Thiol-modified hyaluronan

Abstract

The present invention relates to biscarbodiimides, thiourea derivatives, urea derivatives, and cross-linked hyaluronan derivatives having at least one intramolecular disulfide bond, and methods of preparation thereof. The invention also includes thiolated hyaluronan derivatives and salts thereof having at least one pendant thiol group or a modified pendant thiol group, and methods of preparation thereof. An example of a modified pendant thiol group is a sulfhydryl group linked to a small molecule such as a bioactive agent, for example a drug or pharmaceutically active moiety. A hyaluronan derivative having a sulfhydryl group linked to a pharmaceutically active moiety is useful as a sustained or controlled release drug delivery vehicle. Compositions containing the hyaluronan derivatives of the invention can reversibly viscosify in vivo or in vitro, in response to mild changes in condition, and are thus useful in ophthalmic surgery and in tissue engineering.

1 . (canceled)  
   
   
       2 . A biscarbodiimide having an intramolecular disulfide bond, the biscarbodiimide formed by a method comprising: 
 (a) reacting an isothiocyanate with a disulfide selected from cystamine, 2-aminophenyl disulfide and 4-aminophenyl disulfide, thereby forming a thiourea derivative, or reacting an isocyanate with cystamine, thereby forming a urea derivative; and    (b) reacting the thiourea derivative with an oxidizing agent or a dehydrosulfuration agent, or reacting the urea derivative with a dehydrating agent, thereby forming a biscarbodiimide having an intramolecular disulfide bond.    
   
   
       3 . (canceled)  
   
   
       4 . The biscarbodiimide of  claim 2 , wherein the isothiocyanate is selected from the group consisting of ethyl isothiocyanate, propyl isothiocyanate, butyl isothiocyanate, tert-butyl isothiocyanate, and phenyl isothiocyanate.  
   
   
       5 . (canceled)  
   
   
       6 . The biscarbodiimide of  claim 2 , wherein the oxidizing agent is selected from the group consisting of a hypochlorite, N-bromosuccinimide, 1-chlorobenzothiazole, and N-chloroamidines.  
   
   
       7 . (canceled)  
   
   
       8 . The biscarbodiimide of  claim 2 , wherein the dehydrosulfuration agent is at least one member selected from the group consisting of mercury II oxide, phosgene, diethyl azodicarboxylate-triphenylphosphine, lead oxide, silver oxide, activated aluminum oxide, quinones, thionyl chloride, sulfenyl chloride, chlorosulfonic acid, SCl 2 , S 2 Cl 2 , and phosphorus halides.  
   
   
       9 . (canceled)  
   
   
       10 . The biscarbodiimide of  claim 2 , the biscarbodiimide formed by a method comprising: 
 (a) reacting the isocyanate with cystamine, thereby forming the urea derivative; and    (b) reacting the urea derivative with the dehydrating agent, thereby forming the biscarbodiimide having an intramolecular disulfide bond.    
   
   
       11 . The biscarbodiimide of  claim 10 , wherein the dehydrating agent is at least one member selected from the group consisting of p-toluenesulfonyl chloride in pyridine, POCl 3 , PCl 5 , P 2 O 5  in pyridine, p-toluenesulfonyl chloride in a phase-transfer catalysis system, and Ph 3 PBr 2 -Et 3 N.  
   
   
       12 . (canceled)  
   
   
       13 . The biscarbodiimide of  claim 3 , represented by Structural Formula (1):  
     
       
         
         
             
             
         
       
     
   
   
       14 . The biscarbodiimide of  claim 2 , the biscarbodiimide formed by a method comprising: 
 (a) reacting the isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming the thiourea derivative; and    (b) reacting the thiourea derivative with the oxidizing agent or the dehydrosulfuration agent, thereby forming a 1,1′dithiophenylene bis(ethylcarbodiimide).    
   
   
       15 . The biscarbodiimide of  claim 14 , wherein the isothiocyanate is at least one member selected from the group consisting of ethyl isothiocyanate, propyl isothiocyanate, butyl isothiocyanate, tert-butyl isothiocyanate, and phenyl isothiocyanate.  
   
   
       16 . (canceled)  
   
   
       17 . The biscarbodiimide of  claim 14 , wherein the oxidizing agent is at least one member selected from the group consisting of a hypochlorite, N-bromosuccinimide, 1-chlorobenzothiazole, and N-chloroamidines.  
   
   
       18 . (canceled)  
   
   
       19 . The biscarbodiimide of  claim 14 , wherein the dehydrosulfuration agent is at least one member selected from the group consisting of mercury II oxide, phosgene, diethyl azodicarboxylate-triphenylphosphine, lead oxide, silver oxide, activated aluminum oxide, quinones, thionyl chloride, sulfenyl chloride, chlorosulfonic acid, SCl 2 , S 2 Cl 2 , and phosphorus halides.  
   
   
       20 . (canceled)  
   
   
       21 . (canceled)  
   
   
       22 . The biscarbodiimide of  claim 14 , represented by Structural Formula (2) or (3):  
     
       
         
         
             
             
         
       
     
   
   
       23 . A thiourea derivative having an intramolecular disulfide bond, the thiourea derivative formed by a method comprising the step of reacting an isothiocyanate with a disulfide selected from cystamine, 2-aminophenyl disulfide and 4-aminophenyl disulfide, thereby forming the thiourea derivative having an intramolecular disulfide bond.  
   
   
       24 . The thiourea derivative of  claim 23 , wherein the isothiocyanate is at least one member selected from the group consisting of ethyl isothiocyanate, propyl isothiocyanate, butyl isothiocyanate, sec-butyl isothiocyanate, tert-butyl isothiocyanate, and phenyl isothiocyanate.  
   
   
       25 . (canceled)  
   
   
       26 . The thiourea derivative of  claim 23 , represented by Structural Formula (4):  
     
       
         
         
             
             
         
       
     
   
   
       27 . The thiourea derivative of  claim 23 , formed by a method comprising the step of reacting an isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming the thiourea derivative having an intramolecular disulfide bond.  
   
   
       28 . The thiourea derivative of  claim 27 , wherein the isothiocyanate is at least one member selected from the group consisting of ethyl isothiocyanate, propyl isothiocyanate, butyl isothiocyanate, sec-butyl isothiocyanate, tert-butyl isothiocyanate, and phenyl isothiocyanate.  
   
   
       29 . (canceled)  
   
   
       30 . The thiourea derivative of  claim 23 , represented by Structural Formula (5) or (6):  
     
       
         
         
             
             
         
       
     
   
   
       31 . A urea derivative having an intramolecular disulfide bond, the urea derivative formed by a method comprising the step of reacting an isocyanate with cystamine, thereby forming the urea derivative having an intramolecular disulfide bond.  
   
   
       32 . The urea derivative of  claim 31 , wherein the isocyanate is at least one member selected from the group consisting of ethyl isocyanate, propyl isocyanate, butyl isocyanate, sec-butyl isocyanate, tert-butyl isocyanate, and phenyl isothiocyanate.  
   
   
       33 . (canceled)  
   
   
       34 . The urea derivative of  claim 31 , represented by Structural Formula (7):  
     
       
         
         
             
             
         
       
     
   
   
       35 . (canceled)  
   
   
       36 . (canceled)  
   
   
       37 . (canceled)  
   
   
       38 . (canceled)  
   
   
       39 . (canceled)  
   
   
       40 . (canceled)  
   
   
       41 . (canceled)  
   
   
       42 . (canceled)  
   
   
       43 . (canceled)  
   
   
       44 . (canceled)  
   
   
       45 . (canceled)  
   
   
       46 . (canceled)  
   
   
       47 . (canceled)  
   
   
       48 . (canceled)  
   
   
       49 . (canceled)  
   
   
       50 . (canceled)  
   
   
       51 . A method of preparing a biscarbodiimide compound represented by Structural Formula (1),  
     
       
         
         
             
             
         
       
       comprising:  
       (a) reacting ethyl isothiocyanate with cystamine, thereby forming a thiourea intermediate, 2,2′dithiobis(N-ethyl(N′-ethylthiourea)), having Structural Formula (4),  
       
         
           
           
               
               
           
         
       
       or, reacting ethyl isocyanate with cystamine, thereby forming a urea intermediate, 2,2′dithiobis(N-ethyl(N′-ethylurea)), having Structural Formula (7),  
       
         
           
           
               
               
           
         
       
       (b) reacting the thiourea intermediate with an oxidizing agent or a dehydrosulfuration agent, or reacting the urea derivative with a dehydrating agent, thereby forming a biscarbodiimide compound represented by Structural Formula (1).  
     
   
   
       52 . The method of  claim 51 , wherein the oxidizing agent is at least one member selected from the group consisting of a hypochlorite, N-bromosuccinimide, 1-chlorobenzothiazole, and N-chloroamidines.  
   
   
       53 . (canceled)  
   
   
       54 . The method of  claim 51 , wherein the dehydrosulfuration agent is at least one member selected from the group consisting of mercury II oxide, phosgene, diethyl azodicarboxylate-triphenylphosphine, lead oxide, silver oxide, activated aluminum oxide, quinones, thionyl chloride, sulfenyl chloride, chlorosulfonic acid, SCl 2 , S 2 Cl 2 , and phosphorus halides.  
   
   
       55 . (canceled)  
   
   
       56 . (canceled)  
   
   
       57 . The method of  claim 51 , comprising: 
 (a) reacting ethyl isocyanate with cystamine, thereby forming the urea intermediate, 2,2′dithiobis(N-ethyl(N′-ethylurea)), having Structural Formula (7),                          (b) reacting the urea intermediate with the dehydrating agent, thereby forming a biscarbodiimide compound represented by Structural Formula (1).    
   
   
       58 . The method of  claim 57 , wherein the dehydrating agent is at least one member selected from the group consisting of p-toluenesulfonyl chloride in pyridine, POCl 3 , PCl 5 , P 2 O 5  in pyridine, p-toluenesulfonyl chloride in a phase-transfer catalysis system, and Ph 3 PBr 2 -Et 3 N.  
   
   
       59 . (canceled)  
   
   
       60 . A method of preparing a biscarbodiimide compound represented by Structural Formula (2) or (3),  
     
       
         
         
             
             
         
       
       comprising:  
       (a) reacting ethyl isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming a thiourea intermediate having Structural Formula (5) or (6),  
       
         
           
           
               
               
           
         
       
       (b) reacting the thiourea intermediate with an oxidizing agent or a dehydrosulfuration agent, thereby forming 1,1′dithio-o-phenylene bis(ethylcarbodiimide), having Structural Formula (2),  
       
         
           
           
               
               
           
         
       
       or 1,1′dithio-p-phenylene bis(ethylcarbodiimide), having Structural Formula (3),  
       
         
           
           
               
               
           
         
       
     
   
   
       61 . The method of  claim 60 , wherein the oxidizing agent is at least one member selected from the group consisting of hypochlorite, N-bromosuccinimide, 1-chlorobenzothiazole, and N-chloroamidines.  
   
   
       62 . (canceled)  
   
   
       63 . The method of  claim 60 , wherein the dehydrosulfuration agent is at least one member selected from the group consisting of mercury II oxide, phosgene, diethyl azodicarboxylate-triphenylphosphine, lead oxide, silver oxide, activated aluminum oxide, quinones, thionyl chloride, sulfenyl chloride, chlorosulfonic acid, SCl 2 , S 2 Cl 2 , and phosphorus halides.  
   
   
       64 . (canceled)  
   
   
       65 . (canceled)  
   
   
       66 . (canceled)  
   
   
       67 . A method of cross-linking pendant thiol groups on a thiolated hyaluronic acid derivative to form a hydrogel, the method comprising the step of: 
 reacting a thiolated hyaluronan derivative of structural formula (9),                          with a homobifunctional cross-linker.    
   
   
       68 . The method of  claim 67 , wherein the homobifunctional cross-linker is at least one member selected from the group consisting of 5-thio-2-nitrobenzoic acid, bis-thiosulfonates, bis-alkylhalides, and bis-maleimide derivatives, dithiobis(succinimidylproprionate), 3,3′-dithiobis(sulfosuccinimidylproprionate), 1,4-di-(3′-(2′-pyridyldithio)propionamido)butane, and bismaleimidohexane.  
   
   
       69 . (canceled)  
   
   
       70 . (canceled)  
   
   
       71 . (canceled)  
   
   
       72 . (canceled)