Patent Abstract:
cancer cells are blocked from entering differentiation pathways because of abnormal methylation enzymes , which are responsible for keeping cancer cells in cycling state . effective differentiation inducers are those capable of acting directly or indirectly to convert abnormal methylation enzymes into normal enzymes , thereby enabling cancer cells to undergo terminal differentiation . differentiation employing inducer alone often can not reach completion because of the damage created by the inducer . such damage can be prevented if differentiation is induced in the presence of helper inducers , which are basically inhibitors of the component enzymes of methylation . thus , differentiation induced in the presence of helper inducers is more likely to reach completion . therefore , helper inducers are essential components of differentiation therapy , not just merely to potentiate the activity of differentiation inducers . the present inventors discover that alkyl phenylacetamides , alkyl phenylacetate , 2 , 4 - dichlorophenylacetate , and indole acetate are potent helper inducers .

Detailed Description:
although named as helper inducers , their application in the differentiation therapy of cancer is beyond helping role to potentiate the therapeutic efficacy of differentiation inducers . helper inducers are actually essential components to achieve differentiation therapy . we have noticed that differentiation induced in the presence of helper inducers could reach greater extent of completion as compared to inducers alone ( 40 ). with inducers alone , nbt + cells rarely exceeded 85 %. there was always a small fraction of uninduced cells . this small fraction of uninduced cells is the reason why the remission resulting from ra therapy is so short ( 44 ). differentiation is a long process which must go through two cell cycles of uninterrupted dna hypomethylation ( 45 - 47 ). if cells are damaged during this process , dna synthesis can not progress to completion . these damaged cells if repaired are likely to revert back to the original malignant state . and the symptom reappears . malignant cells have elevated levels of methylation enzymes . these enzymes are dependent upon cancer specific protein factor to maintain stability as ternary enzyme complexes . once that cancer specific protein factor is antagonized by antineoplastons or antineoplaston - like factors induced by differentiation inducers , ternary methylation complexes will dissociate to give rise to individual component enzymes . some of methyltransferases in monomeric forms may become nucleases , better known as latent nucleases , to cause cell damage ( 10 ), resulting in the disruption of differentiation . the employment of helper inducers is designed to control the damage attributable to latent nucleases so that differentiation can reach completion . it is clear that helper inducers are also indispensable components of differentiation therapy . helper inducers can facilitate differentiation and reduce the chance of recurrence . the establishment of phenylacetic acid as helper inducer ( 40 ) and the effectiveness of phenylacetic acid in the treatment of astrocytoma ( 30 , 43 ) have been well documented . it is , however , not a good helper inducer . it requires a few mm quantities to show biological activity as helper inducer . besides , the odor is offensive . there are rooms for improvement . we have discovered that following derivatives of phenylacetic acid were much better on the basis of activity and the odor they generated . n - substituted phenylacetamides are all known compounds listed in reference 51 . however , we have designed a procedure shown in scheme 1 to prepare these derivatives . ## str1 ## phenylacetyl chloride was reacted with methylamine or ethylamine in benzene . the products were purified by column chromatography and recrystallization . dissolved 15 g ( 0 . 1 mol ) of phenylacetyl chloride in 100 ml of benzene . while stirring on a magnetic stirrer , introduced ammonia gas into the benzene solution at 20 °- 30 ° c . after reaction , the solid was collected by filtration , washed with water , and dried . the filtrate was washed in a separators funnel with water , and dehydrated with anhydrous magnesium sulfate . the solution was evaporated to dryness in a rotary evaporator . the residue together with the solid above obtained was purified by column chromatography employing silica gel - chloroform system , and recrystallization with chloroform to yield white crystal as compound 3 - 1 . yield , 11 g ( 80 %). mp . : 153 °- 155 ° c . ; ir ( kbr ) v max : 3300 , 3120 ( nh ), 1670 ( c ═ o ) cm - 1 ; 1 h - nmr ( cdcl 3 3 . 58 ( s , 2h ,-- ch 2 ), 5 . 60 ( br , 2h ,-- nh 2 ), 7 . 24 - 7 . 30 ( m , 5h ,-- c 6 h 5 ), ms , m / z : 135 ( m + ); element analysis : c 8 h 9 no , calculated value : c : 71 . 09 , h : 6 . 71 , n : 10 . 36 , found value : c : 17 . 15 , h : 6 . 93 , n : 10 . 26 dissolved 15 g ( 0 . 1 mol ) of phenylacetyl chloride in 100 ml of benzene . while stirring on a magnetic stirrer , introduced methylamine into the benzene solution at 20 °- 30 ° c . followed the procedure of compound 3 - 1 for the purification of the product to yield n - methyl phenylacetamide as light yellowish crystal . yield , 12 g ( 80 %). mp . : 56 °- 59 ° c . ; ir ( kbr ) v max : 3340 ( nh ), 1630 ( c ═ o ) cm - 1 ; 1 h - nmr ( cdcl 3 ) δ : 2 . 75 ( s , 3h ,-- ch 3 ), 3 . 59 ( s , 2h ,-- ch 2 ), 5 . 59 ( br , 1h ,-- nh ), 7 . 28 - 7 . 31 ( m , 5h ,-- c 6 h 5 ); ms , m / z : 149 ( m + ); element analysis : c 9 h 11 no , calculated value : c : 70 . 04 , h : 8 . 08 , n : 10 . 21 , found value : c : 70 . 18 , h : 8 . 05 , n : 10 . 07 dissolved 15 g ( 0 . 1 mol ) of phenylacetyl chloride in 100 ml of benzene . while stirring on a magnetic stirrer , added 25 g of ethylamine ( 0 . 5 mol ) into the benzene solution at 20 °- 30 ° c . after reaction , the solution was washed with water , dehydrated with anhydrous magnesium sulfate . the solvent was removed by rotary evaporator and the residue was purified by column chromatography as compound 3 - 1 to yield n - ethyl phenylacetamide as yellowish crystal . yield , 14 g ( 85 %). mp . : 72 ° 14 74 ° c . ; ir ( kbr ) v max : 3360 ( nh ), 1630 ( c ═ o ) cm - 1 ; 1 h - nmr ( cdcl 3 ) δ : 1 . 02 ( t , 3h ,-- ch 3 ), 3 . 25 ( m , 2h ,-- n -- ch 2 ), 3 . 50 ( s , 2h ,-- ch 2 -- co --), 7 . 20 - 7 . 33 ( m , 5h ,-- c 6 h 5 ); ms , m / z : 163 ( m + ); element analysis : c 10 h 13 no , calculated value : c : 73 . 59 , h : 8 . 03 , n : 8 . 58 , found value : c : 73 . 67 , h : 8 . 00 , n : 8 . 41 esters of phenylacetic acid are listed in references 52 and 53 . we prepared these derivatives according to the standard procedure shown in scheme 2 . ## str2 ## in the presence of lewis acid , phenylacetic acid was allowed to react with alcohol to yield respective ester . dissolved 13 g ( 0 . 1 mol ) of phenylacetic acid in 500 ml of anhydrous methanol . introduced hc1 gas , while refluxing for 3 hours . the solvent was then removed by evaporation . dissolved the ester in benzene , which was successively washed with water and 10 % naoh . the organic layer was dehydrated with anhydrous magnesium sulfate . the solvent was again removed by evaporation . methyl phenylacetate was finally purified by column chromatography employing silica gel chloroform system . the solvent was removed by evaporation to yield yellowish liquid . yield , 6 g ( 40 % ). bp . : 218 ° c . ; ir ( kbr ) v max : 1700 ( c ═ o ) cm - 1 ; 1 h - nmr ( cdcl 3 ) δ : 3 . 62 ( s , 2h ,-- ch 2 ), 3 . 70 ( s , 3h ,-- ch 3 ), 7 . 30 - 7 . 32 ( m , 5h ,-- c 6 h 5 ); ms , m / z : 150 ( m + ) dissolved 13 g ( 0 . 1 mol ) of phenylacetic acid in 500 ml of anhydrous ethanol . the reaction and purification of the product were as those described for methyl phenylacetate . ethyl phenylacetate was a yellowish liquid . yield , 6 . 5 g ( 40 % ). bp . : 227 ° c . ; ir ( kbr ) v max : 1700 ( c ═ o ) cm - 1 ; 1 h - nmr ( cdcl 3 ) δ : 1 . 24 ( s , 3h ,-- ch 3 ), 3 . 61 ( s , 2h ,-- ch 2 -- co --), 4 . 15 ( s , 2h ,-- och 2 ), 7 . 29 - 7 . 32 ( m , 5h ,-- c 6 h 5 ); ms , m / z : 164 ( m + ) i - 3 . other helper inducers we have so for discovered such as 2 , 4 - dichlorophenyl acetic acid , and indole - 3 - acetic acid were obtained from sigma chemical company . experimental materials herein described such as all trans - retinoic acid , phenylacetic acid , phenylacetyl chloride , butyric acid , and hexanoic acid were obtained from e . merck darmstadt . the procedure developed by liau et al . ( 40 ) was employed for the determination of the activity of helper inducers . the procedure was based on the nbt assay of the induced differentiation of hl - 60 cells . hl - 60 cells were subcultured at an initial concentration of 1 . 5 × 10 5 cells / ml . each flask contained 10 ml . flasks were divided into several sets of 5 flasks containing ra from 0 to 0 . 125 μm . ra was dissolved in methanol . the volume of methanol added was limited to 2 % so that the growth and differentiation of hl - 60 cells were not appreciably affected . one set served as control , while helper inducers of the indicated amounts were added to other sets . after 96 hours cell number was counted , and nbt assay was conducted as previously described ( 40 ). nbt + cells of the control without any addition were always below 4 %. in the presence of helper inducers alone , the control numbers were in general below 10 %. the respective control value was subtracted from each experimental value . ed 50 values which are defined as effective dosages to cause induction of 50 % nbt + cells , can be obtained from plots of nbt + values versus concentrations of ra in the absence and presence of helper inducers . as shown in fig1 ed 50 of ra is 0 . 12 μm . in the presence of 1 μm or 2 μm ethidium bromide as helper inducer , this value is reduced to 0 . 056 μm or 0 . 03 μm , respectively . the reductive index is defined as ed 50 in the presence of helper inducer divided by ed 50 in the absence of helper inducer . this value bears an inverse relationship with the effectiveness of the helper inducer . the biological activity of helper inducers is greatly influenced by the chemical structure . as shown in fig2 butyric is the most active helper inducer among competitive inhibitors of mat . acetic acid is completely ineffective . the addition of a phenyl group to acetic acid restores some activity . hexanoic acid is much less active compared to butyric acid . although butyric acid is a very active helper inducer , it is not a suitable therapeutic drug because it is quickly metabolized . in addition , the odor is very offensive . phenylacetic acid is a stable chemical metabolically , but the activity is not that good . the odor of phenylacetic acid is offensive too . through a systemic search , we have discovered the following compounds to serve as excellent helper inducers . n - methyl phenylacetamide was dissolved in methanol for the determination of its activity as helper inducer . as shown in fig3 it takes 0 . 65 mm to reach a reductive index of 0 . 5 . the corresponding concentration of phenylacetic acid is 4 mm . therefore , it is a good improvement . the growt of hl - 60 was not affected by this compound below 2 mm . it does not have offensive odor . it is a great improvement in this respect too . the only disadvantage is that it is not soluble in water . it can not be formulated as a parenteral preparation . it is perfectly all right to be formulated as oral preparations . ethyl phenylacetamide is very much alike methyl phenylacetamide with respect to chemical properties and biological activity . it is slightly less active , requiring 0 . 87 mm to reach a reductive index of 0 . 5 as shown in fig4 . this is a yellowish liquid with pleasant smell . it was dissolved in methanol for the determination of its activity as helper inducer . as shown in fig5 it is a very active helper inducer . the activity is 10 - fold greater than phenylacetic acid , requiring 0 . 4 mm to reach a reductive index of 0 . 5 . it is very easily hydrolyzed in contact with acid . therefore , the soft gel preparation must have an enteric coating to avoid acid hydrolysis . this is a colorless crystal with pleasant smell . it was dissolved as sodium salt for the determination of its activity as helper inducer . as shown in fig6 it is also a very active helper inducer , requiring 0 . 3 mm to reach a reductive index of 0 . 5 . because of a chlorine - substituted derivative , its toxicity must be checked in order to consider it for clinical application . it certainly can be considered for short - term application of terminal cancer patients . replacement of phenol group with indole group greatly increases the activity as shown in fig7 it takes 0 . 25 mm to reach a reductive index of 0 . 5 . a very active help inducer often by itself is also a formidable inducer . it is not surprising that indole acetic acid is active as inducer at concentration above 0 . 5 mm as shown in fig8 . 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