Pyridinium Cationic Lipids as Gene Transfer Agents

Abstract

Pyridinium cationic lipids useful as non-viral gene delivery agents are disclosed. The agents are prepared by reaction of pyrylium salts with primary amines. Also disclosed are methods of trasfectind cells using the pyridinium cationic lipids as gene transfer agents.

1 . A compound useful as a gene transfer agent, the compound having Formula I: 
     
       
         
         
             
             
         
       
       wherein R 1  is selected from the group consisting of C 1-25  alkyl; C 2-25  alkenyl; C 2-25  alkynyl; C 1-25  alkoxy; C 1-25  alkylaminomethyl; C 1-25  dialkylaminomethyl; phenyl; phenyl substituted with one or more substitutents selected from the group consisting of C 1-5  alkyl, C 2-5  alkenyl, C 2-5  alkynyl, C 1-5  alkoxy, and C 1-5  amino; styryl; and styryl substituted with one or more substitutents selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, C 1-25  alkoxy, and C 1-25  dialkylamino; 
       R 2  and R 3  are C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl; 
       wherein X is selected from the group consisting of halogen, perchlorate, tetrafluoroborate, trifluoromethanesulfonate, methanesulfonate, p-toluenesulfonate, methosulfate, sulfoacetate, acetate, trifluoroacetate, hemisuccinate and hexafluorophosphate, and 
       wherein Z is selected from the group consisting of: 
     
     
       
         
         
             
             
         
       
     
     wherein R 4  and R 5  are independently selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl; C 1-25  acyl, perfluoro C 1-25  alkyl; perfluoro C 1-25  acyl; poly(ethyleneoxy)alkyl, poly(ethyleneoxy)acyl, and cholesteryloxycarbonyl; 
     
       
         
         
             
             
         
       
     
     wherein m is 0-25, X is selected from the group consisting of halogen, perchlorate, tetrafluoroborate, trifluoromethanesulfonate, methanesulfonate, p-toluenesulfonate, methosulfate, sulfoacetate, acetate, trifluoroacetate, hemisuccinate and hexafluorophosphate, R 6  is C 1-25  alkyl, and R 7  and R 8  are independently C 1-25  alkyl; 
     and
   —(CH 2 )mMR 4    
 
     wherein m is 0-25 and M is O, S, or NH and R 4  is selected from the group consisting of C 1-25  alkyl, C 1-25  alkenyl, C 1-25  alkynyl, C 1-25  acyl, perfluoro C 1-25  alkyl; perfluoro C 1-25  acyl; poly(ethyleneoxy)alkyl, poly(ethyleneoxy)acyl, and cholesteryloxycarbonyl. 
   
   
       2 - 4 . (canceled) 
   
   
       5 . The compound of  claim 1 , wherein Z is 
     
       
         
         
             
             
         
       
       wherein m is 2-8, R 6  and R 7  are methyl and R 8  is C 14  alkyl. 
     
   
   
       6 . The compound of  claim 5 , wherein m is 2 and R 8  is C 14  alkyl. 
   
   
       7 - 8 . (canceled) 
   
   
       9 . The compound of  claim 1 , wherein Z is
   —(CH 2 )mMR 4      wherein m is 2, M is O or NH, and R4 is C 1-14  acyl.   
   
   
       10 . The compound of  claim 1 , wherein Formula I is selected from the group of compounds consisting of 1-(2,3-dihydroxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(2,3-dilauroyloxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(2,3-dimyristoyloxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(2,3-dipamitoyloxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(2,3-distearoyloxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(2,3-dioleoyloxypropyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-dihydroxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-dilauroyloxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-dimyristoyloxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-dimyristoyloxypropane-2-yl)-2,4,6-trimethylpyridinium perchlorate, 1-(1,3-dimyristoyloxypropane-2-yl)-2,4,6-trimethylpyridinium tetrafluoroborate, 1-(1,3-dimyristoyloxypropane-2-yl)-2,4,6-trimethylpyridinium chloride, 1-(1,3-dimyristoyloxypropane-2-yl)-2,4,6-trimethylpyridinium iodide, 1-(1,3-dipamitoyloxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-distearoyloxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(1,3-dioleoyloxypropane-2-yl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-dihydroxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-dioctanoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-didecanoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-dilauroyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-dimyristoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-dipalmitoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, 1-(3,4-distearoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate, and 1-(3,4-dioleoyloxyphenylethyl)-2,4,6-trimethylpyridinium hexafluorophosphate. 
   
   
       11 . A method of preparing a compound according to  claim 1 , the method comprising: reacting a compound having the formula: 
     
       
         
         
             
             
         
       
       wherein R 1  is selected from the group consisting of C 1-25  alkyl; halogeno C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl; C 1-25  alkoxy; C 1-25  alkylaminomethyl; C 1-25  dialkylaminomethyl; phenyl; phenyl substituted with one or more substitutents selected from the group consisting of C 1-5  alkyl, C 2-5  alkenyl, C 2-5  alkynyl, C 1-5  alkoxy, and C 1-5  amino; styryl; and styryl substituted with one or more substitutents selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, C 1-25  alkoxy, and C 1-25  dialkylamino; 
       R 2  and R 3  are C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl; 
       wherein X is selected from the group consisting of halogen, perchlorate, tetrafluoroborate, trifluoromethanesulfonate, sulfoacetate, and hexafluorophosphate; with a primary amine. 
     
   
   
       12 . The method of  claim 11 , wherein the primary amine contains one or more hydroxyl groups and wherein the method further comprises alkylating or acylating the one or more hydroxyl groups. 
   
   
       13 . The method of  claim 11 , wherein the primary amine is selected from the group consisting of: 
     
       
         
         
             
             
         
       
       wherein m is 1-25 
     
     
       
         
         
             
             
         
       
     
     wherein n is 0-25, and 
     
       
         
         
             
             
         
       
     
     wherein n, m, and o are independently 0-25, 
     and wherein the method further comprises acylating the one or more hydroxyl groups with an acylhalide having the formula R 4 C(O)X, wherein R 4  is selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, C 1-25  acyl, perfluoro C 1-25  alkyl; perfluoro C 1-25  acyl; poly(ethyleneoxy)alkyl, poly(ethyleneoxy)acyl, and cholesteryloxycarbonyl. 
   
   
       14 . The method of  claim 11 , wherein the primary amine comprises one or more hydroxyl groups and the method further comprises forming the tosylate or trifluoromethanesulfonate ester of the one or more hydroxyl groups and reacting the tosylate or trifluoromethanesulfonate ester with an alcohol having the formula R 4 OH, wherein R 4  is selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, perfluoro C 1-25  alkyl, poly(ethyleneoxy)alkyl, in the presence of a strong base such as potassium t-butoxide or sodium hydride. 
   
   
       15 . The method of  claim 11 , wherein the primary amine contains two or more primary amine groups, and wherein the method further comprises acylating the one or more amino groups with an acylhalide having the formula R 4 C(O)X, wherein R 4  is selected from the group consisting of C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, C 1-25  acyl, perfluoro C 1-25  alkyl; perfluoro C 1-25  acyl; poly(ethyleneoxy)alkyl, poly ethyleneoxy)acyl, and cholesteryloxycarbonyl. 
   
   
       16 . The method of  claim 11 , wherein two equivalents of a compound having Formula II are reacted with a primary amine having two amine groups. 
   
   
       17 . The method of  claim 11 , wherein two equivalents of a compound having Formula II are reacted with a primary amine having the formula NH 2 (CH 2 )mNH 2 , wherein m is 0-25, to yield a compound having the formula 
     
       
         
         
             
             
         
       
     
   
   
       18 . The method of  claim 11 , wherein two equivalents of a compound having Formula II are reacted with a primary amine having the formula NH 2 [(CH 2 ) m NH] k (CH 2 ) o NH 2 , wherein m, k, and o are independently 0-25, to yield a compound having the formula 
     
       
         
         
             
             
         
       
     
   
   
       19 . The method of  claim 11 , wherein two equivalents of a compound having Formula II are reacted with a compound having the formula NH 2 [(CH 2 ) m N(R 9 )] k (CH 2 ) o NH 2 , wherein m, k, and o are independently 0-25 and R9 is selected from the group consisting of C 1-25  alkyl, hydroxy C 1-25  alkyl, amino C 1-25  alkyl, C 2-25  alkenyl; C 2-25  alkynyl, C 1-25  acyl, hydroxy C 1-25  acyl, amino C 1-25  acyl, C 1-25  alkyloxycarbonyl, t-butyloxycarbonyl, adamantyloxycarbonyl, perfluoro C 1-25  alkyl; perfluoro C 1-25  acyl; poly(ethyleneoxy)alkyl, poly(ethyleneoxy)acyl, and cholesteryloxycarbonyl, to yield a compound having the formula 
     
       
         
         
             
             
         
       
     
   
   
       20 . A method of transfecting eukaryotic cells comprising mixing at least one plasmid or polynucleotide with the compound of  claim 1 . 
   
   
       21 - 29 . (canceled) 
   
   
       30 . The method of  claim 20 , wherein said at least one plasmid or polynucleotide is selected from the group consisting of DNA, RNA, oligonucleotides, and truncated oligonucleotides. 
   
   
       31 . The method of  claim 20 , further comprising mixing a helper lipid with the mixture of said at least one plasmid or polynucleotide and said compound, wherein said helper lipid is cholesterol or dioleolyphosphatidylethanolamine. 
   
   
       32 . The method of  claim 31 , wherein the ratio of said compound to said helper lipid is about 1.0:0.1 to about 1.0:1.5.