Patent Application: US-93425401-A

Abstract:
novel non - steroidal estrogen receptor ligands and methods of synthesis are disclosed . the novel molecules are intended for use in therapeutic preparations for the treatment of estrogen receptor related disease states . the compounds specified are tetra - cyclic derivatives and have been shown to be antiproliferative against human estrogen - dependent cancer cells and to have good binding affinity for the estrogen receptor .

Description:
the present invention relates to non - steroidal compounds that demonstrate high - binding affinity for the estrogen receptor , while being devoid of any agonistic effects on reproductive tissues and to non - steroidal compounds that have a high binding affinity for the estrogen receptor while also having some agonistic activity specific to the skeletal and cardiovascular systems . in one preferred embodiment of the non - steroidal compounds , as described by formula i , displaying high binding affinity for the estrogen receptor , the a , b , c and d rings are aromatic . r 1 is 1 - piperidinyl , r 2 is c ═ och ( ch 3 ) 2 , r 3 is ch 3 , r 4 is hydrogen , r 5 is hydroxyl and n = 2 . preferably at least one embodiment is represented by the following formula 3 , or a pharmaceutically acceptable salt thereof : in another preferred embodiment of the invention , the a , b , c and d rings are aromatic . r 1 is 1 - piperidinyl , r 2 is c ═ och 3 , r 3 is ch 3 , r 4 is hydrogen , r 5 is hydroxyl and n = 2 . preferably at least one embodiment is represented by the following formula 4 , or a pharmaceutically acceptable salt thereof : in another preferred embodiment of the invention , the a , b , c and d rings are aromatic . r 1 is 1 - piperidinyl , r 2 is ch 3 c ═ ch 2 , r 3 is ch 3 , r 4 is hydrogen , r 5 is hydroxyl and n = 2 . preferably at least one embodiment is represented by the following formula 5 , or a pharmaceutically acceptable salt thereof : in another preferred embodiment of the invention , the a , b , c and d rings are aromatic . r 1 is 1 - piperidinyl , r 2 is c ( ch 3 )═ c ( ch 3 ) 2 , r 3 is ch 3 , r 4 is hydrogen , r 5 is hydroxyl and n = 2 . preferably at least one embodiment is represented by the following formula 6 , or a pharmaceutically acceptable salt thereof : in yet another preferred embodiment of the invention , the a , b , c and d rings are aromatic . r 1 is 1 - piperidinyl , r 2 is ch ( oh ) ch 3 , r 3 is ch 3 , r 4 is hydrogen , r 5 is hydroxyl and n = 2 . preferably at least one embodiment is represented by the following formula 7 , or a pharmaceutically acceptable salt thereof : set forth below is a preferred synthesis scheme for the preparation of certain preferred embodiments of the anti - estrogens in accordance with the invention . the synthetic steps set forth below are set forth merely by way of examples . those skilled in the art will readily recognize alternative synthetic pathways and variations capable of producing a variety of anti - estrogens in accordance with the present invention . compounds of formula 1 where z ═ o and the b ring is non - aromatic are prepared as depicted in scheme 1 . compounds 1 - 1 are made by acylation of substituted methyl 5 - acetyl salicylates using trifluoromethanesulfonic anhydride . dimethoxyphenylboronic acids dissolved in a polar protic organic solvent , ethanol for example , are added to a solution of 1 - 1 dissolved in an organic solvent , 1 , 2 - dimethoxyethane for example . to this mixture an inorganic base , such as potassium carbonate , and a catalytic amount of tetrakis ( triphenylphosphine ) palladium were added and the mixture refluxed to give the ester biphenyls 1 - 2 ( suzuki coupling ). alkylation of 1 - 2 using a strong base , lithium diisopropylamide ( lda ) for example , and an alkyl halide gives further alkylated biphenyls 1 - 3 . the saponification of 1 - 3 in aqueous base , such as potassium hydroxide , at reflux , affords the free acid biphenyls 1 - 4 . cyclization to the dibenzo lactones 1 - 5 occurs by treatment of 1 - 4 with thionyl chloride followed by aluminum trichloride in a refluxing organic solvent such as 1 , 2 - dichloroethane . treatment of 1 - 5 with a diol , ethylene glycol , for example , in the presence of a catalytic amount of an acid such as p - toluenesulfonic acid in a refluxing organic solvent affords the ketone protected dibenzo [ c ] chromen - 6 - ones 1 - 6 . transmetallation of 4 -[ 2 -( 1 - amino )- alkoxy ]- bromobenzenes using n - butyl lithium or magnesium metal yields the corresponding lithium or magnesium reagents which were subsequently reacted with 1 - 6 to afford the protected dibenzopyran hemiketals 1 - 7 . treatment of 1 - 7 with triethylsilane , followed by either boron trifluoride - etherate or trifluoroacetic acid , reduces the hemiketal with concomitant deprotection of the ketone , to give the substituted dibenzopyran ethers 1 - 8 . treatment of 1 - 8 with either boron tribromide or concentrated hydrogen bromide ( 48 %) yields compounds of formula 1 ( depicted by structure 1 - 9 ), more specifically compounds of formula 1 wherein z ═ o , the b - ring is non - aromatic , wherein r 2 is represented by c ═ or 6 and wherein r 6 is as previously defined . it is readily recognized by those skilled in the art that compounds of general structure 1 - 9 can be further reacted with a variety of grignard reagents , followed by heating in a non - aqueous acidic medium such as glacial acetic acid containing sulfuric acid , giving rise to the corresponding alkene products . it is also readily recognized by those skilled in the art that compounds of general structure 1 - 9 can be further reacted with a reducing agent such as hydrogen gas over palladized charcoal or with a hydride reducing agent such as nabh 4 , giving rise to the corresponding alcohols . it is readily recognized that these alcohols can be reacted in a non - aqueous medium such as glacial acetic acid containing sulfuric acid , to yield the corresponding alkene products . compounds of formula 1 where z ═ c and wherein the b ring and c - ring are aromatic or where z ═ c and wherein the b - ring is not aromatic and the c - ring is aromatic are prepared as shown in scheme 2 . substituted anisoles are converted to methoxy benzaldehydes 2 - 1 , by treatment with hydrogen chloride and zinc cyanide , followed by aluminum trichloride . treatment of 2 - 1 with p - methoxyacetophenone and a base , such as sodium hydroxide in ethanol , under aldol condensation conditions , yields the enones 2 - 2 . treatment of 2 - 2 in a one - pot reaction with bromine , followed by potassium acetate / acetic acid , followed by heating with dbu yields the ynones 2 - 3 . the biphenyl ketones 2 - 4 are obtained via a diels - alder reaction , by heating 2 - 3 in the presence of substituted cyanopyrones . compounds 2 - 4 are cyclized in a one pot intramolecular wittig reaction to the cyano phenanthrenes 2 - 5 by bromination with nbs , followed by conversion to the triphenylphosphonium salt and then by conversion to the ylide with a base such as sodium hydroxide . treatment of 2 - 5 with a grignard reagent in the presence of a catalytic amount of cuprous bromide , followed by stirring in aqueous acid such as 10 % sulfuric acid , gives the keto phenanthrenes 2 - 6 . compounds 2 - 6 are demethylated with either boron tribromide or with concentrated hydrogen bromide ( 48 %) to the diphenolic phenanthrenes 2 - 7 which are subsequently treated with chloroalkylamines in the presence of a base such as sodium hydroxide , to yield compounds of formula 1 ( depicted by structure 2 - 8 ), more specifically compounds of formula 1 wherein z ═ c , the b - ring and the c - ring are aromatic and wherein r 6 is as previously defined . compounds of general structure 2 - 8 can be treated with a second grignard reagent , to provide the tertiary alcohol phenanthrenes 2 - 9 . it should be readily recognized by those skilled in the art , that a variety of grignard reagents can be employed in the formation of the tertiary alcohol phenanthrenes 2 - 9 . it should also be readily recognized that the tertiary alcohol phenanthrenes 2 - 9 can be further reacted by stirring in acetic acid at elevated temperatures , preferably 75 ° c ., to yield the corresponding alkene products . compounds of general structure 2 - 8 can be converted to the corresponding alcohols 2 - 10 , via hydrogenation using hydrogen gas over palladized charcoal . the corresponding alcohols 2 - 10 can also be obtained by using a “ hydride reducing agent such as sodium borohydride . alternatively , compounds of general structure 2 - 8 can also be reduced via “ dissolved metal reduction ” by using for example , li / nh 3 providing compounds of general structure 2 - 12 . it is readily recognized by those skilled in the art that the carbonyl functionality of compounds of general structure 2 - 8 can be protected prior to reaction with li / nh 3 and then deprotected , in order to maintain the carbonyl functionality providing compounds of general structure 2 - 11 . it is also readily recognized that compounds of general structure 2 - 11 can be further reacted with a variety of grignard reagents , followed by heating in a non - aqueous acidic medium such as glacial acetic acid containing sulfuric acid , giving rise to the corresponding alkene products . it is also readily recognized that compounds of general structure 2 - 11 can be further reacted with a reducing agent such as hydrogen gas over palladized charcoal or with a hydride reducing agent such as nabh 4 , giving rise to the corresponding alcohols . it should also be readily recognized that these alcohols can be reacted in a non - aqueous medium such as glacial acetic acid containing sulfuric acid , to yield the corresponding alkene products . the present invention is illustrated in further detail by the following non - limiting examples . methyl 5 - acetylsalicylate ( 25 . 0 g , 129 mmol ) was mixed with ch 2 cl 2 ( 250 ml ) and pyridine ( 60 ml ) under argon at 0 ° c . trifluormethanesulfonic anhydride ( 37 . 9 g , 133 mmol ) was added over 20 min . the reaction was stirred for an additional 30 min and then quenched with water ( 500 ml ). the organic layer was separated and washed three times with 5 % hcl ( 80 ml ). after removing the solvent , the solid obtained was dried under vacuum to yield 40 . 3 g ( 96 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 56 ( 3h , s , coch 3 ), 3 . 89 ( 3h , s , och 3 ), 7 . 32 ( 1h , d , arh ), 8 . 12 ( 1h , d , arh ), 8 . 52 ( 1h , s , arh ). 2 , 4 - dimethoxyphenylboronic acid ( 24 . 0 g , 134 mmol ) was dissolved in ethanol ( 250 ml ). the compound from step a ( 21 g , 67 mmol ) was dissolved in 1 , 2 - dimethoxyethane ( 375 ml ). the two solutions were mixed with tetrakis ( triphenylphosphine ) palladium ( 1 g , 0 . 9 mmol ) and k 2 co 3 ( 8 . 9 g , 64 mmol ). the resulting suspension was refluxed for 2 h and then poured into saturated nahco 3 ( 1 l ). the mixture was extracted three times with ch 2 cl 2 ( 400 ml ) and dried over na 2 so 4 . the solvent was removed to yield 35 . 6 g of crude product which was chromatographed on silica gel with hexane / ethyl acetate ( 2 : 1 ) followed by hexane / ethyl acetate ( 1 : 1 ) to yield 18 . 3 g ( 87 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 68 ( 3h , s , coch 3 ), 3 . 73 ( 3h , s , och 3 ), 3 . 76 ( 3h , s , och 3 ), 3 . 88 ( 3h , s , co 2 ch 3 ), 6 . 52 ( 1h , s arh ), 6 . 62 ( 1h , d , arh ), 7 . 23 ( 1h , d , arh ), 7 . 45 ( 1h , d , arh ), 8 . 13 ( 1h , d , arh ), 8 . 42 ( 1h , s , arh ). optionally , this compound can be dissolved in dme under argon and cooled to − 20 ° c . and reacted with lda and the resulting enolate trapped with an appropriate alkyl halide such as for example methyl iodide . the compound from step b ( 9 . 3 g , 29 . 6 mmol ) was mixed with distilled water ( 500 ml ) and koh ( 3 . 3 g , 59 mmol ) added . the mixture was refluxed for 3 h and then acidified to ph 1 with concentrated hcl . the resulting precipitate was filtered and dried under vacuum at 45 ° c . for 3 h to yield 8 . 1 g ( 90 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 71 ( 3h , s , coch 3 ), 3 . 92 ( 3h , s , och 3 ), 3 . 96 ( 3h , s , och 3 ), 6 . 94 ( 1h , d , arh ), 6 . 96 ( 1h , s , arh ), 7 . 55 ( 1h , d , arh ), 8 . 17 ( 1h , d , arh ), 8 . 51 ( 1h , s , arh ). the compound from step c was mixed with 1 , 2 - dichloroethane ( 60 ml ) and socl 2 ( 1 . 7 ml , 23 mmol ) was added . the mixture was refluxed for 90 min and then cooled to 0 ° c . alcl 3 ( 2 . 3 g , 17 . 3 mmol ) was added and the reaction stirred at room temperature overnight . the solvent was removed and the crude product chromatographed on silica gel with hexane / ethyl acetate ( 1 : 1 ) to yield 5 . 1 g ( 90 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 74 ( 3h , s , coch 3 ), 3 . 94 ( 3h , s , och 3 ), 6 . 92 ( 1h , s , arh ), 6 . 99 ( 1h , d , arh ), 8 . 02 ( 1h , d , arh ), 8 . 11 ( 1h , d , arh ), 8 . 41 ( 1h , d , arh ), 8 . 91 ( 1h , s , arh ). the compound from step d ( 5 . 2 g , 19 mmol ), ethylene glycol ( 4 . 4 g , 71 mmol ) and a catalytic amount of p - toluenesulfonic acid ( 0 . 2 g ) were dissolved in benzene ( 300 ml ) and refluxed for 28 h . the solvent was removed and the crude product chromatographed on silica gel with hexane / ethyl acetate ( 2 : 1 ) to yield 3 . 6 g ( 60 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 75 ( 3h , s , ch 3 ), 3 . 82 ( 2h , t , och 2 ), 3 . 91 ( 3h , s , och 3 ), 4 . 10 ( 2h , t , och 2 ), 6 . 93 ( 1h , s , arh ), 6 . 98 ( 1h , d , arh ), 7 . 47 ( 3h , m , arh ), 8 . 04 ( 1h , s , arh ). 4 - bromophenol ( 5 . 2 g , 30 mmol ), k 2 co 3 ( 10 . 4 g , 75 mmol ) and anhydrous dmf ( 50 ml ) were mixed together under ar and heated at 100 ° c . 1 -( 2 - chloroethyl )- piperidine hydrochloride ( 5 . 5 g , 30 mmol ) was added in portions over 10 min and the reaction maintained at 100 ° c . for 1 . 5 h . after cooling the reaction , the solid was filtered off and the solvent removed under vacuum . the crude product was chromatographed on silica gel with methanol / ethyl acetate ( 3 : 1 ) to yield 6 . 7 g ( 78 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 44 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 59 , ( 4h , m , nch 2 c h 2 ), 2 . 48 ( 4h , m , nc h 2 ch 2 ), 2 . 73 ( 2h , t , nc h 2 ch 2 o ), 4 . 03 ( 2h , t , nch 2 c h 2 o ), 6 . 76 ( 2h , d , arh ), 7 . 33 ( 2h , d , arh ). 1 -[ 2 -( 4 - bromo - phenoxy )- ethyl ]- piperidine ( 0 . 76 g , 2 . 7 mmol ) from step f was dissolved in thf ( 15 ml ) under ar and cooled to − 78 ° c . n - butyl lithium ( 10 . 0 m , 0 . 26 ml , 2 . 6 mmol ) was added and the mixture was stirred at − 78 ° c . for 1 hour . the compound from step e ( 1 . 11 g , 3 . 56 mmol ) was dissolved in thf ( 35 ml ) under ar and cooled to − 78 ° c . to which the freshly prepared lithium salt of 1 -( 2 -( 4 - bromo - phenoxy )- ethyl )- piperidine was transferred via a cannula . the reaction was stirred for 2 h . water ( 150 ml ) was added and the reaction extracted three times with ethyl acetate ( 150 ml ). after removing the solvent the crude product was chromatographed on silica gel with 7 % ch 3 oh / ch 2 cl 2 to yield 0 . 56 g ( 48 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 44 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 59 ( 4h , m , nch 2 c h 2 ch 2 ) 1 . 61 ( 3h , s , ch 3 ), 2 . 25 ( 4h , m , nc h 2 ch 2 ch 2 ), 2 . 83 ( 2h , m , nc h 2 ch 2 o ), 3 . 78 ( 2h , m , oc h 2 ), 3 . 85 ( 3h , s , och 3 ), 4 . 01 ( 2h , m , och 2 ), 4 . 18 ( 2h , m , nch 2 c h 2 o ), 6 . 69 ( 1h , s , arh ), 6 . 73 ( 1h , d , arh ), 6 . 96 ( 2h , d , arh ), 7 . 17 ( 1h , s , arh ), 7 . 56 ( 1h , m , arh ), 7 . 60 ( 2h , d , arh ), 7 . 76 ( 2h , m , arh ). the compound from step g ( 1 . 66 g , 3 . 2 mmol ) was dissolved in ch 2 cl 2 ( 90 ml ) and cooled to − 78 ° c . et 3 sih ( 3 . 1 ml , 19 . 2 mmol ) and bf 3 - et 2 o ( 2 . 6 ml , 19 . 2 mmol ) were added slowly under ar . the mixture was stirred at − 78 ° c ., then at room temperature overnight . saturated nahco 3 ( 100 ml ) was added and the mixture extracted three times with ethyl acetate ( 150 ml ). the combined organic layer was washed with brine ( 100 ml ) and dried over mgso 4 . the solvent was removed and the crude product was chromatographed on silica gel with 7 % ch 3 oh / ch 2 cl 2 to yield a fraction containing a mixture of the title compound along with side products and a fraction containing 0 . 52 g ( 32 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 42 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 59 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 50 ( 7h , m , nc h 2 ch 2 ch 2 and coch 3 ), 2 . 76 ( 2h , m , nc h 2 ch 2 o ), 3 . 77 ( 3h , s , och 3 ), 4 . 08 ( 2h , m , nch 2 c h 2 o ), 6 . 14 ( 1h , s , ph 2 c h o ), 6 . 50 ( 1h , s , arh ), 6 . 61 ( 1h , d , arh ), 6 . 86 ( 2h , d , arh ), 7 . 22 ( 2h , d , arh ), 7 . 45 ( 1h , s , arh ), 7 . 66 ( 1h , d , arh ), 7 . 72 ( 1h , d , arh ), 7 . 93 ( 1h , d , arh ). the mixture containing the compound from step h ( 0 . 25 g , 0 . 565 mmol ) was dissolved in ch 2 cl 2 ( 40 ml ) and cooled to − 78 ° c . and bbr 3 in ch 2 cl 2 ( 1m , 11 ml ) was added . the mixture was kept at − 78 ° c . and allowed to warm to 15 ° c . overnight . the reaction was quenched with saturated nahco 3 ( 5 ml ) and extracted with ch 2 cl 2 ( 20 ml ). the organic layer was washed with saturated nahco 3 , water and dried over na 2 so 4 . the solvent was removed and the crude product purified by preparative tlc plates . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 55 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 80 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 56 ( 3h , s , coch 3 ), 2 . 81 ( 4h , m , nc h 2 ch 2 ch 2 ), 3 . 01 ( 2h , m , nc h 2 ch 2 o ), 4 . 22 ( 2h , m , nch 2 c h 2 o ), 6 . 13 ( 1h , s , ph 2 c h o ), 6 . 50 ( 1h , s , arh ), 6 . 60 ( 1h , d , arh ), 6 . 80 ( 2h , d , arh ), 7 . 21 ( 2h , d , arh ), 7 . 49 ( 1h , s , arh ), 7 . 65 ( 1h , d , arh ), 7 . 74 ( 1h , d , arh ), 7 . 97 ( 1h , d , arh ). 3 , 5 - dimethylanisole ( 25 . 3 g , 0 . 186 mol ) was added to ( chcl 2 ) 2 ( 180 ml ) and maintained at 17 ° c . zn ( cn ) 2 ( 37 . 1 g , 0 . 316 mol ) was added and hcl gas was bubbled through the mixture with stirring . the rate of hcl gas addition was adjusted to allow for hcl absorption . after 1 hour of hcl gas addition the rate of absorption significantly decreased and alcl 3 ( 37 . 2 g , 0 . 279 mol ) was added . a slow rate of hcl gas flow was maintained . the temperature was increased to 55 ° c . and the reaction maintained at this temperature for 3 h . the reaction mixture was then poured onto a mixture of ice ( 800 ml ) and concentrated hcl ( 800 ml ). the content of the reaction vessel was then rinsed twice with chcl 3 ( 200 ml ) and added to the aqueous layer . the resulting biphasic system was stirred at 60 - 65 ° c . overnight . the organic layer was separated and the aqueous layer washed with 200 ml and 150 ml of chcl 3 . the combined extract washed with deionized water ( 3 × 200 ml ) and the organic solvents removed by evaporation . the concentrate was transferred to a distillation flask equipped with a vigreux column and distilled at 110 ° c . ( 0 . 7 mm hg ) to give a distillate ( 29 . 2 g ) containing residual ( chcl 2 ) 2 and a mixture composed of the desired 4 - methoxy - 2 , 6 - dimethylbenzaldehyde isomer and some of the unwanted 6 - methoxy - 2 , 4 - dimethylbenzaldehyde isomer in a 2 : 1 ratio . the distillate was added to methyl t - butyl ether and crystallized overnight at 4 ° c . the crystals were filtered and washed with 5 ml of a mixture of ethyl acetate : hexane ( 1 : 12 ) to give 7 . 84 g of the title compound . the mother liquor was concentrated and chromatographed on silica gel with ethyl acetate / hexane ( 1 : 12 ) to give an additional 10 . 74 g of the title compound ( 18 . 58 g total , 61 %). 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 62 ( 6h , s , ch 3 ), 6 . 59 ( 2h , s , arh ), 10 . 48 ( 1h , s , cho ). the compound from step a ( 18 . 58 g , 0 . 113 mol ) and 4 - methoxyacetophenone ( 17 . 42 g , 0 . 116 mol ) were added to anhydrous ethanol ( 110 ml ) and naoh ( 2 . 5 g ) and stirred overnight at room temperature . the precipitate that formed was filtered , washed with water ( 3 × 50 ml ) and dried overnight under high vacuum to give 29 . 8 g of the title compound . the filtrate was stirred overnight to give and additional 1 . 1 g ( total 30 . 9 g , 92 %). 1 h - nmr ( 400 mhz , cdcl 3 ) δ2 . 44 ( 6h , s , ch 3 ), 3 . 83 ( 3h , s , och 3 ), 3 . 90 ( 3h , s , och 3 ), 6 . 66 ( 2h , s , arh ), 6 . 99 ( 2h , d , arh ), 7 . 16 , ( 1h , d , arch ), 7 . 96 ( 1h , d , coch ), 8 . 01 ( 2h , d , arh ). to a solution of the compound from step b ( 30 . 9 g , 0 . 104 mol ), dissolved in ch 2 cl 2 ( 200 ml ) and cooled in an ice bath , was added a solution of br 2 ( 16 . 7 g , 5 . 37 ml , 0 . 104 mol ) in ch 2 cl 2 ( 100 ml ) over 105 min . with additional stirring for 2 h . a second portion of br 2 ( 1 . 2 ml ) in ch 2 cl 2 ( 50 ml ) was added over 30 min and the reaction left at room temperature overnight , followed by the evaporation of the solvent to give the title compound that was used without further purification . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 51 ( 3h , s , ch 3 ), 2 . 71 ( 3h , s , ch 3 ), 3 . 81 ( 3h , s , och 3 ), 3 . 92 ( 3h , s , och 3 ), 6 . 16 ( 1h , d , cochbr ), 6 . 24 ( 1h , d , arh ), 6 . 34 ( 1h , d , archbr ), 6 . 66 ( 1h , d , arh ), 7 . 03 ( 2h , d , arh ), 8 . 07 ( 2h , d , arh ). the crude compound from step i was added to acetic acid ( 550 ml ) and koac ( 12 . 5 g , 0 . 13 mol ) and stirred for 6 hours . additional koac ( 3 . 0 g ) was added and the mixture stirred overnight . the hoac was evaporated and the residue dissolved in water ( 300 ml ), extracted with chcl 3 ( 300 ml ), the chcl 3 extract washed with water ( 3 × 150 ml ) and concentrated to give the title compound that was used without further purification . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 88 ( 3h , s , coch 3 ), 2 . 58 ( 3h , s , ch 3 ), 2 . 65 ( 3h , s , ch 3 ), 3 . 80 ( 3h , s , och 3 ), 3 . 91 ( 3h , s , och 3 ), 5 . 76 ( 1h , d , cochbr ), 6 . 60 ( 2h , s , arh ), 6 . 87 ( 1h , d , archo ), 6 . 66 ( 1h , d , arh ), 7 . 02 ( 2h , d , arh ), 8 . 04 ( 2h , d , arh ). dbu ( 36 . 2 g , 0 . 238 mol ) and the crude compound from step ii were added to thf ( 350 ml ) and heated to 55 ° c . overnight . the reaction mixture was filtered and the precipitate washed with thf ( 2 × 100 ml ). the filtrate was evaporated and the residue dissolved in chcl 3 ( 300 ml ), washed with water ( 2 × 150 ml ), 8 % hcl ( ph 2 , 150 ml ), and water ( 2 × 150 ml ). it was noted that the final separation was made easier if an aqueous solution of nahco 3 ( 15 ml ) was added to the last extraction . after evaporation of the solvent and drying overnight under high vacuum , the crude material was crystallized from anhydrous toluene ( 30 ml ) by cooling to room temperature and then to 4 ° c . to give 22 . 4 g , ( 73 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 55 ( 6h , s , ch 3 ), 3 . 82 ( 3h , s , och 3 ), 3 . 89 ( 3h , s , och 3 ), 6 . 65 - 8 . 22 ( 6h , arh ). the compound from step c , iii ( 4 . 20 g , 15 . 7 mmol ) and 5 - cyanopyrone ( 1 . 90 g , 15 . 7 mmol ) ( helv . chim . acta ( 1990 ) 73 , 883 ) were heated at 190 ° c . for 17 h . the crude reaction was chromatographed on silica gel with ethyl acetate / hexane ( 1 : 6 ) then ethyl acetate / hexane ( 1 : 2 ) to give 4 . 4 g ( 75 . 5 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 96 ( 6h , s , ch 3 ), 3 . 75 ( 3h , s , och 3 ), 3 . 88 ( 3h , s , och 3 ), 6 . 54 - 7 . 83 ( 9h , arh ). to a refluxing solution of the compound from step d ( 45 . 5 g , 122 mmol ) and aibn ( 1 . 0 g ) in ccl 4 ( 2 . 5 l ) was added a mixture of nbs ( 23 . 0 g , 129 mmol ) and aibn ( 1 . 0 g ) in ten equal portions . the reaction mixture was refluxed for 2 h after the addition of the last portion of nbs / aibn . the reaction mixture was concentrated to approximately 1 l and washed 3 times with water ( 1 l ). the organic layer was dried with anhydrous na 2 so 4 , filtered and the solvent removed . toluene ( 200 ml ) was added to the residue and then evaporated . repeating this procedure gave 48 g of the title compound that was used without further purification . the crude mixture from i was dissolved in dmf ( 200 ml ), triphenyphosphine ( 48 g ) added and the reaction stirred at 100 ° c . for 5 h . the hot reaction mixture was slowly poured into vigorously stirring methyl t - butyl ether ( 4 l ), stirred for an additional 30 min and filtered . the residue was washed twice with methyl t - butyl ether ( 200 ml ) and immediately dissolved in ch 2 cl 2 ( 1 l ). the crude product from ii was added over 5 h to a vigorously stirred mixture of ch 2 cl 2 ( 2 l ) and 50 % aqueous naoh ( 1 l ) at 35 ° c . stirring was continued for an additional 3 h at 35 - 40 ° c . the organic layer was separated and washed consecutively with 1 n hcl ( 200 ml ), water ( 200 ml ), and saturated nahco 3 ( 200 ml ). after drying the organic layer over anhydrous na 2 so 4 and removing the solvent the residue was crystallized from ch 2 cl 2 / hexanes / ethanol ( 80 / 60 / 10 , 150 ml ) to give 24 g of the title compound . the filtrated was concentrated and chromatographed on silica using ch 2 cl 2 / hexanes ( 50 / 50 ) to give an additional 5 . 5 g ( total 29 . 5 g , 68 . 5 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 3 . 12 ( 3h , s , ch 3 ), 3 . 94 ( 3h , s , och 3 ), 3 . 97 ( 3h , s , och 3 ), 7 . 07 - 8 . 94 ( 10h , arh ). the compound from step e , iii ( 10 . 0 g , 28 . 3 mmol ) was added to anhydrous thf ( 200 ml ) and isopropyl magnesium chloride ( 2 . 0 m ) in thf ( 15 . 6 ml ) was added with stirring under ar . after 10 min , crystalline cubr ( 72 mg ) was added and the reaction stirred for 9 h . tlc analysis indicated that there was still starting material present and an additional 8 ml of the grignard reagent was added and the reaction stirred overnight . to the reaction was then added 10 % h 2 so 4 ( 150 ml ) and the mixture placed on a rotary evaporator to remove the thf . the remaining aqueous layer was stirred overnight at room temperature , extracted with chcl 3 ( 3 × 250 ml ) and washed with water ( 3 × 150 ml ). after removing the chcl 3 the residue was dried under high vacuum to yield 11 . 2 g ( quantitative yield ) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 22 ( 6h , d , ch 3 ), 3 . 15 ( 3h , s , ch 3 ), 3 . 54 ( 1h , septet , coch ), 3 . 94 ( 3h , s , och 3 ), 3 . 96 ( 3h , s , och 3 ), 7 . 09 - 8 . 93 ( 10h , arh ). the compound from step f ( 5 . 53 g , 13 . 9 mmol ) and 48 % hbr ( 44 ml ) and acetic acid ( 55 ml ) were heated ( oil bath at 126 ° c .) in a closed thick walled pressure flask for 3 . 5 h . the reaction was then poured into water ( 300 ml ) and extracted with ethyl acetate ( 3 × 250 ml ). the combined extracts were then washed with water ( 3 × 150 ml ). after removing the solvent , the residue was dried under high vacuum to yield the title compound quantitatively . 1 h - nmr ( 400 mhz , dmso - d 6 ) δ h 1 . 10 ( 6h , d , ch 3 ), 3 . 03 ( 3h , s , ch 3 ), 3 . 56 ( 1 h , septet , coch ), 6 . 96 - 8 . 87 ( 10h , arh ), 9 . 65 ( 1h , br , oh ), 10 . 1 ( 1h , br , oh ). the compound from step g ( 1 . 1 g , 3 . 0 mmol ), aliquat ( 3 . 03 g , 7 . 5 mmol ), naoh ( 1 . 2 g , 30 mmol ) and 1 -( 2 - chloroethyl ) piperidinium hydrochloride ( 0 . 552 g , 3 . 0 mmol ) were added to tmu ( 70 ml ) and heated at 50 ° c . for 11 h . the reaction mixture was added to water ( 500 ml ), acidified with concentrated hcl ( 3 ml ) and then neutralized with an excess of nahco 3 until ph 8 . the reaction was extracted with ethyl acetate ( 3 × 250 ml ) and the combined extracts washed with water ( 2 × 150 ml ) and then concentrated . the residue was chromatographed on silica gel with acetone / hexane ( 1 : 2 ) to give 0 . 383 g ( 26 . 5 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 15 ( 6h , d , ch 3 ), 1 . 53 ( 2h , br , nch 2 ch 2 c h 2 ), 1 . 75 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 71 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 92 ( 2h , t , och 2 c h 2 n ), 3 . 07 ( 3h , s , ch 3 ), 3 . 44 ( 1h , septet , coch ), 4 . 23 ( 2h , t , oc h 2 ch 2 n ), 6 . 83 - 8 . 88 ( 10h , arh ). to the compound from example 2 , step h ( 0 . 57 g , 1 . 2 mmol ) was added methyl magnesium iodide ( 3 n ) in thf ( 3 . 0 ml ) and the mixture stirred overnight at room temperature . after removing the thf , chcl 3 ( 50 ml ) was added to the residue along with h 2 so 4 ( 0 . 675 g ) dissolved in water ( 20 ml , ph 1 ). the residue completely dissolved . excess nahco 3 was added and the organic layer separated , evaporated and dried under high vacuum to give the title compound in quantitative yield . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 0 . 77 ( 3h , d , ch 3 ), 0 . 83 ( 3h , d , ch 3 ), 1 . 47 ( 3h , s , ch 3 ), 1 . 53 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 72 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 01 ( 1h , septet , c h ( ch 3 ) 2 ), 2 . 67 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 90 ( 2h , t , och 2 c h 2 n ), 3 . 09 ( 3h , s , ch 3 ), 4 . 21 ( 2h , t , oc h 2 ch 2 n ), 6 . 88 - 8 . 80 ( 10h , arh ). the compound from step a ( 0 . 587 g , 1 . 2 mmol ) was dissolved in glacial acetic acid ( 7 . 0 ml ) and concentrated h 2 so 4 ( 1 drop ) was added . the mixture was heated for 20 min at 75 ° c . with stirring . the reaction was neutralized with nahco 3 ( 10 . 5 g in 25 ml of water ) and extracted with methyl t - butyl ether ( 3 × 25 ml ), evaporated and dried under high vacuum . the residue was chromatographed on silica gel with acetone / hexane ( 1 : 2 ) to give 0 . 407 g ( 85 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 0 . 77 ( 3h , d , ch 3 ), 0 . 83 ( 3h , d , ch 3 ), 1 . 47 ( 3h , s , ch 3 ), 1 . 55 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 76 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 72 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 95 ( 2h , t , och 2 c h 2 n ), 3 . 11 ( 3h , s , ch 3 ), 4 . 24 ( 2h , t , oc h 2 ch 2 n ), 6 . 87 - 8 . 78 ( 10h , arh ). following the procedure outlined in example 2 , step f , 7 - methoxy - 10 -( 4 - methoxyphenyl )- 5 - methylphenanthrene - 2 - carbonitrile ( 1 . 22 g , 3 . 46 mmol ) and methyl magnesium bromide ( 3m ) in diethyl ether ( 2 . 5 ml ) were reacted to give 1 . 42 g ( quantitative yield ) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 61 ( 3h , s , ch 3 ), 3 . 15 ( 3h , s , coch 3 ), 3 . 94 ( 3h , s , och 3 ), 3 . 97 ( 3h , s , och 3 ), 7 . 08 - 8 . 94 ( 10h , arh ). bbr 3 ( 100 ml , 1m in ch 2 cl 2 ) was added to the compound from step a ( 6 . 76 g , 18 . 3 mmol ) dissolved in ch 2 cl 2 ( 200 ml ) at − 60 to − 70 ° c . the reaction was stirred overnight at − 60 to 3 ° c . and then poured into water / ice ( 500 ml ) and extracted with chcl 3 / acetone ( 2 . 5 l , 5 / 1 ). after evaporating the solvent , the residue was chromatographed on silica gel using a gradient of ethyl acetate : methanol ( 95 : 5 ) to ethyl acetate : methanol ( 80 : 20 ) to give the title compound . 1 h - nmr ( 400 mhz , dmso - d 6 ) δ h 2 . 16 ( 3h , s , ch 3 ), 2 . 67 ( 3h , s , coch 3 ), 6 . 52 - 8 . 48 ( 10h , arh ), 8 . 81 ( 1h , br , oh ), 9 . 91 ( 1h , br , oh ). the compound for step b ( 0 . 135 g , 0 . 394 mmol ) was dissolved in anhydrous dmf ( 5 ml ). nah ( 75 mg , 60 % in oil ) was added and the reaction stirred for 10 min at room temperature . a solution of 1 -( 2 - chloroethyl )- piperidine hydrochloride ( 75 mg , 0 . 40 mmol ) in dmf ( 4 . 5 ml ) was added slowly over 2 h . after the addition was complete the reaction was stirred for 2 h at 40 - 50 ° c . and then overnight at room temperature . the reaction mixture was added to water ( 50 ml ), extracted with ethyl acetate ( 75 ml ), dried , concentrated and chromatographed on silica using ethyl acetate : methanol ( 9 : 1 ) to give the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 53 ( 2h , br , nch 2 ch 2 c h 2 ), 1 . 76 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 51 ( 3h , s , coch 3 ), 2 . 72 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 93 ( 2h , t , och 2 c h 2 n ), 3 . 10 ( 3h , s , ch 3 ), 4 . 24 ( 2h , t , oc h 2 ch 2 n ), 6 . 83 - 8 . 87 ( 10h , arh ). the compound from example 4 , step c ( 0 . 65 g , 1 . 4 mmol ) in anhydrous thf ( 150 ml ) was cooled to 0 ° c . methyl magnesium bromide ( 3m ) in diethyl ether ( 4 . 8 ml , 14 mmol ) was added . the reaction was stirred overnight at 0 ° c ., followed by the addition of cold 1n hcl ( 35 ml ). ethyl acetate ( 100 ml ) was added and the aqueous phase saturated with na 2 co 3 the organic layer was separated , dried and the solvent removed to yield a yellow , glassy residue ( 0 . 83 g ) of the title compound that was used without further purification . the crude compound form step a was dissolved in ch 2 cl 2 ( 20 ml ), 10 - camphorsulphonic acid ( 1 g ) was added and the mixture refluxed for 1 h . after removing the solvent the residue was dissolved in ethyl acetate ( 100 ml ) and a saturated solution of na 2 co 3 ( 25 ml ) was added and the mixture stirred for 15 min . the organic layer was separated , dried , concentrated and the residue chromatographed on silica gel using hexanes : acetone ( 60 : 40 ) to yield 0 . 412 g ( 65 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 52 ( 2h , br , nch 2 ch 2 c h 2 ), 1 . 75 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 05 ( 3h , s , ch 3 ), 2 . 71 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 92 ( 2h , t , och 2 c h 2 n ), 3 . 10 ( 3h , s , ch 3 ), 4 . 24 ( 2h , t , oc h 2 ch 2 n ), 5 . 05 ( 1h , s , ═ ch ), 5 . 36 ( 1h , s , ═ ch ), 6 . 84 - 8 . 78 ( 10h , arh ). the compound from example 4 , step c ( 0 . 550 g , 1 . 2 mmol ) was dissolved in methanol ( 45 ml ). nabh 4 ( 0 . 175 g , 4 eq .) was added in portions over 1 h at − 5 to − 3 ° c . the reaction was brought to room temperature and stirred for 2 h . the solvent was removed and water and ethyl acetate were added . the mixture was acidified to ph 2 with concentrated hcl and then excess na 2 co 3 was added . the organic layer was separated and the aqueous layer extracted with ethyl acetate . the combined organic layers were washed with brine , dried and concentrated . the residue was chromatographed on silica with hexanes : acetone ( 1 : 1 ) to yield 0 . 447 g , ( 81 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 34 ( 3h , d , ch 3 ), 1 . 40 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 52 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 47 ( 3h , s , ch 3 ), 2 . 47 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 71 ( 2h , t , och 2 c h 2 n ), 3 . 01 ( 3h , s , ch 3 ), 4 . 16 ( 2h , t , oc h 2 ch 2 n ), 4 . 79 ( 1h , dq , ch ), 5 . 19 ( 1h , d , oh ), 7 . 05 - 8 . 76 ( 10h , arh ), 9 . 75 ( 1h , s , oh ). the compound from example 4 , step c ( 0 . 091 g , 0 . 2 mmol ) was dissolved in dry thf ( 5 ml ). liquid nh 3 ( 6 ml ) was added via a cannula at − 78 ° c . lithium ( 4 mg ) was then added and the reaction stirred for 30 min . additional li ( 10 mg ) was added and the reaction stirred for an additional 1 h . the reaction was quenched with solid nh 4 cl ( 3 . 3 g ). the nh 3 was allowed to boil off and the residue partitioned between ch 2 cl 2 and water . the water layer was separated , extracted with ch 2 cl 2 and the combined organic layers dried , concentrated and chromatographed on silica gel using a gradient of methanol : ch 2 cl 2 ( 5 : 95 to 30 : 70 ) to give 0 . 035 g ( 40 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 16 ( 3h , t , ch 3 ), 1 . 47 ( 2h , m , nch 2 ch 2 c h 2 ), 1 . 67 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 54 ( 2h , q , ch 2 ), 2 . 55 ( 3h , s , ch 3 ), 2 . 56 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 82 ( 2h , t , och 2 c h 2 n ), 2 . 87 ( 1h , dd , arch ), 3 . 00 ( 1h , dd , arch ) 3 . 92 ( 1h , dd , archar ), 4 . 10 ( 2h , m , oc h 2 ch 2 n ), 6 . 50 - 7 . 56 ( 9h , arh ). 3 - methyl - anisole ( 36 . 6 g , 300 mmol ) was reacted as described in example 2 ( step a ) to give 41 . 8 g ( 93 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 65 ( 3h , s , ch 3 ), 3 . 87 ( 3h , s , och 3 ), 6 . 74 ( 1h , d , arh ), 6 . 85 ( 1h , dd , arh ), 7 . 76 ( 1h , d , arh ), 10 . 11 ( 1h , s , cho ). the compound from step a ( 41 . 8 g , 279 mmol ) was reacted as described in example 2 ( step b ) to give 51 g ( 65 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 48 ( 3h , s , ch 3 ), 3 . 85 ( 3h , s , och 3 ), 3 . 90 ( 3h , s och 3 ), 6 . 76 - 6 . 82 ( 2h , s , arh ), 6 . 99 ( 2h , d , arh ), 7 . 40 , ( 1h , d , coch ), 7 . 70 ( 1h , d , arh ), 8 . 08 ( 1h , d , arch ), 8 . 50 ( 2h , d , arh ). the compound from step b ( 51 . 0 g , 182 mmol ) was reacted as described in example 2 ( step c ) to give the title compound that was used without further purification . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 48 ( 3h , s , ch 3 ), 3 . 84 ( 3h , s , och 3 ), 3 . 92 ( 3h , s , och 3 ), 5 . 90 ( 1h , d , archbr ), 6 . 00 ( 1h , d , cochbr ), 6 . 74 ( 1h , d , arh ), 6 . 88 ( 1h , dd , arh ), 7 . 03 ( 2h , d , arh ), 7 . 54 ( 1h , d , arh ), 8 . 07 ( 2h , d , arh ). the crude compound from step i was reacted as described in example 2 ( part c ) to give 79 g of the title compound that was used without further purification . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 88 ( 3h , s , coch 3 ), 2 . 60 ( 3h , s , ch 3 ), 3 . 81 ( 3h , s , och 3 ), 3 . 91 ( 3h , s , och 3 ), 5 . 36 ( 1h , d , cochbr ), 6 . 60 ( 1h , d , cochar ), 6 . 36 ( 1h , d , arh ), 6 . 81 ( 1h , dd , arh ), 7 . 00 ( 2h , d , arh ), 7 . 35 ( 1h , d , arh ), 8 . 05 ( 2h , d , arh ). the crude compound from step ii was reacted as described in example 2 ( part c ) to give 13 g ( 26 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 59 ( 3h , s , ch 3 ), 3 . 85 ( 3h , s , och 3 ), 3 . 91 ( 3h , s , och 3 ), 6 . 82 - 8 . 21 ( 7h , arh ). the compound from step c , iii ( 5 . 61 g , 20 . 0 mmol ) was reacted as described in example 2 ( part d ) to give the title compound in quantitative yield . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 14 ( 3h , s , ch 3 ), 3 . 74 ( 3h , s , och 3 ), 3 . 84 ( 3h , s , och 3 ), 6 . 56 - 7 . 79 ( 10h , arh ). the compound from step d ( 4 . 6 g , 13 mmol ) was reacted as described in example 2 ( part e ) to give the title compound that was used without further purification . the crude mixture from i was reacted as described in example 2 ( part e ) to give the title compound that was used without further purification . the crude product from ii was reacted as described in example 2 ( part e ) to give 2 . 8 g ( 64 %) of the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 3 . 94 ( 3h , s , och 3 ), 3 . 99 ( 3h , s , och 3 ), 7 . 08 - 8 . 73 ( 11h , arh ). the compound from step e , iii ( 2 . 8 g , 8 . 3 mmol ) and methyl magnesium bromide ( 3m ) in diethyl ether ( 6 . 1 ml , 18 mmol ) were reacted as described in example 2 ( part f ) to give the title compound . 1 h - nmr ( 400 mhz , cdcl 3 ) δ h 2 . 61 ( 3h , s , coch 3 ) 3 . 94 ( 3h , s , och 3 ), 3 . 99 ( 3h , s , och 3 ), 7 . 07 - 8 . 73 ( 11h , arh ). the compound from step f was reacted as described in example 2 ( part g ) to give the title compound . 1 h - nmr ( 400 mhz , dmso - d 6 ) δ h 2 . 59 ( 3h , s , coch 3 ), 6 . 95 - 8 . 87 ( 11h , arh ), 9 . 7 ( 1h , br , oh ), 10 . 1 ( 1h , br , oh ). the compound from step g ( 1 . 35 g , 4 . 12 mmol ) was reacted as described in example 2 ( part h ) to give 0 . 175 g ( 9 . 7 %) of the title compound . 1 h - nmr ( 400 mhz , dmso - d 6 ) δ h 1 . 40 ( 2h , br , nch 2 ch 2 c h 2 ), 1 . 53 ( 4h , m , nch 2 c h 2 ch 2 ), 2 . 49 ( 4h , br , nc h 2 ch 2 ch 2 ), 2 . 57 ( 3h , s , ch 3 ), 2 . 70 ( 2h , t , och 2 c h 2 n ), 4 . 16 ( 2h , t , oc h 2 ch 2 n ), 7 . 11 - 8 . 86 ( 11h , arh ), 10 . 12 ( 1h , br , oh ). effectiveness of compounds of formula 3 and 4 at inhibiting mcf - 7 cell proliferation those skilled in the art will appreciate that several acceptable estrogen receptor - binding assays are known and available for initial screening of the compounds of the present invention . the initial screen chosen was a human cell line assay , namely the mcf - 7 cell proliferation assay for detecting anti - estrogenic / estrogenic activity . the mcf - 7 human breast cancer cell line has been used as an industry standard for the evaluation of estrogen receptor antagonists . mcf - 7 cells are estrogen - receptor positive ( er +) cancer cells that respond to estradiol stimulation . antiestrogenic activity is measured in terms of a test article &# 39 ; s ability to inhibit estradiol stimulated proliferation , implying an antagonistic action on the estrogen receptor and estogenic activity can be inferred from increased proliferation . the following testing procedure was used . mcf - 7 cells were maintained in rpmi medium free of phenol red and supplemented with 5 % charcoal - stripped foetal calf serum , hydrocortisone , bovine insulin , penicillin and streptomycin until they reached 70 % confluence . the cells were kept in a 5 % co 2 atmosphere and prior to treatment were washed twice with ca ++ / mg ++ free hanks balanced salt solution and harvested with 1 mm edta prepared in ca ++ / mg ++ free hanks balanced salt solution . after the washes the cells were re - suspended in medium . cells were seeded in 96 - well plates and incubated for 16 hours in 5 % charcoal - stripped calf serum phenol red - free medium . cells were then treated continuously with estradiol , a test article , or a combination of both using various serum concentrations . cell survival was evaluated after 3 - 6 days , by replacing the culture media with 150 μl of fresh medium containing 10 mm 4 -( 2 - hydroxyethyl )- 1 - piperazineethamsulfonic acid buffer ( ph 7 . 4 ) followed by addition of 50 μl of 2 . 5 mg / ml of 3 -( 4 , 5 - dimethylthiazo - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ( mtt ). after 4 hours of incubation at 37 ° c . the medium and mtt were removed and 200 μl of dimethylsulfoxide ( dmso ) was added to dissolve the precipitate of reduced mtt , followed by addition of 25 μl of glycine buffer ( 0 . 1m glycine plus 0 . 1m nacl , ph 10 . 5 ). plates were shaken for 15 minutes and the absorbance was determined at 570 nm with a microplate reader ( biorad , model 450 ). data are expressed as percent (%) cell growth in comparison with untreated cells . fig1 and 2 show the dose - response curves of estradiol stimulated versus unstimulated mcf - 7 cells in the presence of the compounds depicted by formula 3 and formula 4 respectively . the shifts of the response curves indicate that the test compounds are antagonizing the effect of estradiol on these cells . this is a positive indication that the compounds of the present invention are of potential in the treatment of a wide variety of disease states involving the estrogen receptor . effectiveness of compounds of formulas 3 , 5 , 6 and 7 at displacing estrogen er - alpha and er - beta in human recombinant estrogen receptors the human estrogen receptor occurs in two subtypes , alpha and beta . the stable expression of these individual receptor subtypes in cells provides a rapid and accurate means of quantifying the direct interaction of a test article with the estrogen binding sites . briefly , this assay is conducted in 96 well plates where a series of concentrations of test article are used to displace tritiated estradiol from either estrogen receptor alpha or estrogen receptor beta bearing cell membranes , under equilibrium conditions . the measurement of the displaced tritiated estradiol allows the determination of an ic 50 value ( concentration of test article that inhibits 50 % of the estradiol binding ). this measurement is the primary test for mediation of the estrogen receptor and can also be used to measure the relative selectivity of the test article for either the alpha or beta subtype . the alpha subtype assay measures the binding of [ 3 h ] estradiol to the human recombinant estrogen receptor . the receptor preparation was obtained from panvera corporation and used in an assay that followed the method taught by obourn ( obourn et al ., biochemistry 1993 ; 32 : 6229 - 6236 ) with some minor variations . briefly , after proper dilution , a 4 . 5 ng aliquot of receptor protein in modified tris - hcl ph 7 . 5 buffer is incubated with 0 . 5 nm [ 3 h ] estradiol for 2 hours at 25 ° c . non - specific binding is estimated in the presence of 1 . 0 μm diethylstilbestrol . membranes are filtered and washed 3 times , and the filters are counted to determine [ 3 h ] estradiol specifically bound . under the same conditions the receptor protein is incubated with varied concentrations of test article ranging from 1 nm to 1 μm and the displacement of [ 3 h ] estradiol is measured in duplicate . the measurement of the displaced tritiated estradiol allows the determination of an ic 50 value , a direct measure of the test articles interaction with the estrogen receptor alpha . the beta subtype assay also allowed the determination of an ic 50 value of the test article under the same conditions as for the alpha subtype assay with the exception that a 7 . 5 ng aliquot of receptor protein preparation was used . table 1 shows the ic 50 results of the compounds depicted by formula 3 , 5 , 6 and 7 . the ic 50 values are both below 100 μm range . this is an indication of very high binding affinity for the estrogen receptor with a slight preference for the alpha subtype and of the potential of the class of compounds represented by formula i in the treatment of disease states involving the estrogen receptor . the terms and descriptions used herein are preferred embodiments set forth by way of illustration only , and are not intended as limitations on the many variations which those of skill in the art will recognize to be possible in practicing the present invention . it is the intention that all possible variants whether presently known or unknown , that do not have a direct and material effect upon the way the invention works , are to be covered by the following claims . anstead g m , carlson k e , katzenellenbogen j a . the estradiol pharmacophore : ligand structure - 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