Patent Application: US-91478392-A

Abstract:
the invention relates to novel polybenzamide mustards having anticancer and hypoxia - selective properties , to methods of preparing the novel compounds , and to the use of these compounds as anticancer agents . the compounds have general formula ## str1 ## wherein m and m 1 separately represent h , aziridinyl , nch 2 ch 2 y or n 2 , where y is cl , br , i or oso 2 me ; r and r 1 separately represent up to three of h , no 2 , aza , ch 2 q , so 2 nhq or conhq , where q is h , me , n nme 2 n nhcnh 2 and n ═ 2 - 4 ); x represents conh , nhco , o , ch 2 , nh or s ; and a is n , where n ═ 2 to 4 , or a unit chosen from formulae ## str2 ## wherein z ═ ch 2 q , so 2 nh q or conhq , where q is h , me , n nme 2 n nhcnh 2 and n ═ 2 to 4 ), or an acid addition salt or n - oxide thereof .

Description:
the invention will now be described by reference to the following non - limiting examples , and to the figures , in which : fig1 represents an autoradiograph off strand cleavage patterns obtained from the drug - treated 3 &# 39 ;- end labelled ecorf / bamhi fragment of pbr322 dna . the lanes correspond to control dna samples in the presence of 30 mm na + ( lane 1 ) and 10 mm mg 2 + ( lane 2 ), and drug - treated dna in the presence of 30 mm na + ( lane 3 ) and 10 mm mg 2 + ( lane 4 ) ( ionic strength = 0 . 04 ); fig2 shows densitometer scans of strand cleavage patterns obtained using drug treated dna in 0 . 01 she buffer in the presence of : no salt ( bottom panel ), 30 mm na + ( middle panel ) and 10 mm mg 2 + ( top panel ). is = ionic strength of the reaction mixture ; fig3 shows an autoradiograph obtained from cross - linking experiments using 3 &# 39 ;- end labelled ecorl digested linear pbr322 dna incubated with compound 1 , using drug / bp ratios of 0 . 02 , 0 . 04 , 0 . 06 , 0 . 08 and 0 . 10 ( lanes 1 - 5 respectively ). lane d is control denatured dna subjected to identical denaturing conditions to the drug - treated samples , and lane n is control native dna not subjected to denaturation ; and fig4 shows results of unwinding of closed circular superhelical plasmid pbr322 incubated with compound 1 at drug / bp ratio 0 . 0 , 0 . 10 , 0 . 15 , 0 . 20 , 0 . 25 , 0 . 30 , 0 . 35 and 0 . 40 ( numbered 1 to 8 in the figure ). table 1 gives physicochemical data for six compounds within the general formula ( i ), representative of it , and preparable by the processes of the invention . table 1__________________________________________________________________________physicochemical data for representative compounds within general formula ( 1 ) [ diag ] no . r m r . sub . 1 m . sub . 1 mp formula analyses__________________________________________________________________________1 ch . sub . 2 nme . sub . 2 n ( et ) ch . sub . 2 ch . sub . 2 cl ch . sub . 2 nme . sub . 2 n ( et ) ch . sub . 2 ch . sub . 2 cl 200 c . sub . 34 h . sub . 46 cl . sub . 2 n . sub . 6 o . sub . 2 . 2h . sub . 2 o c , h , n , cl2 ch . sub . 2 nme . sub . 2 n ( et ) ch . sub . 2 ch . sub . 2 cl ch . sub . 2 nme . sub . 2 h 162 - 165 c . sub . 30 h . sub . 38 clh . sub . 5 c , h , n , cl3 ch . sub . 2 nme . sub . 2 n ( et ) ch . sub . 2 ch . sub . 2 cl h h 186 - 189 c . sub . 27 h . sub . 31 cln . sub . 4 c , h , n , cl4 ch . sub . 2 nme . sub . 2 n ( et ) ch . sub . 2 ch . sub . 2 cl h n ( et ) ch . sub . 2 ch . sub . 2 cl & gt ; 250 c . sub . 31 h . sub . 39 cl . sub . 2 n . sub . 5 o . sub . 2 c , h , n , cl5 h n ( et ) ch . sub . 2 ch . sub . 2 cl ch . sub . 2 nme . sub . 2 h 160 - 161 c . sub . 27 h . sub . 31 cln . sub . 4 c , h , n , cl6 ch . sub . 2 nme . sub . 2 h ch . sub . 2 nme . sub . 2 h & gt ; 300 c . sub . 26 h . sub . 30 n . sub . 4 o . sub . 2 . 2h cl c , h , n , __________________________________________________________________________ cl the following examples illustrate the preparation of compounds respective of the general formula ( i ). synthesis of bis - n , n &# 39 ;-[ 3 -( n -( 2 - chloroethyl )- n - ethylamino )- 5 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( compound 1 of table 1 ) by the method of scheme 1 . 1 , 1 - carbonyldiimidazole ( 5 . 42 g , 33 . 48 mmol ) was added in portions to a stirred solution of 3 - acetamido - 5 - nitrobenzoic acid ( iii ; r ═ h ) ( larsenet al ., 1956 ) ( 5 g , 22 . 32 mmol ) in dry dmf ( 20 ml ) at room temperature . themixture was then heated at 40 ° c . for 30 min , cooled to 10 ° c . and treated with a solution of dimethylamine ( 40 % in water , 5 ml , 44 . 64mmol ). the mixture was heated at 40 ° c . for 30 min , solvent was thenremoved under reduced pressure and the residue was worked up to give crude n -[ 5 - nitro - 3 -( n , n - dimethylaminocarbonyl )] phenylacetamide ( iv ; r ═ h ) ( 3 . 95 g , 70 %), mp ( meoh ) 193 °- 196 ° c . max 1705 ( con ( ch 3 ) 2 ), 1625 ( coch 3 ), 1515 cm - 1 ( no 2 ). 1 h nmr ( cd 3 socd 3 ) 2 . 11 ( s , 3h , coch 3 ), 3 . 37 ( s , 6h , n ( ch 3 ) 2 )), 7 . 87 ( dxd , j 1 = 2 . 0 hz , j 2 = 1 . 3hz , 1h , h - 2 ), 7 . 96 ( m , 1h , h - 4 ), 8 . 59 ( m , 1h , h - 6 ), 10 . 55 ( s , 1h , nh ). 13 c nmr 24 . 10 ( coch 3 ; 38 . 94 ( n ( ch 3 ) 2 ); 113 . 63 ( c - 2 ); 115 . 82 ( c - 4 ); 122 . 81 ( c - 6 ); 138 . 22 ( c - 1 ); 140 . 45 ( c - 3 ); 147 . 83 ( c - 5 ); 167 . 72 ( con ( ch 3 ) 2 ); 169 . 30 ( coch 3 ); 251 ( m , 56 %), 250 ( m - h , 21 ); 209 ( 79 ), 165 ( 50 ), 91 ( 29 ), 72 ( 31 ), 43 ( 100 ). anal . found : c , 63 . 1 ; h , 5 . 3 ; n , 17 . 0 %. c 11 h 13 n 3 o 4 requires c , 62 . 6 ; h , 5 . 2 ; n , 16 . 7 %. a stirred solution off the above acetamide ( iv ; r ═ h ) ( 1 g , 4 . 26 mmol ) in dry thf ( 20 ml ) was treated slowly with borane - thf complex ( 2 ml ). after stirring for 12h at room temperature , more borane - thf complex ( 3 ml ) was added and the mixture was refluxed for 1h , then cooled and acidified with dilute aqueous hc1 . volatiles were evaporated under reduced pressure , and the aqueous layer was basified with aqueous naoh ( 10 %) and extracted with ch 2 cl 2 to give the crude product . chromatography on silica gel and elution with etoac / hexanes ( 3 : 7 ) gave n , n - dimethyl -[ 3 -( nethylamino ) - 5 - nitro ] benzylamine ( v ; r ═ h ) ( 0 . 28 g , 30 %), mp ( benzene / hexanes ) 83 °- 86 ° c . ( orange prisms ). max 1535 ( no 2 ), 1325 cm - 1 ( cn ). 1 h nmr ( cdcl 3 ) 1 . 31 ( t , j = 7 . 2 hz , 3h , ch 2 ch 3 ), 2 . 57 ( s , 6h , n ( ch 3 ) 2 ), 3 . 24 ( qxd , j 1 = 7 . 2 hz , j 2 = 5 . 2 hz , 2h , ch 2 ch 3 ), 3 . 94 ( s , 2h , ch 2 n ), 4 . 10 ( t , j = 5 . 2 hz , 1h , nh ), 6 . 88 ( m , 1h , h - 2 ), 7 . 41 ( m , 1h , h - 4 ), 7 . 44 ( m , h - 6 ). 13 c nmr 14 . 44 ( ch 2 ch 3 ); 38 . 36 ( nch 2 ); 50 . 43 ( nch 3 ); 67 . 23 ( ch 2 n ); 106 . 62 ( c - 4 ); 114 . 53 ( c - 6 ); 122 . 26 ( c - 2 ); 133 . 34 ( c - 5 ); 149 . 03 ( c - 1 ); 149 . 41 ( c - 3 ); 223 ( m , 8 %), 180 ( m -- c 2 h 5 n , 100 ), 134 ( 17 ), 58 ( 62 ). in similar reductions using the borane . dimethylsulfide complex , yields of 70 % were obtained . a mixture of the above n - ethyl derivative ( v ; r ═ h ) ( 0 . 3 g ,. 1 . 35 mmol ) and excess oxirane ( 1 ml ) in glacial acoh / thf ( 1 : 1 , 20 ml ) was stirred at room temperature for 72h . solvents were removed under reduced pressure , and the residue was chromatographed on silica gel . elution with etoac gaven , n - dimethyl -[ 3 -( n - ethyl - n -( 2 - hydroxyethyl ) amino ) - 5 - nitro ] benzylamine ( vi ; r ═ h ) ( 0 . 15 g , 42 %) s an orange solid , top . ( ch 2 cl 2 / hexanes ) 102 -° 104 ° c . max 3525 ( oh ), 1530 ( no 2 ), 1325 ( arylcn ) cm - 1 . 1 h nmr ( cdcl 3 ) 1 . 22 ( t , j = 7 . 1 hz , 3h , ch 2 ch 3 ) 2 . 58 ( s , 6h , n ( ch 3 ) 2 ), 3 . 51 ( q , j = 7 . 1 hz , 2h , nch 2 ch 3 ), 3 . 56 ( t , j = 5 . 8 hz , 2h , nch 2 ch 2 oh ), 3 . 86 ( br s , 2h , ch 2 oh ), 3 . 95 ( br s , 2h , ch 2 n ( ch 3 ) 2 ), 7 . 05 ( br s , 1h , h - 2 ), 7 . 41 ( br s , 1h , h - 4 ), 7 . 56 ( t , j = 2 . 1 hz , 1h , h - 6 ). 13 c nmr 11 . 70 ( ch 2 ch 3 ); 45 . 69 ( nch 2 h 3 ); 50 . 63 ( n ( ch 3 ) 2 ); 52 . 40 ( nch 2 ch 2 oh ); 60 . 17 ( ch 2 oh ); 67 . 65 ( ch 2 n ( ch 3 ) 2 ); 106 . 65 ( c - 4 ); 113 . 38 ( c - 6 ); 121 . 59 ( c - 6 ); 133 . 40 ( c - 1 ); 148 . 74 ( c - 5 ); 149 . 34 ( c - 3 ). m / z 267 ( m , 0 . 3 %); 236 ( m -- ch 2 oh , 100 ), 192 ( 27 ), 58 ( 61 ). ( found : c , 53 . 4 ; h , 8 . 3 ; n , 14 . 0 %. c 13 h 21 n 3 o 3 . 1 . 5h 2 o requires c , 53 . 0 ; h , 8 . 2 ; n , 14 . 3 %). a solution of the above n - hydroxyethyl derivative ( vi : r ═ h ) ( 0 . 2 g , 0 . 75 retool ) in ch 2 cl 2 ( 5 ml ) containing net 3 was treated with methanesulfonyl chloride ( 0 . 2 ml , 2 . 62 mmol ), followed by excess licl in dmf ( 5 ml ). workup gave n , n - dimethyl -[ 3 -( n - ethyl - n -( 2 - chloroethyl ) amino )- 5 - nitro ] benzylamine ( vii ; r ═ h ) ( 0 . 11 g , 52 %), mp ( ch 2 cl 2 / hexane ) 169 °- 172 ° c . max 1525 ( no 2 ) 1320 ( cn ), 760 cm - 1 ( ch 2 cl ). 1 h nmr ( cdcl 3 ) 1 . 25 ( t , j = 7 . 1 hz , 3h , ch 2 ch 3 ), 3 . 61 ( q , j = 7 . 1 hz , 2h , ch 2 ch 3 ), 3 . 78 ( m , 2h , nch3 . 83 ( m , 2h , nch 2 ), 4 . 20 ( s , 2h , ch 2 n ( ch 3 ) 2 ), 7 . 44 ( m , 1h , h - 2 ), 7 . 53 ( m , 1h , h - 4 ), 7 . 84 ( dxd , j 1 = 1 . 3 hz , j 2 = 2 . 4 hz , 1h , h - 6 ). 13 c nmr 11 . 92 ( ch 2 ch 3 ); 41 . 22 ( ch 2 ch 3 ); 42 . 62 ( n ( ch 3 ) 2 ); 46 . 0 9 ( ch 2 cl ); 52 . 13 ( nch 2 ); 61 . 19 ( ch 2 n ( ch 3 ) 2 ); 107 . 27 ( c - 4 ); 111 . 68 ( c - 6 ); 119 . 17 ( c - 2 ); 131 . 49 ( c - 1 ); 149 . 18 ( c - 5 ); 149 . 64 ( c - 3 ). m / z 285 ( m , 15 %), 242 ( m - ch 2 nch 3 ) 86 ), 236 ( m - ch 2 cl , 62 ), 193 ( 242 -- ch 2 cl , 37 ), 58 ( 100 ). ( found : c , 48 . 0 ; h , 6 . 8 ; n , 12 . 5 ; cl , 13 . 5 . c 13 h 20 n 3 o . sub . 2 cl . 2h 2 o requires c , 48 . 4 ; h , 7 . 5 ; n , 13 . 0 ; cl , 11 . 0 %). a solution of the above mustard ( vii ; r ═ h ) ( 0 . 57 g , 2 . 00 mmol ) in conc . hcl ( 5 ml ) was stirred vigorously while tin ( ii ) chloride ( 1 . 80 g , 8 . 00 mmol ) was added in portions . the solution was refluxed for 2h , cooled , diluted with water , washed with etoac , basified with conc . ammoniato ph 8 - 9 , extracted with ch 2 cl 2 , and worked up to give 3 -[ n -( 2 - chloroethyl ) - n - ethylamino ]- 5 -[( n , n - dimethylamino ) methyl ] aniline ( viii ; r ═ h ) ( 0 . 34 g , 67 %) as an oil , which was used directly . 1 hbrmr ( cdcl 3 ) 1 . 16 ( m , 3h , ch 2 ch 3 ), 2 . 74 ( s , 6h , n ( ch 3 ) 2 ), 3 . 35 ( m , 2h , ch 2 ch 3 ), 3 . 62 ( s , 4h , nch 2 ch 2 cl ), 3 . 94 ( s , 2h , ch 2 n ( ch 3 ) 2 ) 6 . 22 ( br s , 3h , h - 2 , 4 , 6 ). 1 , 4 - benzenedicarbonyl dichloride ( xviii ; z ═ h ) ( 0 . 14 g , 0 . 67 mmol ) was added in one portion to a stirred solution of the above amine ( viii ) ( 0 . 34g , 1 . 33 retool ) in ch 2 cl 2 ( 5 ml ), and the mixture was stirred atroom temperature for 30 min . the solvent was removed under reduced pressureand the residue was dissolved in water , basified with conc . ammonia to ph 8 - 9 and extracted with ch 2 cl 2 . workup gave the free base of bis - n , n &# 39 ;-[ 3 -( n -( 2 - chloroethyl )- n - ethylamino )- 5 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( 1 ) ( 0 . 31 g , 72 %) as a pale yellow solid , mp ( ch 2 cl 2 / ether ) 200 ° c . ( dec ). max 1645 ( co ), 1610 ( c - c ), 1340 ( cn ) 1155 , 715 cm - 1 ( ch 2 cl ). 1 h nmr ( of dihcl salt ( cd 3 socd 3 ) 1 . 14 ( t , j = 6 . 9 hz , 6h , ch 2 ch 3 ), 2 . 71 ( d , j = 4 . 7 hz , 12h , n ( ch 3 ) 2 ), 3 . 46 ( g , j = 6 . 9 hz , 4h , nch 2 ch 3 ), 3 . 65 ( t , j = 6 . 7 hz , 4h , ch 2 cl ), 3 . 80 ( t , j = 6 . 7 hz , 4h , nch 2 ch 2 cl ), 4 . 18 ( d , j = 5 . 1 hz , 4h , ch 2 n ( ch 3 ) 2 ), 6 . 89 ( s , 2h , h - 4 &# 39 ;, 4 &# 34 ;), 7 . 23 ( s , 2h , h - 2 &# 39 ;, 2 &# 34 ;), 7 . 37 ( s , 2h , h - 6 &# 39 ;, 6 &# 34 ;), 8 . 13 ( s , 4h , h - 2 , 3 , 5 , 6 ), 10 . 47 ( s , 2h , nh ), 10 . 90 ( br s , 2h , hcl ). 13 c nmr 12 . 51 ( ch 2 ch 3 ); 40 . 76 ( ch 2 cl ); 45 . 09 ( n ( ch 3 ) 2 ; 45 . 67 ( nch 2 ch 3 ); 52 . 36 ( nch 2 ch 2 cl ); 64 . 44 ( ch 2 n ( ch 3 ) 2 ); 103 . 09 ( c - 4 &# 39 ;, 4 &# 34 ;); 109 . 10 ( c - 2 &# 39 ;, 2 &# 34 ;); 109 . 17 ( c - 6 &# 39 ;, 6 &# 34 ;); 127 . 51 ( c - 2 , 3 , 5 , 6 ); 137 . 92 ( c - 5 &# 39 ;, 5 &# 34 ;); 139 . 17 ( c - 1 &# 39 ;, 1 &# 34 ;); 139 . 74 ( c - 3 &# 39 ;, 3 &# 34 ;); 147 . 87 ( c - 1 , 4 ); 164 . 92 ( co ). massspectrum ( of free base ) m / z 640 ( m , 28 %), 105 ( 50 ), 58 ( 100 ). ( found : c , 60 . 9 ; h , 7 . 2 ; n , 11 . 9 ; cl , 12 . 7 . c 34 h 46 n 6 o 2 cl 2 . 2h 2 o requires c , 60 . 3 ; h , 7 . 9 ; n , 12 . 4 ; cl , 10 . 5 %). synthesis of n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) - 5 -( n , n - dimethylaminomethyl ) phenyl ]- n 1 -[( 3 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( compound 2 of table 1 ) and n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) - 5 -( n , n - dimethylaminomethyl ) phenyl ]- n 1 - phenyl - 1 , 4 - benzenedicarboxamide ( compound 3 of table 1 ) by the method of scheme 2 . equimolar amounts of 3 -( n , n - dimethylaminomethyl ) aniline ( xvi ; r ═ h ) ( stedman , e . j . chem . soc . 1927 , 1902 ) and 4 -( methoxycarbonyl ) benzenecarbonyl chloride ( xvii ; z ═ h ) were reacted together in pyridineat 0 ° c ., to give methyl 4 -[ 3 -( n , n - dimethylaminomethyl ) phenyl ] carbamoylbenzene - carboxylate ( ix ; r ═ z ═ h ) ( 73 %), mp ( dilsopropyl ether ) 108 °- 109 ° c . 1 h nmr ( cd 3 socd 3 ) 10 . 42 ( s , 1 h , conh ), 8 . 13 ( s , 4 h , h - 2 &# 39 ;, h - 3 &# 39 ;), 7 . 75 ( s , 1h , h - 2 ), 7 . 70 ( d , j = 8 . 2 hz , 1 h , h - 6 ), 7 . 30 ( t , j = 8 . 2 hz , 1 h , h - 3 ), 7 . 04 ( d , j = 8 . 2 hz , 1 h , h - 4 ), 3 . 97 ( s , 3h , cooch 3 ), 3 . 40 ( s , 2 h , ch 2 ), 2 . 18 ( s , 6 h , n ( ch 3 ) 2 ). anal . ( c 18 h . sub . 20 n 2 o 3 ) c , h , n . a solution of ester ( ix ; r ═ z ═ h ) ( 5 . 30 g , 17 mmol ) in meoh ( 10 ml ) was treated with one equivalent of naoh ( 17 . 0 nil of 1 . 0 n aqueous solution ), and the mixture was heated until the meoh had boiled off , and for 1 h under reflux , then cooled and filtered . exact neutralisation ( with17 . 0 ml of 1 . 0 n aqueous hcl ) followed by refrigeration yielded 4 -[ 3 -( n , n - dimethylaminomethyl ) phenyl ] carbamoylbenzene - carboxylic acid ( x ; r ═ z ═ h ) ( 4 . 73 g , 93 %), mp ( meoh / etoac ) 209 °- 210 ° c . 1 h nmr ( cd 3 socd 3 ) 10 . 41 ( s , 1 h , nh ), 8 . 07 ( d , j = 7 . 0 hz , 2 h , h - 2 &# 39 ;, 6 &# 39 ;), 8 . 02 ( d , j = 7 . 0 hz , 2 h , h - 3 &# 39 ;, 5 &# 39 ;), 7 . 82 ( s , 1 h , h - 2 ), 7 . 72 ( d , j = 8 . 05 hz , 1 h , h - 4 ), 7 . 33 ( u , j = 7 . 8 hz , 1 h , h - 5 ), 7 . 09 ( d , j = 7 . 6 h z , 1 h , h - 6 ), 3 . 61 ( s , 2 h , ch 2 ), 2 . 32 ( s , 6 h , n ( ch 3 ) 2 ). anal . ( c 17 h 18 n 2 o 3 ) c , h , n . an ice cold solution of : ( x ; r ═ z ═ h ) ( 0 . 70 g , 2 . 35 retool ) in dry dmf ( 5 me ) containing 1 - methylimidazole ( 0 . 21 g , 2 . 56 mmol ) was added to solid ( viii ; r ═ h ) ( 0 . 61 g , 2 . 38 mmol ) contained in a precooled flask . the mixture was stirred until homogeneous , and then treated dropwise with diethyl cyanophosphonate ( 93 %, 0 . 43 g , 2 . 45 retool ) at 0 ° c . the mixture was stirred at 25 ° c . for 1 . 5 h , then diluted with a largeexcess of 0 . 5 n na 2 co 3 and the resulting solid collected and extracted into ch 2 cl 2 . the ch 2 cl 2 solution was washed twice with water and evaporated , and the residue was chromatographed on a short column of alumina ( activity ii - iii ). elution with etoac gave n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino )- 5 -( n , n - dimethylaminomethyl ) phenyl ]- n 1 - [( 3 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( 2 ) ( 0 . 59 g , 47 %), mp ( etoac / petroleum ether ) mp 162 °- 165 ° c . 1 h nmr ( cdcl 3 ) 8 . 27 & amp ; 8 . 15 ( 2 × s , 2 h , conh , conh ), 7 . 85 ( s , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;, 6 &# 39 ;), 7 . 67 ( d , j = 7 . 9 hz , 1 h , h - 2 &# 34 ;), 7 . 56 ( s , 1 h , h - 6 &# 34 ;), 7 . 31 ( t , j = 7 . 8 hz , 1 h , h - 3 &# 34 ;), 7 . 11 ( s , 1 h , h - 4 &# 34 ;), 6 . 79 ( s , 1 h , h - 4 ), 6 . 45 ( s , 1 h , h - 6 ), 3 . 71 ( s , 4 h , nch 2 ch 2 cl ), 3 . 44 ( q , j = 7 . 0 hz , 2 h , nch 2 ch 3 ), 3 . 42 & amp ; 3 . 45 ( 2 × s , 2 h , 2 x ch 2 n ( ch 3 ) 2 ), 2 . 23 ( s , 12 h , 2 × n ( ch 3 ). sub . 2 ), 1 . 19 ( t , j = 7 . 0 hz , 3 h , nch 2 ch 3 ). anal . ( c 30 h 38 cln 5 o 2 ) c , h , n , cl . similar reaction of 4 - phenylcarbamoylbenzenecarboxylic acid and amine ( viii ; r ═ h ) gave n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) - 5 -( n , n - dimethylaminomethyl ) phenyl ]- n 1 - phenyl - 1 , 4 - benzenedicarboxamide ( 3 ) ( 52 %), mp ( etoac / petroleum ether ) 186 °- 189 ° c . 1 h nmr ( cdcl 3 ) 8 . 08 , 7 . 98 ( 2 × s , 2 h , conh , conh ), 7 . 90 ( s , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;, 6 &# 39 ;), 7 . 67 ( d , j = 7 . 8hz , 2 h , h - 2 &# 34 ;, 6 &# 34 ;), 7 . 38 ( t , j = 7 . 6 hz , 2 h , h - 3 &# 34 ;, 5 &# 34 ;), 7 . 25 ( s , 1 h , h - 2 ), 7 . 18 ( t , j = 7 . 4 hz , h - 4 &# 34 ;), 6 . 75 ( s , 1 h , h - 4 ), 6 . 46 ( s , 1 h , h - 6 ), 3 . 66 ( s , 4 h , nch 2 ch 2 cl ), 8 . 44 ( q , j = 7 . 0 hz , 2 h , nch 2 ch 3 ), 3 . 36 ( s , 2 h , ch 2 n ( ch 3 ) 2 ), 2 . 24 ( s , 6 h , n ( ch 3 ) 2 ), 1 . 21 ( t , j = 7 . 0 hz , 3 h . nch 2 ch 3 ). anal . ( c 27 h 31 cln 4 o 2 ) c , h , n , cl . synthesis of n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) - 5 -( n , n - dimethylaminomethyl ) phenyl ]- n 1 -[ 3 -( n -( 2 - chloroethyl ) - n - ethylamino ) phenyl ]- 1 , 4 - benzenedicarboxamide ( compound 4 of table 1 ) and n -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) phenyl ]- n 1 -[ 3 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( compound 5 of table 1 ) by the method of scheme 3 . a solution of n - ethylaniline ( 13 . 3 g , 80 mmol ) in thf ( 50 nml ) and acoh ( 50nil ) was treated with oxirane ( 15 ml , 0 . 3 mol ), and the mixture was stirredat 20 ° c . for 36 h . additional oxirane ( 15 ml ) was then added , and the mixture was stirred at 20 ° c . for an additional 36 h . solvent was then removed under reduced pressure , and the residue was partitioned between ch 2 cl 2 and 1n aqueous na 2 co 3 . the residue obtained from workup of the organic layer was chromatographed on sio 2 ( etoac / petroleum ether , 1 : 3 ) to give n - ethyl - n -( 2 - hydroxyethyl )- 3 - nitroaniline ( xi ; r ═ h ) ( 12 . 3 g , 75 %), mp ( benzene / petroleum ether ) 43 ° c . 1 h nmr ( cdcl 3 ) 7 . 53 ( t , j = 2 . 3 hz , 1 h , h - 2 ), 7 . 49 ( dd , j = 8 . 2 , 2 . 0 hz , 1 h , h - 4 ), 7 . 30 ( dd , j = 8 . 4 , 8 . 2 hz , 1 h , h - 5 ), 7 . 01 ( dd , j = 8 . 4 , 2 . 6 hz , 1 h , h - 6 ), 3 . 83 ( br s , 1 h , ch 2 oh ), 3 . 54 ( t , j = 5 . 9 hz , 2 h , nch 2 ch 2 oh ), 3 . 49 ( q , j = 7 . 1 hz , 2 h . nch 2 ch 3 ), 1 . 72 ( br s , 1 h , oh ), 1 . 21 ( t , j = 7 . 1 hz , 3 h , ch 3 o . anal . ( c 10 h 14 n 2 o 3 ) c , h , n . a stirred solution of the above alcohol ( xi ; r ═ h ) ( 4 . 0 g , 19 mmol ) in ch 2 cl 2 ( 35 ml ) containing net 3 ( 2 . 91 ml , 21 mmol ) was treated dropwise at 0 ° c . with methanesulfonylchloride ( 1 . 62 ml , 21 mmol ). after being stirred at 0 ° c . for a further 30 min and at20 ° c . for 30 min , the reaction mixture was diluted with ch 2 cl 2 ( 35 ml ) and washed successively with 1n hcl , 1n na 2 co 3 and saturated nacl , and worked up to give the crude mesylate , which was immediately treated with licl ( 2 g ) in dry dmf ( 20 ml ) at 75 ° c . for 30 min . removal of excess solvent under reduced pressure , and chromatography of the residue on sio 2 ( etoac / petroleum ether , 1 : 4 ) gave n -( 2 - chloroethyl )- n - ethyl - 3 - nitroaniline ( xii ; r ═ h ) ( 3 . 58 g , 82 %) as yellow prisms , mp ( petroleum ether ) 56 °- 57 ° c . 1 h nmr ( cdcl 3 ) 7 . 51 ( dd , j = 7 . 9 , 1 . 9 hz , 1 h , h - 4 ), 7 . 48 ( t , j = 2 . 4 hz , 1 h , h - 2 ), 7 . 32 ( m , 2 h , h - 5 ), 6 . 95 ( dd , j = 8 . 4 , 2 . 7 hz , h - 6 ), 3 . 70 ( m , 2 h , nch 2 ch 2 cl ), 3 . 64 ( m , 2 h , ch 2 cl ), 3 . 50 ( q , j = 7 . 1 hz , 2 h , nch 2 ch 3 ), 1 . 22 ( t , j = 7 . 1 hz , 3 h , ch 3 ). anal . ( c 10 h 13 cln 2 o 2 ) c , h , n , cl . a solution of the above nitro mustard ( xii ; r ═ h ) ( 2 . 51 g , 11 mmol ) in 12 n hcl ( 25 ml ) was treated portionwise at 25 ° c . with sncl 2 . 2h 2 o ( 9 . 9 g , 44 mmol ), heated on a steam bath at 90 ° c . for 1 h , then evaporated to dryness under reduced pressure . the residue as shaken vigorously with a mixture of ch 2 cl 2 , 2 n nh 4 oh and ice , and filtered through a celite pad . workup of the organic layer gave essentially pure 3 -[ n -( 2 - chloroethyl ) - n - ethylamino ] aniline ( xiii ; r ═ h ) ( 1 . 92 g , 88 %) as an oil , which was used immediately . a stirred solution of the amine ( xiii ; r ═ h ) ( 2 . 38 g , 12 mmol ) in ch 2 cl 2 ( 30 ml ) containing net 2 ( 1 . 80 ml , 13 mmol ) was treated dropwise at 0 ° c . with a solution of 4 - methoxycarbonylbenzenecarbonyl chloride ( xvii ; z ═ h ) ( 2 . 18 g , 11 mmol ) in ch 2 cl 2 ( 10 ml ). after being stirred for a further 15 min at 0 ° c . and for 15 min at 25 ° c ., the mixture was washed with 1 n na 2 co 3 and water , and the residue from the organic layer was chromatographed on sio 2 ( ch 2 cl 2 ) to givemethyl 4 -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) phenyl ] carbamoylbenzenecarboxylate ( xiv ; r ═ z ═ h ) ( 3 . 36 g , 85 %), mp ( benzene / petroleum ether ) 111 °- 112 ° c . 1 h nmr ( cd 3 socd 3 ) 10 . 25 ( s , 1 h , conh ), 8 . 09 ( m , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;, 6 &# 39 ;), 7 . 16 ( d , j = 1 . 8 hz , 1 h , h - 2 ), 7 . 15 ( m , 2 h , h - 4 , h - 6 ) e 6 . 49 ( m , 1 h , h - 5 ), 3 . 93 ( s , 3 h , cooch 3 ), 3 . 73 ( t , j = 7 . 1 hz , 2 h , nch 2 ch 2 cl ), 3 . 61 ( t , j = 7 . 1 hz , 2 h , ch 2 cl ), 3 . 41 ( t , j = 7 . 0 hz , 2 h , nch 2 ch 3 ), 1 . 12 ( t , j = 7 . 0 hz , 3 h , chs ). anal . ( c 19 h 21 cln 2 o 3 ) c , h , n , cl . a suspension of ( xiv ; r ═ z ═ h ) ( 2 . 88 g , 8 mmol ) in meoh ( 100 ml ) containing koh ( 5 . 6 g ) was stirred at 25 ° c . until homogeneous , andthen for a further 5 h . the mixture was diluted with water , filtered , and acidified with acoh to give 4 -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) phenyl ] carbamoylbenzenecarboxylic acid ( xv ; r ═ z ═ h ) ( 2 . 08 g , 75 %), mp ( etoac ) 203 ° c . ( dec .). 1 h nmr ( cd 3 socd 3 ) 13 . 3 ( br s , 1 h , cooh ), 10 . 21 ( s , 1 h , conh ), 8 . 05 ( q , j = 8 . 50 , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ; 6 &# 39 ;), 7 . 16 ( m , 3 h , h - 2 , 4 , 6 ), 6 . 48 ( m , 1 h , h - 5 ), 3 . 72 ( t , j = 7 . 1hz , 2 h , nch 2 ch 2 cl ), 3 . 60 ( t , j = 7 . 1 hz , 2 h , ch 2 cl ), 3 . 40 ( g , j = 7 . 0 hz , nch 2 ch 3 )-, 1 . 12 ( t , j = 7 . 0 hz , 3 h , ch 3 ). anal . ( c 18 h 19 cln 2 o 3 ) c , h , n , cl . reaction of acid ( xv ; r ═ z ═ h ) and amine ( viii ; r ═ h ) by the method outlined in example 2 gave n -[ 3 -( n -( 2 - chloroethyl ) - n - ethylamino )- 5 -( n , n - dimethylaminomethyl ) phenyl - n 1 -[ 3 -( n -( 2 - chloroethyl )- n - ethylamino ) phenyl ]- 1 , 4 - benzenedicarboxamide ( 4 ) ( 61 %) mp ( etoac / petroleum ether ) & gt ; 250 ° c . 1 h nmr ( cdcl 3 ) 8 . 11 , 8 . 07 ( 2 × s , 2 h , conh , conh ), 7 . 87 ( s , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;, 6 &# 39 ;), 7 . 24 ( s , 2 h , h - 2 , 2 &# 34 ;), 7 . 20 ( m , 1 h , h - 5 &# 34 ;), 6 . 87 ( d , j = 7 . 8 hz , 1 h , h - 4 &# 34 ;), 6 . 77 ( s , 1 h , h - 4 ), 6 . 50 ( dd , j = 8 . 3 , 2 . 4 hz , 1 h , h - 6 &# 34 ;), 6 . 44 ( s , 1 h , h - 6 ), 3 . 62 ( s , 8 h , 2 × ch 2 ch 2 cl ), 3 . 44 ( 2 × q , j = 7 . 0 hz , 4 h , 2 × nch 2 ch 3 ), 3 . 36 ( s , 2 )( ch 2 n ( chs ) 2 ), 2 . 23 ( s , 6 h , n ( ch 3 ) 2 . ), 1 . 99 ( 2 × t , j = 7 . 0 hz , 6 h , 2 × ch 3 ). anal . ( c 31 h 39 cl 2 n 5 o 2 ) c , h , n , cl . similar reaction of amine ( xiii ; r ═ h ) and acid ( x ; r ═ z = h ) ( see example 2 for preparation ) gave n -[ 3 -( n -( 2 - chloroethyl ) - n - ethylamino ) phenyl ]- n 1 -[ 3 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( 5 ) ( 59 %), mp 160 °- 161 ° c . ( etoac / petroleum ether ). 1 h nmr 8 . 35 & amp ; 8 . 26 ( 2 × s , 2 h , conh , conh ), 7 . 80 ( s , 4 h , h - 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;, 6 &# 39 ;), 7 . 66 ( d , j = 8 . 2 hz , 1 h , h - 6 &# 34 ;), 7 . 57 ( s , 1 h , h - 2 &# 34 ;), 7 . 30 ( t , j = 7 . 7 hz , 1 h , h - 5 &# 34 ;), 7 . 18 ( t , j = 8 . 4 hz , 1 h , h - 5 ), 7 . 11 ( d , j = 7 . 7 hz , 1 h , h - 4 &# 34 ;), 6 . 89 ( d , j = 7 . 7 hz , 1 h , h - 4 ), 6 . 49 ( dd , j = 8 . 4 , 2 . 3 hz , 1 h , h - 6 ), 3 . 61 ( s , 4 h , nch 2 ch 2 cl ), 3 . 41 ( g , j = 7 . 0 hz , 2 h , nch 2 ch 3 ), 3 . 39 ( s , 2 h , ch 2 n ( ch 3 ) 2 ), 2 . 24 ( s , 6 h , n ( ch 3 ) 2 ), 1 . 19 ( t , j = 7 . 0 hz , 3h , nch 2 ch 3 ). anal . ( c 27 h 31 cln 4 o 2 ) c , h , n , cl . synthesis of bis - n , n 1 -[ 3 -( n , n - dimethylaminomethyl ) - phenyl ]- 1 , 4 - benzenedicarboxamide ( compound 6 of table 1 ) by the method ofscheme 4 . powdered 1 , 4 - benzenedicarbonyl dichloride ( xviii ; z ═ h ) ( 1 . 01 g , 5 mmol ) was added to a stirred solution of 3 -( n , n - dimethylaminomethyl ) aniline ( 1 . 65 g , 11 mmol ) in dry dmf ( 20 ml ), and the reaction mixture was stirredat 25 ° c . for 30 min , and at 90 ° c . for 15 min . the cooled mixture was diluted with 1 n na 2 co 3 to give bis - n , n 1 -[ 3 -( n , n - dimethylaminomethyl ) phenyl ]- 1 , 4 - benzenedicarboxamide ( 6 ). 1 h nmr ( free base in cdcl 3 ) [ symmetric molecule ] δ8 . 40 ( s , 1 h , conh ), 7 . 79 ( s , 2 h , h - 2 &# 39 ;, 6 &# 39 ;), 7 . 65 ( d , j = 8 . 0 hz , 1 h , h - 6 ), 7 . 57 ( s , 1 h , h - 2 ), 7 . 29 ( m , 1 h , h - 5 ), 7 . 11 ( d , j = 7 . 6 hz , 1 h , h - 4 ), 3 . 48 ( s , 2 h , ch 2 n ( ch 3 ) 2 ), 2 . 23 ( s , 6 h , n ( ch 3 ) 2 )- dihydrochloride salt , mp & gt ; 300 ° c . ( meoh / etoac ). anal . ( c 26 h 30 n 4 o 2 . 2hcl ) c , h , n , cl . i ) preparation of labelled dna fragment a 375 base pair ecori to bamhi fragment of pbr322 dna was 3 &# 39 ;- end labelled at the ecori site using klenow fragment and 32 p - datp . the resulting labelled fragment was isolated on a 4 % non - denaturing polyacrylamide gel . given below is a partial sequence of the fragment from base pair 31 to 140 : ## str9 ## labelled dna ( ca . 30 , 000 cpm ) was incubated with the alkylating agent compound 1 in the presence of 1 μg of calf thymus dna in 100 μl of 0 . 01 she buffer ( ph = 7 . 3 , ionic strength = 0 . 01 ) at 37 ° c . for 30 minutes . the drug to base pair ratio was adjusted to 0 . 20 against the carrier dna . although at this ratio more than one alkylation per labelled fragment occurred ( prakash et al ., 1990 ), further experiments showed that at the lower ratio of 0 . 1 , where less than one alkylation per fragment occurred , the alkylation pattern was nearly identical to that at 0 . 02 shown in fig1 . for experiments with mg 2 + , the reactions were carried out either in the presence of 30 mm nacl or 10 mm mgcl 2 ( final ionic strength = 0 . 04 ). the reaction mixture was chilled in ice and modified dna was precipitated with ethanol and lyophilised . the modified dna pellet was dissolved in 100 μl of 0 . 01 she ( ph = 7 . 3 ) andheated for 10 minutes at 90 ° c . then 11 1 of 10 m piperidine was added to the solution and the reaction mixture was further heated for 10 minutes at 90 ° c . the sample was then lyophilised overnight , precipitated with ethanol and dissolved in 3 μls of sequencing dye madeof 80 % deionised formamide , 1 % xylene cyanol and 1 % bromophenol blue . the sample was denatured at 90 ° c . for two minutes prior to loading on a sequencing gel . the polyacrylamide gels were run as described previously ( prakash et al ., 1990 ). the strand cleavage pattern obtained by chemical treatment of the drug - treated dna is shown in fig1 . adenines in runs of as ( 33 - 35 , 46 - 48 ) show strong bands , with weaker bands also seen for adenines in ta and at sequences ( 57 , 61 , 85 , 92 and 94 ). very little guanine alkylation is observed , after taking into account the control and the loading variations . fig2 shows the densitometer scans of the alkylation patternsobtained under different ionic strengths ( panels middle and bottom ) and in the presence of mgcl 2 ( top panel ). changing the ionic strength from 0 . 01 to 0 . 04 does not affect the band distribution , but using the divalentcation mg 2 + instead of the monovalent na + leads to an increase in the intensities of bands at 57 and 61 . preliminary investigation using aniline mustards with varying drug / base pair ratios up to 0 . 10 showed that , on average , one alkylation or less per 375 - bp labelled strand occurs ( prakash et al ., 1990 ), implying that roughly one out of 20 molecules of drug available on average per strand eventually alkylates the dna . fig2 and 3 show that compound 1 preferentially alkylates adenines occurring in runs of as , and to a much smaller extent in 5 &# 39 ;- at and 5 &# 39 ;- ta sequences . we have previously shown that mg 2 + apparently inhibits adenine alkylation by dna intercalator - targeted mustards in the major groove , but not in the minor groove ( prakash et al ., 1990 ). the results summarized in fig3 are thus consistent with compound 1 alkylating adenines at minor groove sites ( presumably n3 ), since addition of mg 2 + fails to diminish the band intensities , and in fact increases the degree of alkylation at adenines occurring in at and ta sequences ( eg bands 57 and 61 ). this may be due to the unusual conformation present in the atz and ta junctions , which may be further affected by the presence ofmg 2 + in the major groove . crosslinking experiments show that compound1 gives rise to about one interstrand crosslink from every 10 alkylation events ; a much better ratio than those ( ca . 1 in 20 ) which we have previously found with dna intercalator - targeted aniline mustards ( prakash et al ., 1990 ). these experiments were carried out as described previously ( prakash et al ., 1990 ). fig3 shows the crosslinking of dna by compound 1 at various concentrations . at a drug : bpr ratio of 0 . 04 , on average one crosslink per 4362 - bp dna strand has occurred . this is based on a statistical model , in which the natural log ratio of the band intensities of the sample bands and that for the denatured dna are used to determine the number of cross - links per fragment ( prakash et al ., 1990 ). at the same input ratio there are not more than about 10 alkylation events per 4362 - bp fragment [( 4362bp / 375bp )×( 0 . 04 d per bp / 0 . 10 d per bp ) x ( 2 strands per fragment )= ca . 10 ], suggesting a cross - link to mono - adduct ratio of about 0 . 1 . fig4 shows the results from the helix unwinding assay . the drug does not completely remove the superhelical turns from supercoiled dna even at a drug to basepair ratio of 0 . 40 , indicating that the drug is not intercalated into dna . the negative results of the helix unwinding studies ( fig4 ) indicate that compound 1 does not intercalate into dna . cpk models of compound 1 bindingto dna in the minor groove suggest that the molecule spans a distance of about 3 basepairs when the alkylating moieties are in closest proximity tothe n3 sites of adenines on opposite strands . with such a binding mode , crosslinking would be most likely to take place in sequences aat , att , taaand tta . since we have shown here that alkylation is strongest in runs of as , the weak alkylation observed at at junctions ( eg at bases 57 and 61 which have runs of ts preceding or following the a ) may in fact be due to initial alkylation of adenines in the opposite strand followed by interstrand crosslinking . growth inhibition studies were performed as described in detail elsewhere ( wilson et al ., 1989 ; finlay et al ., 1984 ). ic 50 s were determined asthe drug concentration needed to reduce the cell mass ( protein content , measured after 72 - 78h by staining with methylene blue and measuring absorbance in a microplate photometer ) to 50 % of the mean value for 8 control cultures on the same 96 - well plate . the ratio of ic 50 values against aa8 and uv4 cell lines is defined as hf = ic 50 ( aa8 )/ ic 50 ( uv4 ). compound ( 1 ) had an ic 50 against p388 leukaemia cells of ca . 0 . 05 nm , and against aa8 cells of ca . 800 nm . it had an hf of 15 , and showed significant activity against p388 leukaemia in vivo ( ils of 37 % at an optimal dose of 9 mg / kg , when given as a single intraperitoneal dose ). it is clear from these data that compound 1 , a representative example of the compounds of general formula ( i ), is a potent cytotoxic agent , with invivo anticancer activity . the present invention therefore also provides pharmaceutical compositions having antitumour activity and comprising at least one compound represented by the general formula ( i ), and one or morepharmaceutically - acceptable carriers or diluents . it will be clearly understood that the invention in its general aspects is not limited to the specific details referred to herein . braithwaite , a . w . & amp ; 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