WO1990008142A1 - SYNTHESIS AND APPLICATION OF 1,3,6,8- AND 2,4,6,8-SUBSTITUTED DIBENZOFURANS AND DIBENZO-p-DIOXINS AS ANTITUMORIGENIC AGENTS - Google Patents

Abstract

Novel 2,4,6,8- and 1,3,6,8-substituted dibenzofurans and dibenzo-p-dioxins, the preparation thereof and the use thereof as antiestrogens in the treatment of estrogen dependent tumors.

Description

Synthesis and Application of 1,3,6,8-And 2,4,6,8-Substituted Dibenzofurans and Dibenzo-p-Dioxins as Antitumorigenic Agents

This invention relates to a series of 1,3,6,8 and 2,4,6,8-substituted dibenzofurans and dibenzo-p-dioxins and to methods of preparing such compounds. It further relates to the use of the compounds as antiestrogenics and to pharmaceutical compositions containing said compounds.

Antiestrogens are a class of chemicals which inhibit estrogens from eliciting their full response in target tissues. They can be used to explore the mechanisms of action of estrogens and to provide treatment for estrogen-dependent diseases (e.g. tumors). An antiestrogenic compound currently being utilized in the treatment of mammary cancer is tamoxifen. Progesterone and related progestins have also been used extensively to treat mammary cancer in laboratory animals and humans. Numerous other antiestrogens have been disclosed in recent years including inhibitors of aromatase (Bednarski Pat. No. 4,745,109), antiestrogenic hydrazones (Morgan Pat. No. 4,732,904) and antiestrogenic benzothiophenes (Jones Pat. No. 4,418,068). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic man-made chemicals. It acts through initial binding to the aryl hydrocarbon (Ah) receptors. Research in the past has focused on the identification and mechanisms of the toxic effects of TCDD in the body and particularly on its induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin 0-deethylase (EROD). (Mason et al. Toxicol. 41, 21-31 (1986); Denomme et al. Chem. Biol. Interactions, 57_r 175-187 (1986).)

Recent studies in several laboratories have reported the activity of TCDD as an antiestrogen in rats, mice and MCF-7 human breast cancer cell lines in culture. For example, TCDD treatment resulted in decreased uterine weights in weanling female C57BL/6 mice and female Long Evans rats and TCDD partially blocked the estrogenic effects of 17B-estradiol on uterine weights. (Romkes et al., Toxicol. and Applied Pharm. 87, 306-314 (1987)). TCDD also decreased constitutive and 17B-estradiol induced uterine and hepatic estrogen and progesterone receptor levels in the female rat and suppressed the estrogen mediated excretion of tissue plasminogen activator activity in MCF-7 human breast cancer cells in culture. (Romkes et al., Toxicol. and Appl. Pharmacol. 92, 368-380 (1988); Gierthy et al.. Cancer Research 47, 6198-6203 (1987).) Kociba and coworkers reported that after long term feeding studies, TCDD significantly decreased the spontaneous development of mammary and uterine tumors in Female Spraque-Dawley rats (Kociba, Toxicol. and Appl. Pharmacol. 46, 279-303 (1978)). However, TCDD is so hepatocarcinogenic that it has not been seriously considered for use as a therapeutic antiestrogenic.

TCDD does not bind to the estrogen receptor and studies suggest, although we do not limit ourselves to this theory, that the antiestrogenic activities of TCDD are mediated through the Ah receptor. Many dibenzo-p- dioxin and dibenzofuran analogs of TCDD also exhibit a binding affinity for the Ah receptor. Recent studies have focused on the identification and mechanism of action of these compounds as TCDD antagonists particularly in inhibiting TCDD-mediated induction of AHH and EROD in laboratory animals and mammalian cells in culture. (Keys et al., Toxicol. Letters, 31 (1986) 151-158, Astroff et al.. Molecular Pharmacol. 33:231-236 (1988).) Studies have particularly focused on 1,3,6,8 and 2,4,6,8- substituted dibenzofurans. The activity of the 2,3,6,8- and 1,3,6,8-substituted dibenzofurans and dibenzo-p- dioxins as Ah receptor agonists (e.g. induction of AHH, thymic atrophy, etc.) was low to non-detectable in most biosystems despite their moderate binding affinity for the Ah receptor.

The present invention provides for a class of compounds, 1,3,6,8 and 2,4,6,8 substituted dibenzofurans and dibenzo-p-dioxins, which are nontoxic at biologically active levels and which are antiestrogens. The compounds can be used pharmaceutically for antiestrogen therapy and particularly for the treatment of estrogen-dependent tumors. This invention further provides for pharmaceutical compositions of the antiestrogenic compounds and for methods of administering the compounds to inhibit estrogen activity and to treat estrogen dependent tumors. In addition, this invention provides a method of producing 1,3,6,8 and 2,4,6,8 alkylated halodibenzofurans and alkylated halodibenzo-p-dioxins.

A series of 6-methyl 1,3,8-trichlorodibenzofuran (MCDF) analogs in which each of the positions was replaced with a hydrogen, demonstrated TCDD antagonist activity. For most of the compounds, their partial antagonist potential correlated with their competitive binding affinities for the hepatic AH receptor. Since the presence of a substituent at C-6 is important for antagonist activity, a series of 6-substi-tuted 1,3,8- tricholordibenzofurans were synthesized and their structural activity effects as TCDD antagonists were also determined. All of the 6-substituted acyclic alkyl- substituted analogs were active as TCDD antagonists in vivo and in vitro but showed low agonist activity for AHH and EROD induction. All of these antagonists were at least 10 less active (or toxic) than TCDD and their antagonist activities were observed at subtoxic doses.

Based on previous studies with 6-methyl 1,3,8- tricholordibenzofuran (MCDF) which have indicated that MCDF is an antagonist to the activities of TCDD it was expected that this compound would also antagonize the antiestrogenicity of TCDD. However, like TCDD, MCDF caused a dose response decrease in uterine and hepatic cytosolic and nuclear estrogen and progesterone receptor levels. MCDF was also active as an antiestrogen in inhibiting 17B-estradiol-induced increases in rat uterine wet weight. (Astroff and Safe, Toxicol. and Appl. Pharmacol. 95, p.435-443 (1988)).

The antiestrogenic activities of MCDF were completely unexpected and the reasons that MCDF is .a poor agonist for the traditional "Ah receptor mediated response" but a good agonist for the modulation of steroid hormone receptor binding levels are unknown. Therefore, MCDF represents a new class of halogenated aromatic antiestrogens which exhibit relatively high Ah receptor binding activity and low toxicity.

The invention will be described in terms of preferred embodiments which represent the best mode known to the applicants at that time of this application. Synthesis of 1,3,6,8 and 2,4,6,8 Substituted Dibenzofurans

The starting reactants for 1,3,6,8 and 2,4,6,8- substituted dibenzofuran are substituted phenols and substituted 6-haloanilines. The composition of the substituted dibenzofuran end product is dependent upon the positions of the substituents on the substituted phenol and the substituted haloaniline. Because the substituted dibenzofuran is symmetrical through the vertical axis, the end product and the beginning reactants could be renamed for their mirror images. To be consistent, this discussion will discuss the reactants and the end product as pictured in diagram 1.

Possible substituents are bromine, chlorine, fluorine and/or a linear or branched alkyl group of one to four carbons_* The substituted positions may also be individually and independently occupied by a hydrogen instead of a substituent. In the preferred mode the halogen substituents will be chlorine or bromine and the substituents of the reactants will be such that the final substituted dibenzofuran will be substituted with an alkyl group at position 6. In addition, no more than one of the positions of the end product which could be occupied by a substituent or a hydrogen should be occupied by a hydrogen and at least two of the positions should be occupied by halogen substituents.

The substituted phenol (100 mmol) and substituted 6- haloaniline (20 mmol) are mixed and heated with stirring at 120-130°C. Isoa yl nitrite (60 mmol) is added dropwise over thirty minutes (30 in) and the mixture is stirred for 18-24 hours at 120-130°C. The excess phenol is removed by evaporation and the intermediate product is recovered on a silica gel column using an appropriate solvent such as petroleum spirit/acetone (9:1). The solvent is evaporated and the residue is dissolved in an appropriate diluent such as dimethyl sulfoxide or hexamethylphosphoramide (HMPA) . Andydrous potassium carbonate (60 mmol) (or another appropriate base such as sodium carbonate) is added and the mixture is stirred for 1 to 2 hours at 180-200°C. The substituted dibenzofuran is recovered from a silica gel column and is recrystallized in an appropriate solvent such as anisole/methanol (1:9) or chloroform/ ethanol (1:9). A single or repeated (2x) recrystallization gives the pure product (> 98% as determined by gas chromatography) .

Synthesis of 1,3,6,8 or 2,4,6,8- Substituted Dibenzo-p-Dioxin

Two different methods for the synthesis of 1,3,6,8 or 2,4,6,8-substituted dibenzo-p-dioxins will be discussed. As with the dibenzofurans the appropriate substituted dibenzo-p-dioxins are obtained by the positioning of the appropriate substituents on the reactants or starting materials. Because the dibenzo-p-dioxin is symmetrical on two axis the beginning reactants and the final product could be characterized in many different ways. For the sake of consistency all of the reactants and the final product will be named as depicted in diagrams 2 and 3.

Diagram 2

Diagram 3

Possible substituents are chlorine, fluorine and bromine and linear or branched alkyl groups of one to four carbons. The substituted positions may also be independently and individually occupied by a hydrogen instead of a substituent. In the preferred mode, the halogen substituents will be chlorine or bromine and the substituents on the reactants will be such that the final substituted dibenzo-p-dioxin will be substituted with an alkyl group at position six. In addition, no more than one of the positions of the end product which may be filled by either a substituent or a hydrogen will be occupied by a hydrogen and at least two of the substituents will be halogens.

The preferred method of synthesis utilizes 2,4,6- substituted phenols as the starting reactants. The sodium salt of the appropriately substituted^* phenols in the presence of an appropriate diluent such as dimethyl sulfoxide are heated at 220-250° for 16-24 hours. The mixture is adsorbed on silicic acid and is recovered on a silica gel column using an appropriate solvent such as petroleum spirit. The solvent is removed and the residue is recrystallized from an appropriate solvent such as anisole/methanol (1:9) or chloroform/methanol (1:9) yielding 1,3,6,8-substituted dibenzo-p-dioxins. The minor product 1,3,7,9-substituted dibenzo-p-dioxin (2,4,6,8- tetrachlorodibenzo-p-dioxin) can be recovered from the mother liquors and further purified by high pressure liquid chromolography

Another method which the inventor believes may be used for synthesis of 1,3,6,8 or 2,4,6,8-substituted dibenzo-p-dioxins utilizes a substituted catechol and a substituted halonitrobenzene as the starting reactants. The substituted catechol (3 mmol) and the substituted halonitrobenzene (3 mmol) and anhydrous potassium carbonate (12 mmol) (or another appropriate base) and an appropriate diluent such as dimethyl sulfoxide or HMPA are heated with stirring at 180-190°C for 1-4 hours. The mixture is adsorbed on silicic acid and is recovered on a silica gel column using an appropriate solvent such as petroleum spirit or hexane. The solvent is removed and the residue is recrystallized from an appropriate solvent such as anisole/methanol (1:9) or chloroform/methanol (1:9). Antiestrogenic Effects of 1,3,6,8 and 2,4,6,8-Substituted Dibenzofurans and Dibenzo-p-Dioxins

The 1,3,6,8 and 2,4,6,8-substituted dibenzofurans and dibenzo-p-dioxins synthesized as described above may be used as antiestrogens. MCDF when administered with estradiol will significantly reduce or inhibit 17B- estradiol mediated responses. In comparison to control test animals, MCDF decreases the number of nuclear and cytosolic hepatic and uterine estrogen receptors and decreases uterine wet weights in rats and mice. These effects can be observed using amounts of MCDF which are nontoxic. A detailed description of the antiestrogen effect of MCDF is given in example 3. Results have.also been obtained which suggest these compounds will be useful as antiestrogens in human breast cancer cells (See example 5).

While it is unclear by what mechanism the 1,3,6,8 substituted dibenzofurans act, it appears that the antiestrogenic effect is somehow related to the ability of these compounds to bind to the Ah receptor. It is this binding activity which also allows these compounds to act as antagonists to 2,3,7,8-tetrachlorodibenzo-p-dioxin in its effects as an inducer of aryl hydrocarbon hydroxylase (AHH), other monooxygenase enzymes, and other toxic responses (Keys et al., Toxicol. Letters 31 (1986) 151-158 and Banister et al., Chemosphere, Volume 16 Nos. 8/9 pages 1739-1742 1987). The 2,4,6,8-substituted dibenzofurans and the 1,3,6,8 and 2,4,6,8-substituted dibenzo-p-dioxins will act in the same fashion due to their structural similarity and should exhibit equally low toxicity. The preferred mode for use of these compounds as an antiestrogenic and for treatment of estrogen dependent - tumors is the administration of a 6-substituted 1,3,8- trichlorodibenzofuran or dibenzo-p-dioxin with the substituent at position 6 being a linear or branched alkyl group of one to four carbons. This compound should be administered in a biologically active amount, which in test animals is 50-100 ug/kg, dissolved in a carrier suitable for lipid soluable compounds such as corn oil or soybean oil. An aqueous emulsion or encapsulation could also be used to administer the drug orally.

Example 1

Synthesis of 6-methyl 1,3,8-trichlorodibenzofuran.

20 gms. of 2-methyl-4-chlorophenol (Aldrich Chemicals) and 5 gms. of 2,4,6-trichloroaniline (Aldrich Chemicals) were mixed and heated with stirring to 120°C. 6 mis. of isoamyl nitrate was added dropwise over a thirty minute period and the mixture was stirred for 18 hours at 120°C. The excess phenol was removed by evaporation and the residue was adsorbed on silica gel and added to the top of a silica gel column packed with petroleum spirit/diethylether (3:7). The column was eluted with 500 ml. of the above solvent, the solvent was evaporated, and the residue was dissolved in dimethyl sulfoxide. 500 mg. of anhydrous potassium carbonate was added and the mixture was stirred for two hours at 190°C. It was then adsorbed on 30-40 grams of silica gel. The solvent was evaporated and the silica gel was added to the top of a 20 x 5 cm silica gel column which was packed and equilibrated with petroleum spirit. 6-methyl 1,3,8-trichlorodibenzofuran was eluted from the column with 500 mis. of petroleum spirit and the residue was crystalized from anisole/methanol (1:9). 280 mgs. of the product was recovered and was 99% pure as determined by gas chromatographic analysis and a molecular weight of 284 was confirmed- with a VG 12000 quadrupole mass chromatograph coupled to a Hewlet Packard 500 gas chromatograph. The 220-MHz. proton magnetic resonance spectrum (in deuterochloroform) was determined with a Varian XL200 spectrometer and gave (in CDCL.-): 8.07 (H-9,d,J=1.6 Hz) 7.48, 7.33 (H-l/H-3,d,J=1.6 Hz); 7.29, 7.29ppm (H-7,m).

Example 2

Synthesis of 1,3,6,8-tetrachlorodibenzo-p-dioxin.

This is a prophetic example. 2.5 gms. of the sodium salt of 2,4,6-trichlorophenol and 5 to 7 mis. of dimethylsulfoxide are stirred at 200-220°C for 16-24 hours. The reaction mixture is adsorbed on 10 gms. of silica gel, the solvent is allowed to evaporate and the absorbed reaction mixture is placed on top of a silica gel column (60 gms. silica gel, 4 x 2 mm). The column is eluted with 500 mis. of petroleum spirit. The solvent is removed in vacuo and the residue is recrystallized from 4 is. of anisole/methanol (1:9) to give a 1-10% yield of 1,3,6,8-tetrachlorodibenzo-p-dioxin. The minor product 1,3,7,9-tetrachlorodibenzo-p-dioxin (2,4,6,8- tetrachlorodibenzo-p-dioxin) can be recovered from the mother liquors and further purified by high pressure liquid.

Example 3

Synthesis of 6-methyl 1,3,8-trichlorodibenzo-p- dioxin. This is a prophetic example. 480 mg. of 6-methyl-4- chloro-catechol, 680 mg. of 2,3,5-(or 2,4,6) trichloronitrobenzene, 420 mg. of anhydrous potassium • carbonate and 3 mis. of dimethyl sulfoxide are heated with stirring at 180°C for four hours. The mixture is adsorbed on 30 gms. of silicic acid, the solvent is allowed to evaporate, and the material is placed as a top layer on a 20 x 5 cm silica gel column (60 gms. of silicic gel packed with petroleum spirit). The material is eluted with 350 ml. of petroleum spirit. The solvent is removed in vacuo and the residue is recrystallized from 4 ml. of anisole/methanol (1:9). The crystals are removed by filtration to give 0.15 gms. of 6-methyl 1,3,8- trichlorodibenzo-p-dioxin. This compound may also contain 9-methyl-l,3,7-trichlorodibenzo-p-dioxin as an impurity. This compound is expected to exhibit comparable activity due to the 2-fold axis of symmetry of this molecule.

Example 4

Antiestrogenic activities of 6-methyl 1,3,8- tricholordibenzofuran.

Twenty-five day old female Sprague/Dawley rats with at least four rats in each treatment group were injected with either corn oil (control) 17B-estradiol (5 μM/rat, sid x 2 days), TCDD, MCDF, estradiol plus TCDD or estradiol plus MCDF (see Tables for dose levels). TCDD or MCDF were co-administered with a second dose of estradiol and the animals were killed 48-hours later. A second group of animals was similarly treated with corn oil, TCDD, MCDF, or TCDD plus MCDF, all administered in corn oil at 10 ml/kg and terminated 72 hours later. The animals were terminated by cervical dislocation. Livers were perfused with ice-cold TEDG buffer (lOmM Tris-HCl, 1.5 mM EDTA-4Na, ImM dithiothreitol, 10% glycerol (v/v), pH 7.4). Following perfusion in situ, livers were removed, weighed, and homogenized in ice-cold TEDG buffer. Uteri were also removed, weighed, and homogenized in ice-cold buffer. The homogenates were then centrifuged at 800xg (200 rpm) for 15 minutes and the nuclear pellets were stored at -80°C. The supernatants were further centrifuged at 180,000xg for 1.5 hour. The cytosolic supernatant was also stored at -80°C. Estrogen and progesterone receptor levels were determined within 7 days.

Estrogen and progesterone receptor levels were determined by the hydroxyapatite assay as described by

Clark et al.(J. Steroid, Biochem. , 16, 323-328 (1982)).

3 Briefly, samples were incubated with lOnM [ H] 17B-

3 estradiol or [ H] R5020 with or without 200-fold excess cold DES or progesterone, respectively. Following the incubations, samples were counted and specific binding was calculated by subtraction of nonspecific from total binding. Assuming one steroid molecule binds to one estrogen/progesterone receptor, the number of moles of receptor can be calculated given the specific activity of the radioligand. The data presented in Tables 1-3 are means ± SD using at least four animals per treatment group. Significant differences were determined by ANOVA.

The results of these experiments are shown in Tables 1, 2 and 3 below.

TABLE 1 EFFECTS OF TCDD AND MCDF ON HEPATIC AND UTERINE ER and PR LEVELS IN THE FEMALE RAT

Hormone receptor levels (fmol/mg protein)'

ERn PRc PRn

Uterine levels

Hepatic levels

TABLE 2

EFFECTS OF TCDD AND MCDF ON HEPATIC AND UTERINE ER and PR LEVELS IN FEMALE RATS TREATED WITH ESTRADIOL

Hormone receptor levels (fmol/mg protein)'

Data expressed as means ± SD. * Values significantly different from controls (p <0.01). ** Values significantly different from estradiol.

TABLE 3

Effects of TCDD and MCDF on Uterine Wet Weights In Female Rats Treated With Estradiol

Treatment Uterine Wet Weight (dose, μmol/kg) (as % body-wt)

Corn Oil 0.154 ± 0.010

Estradiol (E-2) 0.322 ± 0.035

TCDD (0.256) 0.086 ± 0.011*

MCDF (100) 0.090 ± 0.013* E-2 + TCDD 0.245 ± 0.026**

E-2 + MCDF 0.255 ± 0.031**

*Significantly different from controls (p <0.01). **Significantly different from estradiol-treated (p <0.01).

Dose levels of 50 and 100 umol/kg MCDF caused a dose dependent decrease in constitutive uterine hepatic estrogen receptors and progesterone receptor levels (Table 1). In addition, the uterine and hepatic estrogen receptors and progesterone receptor levels in rats treated with MCDF plus estradiol were 43, 59, 36 and 75%, respectively, of the corresponding levels observed in rats treated with 17B-estradiol alone (Table 2). As demonstrated in Table 3 MCDF also significantly decreased uterine wet weights compared to the corn oil treated animals, and in the co-treated animals MCDF partially inhibited the estradiol induced increase in uterine wet weight. This reduction of estrogen receptors and uterine wet weight is indicative of antiestrogenic activity. The amounts of these compounds which produce the antiestrogenic activity have not demonstrated any toxicity in parallel toxicity studies.

Example 5

TABLE 4

Antiestrogenic Activity In MCF-7 Cells

Effects of MCDF and Cotreatment of MCDF Plus 2,3,7,8-TCDD on ERn in MCF-7 Cells.

Treatment Coadministered ERn (dose) with (dose) (fmol/mg protein) control 221. 2 (5.6) MCDF (15 μM) 207. 5 (6.6)* MCDF (50 μM) 171. 6 (13.7)* MCDF (75 μM) 141. 8 (13.6)* MCDF (100 μM) 135 . 8 (4.5)* MCDF (150 μM) 114 . 2 (7.9)*

control 225.5 (22.6)

2,3,7,8-TCDD (10 nM) MCDF (15 μM) 147.5 (9.6)* 2,3,7,8-TCDD (10 nM) MCDF (50 μM) 136.8 (14.7)* 2,3,7,8-TCDD (10 nM) MCDF (75 μM) 133.1 (10.4)* 2,3,7,8-TCDD (10 nM) MCDF (100 μM) 123.1 (7.1)* 2,3,7,8-TCDD (10 nM) MCDF (150 μM) 129.3 (2.9)* 2,3,7,8-TCDD (25 nM) MCDF (15 μM) 146.9 (9.6)* 2,3,7,8-TCDD (25 nM) MCDF (50 μM) 85.5 (16.5)* 2,3,7,8-TCDD (25 nM) MCDF (75 μM) 91.3 (9.7)* 2,3,7,8-TCDD (25 nM) MCDF (100 μM) 100.5 (10.4)* 2,3,7,8-TCDD (25 nM) MCDF (150 μM) 108.1 (18.1)*

Significantly different from control (p <0.01) TABLE 5

Interactive Effects of Estradiol, 2,3,7,8-TCDD and MCDF on ERn in MCF-7 Cells

Treatment Coadministered ERn (dose) with (dose) (fmol/mg protein) control (10.2) estradiol (10 nM) (3.9)* estradiol (10 nM) (17.6)* estradiol (10 nM) (7.9)* estradiol (10 nM) (14.3)*

estradiol (10 nM) (5.5)*

estradiol (10 nM) (9.7)*

estradiol (10 nM) (2.1)*

estradiol (10 nM) (5.3)*

estradiol (10 nM) (8.3)*

* Significantly different from control (p< 0.01).

MCF-7 cells, obtained from American Type Culture

Collection (Rockville, Maryland) were maintained at 37° C

2 in closed Corning T-25, 75 or 150 cm flasks (Corning

Glassworks, Corning, New York) and passaged in logarithmic growth phase. Growth medium was Eagle's minimal essential medium containing Hanks' balanced salts, L-glutamine and non-essential amino acids (GIBCO 410-1600EB) , supplemented with 0.006 ug/ml insulin (Sigma), 0.01 M HEPES buffer

(GIBCO), 5.5 M glucose, 0.026 M sodium bicarbonate, 0.1 mM non-essential amino acids (GIBCO), 0.1 mM essential a ino acids (GIBCO), 0.01 mM sodium pyruvate (Sigma), 1.25 mg/liter amphotericin B, 2500 units penicillin/liter, 12.5 mg/liter streptomycin/gentamycin and 5% charcoal-dextran- stripped fetal bovine serum.

For the 2,3,7,8-TCDD antiestrogenicity studies, cells were treated 24 hours prior to harvesting with various concentrations of 2,3,7,8-TCDD (0.1 nM to 100 nM 2,3,7,8- TCDD). Interactive effects between 2,3,7,8-TCDD and estradiol were conducted using these same concentrations of 2,3,7,8-TCDD and 10 nM estradiol. To study possible antagonistic/antiestrogenic effects of 6-methyl-l,3,8- trichlorodibenzofuran (MCDF) , varying concentrations from 15 to 150 μM were added or coadministered to the MCF-7 cells.

MCF-7 cells from a single near confluent T-25 flask were incubated at 37 C for 0.5 hours prior to harvesting

3 with 10 nM 2,4,6,7-[ H]-estradiol. The medium was then decanted and the cells were rinsed once with Hanks' balanced salt solution containing 1 mM EDTA. The cells were collected by centrifugation, washed gently in 2 ml of TEDG buffer and homogenized in 300 ul of TEDG buffer containing protease inhibitors [soybean trypsin inhibitor 5 mg/ml (Sigma), leupeptin 1 mg/ml (Sigma), and phenylmethylsulfonyl fluoride 1 mg/ml (Sigma)] by 40 strokes of a Dounce homogenizer. The homogenate was centrifuged at 800 x g for 10 minutes. The crude nuclear pellet was resuspended in 300 ul of TEDGK buffer (10 mM Tris-HCIL, 1.5 mM EDTA-4Na, 10 mM dithiothreitol, 10%

(v/v) glycerol, 0.8 M KCI, pH 8.5 at 4° C) . The nuclear ER levels were then determined as described below.

The nuclear extract previously obtained was incubated 3 with 10 nM [ H]-estradiol plus or minus radioinert diethylstilbestrol for 1 hour at 0-4° C, with resuspension by agitation every 15 minutes. The sample was then centrifuged at 180,000 x g for 30 minutes, then 200 ul of the supernatant obtained was layered onto linear 1-20% sucrose gradients prepared in TEDGMK buffer. As before, the gradients were centrifuged at 406,000 x g for 2.5 hours in a vertical rotor. Following centrifugation, 30 fractions of 0.16 ul were collected and the radioactivity in each sample was determined.

The dose response data following MCDF treatment are given in Tables 4 and 5. MCDF significantly decreased ERn levels in the MCF-7 cells. The combined effects of MCDF plus 2,3,7,8-TCDD was clearly not additive. The ERn levels for the combined treatment were not significantly different than those observed after treatment with either 2,3,7,8-TCDD or MCDF.

Claims

CLAIMS !

1. Compounds of the formula

wherein X, individually and independently, is a hydrogen or a substituent selected from the group bromine, chlorine, fluorine, or a linear or branched alkyl group of one to four carbons, with no more than one X being a hydrogen;

wherein Y is a substituent selected from the group bromine, chlorine, fluorine, or a linear or branched alkyl group of one to four carbons;

said compound having at least two halogenated substituents; and

excluding 2, ,6,8-tetrachlorodibenzofuran.

2. Compounds of the formula

wherein X, individually and independently, is a hydrogen or a substituent selected from the group bromine, chlorine, fluorine, or a linear or branched alkyl group of one to four carbons, with no more than one X being a hydrogen;

wherein Y is a substituent selected from the group bromine, chlorine, fluorine, or a linear or branched alkyl group of one to four carbons;

said compound having at least two halogenated substituents; and

excluding 1,3,6,8-tetrachlorodibenzofuran.

3. A compound as recited in claim 1 wherein X is a hydrogen or a chlorine.

4. A compound as recited in claim 2 wherein X is a hydrogen or a chlorine.

5. A compound as recited in claim 1 wherein X is a chlorine or a hydrogen and Y is a linear or branched alkyl group of one to four carbons.

6. A compound as recited in claim 2 wherein X is a chlorine or a hydrogen and Y is a linear or branched alkyl group of one to four carbons.

7. A compound as recited in claim 1 wherein X is a chlorine and Y is a branched or linear alkyl group of one to four carbons.

8. A compound as recited in claim 2 wherein X is a chlorine and Y is a linear or branched alkyl group of one to four carbons.

9. A compound as recited in claim 5 wherein X is a chlorine and Y is a methyl.

10. A compound as recited in claim 6 wherein X is a chlorine and Y is a methyl.

11. A method of inhibiting estrogen activity comprising administering a biologically active amount of a compound claimed in claims 1 through 10 or 1,3,6,8 or 2,4,6,8- tetrachlorodibenzofuran.

12. A method of treating estrogen-dependent tumors comprising administering a biologically active amount of a compound claimed in claims 1 through 10 or 1,3,6,8 or 2,4,6,8-tetrachlorodibenzofuran.

13. A composition comprising a biologically active amount of a compound claimed in claims 1 through 10 or 1,3,6,8 or 2,4,6,8-tetrachlorodibenzofuran and an appropriate carrier.

14. A composition as recited in claim 13 wherein the carrier is suitable for a lipid soluble compound.

15. A method of producing alkylated poly halodibenzofurans comprising:

reacting an alkylated halophenol with a haloaniline and amyl nitrite to produce an intermediate;

recovering and purifying said intermediate;

reacting said intermediate in the presence of anhydrous potassium carbonate or other appropriate base to produce the alkylated polyhalodibenzofuran;

recovering and purifying the alkylated polychlorodibenzofuran.

16. The method of claim 15 wherein the alkylated halophenol is 2-methyl-4-chlorophenol, the haloaniline is 2,4,6-trichloroaniline, and the product is 6-methyl- 1,3,8-trichlorodibenzofuran.

17. A method of producing alkylated polyhalodibenzo-p- dioxins comprising:

reacting an alkylated halocatechol with trihalonitrobenzene, in the presence of potassium carbonate or other appropriate base to produce an alkylated polyhalodibenzo-p-dioxin; and recovering and purifying said alkylated polyhalodibenzo-p-dioxin.

18. The method of claim 19 wherein the alkylated catechol is a 6-methyl-4-chlorocatechol, the trinitrobenzene is a 2,3,5-trichloronitrobenzene and the product is 6-methyl- 1,3,8-trichlorodibenzo-p-dioxin.