV-1 cells cotransfected with the ERE-tk-Luc luciferase reporter plasmid and the ER-RXR-alpha and RAR-gamma-VP-16 receptor expression plasmids to distinguish RAR ligands, which are simple antagonists, from those with negative hormonal activity.

We have concluded that: RAR negative hormones mediate the repression of RAR-mediated transcriptional activity by promoting increased interaction between RAR and NCPs. Furthermore, we have demonstrated that AGN 193109 can potentiate the effects of other nuclear receptor agonists in a manner consistent with the sharing of NCPs among members of the steroid superfamily of nuclear receptors. Thus, ligands can be assigned and screened to identify compounds with negative hormonal activity at these non-RAR nuclear receptors.

Our method of RAR negative hormone screening based on CV-1 cells cotransfected with the ERE-tk-Luc luciferase reporter plasmid and the ER-RXR-alpha and RAR-gamma-VP-16 receptor expression plasmids can generally be employed so as to convert the RAR-gamma portion of the RAR-gamma-VP-16 plasmid into the peroxidase proliferator-activated receptor (PPAR), the Vitamin D Receptor (VDR), the thyroid hormone receptor (T3R) or any other steroid superfamily nuclear receptor capable of heterodimerizing with RXR. CV-1 cells co-transfected with these plasmids can express high basal levels of luciferase activity. Ligands capable of binding the ligand binding domain of the receptor that replaces the RAR- γ moiety can be readily screened for negative hormone activity by determining their ability to repress luciferase activity.

For steroid superfamily nuclear receptors that do not heterodimerize with RXR (e.g., glucocorticoid and estrogen receptors), the end result can be achieved using GR-VP-16 or ER-VP-16 receptors and a luciferase reporter plasmid consisting of a suitable glucocorticoid or estrogen response element fused to a heterogeneous promoter element and luciferase or other reporter gene. The basic features of the generalized negative hormone screening test are a method comprising at least a ligand binding domain of a specific nuclear receptor for screening for inverse agonists and a promoter for concentrating the nuclear receptor ligand domain in a reporter gene. This can be achieved by using the natural DNA binding site of the receptor or by constructing a chimeric receptor with a heterogeneous DNA binding domain and correspondingly using a reporter gene under the control of a DNA regulatory element which can be recognized by said heterogeneous DNA binding domain. In a preferred embodiment, plasmids expressing nuclear receptors useful for screening for inverse agonists may express the nuclear receptor as a fusion protein containing a combinatorial activation domain, such as the HSV VP-16 activation domain, in order to provide a permissive high basal activity. This high basal activity effectively increases the sensitivity of the assay, thereby enabling analysis of nuclear receptor ligands that repress basal transcriptional activity in the absence of added nuclear receptor agonists.

The following example describes one method that can be used to screen for compounds having negative hormonal activity at the thyroid hormone receptor.

Example 26

Method for identifying negative hormone of thyroid hormone receptor

CV-1 cells were co-transfected with the luciferase reporter plasmid ERE-tk-Luc and plasmids ER-RXR-alpha and T3R-VP-16. T3R-VP-16 is identical to RAR- γ -VP-16, except that the RAR- γ portion of the RAR- γ -VP-16 receptor has been replaced by thyroid hormone receptor cDNA. Such asThus, T3R-VP-16 expresses a fusion protein with the activation domain of HSV VP-16 in the N-terminal structure of the thyroid hormone receptor. Standard transfection and cell culture methods were used for this purpose. After transfection, cells were rinsed and growth medium containing 10% bovine embryo serum that had been extracted with activated charcoal was provided. With separate vehicle (ethanol), thyroid hormone (10)^-9-10^-10M) or compound TR-1 (10)^-9-10^-6M) treating the cells. TR-1 is a synthetic thyroid hormone receptor ligand that exhibits strong affinity for the thyroid hormone receptor in competitive binding studies, yet it does not activate the transfected thyroid hormone receptor in a transient cotransfection transactivation assay using a thyroid hormone response reporter gene and a thyroid hormone receptor expression plasmid. In addition, TR-1 is capable of antagonizing thyroid hormone-mediated transactivation and is itself a thyroid receptor antagonist.

Analysis of luciferase activity from CV-1 cells transfected with ERE-tk-Luc, ER-RXR- α and T3R-VP-16 showed high basal levels of luciferase reporter activity in vehicle-treated cells. Cells treated with thyroid hormone showed a slight increase in luciferase activity in a dose-dependent manner. Cells treated with TR-1 showed a dose-dependent decrease in luciferase activity. This indicates that TR-1 exhibits thyroid receptor inverse agonist activity, presumably due to increased interaction of NCP with thyroid hormone receptors.

The rate of proliferation of human primary retinal pigment epithelial cells was suppressed by treatment with RAR agonists. The observed therapeutic values have been shown in post-operative treatment with retinoids after retinal reattachment surgery. We have demonstrated above that AGN 193109 RAR negative hormones can sensitize primary RPE cells to the antiproliferative effects of ATRA and 13-cis retinoic acid during co-administration. Furthermore, AGN 193109 was also shown to sensitize RPE cells to the antiproliferative effects of other nuclear receptor agonists. More specifically, AGN 193109 sensitizes RPE cells to the antiproliferative effects of glucocorticoid stimulant, dexamethasone, and thyroid hormone stimulant 3, 3', 5-triiodothyronine (T3). These data are consistent with our working model, in which AGN 193109 regulates the effectiveness of NCPs shared among the nuclear receptor family members. Treatment of RPE cells with the thyroid hormone receptor inverse agonist TR-1 will similarly alter the effectiveness of sharing NCPs, so that co-administration with non-thyroid receptor agonists such as RAR agonist 13-cis retinoic acid will result in enhanced antiproliferative effects on RPE cultures compared to 13-cis retinoic acid treated as the sole drug.

The following example describes one method that may be used to make primary RPE cells more sensitive to the antiproliferative activity of RAR agonists. Remarkably, this example further describes how the activity of RAR agonists is potentiated by co-administration with a negative hormone.

Example 27

Primary retinal pigment fixation by co-administration of TR-1 thyroid hormone inverse agonist Endothelial cells sensitive to antiproliferative effects of RAR agonists

Human primary RPE cells were obtained and cultured according to standard methods. The cultured cells were divided into four groups and treated as follows. Group 1 received vehicle (ethanol) alone. Group 2 was used at a concentration of 10^-11-10^-613-cis retinoic acid treatment of M. The concentration of the compound in group 3 is 10^-11-10^-1And (3) treating the thyroid hormone inverse stimulant TR-1 of M. Group 4 was used at a concentration of 10^-11-10^-6Co-treatment of M with 13-cis retinoic acid and TR-1. Cells were re-supplied with fresh growth medium every two days during the total five day treatment and re-treated with the appropriate compounds. The proliferation rate during the course of the experiment was quantified by measuring the number of cells in culture with an electronic cell counter.

TR-1 treated cells (group 3) showed substantially the same rate of cell proliferation as the control (group 1) cells and this inverse agonist had no effect on the growth rate measured for the cultures. Cells treated with 13-cis retinoic acid (group 2) showed a dose-dependent decrease in cell number. Comparison of the dose-dependent decrease in cell proliferation in group 4 cells (co-administered 13-cis RA and TR-1) with the results obtained in group 3 demonstrates that co-administration of TR-1 thyroid hormone receptor inverse agonist sensitizes RPE cultures to the antiproliferative effect of 13-cis retinoic acid as determined by a shift of the dose response curve of the RAR agonist to the left for group 4 versus group 2 cells.

Claims (44)

1. A compound having the formula:

wherein X is S, O, NR ', wherein R' is H or alkyl of 1 to 6 carbon atoms;

R₂is hydrogen, lower alkyl of 1-6 carbon atoms, F, Cl, Br, I, CF₃Fluorine substituted alkyl with 1-6 carbon atoms, OH, SH, alkoxy with 1-6 carbon atoms or alkylthio with 1-6 carbon atoms;

R₃is hydrogen, lower alkyl of 1 to 6 carbon atoms or F;

m is an integer of 0 to 3;

o is an integer of 0 to 3;

z is-C ≡ C-,

-N＝N-，

-N＝CR₁-, wherein R₁Is H or alkyl of 1 to 6 carbon atoms,

-CR₁＝N-，

-(CR₁＝CR₁)_n-wherein n' is an integer from 0 to 5,

-CO-NR₁-，

-CS-NR₁-，

-NR₁-CO，

-NR₁-CS，

-COO-，

-OCO-，

-CSO-，

-OCS-；

y is phenyl or naphthyl or heteroaryl selected from pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrazolyl, said phenyl and heteroaryl optionally substituted by one or two R₂Is substituted by radicals, or

When Z is- (CR)₁＝CR₁)_n’And n' is 3, 4 or 5, then Y is represented at (CR)₁＝CR₁)_n’A direct bond between the group and B;

a is (CH)₂)_qWherein q is 0 to 5, a lower branched alkyl group having 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and 1 or 2 double bonds, an alkynyl group having 2 to 6 carbon atoms and 1 or 2 triple bonds;

b is hydrogen, COOH or a pharmaceutically acceptable salt thereof, COOR ₈，CONR₉R₁₀，-CH₂OH，CH₂OR₁₁，CH₂OCOR₁₁，CHO，CH(OR₁₂)₂，CHOR₁₃O，-COR₇，CR₇(OR₁₂)₂，CR₇OR₁₃O or tri-lower alkylsilyl, wherein R₇Is alkyl, cycloalkyl or a compound having 1 to 5 carbon atomsAlkenyl of a radical, R₈Is an alkyl group of 1 to 10 carbon atoms or a trimethylsilylalkyl group wherein the alkyl group has 1 to 10 carbon atoms, or a cycloalkyl group of 5 to 10 carbon atoms, or R₈Is phenyl or lower alkylphenyl, R₉And R₁₀Independently hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, or phenyl or lower alkylphenyl, R₁₁Is lower alkyl, phenyl or lower alkylphenyl, R₁₂Is lower alkyl, and R₁₃Is a divalent alkyl radical of 2 to 5 carbon atoms, and

R₁₄is (R)₁₅)_r-phenyl, (R)₁₅)_r-naphthyl or (R)₁₅)_r-heteroaryl, wherein said heteroaryl has 1-3 heteroatoms selected from O, S and N, r is an integer from 0 to 5, and

R₁₅independently H, F, Cl, Br, I, NO₂，N(R₈)₂，NH(R₈)，COR₈，NR₈CON(R₈)₂，OH，OCOR₈，OR₈CN, an alkyl group having 1 to 10 carbon atoms, a fluoroalkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms and 1 to 3 double bonds, an alkynyl group having 1 to 10 carbon atoms and 1 to 3 triple bonds, or a trialkylsilyl or trialkylsiloxy group wherein the alkyl groups independently have 1 to 6 carbon atoms.

2. The compound of claim 1, wherein Y is phenyl, pyridyl, thienyl, or furyl.

3. The compound of claim 1, wherein Y is phenyl.

4. The compound of claim 3 wherein the phenyl ring is 1, 4 (para) substituted.

5. The compound of claim 1, wherein Y is naphthyl.

6. The compound of claim 1, wherein Y is pyridyl.

7. The compound of claim 1, wherein Y is thienyl or furyl.

8. The compound of claim 1, wherein Z is- (CR)₁-CR₁)_n’-and n' is 3, 4 or 5 and Y represents in (CR)₁＝CR₁)_n’Direct bond between group and B.

9. The compound of claim 1, wherein R₂Is H, F or CF₃。

10. The compound of claim 1, wherein R₃Is H or methyl.

11. The compound of claim 1, wherein R₁₄Is (R)₁₅)_r-phenyl.

12. The compound of claim 1, wherein R₁₄Is (R)₁₅)_r-a heteroaryl group.

13. The compound of claim 12, wherein R₁₄Is (R)₁₅)_r-heteroaryl, wherein the heteroaryl is a 5 or 6 membered ring having 1 or 2 heteroatoms.

14. The compound of claim 13, wherein said heteroaryl is selected from the group consisting of 2-pyridyl, 3-pyridyl, 2-thienyl, and 2-thiazolyl.

15. The compound of claim 1, wherein R₁₅Group H, CF₃F, lower alkyl, lower alkoxy, hydroxy or chloro.

16. The compound of claim 1, wherein Z is-C ≡ C-.

17. The compound of claim 1, wherein Z is-N ═ N-.

18. The compound of claim 1, wherein Z is-CO-NR₁-。

19. The compound of claim 1, wherein Z is-CS-NR₁-。

20. The compound of claim 1, wherein Z is-CS-NR₁-。

21. The compound of claim 1 wherein Z is-COO-.

22. The compound of claim 1, wherein Z is- (CR)₁＝CR₁) n '-and n' are 1.

23. A compound having the formula:

wherein X is [ C (R)₁)₂]_nWherein R is₁Is H or alkyl of 1 to 6 carbon atoms, and n is an integer between 0 and 2;

R₂is hydrogen, lower alkyl of 1-6 carbon atoms, F, Cl, Br, I, CF₃Fluorine substituted alkyl with 1-6 carbon atoms, OH, SH, alkoxy with 1-6 carbon atoms or alkylthio with 1-6 carbon atoms;

R₃is hydrogen, lower alkyl of 1 to 6 carbon atoms or F;

m is an integer of 0 to 3;

o is an integer of 0 to 3;

z is- (CR)₁＝CR₁)_n’-wherein n' is 0 or an integer from 2 to 5;

y is phenyl or naphthyl or is selected from pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrazinylHeteroaryl of azolyl, said phenyl and heteroaryl being optionally substituted by one or two R₂Is substituted by radicals, or

When Z is- (CR)₁＝CR₁)_n’And n' is 3, 4 or 5, then Y is represented at (CR)₁＝CR₁)_n’A direct bond between the group and B;

A is (CH)₂)_qWherein q is 0 to 5, a lower branched alkyl group having 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and 1 or 2 double bonds, an alkynyl group having 2 to 6 carbon atoms and 1 or 2 triple bonds;

b is hydrogen, COOH or a pharmaceutically acceptable salt thereof, COOR₈，CONR₉R₁₀，-CH₂OH，CH₂OR₁₁，CH₂OCOR₁₁，CHO，CH(OR₁₂)₂，CHOR₁₃O，-COR₇，CR₇(OR₁₂)₂，CR₇OR₁₃O or tri-lower alkylsilyl, wherein R₇Is alkyl, cycloalkyl or alkenyl having 1 to 5 carbon atoms, R₈Is an alkyl group of 1 to 10 carbon atoms or a trimethylsilylalkyl group wherein the alkyl group has 1 to 10 carbon atoms, or a cycloalkyl group of 5 to 10 carbon atoms, or R₈Is phenyl or lower alkylphenyl, R₉And R₁₀Independently hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, or phenyl or lower alkylphenyl, R₁₁Is lower alkyl, phenyl or lower alkylphenyl, R₁₂Is lower alkyl, and R₁₃Is a divalent alkyl radical of 2 to 5 carbon atoms, and

R₁₄is (R)₁₅)_r-phenyl, (R)₁₅)_r-naphthyl or (R)₁₅)_r-heteroaryl, wherein said heteroaryl has 1-3 heteroatoms selected from O, S and N, r is an integer from 0 to 5, and

R₁₅independently H, F, Cl, Br, I, NO₂，N(R₈)₂，NH(R₈)，COR₈，NR₈CON(R₈)₂，OH，OCOR₈，OR₈CN, an alkyl group having 1 to 10 carbon atoms, a fluoroalkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms and 1 to 3 double bonds, an alkynyl group having 1 to 10 carbon atoms and 1 to 3 triple bonds, or a trialkylsilyl or trialkylsiloxy group wherein the alkyl groups independently have 1 to 6 carbon atoms.

24. The compound of claim 23, wherein n' is 3.

25. The compound of claim 23, wherein n' is 0.

26. The compound of claim 24, wherein n is 1.

27. The compound of claim 26, wherein a is (CH)₂)_qWherein q is O.

28. The compound of claim 27 wherein B is COOH or a pharmaceutically acceptable salt thereof, COOR₈Or CONR₉R₁₀。

29. The compound of claim 25, wherein n is 1.

30. The compound of claim 29, wherein Y is naphthyl.

31. The compound of claim 30, wherein the naphthyl is 2, 6 substituted.

32. The compound of claim 31, wherein a is (CH)₂)_qWherein q is 0.

33. The compound of claim 32 wherein B is COOH or a pharmaceutically acceptable salt thereof, COOR₈Or CONR₉R₁₀。

34. The compound of claim 1, wherein X is S.

35. The compound of claim 34, wherein Z is-C ≡ C-.

36. The compound of claim 35, wherein Y is phenyl.

37. The compound of claim 36, wherein R₁₄Is (R)₁₅)_r-phenyl.

38. The compound of claim 37, wherein R₁₅Is a lower alkyl group.

39. The compound of claim 38, wherein a is (CH)₂)_qWherein q is 0.

40. The compound of claim 39 wherein B is COOH, COOR₈Or a pharmaceutically acceptable salt thereof.

41. The compound of claim 40, wherein m is 0.

42. A compound having the formula:

wherein R is₁₅Is lower alkyl of 1-6 carbon atoms and R₈ ^*Is H, lower alkyl of 1 to 8 carbon atoms or a pharmaceutically acceptable salt of said compound.

43. The compound of claim 42, wherein R₁₅Is methyl.

44. The compound of claim 43, wherein R₈ ^*Is H or a pharmaceutically acceptable salt of said compound。