Patent Application: US-73789500-A

Abstract:
isoflavones are modified by esterification at one or more of the c 4 ′, c 5 , c 6 , and c 7 positions to promote bioavailability , and especially to enhance solubility over the corresponding unesterified isoflavones . preferred modifications produce a carboxylic acid hemiester or a phosphate ester which is biologically hyrolyzable . preferred starting isoflavones are genistin and daidzin , and still more preferably comprises an aglycone form such as genistein or daidzein . esterified isoflavones may be employed therapeutically or prophylactically for a variety of conditions , provided as a dietary supplement , or added to natural or processed food - stuffs .

Description:
using this structure as a reference , known isoflavones include the following ( where the 6 “ position is on the glucose ring ): it is now contemplated that a natural or modified isoflavone may be esterified to provide a pro - compound having increased bioavailability , and in particular enhanced aqueous solubility relative to the unesterified isoflavone . in preferred embodiments one or more of r 7 , r 6 , r 5 and r 4 ′ comprise zooo — or zpo 4 —, where z is selected from the group consisting of a straight or branched aliphatic chain , including an alkyl , alkenyl , alkynyl , alkoxyalkyl , alkylthioalkyl , aminoalkyl group , including substituted derivatives of such groups , a substitute or non - substituted cycloalkyl , and an aromatic group , including aryl , aralkyl , or alkylaryl , and substituted derivatives such as where a ring contains one or more nitrogen , sulfur , oxygen , phosphorous or silicon heteroatoms . such compounds are considered herein to be esterified isoflavones in which an isoflavones is modified by esterification in at least one of the c4 ′, c5 , c6 , and c7 positions . to clarify further , it is contemplated that z may comprise hydrogen ; hydroxyl ; cyano ; nitro ; halo ; alkyl such as methyl , ethyl , butyl , pentyl , octyl , nonyl , tert - butyl , neopentyl , isopropyl , sec - butyl , dodecyl and the like , alkenyl such as 1 - propenyl , 4 - butenyl , 1 - pentenyl , 6 - hexenyl , 1 - heptenyl , 8 - octenyl and the like ; alkoxy such as propoxy , butoxy , methoxy , isopropoxy , pentoxy , nonyloxy , ethoxy , octyloxy , and the like ; alkanoyl such as butanoyl , pentanoyl , octanoyl , ethanoyl , propanoyl and the like ; arylamino and diarylamino such as phenylamino , diphenylamino and the like ; alkylsulfinyl , alkylsulfonyl , alkylthio , arylsulfonyl , arylthio , and the like , such as butylthio , neopentylthio , methylsulfinyl , benzylsulfinyl , phenylsulfinyl , propylthio , octylthio , nonylsulfonyl , octylsulfonyl , methylthio , isopropylthio , phenylsulfonyl , methylsulfonyl , nonylthio , phenylthio , ethylthio , bezylthio , phenethylthio , sec - butylthio , naphthylthio and the like ; alkoxycarbonyl such as methoxycarbonyl , ethoxycarbonyl , butoxycarbonyl and the like ; alkyl amino and dialkylarnino such as dimethylamino , methylamino , diethylamino , ethylamino , dibutylamino , butylamino and the like ; cycloalkyl such as cyclohexyl , cyclopentyl , cyclooctyl , cycloheptanyl and the like ; alkoxyalkyl such as methoxymethylene , ethoxymethylene , butoxymethylene , propoxyethylene , pentoxybutylene and the like ; arylalkylarnino such as methylphenylamino , ethylphenylamino and the like ; aryloxyalkyl and aryloxyaryl such as phenoxyphenylene , phenoxymethylene and the like ; and various substituted alkyl and aryl groups such as 1 - hydroxybutyl , 1 - aminobutyl , 1 - hydroxylpropyl , 1 - hydroxypentyl 1 - hydroxyoctyl , 1 - hydroxyethyl , 2 - nitroethyl , trifluoromethyl , 3 , 4 - epoxy - butyl , cyanomethyl , 3 - chloropropyl , 4 - nitrophenyl , 3 - cyanophenyl , 1 - hydroxymethyl , and the like ; hydroxyl terminated alkyl and aryl groups such as , 2 - hydroxy ethyl , 4 - hydroxy butyl and 4 - hydroxy phenyl ; sulfonic , sulfuric , carboxylic , phosphoric and phosphoric acid terminated alkyl and aryl groups such as ethylsulfonic acid , propylsulfonic acid , butylsulfonic acid , phenylsulfonic acid , and the corresponding carboxylic and phosphoric acids and derivatives of said sulfonic , carboxylic and phosphoric acids as for example salts , esters and the like . of course , those skilled in the art will appreciate that higher solubility in aqueous environments is generally preferred over lower solubility . thus , where r 7 , r 6 , r 5 or r 4 ′ comprise zooo —, z is advantageously selected to have a polar group , and any alkyl segment is advantageously selected to be small . among the dicarboxylic acid groups , hemisuccinate is by far the most common , useful and biocompatible group , and is specifically contemplated for this purpose . glutarate and adipate are also preferred . where r 7 , r 6 , r 5 and r 4 ′ comprise zpo 4 —, a ( oh ) 2 po 2 ester is preferred because it has two polar groups , is a good solubilizer , and has high biological compatibility . any additions to the po 4 ( such as ( ro ) 2 po 2 —) are contemplated to generally reduce aqueous solubility , and are therefore disfavored . with respect to complexes , it is contemplated to employ metal salts of the esterified isoflavones , especially li +, na +, k +, mg ++ and ammonium salts , including nh4 + and low molecular weight mono - or polyalkylammonium . by way of example , and not of limitation , several embodiments of the inventive subject matter have been prepared and characterized . these examples all fall within the group of pro - compounds where at least one of r 7 , r 6 , r 5 and r 4 ′ is h 2 po 4 —, and hooc —( ch 2 ) x — coo — where x = 2 , 3 , or 4 . these new phosphate esters and hemiesters are all water soluble and readily hydrolyzed . they are also stable . in aqueous solutions of the exemplified compounds , for example , hydrolysis occurs to the extent of less than 1 % per day , when stored at ph 7 . 4 and 37 ° c . dry formulations of the same compounds appear to be indefinitely stable . a solution of genistein ( 135 mg , 0 . 5 mmole ) and di - tert - butyl phosphoramidite ( 330 ul , 1 . 0 mmole ) in dmf ( 1 ml ) was stirred under argon while 1h - tetrazole ( 210 mg in 0 . 5 ml of dmf ; 3 . 0 mmole ) was added dropwise . after a few minutes , the solution was cooled to − 20 ° c ., then a solution of m - chloroperbenzoic acid ( 260 mg in 0 . 5 ml of methylene chloride , 1 . 5 mmole ) was added dropwise . after warming to room temperature , the mixture was diluted threefold with ethyl acetate , then washed with 10 % sodium metabisulfie and 10 % sodium bicarbonate . a wash with 5 % sodium carbonate removed a trace of unreacted genistein . the ethyl acetate solution , containing the butyl esters of the genistein phosphates , was washed with 1m hcl and dried over sodium sulfate . after removal of the solvent in vacuo , the residue was treated with 30 % tfa in acetic acid for 90 minutes at room temperature . the solvents were removed in vacuo , and the residue was taken up in ethanol and neutralized with sodium hydroxide to ph 5 . 5 . removal of the solvent in vacuo left a mixture of sodium salts of genistein phosphates , 145 mg . hplc analyses were performed using a chrompack intersil c8 column , 4 . 6 × 250 mm . the solvent was a mixture of 25 % acetonitrile and 75 % 0 . 1m diammonium phosphate , ph 2 . 5 , at a flow rate of 1 ml / min . detection was by uv at a wavelength of 260 nm . hplc analysis of the phosphate mixture showed approximately equal amounts of the 4 ′- phosphate , the 7 - phosphate and the 4 ′, 7 - diphosphate , and only a small amount of the 5 - phosphate . a ) genistein - 7 - tosylate : p - toluenesulfonyl chloride ( 540 mg , 2 . 8 mmoles ) was added during 4 hours to a stirred mixture of genistein ( 730 mg , 2 . 7 mmoles ) and potassium carbonate ( 2 g ) in 25 ml of acetone . after stirring overnight , the mixture was poured into brine and extracted with ethyl acetate . the extract was evaporated under vacuum , and the residue chromatogrammed through silica gel with dichloromethane and chloroform . crystallization from methanol yielded 920 mg ( 80 . 2 % yield ) of genistein - 7 - tosylate . the proton magnetic resonance spectrum was consistent with the expected structure . 4 ′, 5 - di ( methoxymethyl )- genistein - 7 - tosylate : chloromethyl methyl ether ( 90 ul , 1 . 12 mmoles ) was added to a solution of genistein - 7 - tosylate ( 106 mg , 0 . 25 mmoles ) and diisopropylethylamine ( 200 ul , 1 . 15 mmoles ) in 0 . 6 ml of dioxane , under argon atmosphere , and stirred overnight . the mixture was poured into brine , extracted with ethyl acetate , and chromatogrammed through silica gel with dichloromethane . crystallization from methanol yielded 115 mg ( 90 % yield ) of 4 ′, 5 - di ( methoxymethyl )- genistein - 7 - tosylate . the proton magnetic resonance spectrum was consistent with the expected structure . c ) 4 ′, 5 - di ( methoxymethyl )- genistein : potassium carbonate ( 700 mg ) in water ( 5 ml ) was added to a solution of 4 ′, 5 - di ( methoxymethyl )- genistein - 7 - tosylate ( 600 mg , 1 . 17 mmoles ) in 15 ml methanol under argon , and stirred overnight . the mixture was poured into brine , extracted with ethyl acetate , and recrystallized from methanol . the yield of 4 ′, 5 - di ( methoxymethyl )- genistein was 344 mg ( 82 %). the electrospray mass spectrum in negative mode showed ion m / z 357 [ m − 1 ] which confirmed the expected molecular weight of 358 . the proton and carbon magnetic resonance spectra were consistent with the expected structure . d ) genistein - 7 - phosphate : 1h - tetrazole ( 120 mg , 1 . 7 mmoles ) was added to a solution of di - tert - butyl - diethylphosphoramidite ( 180 ul , 0 . 64 mmoles ) and 4 ′, 5 - di ( methoxymethyl )- genistein ( 200 mg , 0 . 56 mmoles ) in 1 . 5 ml of n , n - dimethylacetamide under argon . after 10 minutes at room temperature , the mixture was cooled to − 40 ° c ., and a solution of m - chloroperbenzoic acid ( 130 mg , 0 . 75 mmoles ) in dichloromethane was added rapidly . after warming to room temperature , the mixture was diluted with ether and washed with brine containing sodium bicarbonate . the solvent was removed , and the residue treated with 40 % trifluoroacetic acid in acetic acid for 30 minutes . the volatile acids were removed under vacuum , and the residue dissolved in 2 - propanol ( 4 ml ) containing 0 . 2 ml 6n hcl and left overnight . the mixture was poured into brine and extracted with ethyl acetate . the solvent was removed , and the residue was dissolved in ethanol ( 3 ml ) and adjusted to ph 5 . 5 with naoh . after evaporation , the residue was crystallized from ethanol , yielding 155 mg ( 75 % yield ) of genistein - 7 - phosphate as the sodium salt . the electrospray mass spectrum in negative mode showed ion m / z 349 [ m − 1 ] which confirmed the expected molecular weight of 350 . the nuclear magnetic resonance spectra were consistent with the expected structure . a solution of genistein ( 135 mg , 0 . 5 mmole ) in 2 . 0 ml of pyridine was stirred at room temperature while succinic anhydride ( 100 mg , 1 . 0 mmole ) was added in several portions . after stirring overnight at room temperature , the solvent was removed in vacuo . the gummy residue was taken up in water , adjusted to ph 3 . 0 , and extracted three times with ethyl acetate . the ethyl acetate extracts were washed with water , then evaporated to dryness in vacuo . the crude mixture of mixed hemisuccinic acid esters weighed 205 mg . thin layer chromatography of the product showed principally the presence of mixed mono - and disuccinate esters of genistein . the product was completely soluble in phosphate buffer at ph 7 . to a solution of 4 ′, 5 - di ( methoxymethyl )- genistein ( see example 2c ) ( 100 mg , 0 . 28 mmole ) in 1 . 5 ml of pyridine was added succinic anhydride ( 50 mg , 0 . 5 mmole ) with stirring at room temperature . after stirring overnight , the solvent was removed in vacuo . the residue was taken up in water containing one drop of glacial acetic acid , and again evaporated to dryness in vacuo . the residue was chromatogrammed through silica gel with dichloromethane and ethyl acetate . the yield of the 7 - hemisuccinic ester of 4 ′, 5 - di ( methoxymethyl )- genistein was 102 mg ( 78 %). the product was dissolved in 2 - propanol ( 3 ml ) containing 0 . 2 ml 6n hcl and left overnight . the solution was evaporated to dryness . the residue taken up in 1 ml of ethyl acetate and crystallized by the addition of hexane . the yield of genistein - 7 - hemisuccinate was 52 mg ( 50 %). hplc analysis was conducted using a partisil ods - 3 column ( 9 . 5 × 250 mm ), with methanol as the mobile phase , and uv detection at 260 nm . a solution of genistein - 7 - phosphate ( 2 . 5 mg ) in 5 ml of phosphate - buffered saline ( 0 . 1 m ) at ph 7 . 4 was incubated at 37 ° for 10 days . analysis by hplc showed that absence of free genistein . a solution of genistein - 7 - hemisuccinate ( 2 . 5 mg ) in 5 ml of phosphate - buffered saline ( 0 . 1 m ) at ph 7 . 4 was incubated at 37 ° for 10 days . analysis by hplc showed a conversion of about 4 % of the hemisuccinate ester to free genistein . in each of these experiments , free genistein was extracted with a 1 : 1 mixture of ethyl acetate and hexane , then analyzed by hplc under the conditions described in example 5 . a ) in human serum ( sierra biologicals ) at 37 ° c ., the half - life for hydrolysis to free genistein was about 5 hours . b ) in human blood ( sierra biologicals ) at 37 ° c ., the half - life for hydrolysis to free genistein was about 6 hours . c ) in rat blood ( sierra biologicals ) at 37 ° c ., the half - life for hydrolysis to free genistein was about 30 minutes . d ) in human serum ( sierra biologicals ) at 37 ° c ., the half - life for hydrolysis to free genistein was about 5 hours . e ) in alkaline phosphatese type vii - s at 37 ° c ., the initial rate of hydrolysis to free genistein was 0 . 08 % per minute . this enzyme is from bovine intestinal mucosa ( sigma cat no . p5521 ). 0 . 5 dea units were dissolved in 1 . 0 ml of 0 . 1m glycine buffer ph 10 . 4 , and the initial substrate concentration was 1 . 07 mm . f ) in alkaline phosphatese type xxiv at 37 ° c ., the initial rate of hydrolysis to free genistein was 0 . 05 % per minute . this enzyme is from human placenta ( sigma cat no . p3895 ). 0 . 1 glycine units were dissolved in 1 . 0 ml of 0 . 1m glycine buffer ph 10 . 4 , and the initial substrate concentration was 1 . 07 mm . it is contemplated that esterified isoflavones will be readily converted to free isoflavone in biological media such as gastrointestinal fluid and blood . among other things , gastrointestinal fluids often have enzymes and sufficiently high ph to hydrolyze ester bonds , and blood generally contains enzymes such as phosphatases and esterases which can hydrolyze phosphate ester and carboxylate ester bonds . contemplated uses of esterified isoflavones include any presently known or later discovered uses for isoflavones or isoflavonoids . among other things , it is contemplated that esterified isoflavones can be administered to ( which term is used herein to include “ taken by ”) a person for any of the beneficial effects for which a natural isoflavonoid is thought to be advantageous . this specifically includes any of the effects listed above or described in any of the literature cited herein , and includes uses where the desired effect is antiangiogenic , antihemolytic , antiischemic , antileukemic , antimitogenic , antimutagenic , antioxidant , fungicidal , pesticidai , mao - inhibition , phytoalexin , and tyrosine kinase inhibition . it is especially contemplated that esterified isoflavones can be used to treat osteoporosis and other symptoms of estrogen deficiency in postmenopausal women . also , it is contemplated that the compounds of the present invention can be used to prevent osteoporosis and consequent fractures that result from osteoporosis , which are major contributors to morbidity and mortality in the elderly . still further , it is contemplated that esterified isoflavones can be used prophylactically to provide uv protection and in other ways to improve general skin health , to stimulate the immune system , and to reduce undesirable effects of oxidation ( i . e ., provide antioxidant benefits ). those skilled in the art will recognize that esterified isoflavones may be employed in many different ways . when taken orally , esterified isoflavones may be incorporated into food or beverage material , for nutritional , therapeutic , prophylactic value , or any combination of these . esterified isoflavones may also be administered by any appropriate form of in vivo delivery , which is defined herein to include oral , intravenous , subcutaneous , intraperitoneal , intrathecal , intramuscular , intracranial , inhalational , topical , transdermal , suppository ( rectal ), pessary ( vaginal ), administration and the like . thus , delivery may occur through foods , pills , capsules or liquids as a nutritional supplement , or as a pharmaceutical by way of example , it is contemplated that compounds according to the present invention can be administered alone , or formulated in admixture with a pharmaceutically acceptable carrier . for example , the compounds of the present invention can be administered orally as pharmacologically acceptable salts . because preferred compounds of the present invention are relatively water soluble , they can be administered intravenously in physiological saline solution ( e . g ., buffered to a ph of about 7 . 2 to 7 . 5 ). conventional buffers such as phosphates , bicarbonates or citrates can be used for this purpose . of course , one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity . it is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients . one of ordinary skill in the art will also take advantage of favorable pharmacokinetic parameters of the pro - drug forms , where applicable , in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound . in addition , compounds according to the present invention may be administered alone or in combination with other agents for the treatment of the above mentioned diseases or conditions . combination therapies according to the present invention may comprise the administration of at least one compound of the present invention or a functional derivative thereof , and at least one other pharmaceutically active ingredient . the active ingredient ( s ) and pharmaceutically active agents may be administered separately or together and when administered separately this may occur simultaneously or separately in any order . the amounts of the active ingredient ( s ) and pharmaceutically active agent ( s ) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect . to prepare the pharmaceutical compositions according to the present invention , a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose . a carrier may take a wide variety of forms depending on the form of preparation desired for administration , e . g ., oral or parenteral . in preparing pharmaceutical compositions in oral dosage form , any of the usual pharmaceutical media may be used . thus , for liquid oral preparations such as suspensions , elixirs and solutions , suitable carriers and additives including water , glycols , oils , alcohols , flavouring agents , preservatives , colouring agents and the like may be used . for solid oral preparations such as powders , tablets , capsules , and for solid preparations such as suppositories , suitable carriers and additives including starches , sugar carrier , such as dextrose , mannitol , lactose and related carriers , diluents , granulating agents , lubricants , binders , disintegrating agents and the like may be used . if desired , the tablets or capsules may be enteric - coated or sustained release by standard techniques . thus , specific embodiments and applications of esterified isoflavones have been disclosed . it should be apparent , however , to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein . the inventive subject matter , therefore , is not to be restricted except in the spirit of the appended claims . 1 . phytochemical database of the usda agricultural research service , stephen m . beckstrom - sternberg & amp ; james a . duke , internet address : www . ars - grin . gov /˜ ngrlsb / 2 . a comparative survey of leguminous plants as sources of the isoflavones , genistein and daidzein : implications for human nutrition and health . kaufman p b ; duke j a ; brielmann h ; boik j ; hoyt j e , j altern complement med , 1997 , vol . 3 ( 1 ) p7 - 12 . 3 . the merck index , 12th edition ( 1996 ), merck & amp ; co ., inc , whitehouse station , n . j ., genistein , genistin , biochanin a : entry no . 4395 sophoricoside : entry no . 8867 4 . genistein inhibits growth of b16 melanoma cells in vivo and in vitro and promotes differentiation in vitro . record i r ; broadbent j l ; king r a ; dreosti i e ; head r j ; tonkin a l . int . j . cancer , 1997 , vol 72 ( 5 ) p860 - 4 5 . genistein inhibits proliferation and in vitro invasive potential of human prostatic cancer cell lines . santibanez j f ; navarro a ; martinez j . anticancer res , 1997 , vol 17 ( 2a ) p1199 - 204 6 . action of genistein and other tyrosine kinase inhibitors in preventing osteoporosis . presented by h . c . blair at : second international symposium on the role of soy in preventing and treating chronic disease , sep . 15 - 18 , 1996 , brussels , belgium . 7 . inhibitory effect of genistein on bone resorption in tissue culture . yamaguchi m ; gao y h . biochem pharmacol , 1998 , vol 55 ( 1 ) p71 - 6 . 8 . effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance . tikkanen m j et al ., proc natl acad sci ( usa ), march 1998 , vol 95 ( 6 ), p3106 - 10 . 9 . genistein , the dietary - derived angiogenesis inhibitor , prevents ldl oxidation & amp ; protects endothelial cells from damage by atherogenic ldl . kapiotis s , et al . arterioscler thromb vasc biol ( us ), november 1997 , vol 17 ( 11 ), p2868 - 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