Novel compound with effects of thrombolysis, free radical scavenging and thrombus-targeting as well as preparation method and use thereof

Abstract

The present invention discloses a novel compound with effects of thrombolysis, free radical scavenging and thrombus-targeting, as well as a preparation method and use thereof. The compound is a ternary conjugate formed by conjugating a thrombolytic peptide, a free radical scavenger and a thrombus-targeting/antithrombotic peptide together via a linking arm. The present invention also discloses a pharmaceutical composition containing the compounds, wherein the compounds form a nanospherical structure.

1 . A ternary conjugate consisting of
 an imidazoline having NO free radical scavenging activity,   a peptide having thrombolytic activity, and   a thrombus-targeting peptide,   which are linked together via a suitable linking arm.   
     
     
         2 . The ternary conjugate according to  claim 1 , represented by formula I: 
       
         
           
           
               
               
           
         
       
       wherein NN represents an imidazoline having NO free radical scavenging activity; AA 1  represents a linking arm having at least three groups for linking; AA 2  represents a peptide having thrombolytic activity; and AA 3  represents a thrombus-targeting peptide. 
     
     
         3 . The ternary conjugate according to  claim 1 , wherein the imidazoline having NO free radical scavenging activity is 1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline. 
     
     
         4 . The ternary conjugate according to  claim 1 , wherein the groups for linking are selected from the group consisting of a carboxyl group and an amino group. 
     
     
         5 . The ternary conjugate according to  claim 4 , wherein the linking arm is a natural amino acid, particularly L-Lys, L-Asp or L-Glu. 
     
     
         6 . The ternary conjugate according to  claim 1 , wherein the peptide having thrombolytic activity is an oligopeptide comprising a PAK (Pro-Ala-Lys) sequence, an AKP (Ala-Lys-Pro) sequence or a KAP (Lys-Ala-Pro) sequence, or a peptide having repeating units of the PAK sequence, the AKP sequence or the KAP sequence. 
     
     
         7 . The ternary conjugate according to  claim 1 , wherein the thrombus-targeting peptide is an oligopeptide comprising an RGD (Arg-Gly-Asp) sequence. 
     
     
         8 . The ternary conjugate according to  claim 1 , wherein the thrombus-targeting peptide is a polypeptide obtained from conjugating modification of an RGD (Arg-Gly-Asp) peptide with a YIGS (Tyr-Ile-Gly-Ser) peptide. 
     
     
         9 . The ternary conjugate according to  claim 1 , wherein the imidazoline having NO free radical scavenging activity is 1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline, the linking arm is L-Lys, L-Asp or L-Glu, the peptide having thrombolytic activity is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the thrombus-targeting peptide is an oligopeptide comprising an RGD sequence (Arg-Gly-Asp). 
     
     
         10 . A pharmaceutical composition comprising the ternary conjugate according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         11 . The pharmaceutical composition according to  claim 10 , wherein the ternary conjugate is in the form of a nanospherical structure. 
     
     
         12 . The pharmaceutical composition according to  claim 10  for use as a thrombolytic drug, an NO free radical scavenging drug, or an anti-thrombus drug. 
     
     
         13 . The pharmaceutical composition according to  claim 10  for use as a drug in treating stroke or cerebral infarction. 
     
     
         14 . The pharmaceutical composition according to  claim 13  for use in treating stroke or cerebral infarction beyond 3 hours from the onset of symptoms. 
     
     
         15 . A method for preparation of the ternary conjugate according to  claim 1 , wherein the ternary conjugate is represented by formula I, 
       
         
           
           
               
               
           
         
         wherein NN represents an imidazoline having NO free radical scavenging activity; AA 1  represents a linking arm having at least three groups for linking; AA 2  represents a peptide having thrombolytic activity; and AA 3  represents a thrombus-targeting peptide; 
       
       said method comprising the steps of:
 (1) providing the imidazoline having NO free radical scavenging activity (NN), the linking arm having at least three groups for linking (AA 1 ), the peptide having thrombolytic activity (AA 2 ) and the thrombus-targeting peptide (AA 3 ), wherein the linking arm has a first group for linking, a second group for linking, and a third group for linking; 
 (2) linking the imidazoline having NO free radical scavenging activity (NN) to the first group for linking on the linking arm (AA 1 ), to form a compound of general formula IM-1:
   NN-AA 1   (IM-1);
 
 
 (3) linking the peptide having thrombolytic activity (AA 2 ) to the compound of general formula IM-1, wherein one end of the peptide having thrombolytic activity is linked to the second group for linking on the linking arm, to form a compound of general formula IM-2:
   NN-AA 1 -AA 2   (IM-2); and
 
 
 (4) linking the thrombus-targeting peptide (AA 3 ) to the compound of general formula IM-2, wherein one end of the thrombus-targeting peptide is linked to the third group for linking on the linking arm, to form the compound of formula I; 
 wherein the order of steps (3) and (4) may be reversed. 
 
     
     
         16 . The method according to  claim 15 , wherein step (1) further comprises protecting the second and the third groups for linking on the linking arm (AA 1 ) with protecting groups, and protecting active groups of the peptide having thrombolytic activity (AA 2 ) and of the thrombus-targeting peptide (AA 3 ), other than the end to be used for linking, with protecting groups; step (3) further comprises deprotecting the protected second group for linking first, and then linking the peptide having thrombolytic activity to the deprotected second group for linking; step (4) further comprises deprotecting the protected third group for linking first, and then linking the thrombus-targeting peptide to the deprotected third group for linking; and after step (4), there is further a step of deprotecting the protected active groups of the peptide having thrombolytic activity (AA 2 ) and of the thrombus-targeting peptide (AA 3 ). 
     
     
         17 . The method according to  claim 15 , wherein the first group for linking is an amino group, and the second and the third groups for linking are selected from the group consisting of a carboxyl group and an amino group. 
     
     
         18 . The method according to  claim 17 , wherein the linking arm is a natural amino acid, particularly L-Lys, L-Asp or L-Glu. 
     
     
         19 . The method according to  claim 17 , wherein the peptide having thrombolytic activity is an oligopeptide comprising a PAK (Pro-Ala-Lys) sequence, an AKP (Ala-Lys-Pro) sequence or a KAP (Lys-Ala-Pro) sequence, or a peptide having repeating units of the PAK sequence, the AKP sequence or the KAP sequence. 
     
     
         20 . The method according to  claim 17 , wherein the thrombus-targeting peptide is an oligopeptide comprising an RGD (Arg-Gly-Asp) sequence. 
     
     
         21 . The method according to  claim 15  or  16 , wherein the imidazoline having NO free radical scavenging activity is 1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline, the linking arm is L-Lys, L-Asp or L-Glu, the peptide having thrombolytic activity is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the thrombus-targeting peptide is an oligopeptide comprising an RGD sequence (Arg-Gly-Asp).