in this invention , linear polyethylene glycol and 2 , 2 - bis ( hydroxymethyl ) propionic anhydride are used as starting materials . diethylenetriaminepentacetic acid ( dtpa ) is the chelator to stabilize gd . a . preparation of the first generation benzylidene protected chelate p - d 1 -( o 2 bn ) peg diol ( mw 4000 da , 9 . 2 g , 2 . 3 mmol , 1 eq ) and dmap ( 0 . 1670 g , 0 . 39 mmol ) are mixed in a round - bottom conical vial . the mixture is dissolved in a 25 ml of dcm and then the vial is filled with nitrogen gas . benzylidene - 2 , 2 - bis ( oxymethyl ) propionic anhydride ( 4 . 27 g ( 10 mmol )) is dissolved in another vial , and then slowly dripped into the reaction vial . after 24 hours of stirring and reacting in room temperature , 10 ml methanol is added and the reaction is kept for another 6 hours for removing the un - reacted benzylidene - 2 , 2 - bis ( oxymethyl ) propionic anhydride . an excessive amount of ethyl ether ( 700 ml ) is added and the mixture is stirred until white precipitates are released , and the yield rate is about 90 %. among the products , the d n is d 1 with the structure of ( i ) as follows : 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 06 ( s , 6 ), 3 . 55 ( t , 6 ), 3 . 61 ( bs ), 3 . 68 ( t , 6 ), 4 . 32 ( t , 4 ), 4 . 64 ( d , 4 ), 5 . 43 ( s , 2 ), 7 . 28 ( m , 6 ), 7 . 42 ( m , 4 ). after the above product ( 11 . 8 g ) is dissolved in 40 ml of 1 : 2 ch 2 cl 2 / meoh solution , 1 . 18 g of pd / c is added , and the mixture is stirred for 24 hours under a hydrogen - saturated environment . when the reaction ends , pd / c is filtrated from dcm , and as described above , an excessive amount of ethyl ether 600 ( ml ) is added to release the white precipitates . the yield rate of the product after freeze - drying is approximately 90 %. 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 08 ( s , 6 ), 3 . 67 ( bs ), 4 . 31 ( t , 4 ). p - d 1 - oh ( 2 . 0 g , 0 . 4618 mmol ) and diethylenetriaminepentaacetic acid mono - n - hydroxysuccinimide ester ( dtpa - hsie ) ( 1 . 0871 g , 2 . 2 mmol ) are mixed in a 50 ml round - bottom conical vial and vacuum dried for 3 hours . anhydrous dmso ( 10 ml ) and triethylamine ( 224 μl ) are injected into the mixture and stirred for 48 hours at room temperature under saturated nitrogen gas . acetonitrile / acetone is used to release white residue ; the white solid product after centrifugation and freeze - drying is p - d 1 - dtpa . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 14 ( s , 6 ), 3 . 1 ( t , 16 ), 3 . 4 ( t , 16 ), 3 . 57 ( bs ), 3 . 75 ( s , 8 ), 3 . 8 ( s , 32 ). the principle of preparing the second generation p - d 2 -( o 2 bn ) product is approximately the same as that of the first generation . p - d 1 -( o 2 bn ) ( 95 . 6 g , 0 . 83 mmol , 1 equiv ) and dmap ( 0 . 326 g , 2 . 6 mmol , 3 . 2 equiv ) is mixed and then dissolved in 25 ml dcm . after benzylidene - 2 , 2 - bis ( oxymethyl ) propionic anhydride ( 13 . 3 mmol , 16 equiv , 5 . 69 g ) is added , the mixture is stirred at room temperature for 24 hours . un - reacted benzylidene - 2 , 2 - bis ( oxymethyl ) propionic anhydride is removed with 15 ml methanol . then ethyl ether is used to release the white precipitate at a yield rate of 80 % after freeze - drying . the product d n is d 2 with the structure ( ii ) as follows : 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 03 ( s , 12 ), 1 . 26 ( s , 6 ), 3 . 63 ( bs ), 3 . 78 ( t , 4 ), 4 . 03 ( t , 4 ), 4 . 38 ( s , 8 ), 4 . 56 ( d , 8 ), 5 . 41 ( s , 4 ), 7 . 19 ( m , 12 ), 7 . 38 ( m , 8 ). the product ( 5 . 5 g ) from the above process is dissolved in 45 ml of 1 : 2 dcm / meoh solution . the de - protection procedure in step b . of example 1 is repeated , and the final yield rate is about 88 %. 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 03 ( s , 12 ), 1 . 19 ( s , 6 ), 3 . 43 ( m , 8 ), 3 . 64 ( bs ), 4 . 08 ( m , 8 ), 4 . 40 ( d , 4 ). the method of synthesizing the second generation p - d 2 - dtpa is approximately the same as that of first generation . p - d 2 - oh ( 0 . 265 mmol , 1 . 3965 g ) and 1 . 2482 g ( 2 . 54 mmol ) dtpa - hsie is mixed in a 50 ml round bottom conical vial and vacuum dried for 4 hours . anhydrous dmso ( 10 ml ) and 350 μl triethylamine is injected into the mixture , and stirred for 48 hours at room temperature under saturated nitrogen gas . acetonitrile / acetone solution is used to release the white precipitate , and p - d 2 - dtpa is produced after centrifugation and freeze - drying . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 04 ( m ), 1 . 18 ( m ), 3 . 07 ( t , 16 ), 3 . 21 ( t , 16 ), 3 . 58 ( bs ), 3 . 68 ( m ), 3 . 79 ( d ), 4 . 21 ( bs ). the preparation process of the third generation p - d 3 -( o 2 bn ) is similar to that of the first and second generations . the product from step b . in example 2 ( 2 . 88 g , 0 . 40 mmol , 1 equiv ), benzylidene - 2 , 2 - bis ( oxymethyl ) propionic anhydride ( 5 . 48 g , 12 . 8 mmol , 32 equiv ), and dmap ( 0 . 3151 g , 2 . 57 mmol , 6 . 4 equiv ) are dissolved in 35 ml dcm at room temperature and reacted for 24 hours . the extracting procedure in step a . of example 2 is repeated , and the final product yield rate is about 89 %. the d n product is d 3 with the structure of ( iii ) as follows . 1 h nmr ( 400 mhz , cdcl 3 ): δ 0 . 89 ( s , 24 ), 1 . 16 ( s , 6 ), 1 . 17 ( s , 12 ), 3 . 57 ( t , 6 ), 3 . 67 ( bs ), 3 . 77 ( t , 3 ), 4 . 15 ( q , 6 ), 4 . 28 ( t , 3 ), 4 . 33 ( m , 16 ), 4 . 55 ( d , 16 ), 5 . 37 ( s , 8 ), 7 . 30 ( m , 24 ), 7 . 35 ( m , 16 ). the product ( 4 g ) from step a . is dissolved in the mixture of dcm and meoh ( 1 : 1 ). pd / c catalyst ( 0 . 4 g ) is added , and stirred for 24 hours under a hydrogen - saturated environment . white powder product ( 1 . 8 g ) is produced after filtering and freeze - drying . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 07 ( s , 24 ), 1 . 27 ( s , 6 ), 1 . 34 ( s , 12 ), 3 . 47 ( t ), 3 . 64 ( bs ), 3 . 76 ( m ), 4 . 26 ( m ), 4 . 32 ( dd , 10 ). the method of synthesizing the third generation p - d 3 - dtpa is the same as aforementioned . p - d 3 - oh ( 1 . 097 g , 0 . 1938 mmol ) and 1 . 814 g ( 3 . 6 mmol ) dtpa - hsie are mixed in a 50 ml round bottom conical vial and vacuum dried for 4 hours . anhydrous dmso ( 10 ml ) and 515 μl triethylamine are injected into the mixture , and stirred for 64 hours at room temperature under saturated nitrogen gas . after white precipitate is released by means of acetonitrile / acetone solution , the white solid product , p - d 3 - dtpa , is produced after centrifugation and freeze - drying . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 03 ( s ), 1 . 25 ( s ), 1 . 29 ( s ), 2 . 7 ( m ), 3 . 16 ( t ), 3 . 46 ( t ), 3 . 79 ( bs ), 3 . 80 ( m ), 3 . 97 ( bs ), 4 . 21 ( m ). in the p - d n - dtpa dendritic compounds of the first , second and third generation , the resonant frequency in nmr spectrum of the methyl on hydrogen is decreased as the generation extends . therefore , it is proved that the dendritic compound of p - d n - dtpa in the present example is the dendritic compound of the first , second and third generations . in addition , infrared spectrum can be applied to verify the structure of dtpa ; in other words , the original c — o bond of dtpa is disappeared at the peak of 1200 cm − 1 , and instead , the carbonyl peak is appeared between signals of 1638 and 1598 cm − 1 . as a result , it is proved that the dendritic compound in the present example possesses the dtpa to chelate gd . p - d 3 - dtpa ( 0 . 066 g , 0 . 005 mmol ) is dissolved in 10 ml water , and a 16 - time amount of gdcl 3 . 6h 2 o ( 0 . 031 g , 0 . 08 mmol ) is added . the ph value is adjusted in neutral ( ph7 ) by 0 . 1 n sodium hydroxide solution . the result of the reaction is verified by ftir and white solid product is produced after freeze - drying . by evaluating the effectiveness of the reacted dendritic compound p - d 3 - dtpa - gd 3 + of the third generation as an agent of enhancing image , it can be compared with the magnetizing relaxation of dtpa - gd ( magnevist ™) by directly comparing the magnetizing relaxation rate via nmr . the results of comparison are showed in table 1 in which r 1 and r 2 respectively represent vertical and horizontal relative relaxation times , and b 0 as the internal magnetic intensity of nmr . the greater volumes of r 1 and r 2 , the stronger image signal will be . therefore , table 1 is clearly indicated the chelating gd element of third generation dendritic compound provides a superior imaging result compared to dtpa - gd ( magnevist ™), and demonstrated distinct progress in nmr imaging . furthermore , conventional imaging agents require high concentration of gd to achieve the ideal image , and it is also a challenge to accumulate the imaging agent on a certain location . the dendritic compound in this invention provides a vivid and clear image with no need to accumulate the image agent on one position and this situation promotes a more suitable clinical application . in addition , each dendritic compound in the present invention contains protected oh functional groups , which extend to a higher generation and possesses the magnifying ability by times . therefore , compared to known imaging agents , a better imaging contrast is provided with the same amount . it is also known that chelator with small molecule weight penetrates vascular endothelial cells easily such that it disperses while circulating ; the dendritic compound is a high molecule carrier which decreases the possibility of being drained away during blood circulation . the “ core ” of the dendritic compound of the present invention is polyethylene glycol and its derivatives . polyethylene glycol , a bio - compatible polymer certified by the fda in the usa , is usually applied to biomedical polymers and can be eliminated spontaneously via circulation . therefore , the dendritic compound of the present invention can be an imaging agent with low toxicity .
a dendritic compound of the following structure : pd n - z - l is disclosed . in the structure above , p is x — r —, r is an integer ranging from 1000 to 4000 , x is oh , nh 2 , or or , r is c 1 to c 10 alkyl , d n is a residue of branched c 3 to c 30 polyol compounds , n is the quantity of layers of the residue of branched compounds and is an integer equal to or greater than 1 , l is a metal cation , z is the residue of a c 3 to c 30 compound with multi functional groups . the functional groups illustrated above can be carboxylic groups , amino groups , amide groups , or chelating groups . the carboxylic groups , ester groups , amino groups , or amide groups bind to d n , and the chelating groups bind to the metal cations .