Patent Application: US-93408601-A

Abstract:
molecules demonstrating anti - proliferative effects against epithelial cancer cell lines and endothelial cells and methods of synthesis are disclosed . the molecules are intended for use in therapeutic preparations for the treatment of cancer through either anti - angiogenesis or other anti - cancer mechanisms . the compounds specified are 6h - dibenzopyran - 6 - one derivatives and have been shown to be antiproliferative against human endothelial cells .

Description:
the present invention is further illustrated by a series of new chemical agents that demonstrate anti - proliferative effects against human endothelial cells in addition to demonstrating an inhibitory effect directly on cancer cells for the treatment of solid tumors . in one preferred embodiment of the invention , r 1 is or 3 , r 2 is r 3 is ch 3 and r 4 is a methoxy group . preferably at least one embodiment is represented by the following formula ii , or a pharmaceutically acceptable salt thereof : in yet another preferred embodiment of the invention , r 1 is or 3 , r 2 is r 3 is ch 3 and r4 is a methoxy group . preferably at least one embodiment is represented by the following formula ii , or a pharmaceutically acceptable salt thereof : set forth below is a preferred synthesis scheme for the preparation of certain preferred embodiments of the anti - cancer molecules in accordance with the invention . the 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 the dibenzo [ c ] chromen - 6 - one derivatives in accordance with the present invention . a general process for the preparation of compounds of formula i , is depicted in schemes 1 and 2 . a process for the preparation of certain preferred embodiments for compounds of formula i , is depicted in scheme 3 . as depicted in scheme 3 , compound 1 . 1 is made by acylating methyl 5 - acetyl salicylate using trifluoromethanesulfonic anhydride . dimethoxyphenylboronic acid dissolved in a polar protic organic solvent , ethanol for example , was added to 1 . 1 , dissolved in an organic solvent such as for example , 1 , 2 - dimethoxyethane . an inorganic base such as potassium carbonate and a catalytic amount of tetrakis ( triphenylphosphine ) palladium was added and the mixture refluxed to give the biphenyl product 1 . 6 . the formation of compound 1 . 7 can be accomplished via the alkylation of 1 . 6 , using a strong base such as for example lithium diisopropylamide ( lda ) and an alkyl halide . the saponification of 1 . 7 in an aqueous base such as potassium hydroxide at reflux affords the free acid biphenyl product 1 . 8 . cyclization to lactone 1 . 9 , an example of one of the compounds represented by formula i , occurs by treating 1 . 8 with thionyl chloride followed by treatment with aluminum trichloride , in a refluxing organic solvent such as 1 , 2 - dichloroethane . treatment of 1 . 9 with a diol such as 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 1 . 10 , which is another example of one of the compounds represented by formula i . the present invention which is defined by the claims , is further illustrated by the following non - limiting examples . methyl 5 - acetylsalicylate ( 25 g , 129 mmol ) was dissolved in ch 2 cl 2 ( 250 ml ) and pyridine ( 60 ml ) under argon at 0 ° c . trifluoromethanesulfonic anhydride ( 37 . 9 g , 133 mmol ) was then 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 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 reaction 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 ). the compound from step b ( 1 . 03 g , 3 . 2 mmol ) was dissolved in dme ( 40 ml ) under argon and cooled to − 20 ° c . and lda ( 1 . 6 ml , 3 . 2 mmol ) was added . the mixture was then warmed to room temperature and stirred for 30 min . methyl iodide ( 0 . 2 ml , 3 . 2 mmol ) was slowly added over 15 min . to give a cloudy suspension . the mixture was again cooled to − 20 c . and a second portion of lda ( 1 . 6 ml , 3 . 2 mmol ) was added . after the mixture was warmed to room temperature and stirred for 30 min a second portion of methyl iodide ( 0 . 2 ml , 3 . 2 mmol ) was added and the mixture stirred for 60 h . the mixture was added to cold nahco 3 ( 100 ml ) and extracted with ethyl acetate ( 3 × 100 ml ). the organic layer was washed with 5 % hcl ( 3 × 25 ml ) then dried and evaporated . the residue was chromatographed on silica gel with hexane / ethyl acetate ( 2 : 1 ) to give 0 . 208 g ( 24 %) of the title compound . 1h - nmr ( 400 mhz , cdcl 3 ) δ h 1 . 24 ( 6h , t , ch ( c h 3 ) 2 ), 3 . 60 ( 1h , m , c h ( ch 3 ) 2 , 3 . 71 ( 3h , s , och 3 ), 3 . 89 ( 3h , s , och 3 ), 3 . 93 ( 3h , s , och 3 ), 6 . 86 - 8 . 32 ( 6h , arh ). the compound from step c ( 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 d was dissolved in 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 . at which point alcl 3 ( 2 . 3 g , 17 . 3 mmol ) was added . the resulting reaction mixture was stirred overnight at room temperature . 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 , 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 e ( 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 ). effectiveness of 3 - methoxy - 8 -( 2 - methyl -[ 1 , 3 ]- dioxolane - 2 - yl )- benzo [ c ] chromen - 6 - one against endothelial cells and epithelial cancer cell lines . those skilled in the art will appreciate that several acceptable cell proliferation assays are known and available for demonstrating the activity of the compounds of the present invention . the proliferation of endothelial cells is an important step in the process of blood vessel formation . therefore , cells derived from the endothelium are useful in the study of angiogenesis and in vitro model systems utilising endothelial cells have the additional advantage of simplicity . two model endothelial cell lines are the human umbilical vein endothelial cells ( huvec ) and the bovine brain - derived capillary endothelial cells ( bbce ). both have been used extensively to study the biology of endothelial cells . the following testing procedures were used . bbce are maintained in a regular medium containing dmem plus 10 % new - born calf serum and 2 . 5 mg / ml bfgf added every second day . sub - confluent cells are collected , diluted to 5 , 000 cells per ml and seeded in 1 ml aliquots per well into 12 - well cluster dishes . cells are treated with drug candidates or the vehicle every second day . 2 - methoxyestradiol is used as a positive control . after six days , the cells are washed and counted using a coulter particle counter . the results are expressed as ic 50 values , that is , the concentration of the respective test compound resulting in half the number of cells that are obtained in controls . cancer cells ( mcf - 7 ; mda - mb - 435 ; hct - 116 ; b16 ) & amp ; human umbilical vein endothelial cells ( huvec ) huvec are maintained in a m199 medium , supplemented with 90 mg / ml heparin , 2 mm l - glutamate , 10 % foetal bovine serum ( fbs ), 90 mg / ml heparin sulphate , 20 mg / mi endothelial cell growth supplement , 100 mg / ml penicillin and 100 mg / ml streptomycin . the mcf - 7 , mda - mb - 435 , hct - 116 and b16 cells are cultured in rpmi , d - mem , rpmi and mccoy &# 39 ; s 5r medium , supplemented with 10 % glutamine , 1 % non - essential amino acids ( 10 mm ) and 1 % sodium pyruvate ( 100 mm ) respectively . all culture mediums are supplemented with 10 % fbs . all cells are maintained in an atmosphere of 5 % co 2 . exponentially growing cells are seeded in 96 - well plates and incubated for 16 hours . cells are then treated continuously with the test articles and cell survival is evaluated 96 hours later by replacing the culture media with 150 μl fresh medium , containing 10 mm 4 -( 2 - hydroxyethyl )- 1 - piperazineethanesulphonic acid buffer ( ph 7 . 4 ). next , 50 μl of 2 . 5 mg / ml of 3 -( 4 , 5 - dimethylthiazo - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ( mtt ) in phosphate buffer solution ( pbs ) ( ph 7 . 4 ), is added . after 3 - 4 hours of incubation at 37 ° c ., the medium and mtt are removed and 200 μl of dimethylsulfoxide ( dmso ) is added to dissolve the precipitate of reduced mtt , followed by the addition of 25 μl glycine buffer ( 0 . 1m glycine plus 0 . 1m nacl , ph 10 . 5 ). the absorbance is determined at 570 nm with a microplate reader ( biorad ). [ 0081 ] fig1 shows the effects of the compound depicted by formula ii on the proliferation of four epithelial cancer cell lines . this is an indication that the compounds of the present invention are of potential in the treatment of a wide variety of cancers . [ 0082 ] fig3 shows the effects of the compound depicted by formula ii on the proliferation of huvec cells . this is an indication that the compounds of the present invention are of potential in the treatment of diseases characterized by the undesired proliferation of endothelial cells . [ 0083 ] fig4 shows the effects of the compound depicted by formula ii on the proliferation of bbce cells . this is also an indication that the compounds of the present invention are of potential in the treatment of diseases characterized by the undesired proliferation of endothelial cells . effectiveness of 8 - acetyl - 3 - methoxy - benzo [ c ] chromen - 6 - one against endothelial cells and epithelial cancer cell lines the compound depicted by formula iii was tested using the same procedures as disclosed in example 2 above . [ 0085 ] fig2 shows the effects of the compound depicted by formula iii on the proliferation of four epithelial cancer cell lines . this is an indication that the compounds of the present invention are of potential in the treatment of a wide variety of cancers . [ 0086 ] fig3 shows the effects of the compound depicted by formula iii on the proliferation of huvec cells . this is an indication that the compounds of the present invention are of potential in the treatment of diseases characterized by the undesired proliferation of endothelial cells . [ 0087 ] fig4 shows the effects of the compound depicted by formula iii on the proliferation of bbce cells . this is also an indication that the compounds of the present invention are of potential in the treatment of diseases characterized by the undesired proliferation of endothelial cells . 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 skilled 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 . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims . auerbach w and auerbach r ; 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