Amine-Containing Transfection Reagents and methods for making and using same

Abstract

There are provided for herein novel amine-containing transfection compounds and methods for making and using same. The compounds are generally obtained by reacting a primary amine with an unsaturated compound. Transfection complexes made using the amine-containing transfection compounds in combination with additional compounds to encapsulate biologically active agents such as nucleic acids are also provided for herein. Methods of using the transfection complexes for the in vivo or in vitro delivery of biologically active agents are also described. The transfection complexes of the present invention are highly potent, thereby allowing effective modulation of a biological activity at relatively low doses compared to analogous transfection compounds known in the art.

1 . A compound having the general structure I, or pharmaceutically acceptable salts thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         each of X 1  and X 2  is a moiety independently selected from the group consisting of O S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20  hydrocarbon chain; 
         each of Y and Z is a moiety independently selected from the group consisting of C═O, C═S, S═O, CH—OH and SO 2 ; each of R 1 , R 2  and R 3 , R 4 , R 5 , R 6  and R 7  is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched aryl group, a substituted or unsubstituted, branched or unbranched heteroaryl group; 
         x is an integer independently having the value between 1 and 10, inclusively; 
         n is an integer independently having the value between 1 and 3, inclusively, m is an integer independently having the value between 0 and 20, inclusively, p is an integer independently having the value of 0 or 1, wherein if m=p=0, then R 2  is hydrogen, 
         with the further proviso that if at least one of n or m has the value of 2, then R 3  and nitrogen in structure I form a moiety selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
         wherein each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I. 
       
     
     
         2 . The compound of  claim 1 , or pharmaceutically acceptable salts thereof, wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         3 . The compound of  claim 2 , or pharmaceutically acceptable salts thereof, wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         4 . The compound of  claim 1 , or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R 3  is selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3  to the nitrogen atom in structure I, wherein each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure I. 
       
     
     
         5 . The compound of  claim 1 , or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6. 
     
     
         6 . The compound of  claim 1  having the general structure II or pharmaceutically acceptable salts thereof: 
       
         
           
           
               
               
           
         
       
     
     
         7 . The compound of  claim 6 , or pharmaceutically acceptable salts thereof, wherein at east one X 1  is NH. 
     
     
         8 . The compound of  claim 6 , or pharmaceutically acceptable salts thereof, wherein at least one X 1  is O. 
     
     
         9 . The compound of  claim 6 , or pharmaceutically acceptable salts thereof, wherein each R 1  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         10 . The compound of  claim 9 , or pharmaceutically acceptable salts thereof, wherein each of R 1  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         11 . The compound of  claim 6 , or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R 3  is selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3  to the nitrogen atom in structure II, wherein each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure II. 
       
     
     
         12 . The compound of  claim 6 , or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6. 
     
     
         13 . The compound of  claim 1 , or pharmaceutically acceptable salts thereof, the compound being selected from the group consisting of compounds 1-87 or isomers thereof: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         14 . A method for synthesizing a compound having the structure I, or pharmaceutically acceptable salts thereof, 
       
         
           
           
               
               
           
         
         the method comprising reacting: 
         (a) one or more equivalents of an unsaturated component comprising at least two compounds selected from the group consisting of the first intermediate having the structure R 1 —X 1 —Y—(CR 4 R 5 ) n Br and the second intermediate having the structure R 2 —X 2 —Z—(CR 6 R 7 ) m —Br, wherein in (CR 4 R 5 ) n  and (CR 6 R 7 ) m  portions of the structures, each R 4  is the same or different, each R 5  is the same or different, each R 6  is the same or different, and each R 7  is the same or different, wherein the first and the second intermediates are the same or different, 
         with 
         (b) one equivalent of an amino component comprising a primary amine NH 2 —R 3 , a diamine, a polyamine or a combination thereof, to thereby obtain the compound having the structure I, 
         wherein: 
         each of X 1  and X 2  is a moiety independently selected from the group consisting of O S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20  hydrocarbon chain; 
         each of Y and Z is a moiety independently selected from the group consisting of C═O, C═S, S═O, CH—OH and SO 2 ; each of R 1 , R 2  and R 3 , R 4 , R 5 , R 6  and R 7  is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched aryl group, a substituted or unsubstituted, branched or unbranched heteroaryl group; 
         x is an integer independently having the value between 1 and 10, inclusively; 
         n is an integer independently having the value between 1 and 3, inclusively, m is an integer independently having the value between 0 and 20, inclusively, p is an integer independently having the value of 0 or 1, wherein if m=p=0, then R 2  is hydrogen, 
         with the further proviso that if at least one of n or m has the value of 2, then R 3  and nitrogen in structure I form a moiety selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
         wherein each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I. 
       
     
     
         15 . The method of  claim 14 , wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         16 . The method of  claim 14 , wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         17 . The method of  claim 16 , wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         18 . The method of  claim 14 , wherein each of n and m independently has the value of 1 or 3, and the primary amine NH 2 —R 3  is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3  to the amino group in the primary amine NH 2 —R 3 , wherein each H on any * position can be replaced to achieve the attachment to the nitrogen atom in the primary amine NH 2 —R 3 . 
     
     
         19 . The method of  claim 14 , wherein the amino component is a primary amine NH 2 —R 3 , and the molar ratio between the unsaturated component and the primary amine is between about 1:1 and about 6:1. 
     
     
         20 . The method of  claim 14 , wherein each of R 4 , R 5 , R 6  and R 7  is hydrogen and each of Y and Z is C═O. 
     
     
         21 . The method of  claim 20 , or pharmaceutically acceptable salts thereof, wherein each R 1  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         22 . The method of  claim 21 , or pharmaceutically acceptable salts thereof, wherein each of R 1  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         23 . A transfection complex comprising an amine-containing transfection reagent having the general structure I, or pharmaceutically acceptable salts thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         each of X 1  and X 2  is a moiety independently selected from the group consisting of O S, N-A and C-A, wherein A is selected from the group consisting of hydrogen and a C 1 -C 20  hydrocarbon chain; 
         each of Y and Z is a moiety independently selected from the group consisting of C═O, C═S, S═O, CH—OH and SO 2 ; each of R 1 , R 2  and R 3 , R 4 , R 5 , R 6  and R 7  is a moiety independently selected from the group consisting of hydrogen, a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched aryl group, a substituted or unsubstituted, branched or unbranched heteroaryl group; 
         x is an integer independently having the value between 1 and 10, inclusively; 
         n is an integer independently having the value between 1 and 3, inclusively, m is an integer independently having the value between 0 and 20, inclusively, p is an integer independently having the value of 0 or 1, wherein if m=p=0, then R 2  is hydrogen, 
         with the further proviso that if at least one of n or m has the value of 2, then R 3  and nitrogen in structure I form a moiety selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
         wherein each of g, e and f is an integer independently having the value between 1 and 6, inclusively, “HCC” symbolizes a hydrocarbon chain, and each * indicates the nitrogen atom in structure I. 
       
     
     
         24 . The transfection complex of  claim 23 , or pharmaceutically acceptable salts thereof, wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         25 . The transfection complex of  claim 24 , or pharmaceutically acceptable salts thereof, wherein each of R 1  and R 2  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         26 . The transfection complex of  claim 23 , or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R 3  is selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3  to the nitrogen atom in structure I, wherein each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure I. 
       
     
     
         27 . The transfection complex of  claim 23 , or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6. 
     
     
         28 . The transfection complex of  claim 23 , wherein the amine-containing transfection reagent has the general structure II, or pharmaceutically acceptable salts thereof: 
       
         
           
           
               
               
           
         
       
     
     
         29 . The transfection complex of  claim 28 , or pharmaceutically acceptable salts thereof, wherein at east one X 1  is NH. 
     
     
         30 . The transfection complex of  claim 28 , or pharmaceutically acceptable salts thereof, wherein at least one X 1  is O. 
     
     
         31 . The transfection complex of  claim 28 , or pharmaceutically acceptable salts thereof, wherein each R 1  is independently selected from the group consisting of substituted or unsubstituted, branched or unbranched alkyl or alkenyl groups having between 3 and about 20 carbon atoms, and between 0 and 4 double bonds. 
     
     
         32 . The transfection complex of  claim 31 , or pharmaceutically acceptable salts thereof, wherein each of R 1  is independently selected from the group consisting of substituted or unsubstituted, unbranched alkyl or alkenyl groups having between 8 and about 18 carbon atoms, and between 0 and 2 double bonds. 
     
     
         33 . The transfection complex of  claim 28 , or pharmaceutically acceptable salts thereof, wherein each of n and m independently has the value of 1 or 3, and R 3  is selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein each “HCC” symbolizes a hydrocarbon chain, and each * shows a potential point of attachment of R 3  to the nitrogen atom in structure II, wherein each H on any * position can be replaced to achieve the attachment to the nitrogen atom in structure II. 
       
     
     
         34 . The transfection complex of  claim 28 , or pharmaceutically acceptable salts thereof, wherein x has the value between 1 and 6. 
     
     
         35 . The transfection complex of  claim 23 , or pharmaceutically acceptable salts thereof, the compound being selected from the group consisting of compounds 1-87 or isomers thereof: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         36 . The transfection complex of  claim 23 , further comprising at least one helper lipid. 
     
     
         37 . The transfection complex of  claim 36 , wherein the helper lipid is selected from the list consisting of BMOP (N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide), DDPES (Dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide), DSPC, CTAB:DOPE (formulation of cetyltrimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (dioleoylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine), DMPE (Dimyristoylphospatidylethanolamine), DOMG, DMA, DOPC (Dioleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), DPEPC (Dipalmitoylethylphosphatidylcholine), DODAC (dioleoydimethylammonium chloride), DOSPER (1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-Propylamid), DOTMA (N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride), DDAB (didoceyl methylammonium bromide), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate), DOTAP.Cl, DC-chol (3,13-N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOSPA (2-(sperminecarboxamido)ethyl)-N,N-dimethyl-lammonium trifluoroacetate), DC-6-14 (O,O′-Ditetradecanoyl-N-(alphatrimethylammonioacetyl) diethanolamine chloride), DCPE (Dicaproylphosphtidylethanolamine), DLRIE (dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide), DODAP (1,2-Dioleoyl-3-dimethylammonium-propane), Ethyl-PC, DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamidoethyl]-N,N-di-met-hyl-1-propanaminium trifluoroacetate), DOGS (dioctadecylamidoglycyl carboxyspermine), DMRIE (N-[1-(2,3 dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide), DOEPC (Dioleoylethyl-phosphocholine), DOHME (N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,Ndimethylammonium iodide), GAP-DLRIE:DOPE (N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine), DPPC (Dipalmitoylphosphatidylcholine), DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol)).Cl), N-lauroylsarcosine, (R)-(+)-limonene, lecithins (and derivatives thereof); phosphotidylethanolamine (and derivatives thereof); phosphatidylethanolamines, dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), DPPE dipalmitoylphosphatidylethanolamine), dipalmiteoylphosphatidylethanolamine, O-Chol (3 beta[1-ornithinamidecarbamoyl]cholesterol), POPE (palmitoyloleoylphosphatidylethanolamine) and distearoylphosphatidylethanolamine; phosphotidylcholine; phosphatidylcholines, DPPC (dipalmitoylphosphatidylcholine) POPC (palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine; phosphatidylglycerol; piperazine-based cationic lipids, phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol; phosphatidylserine (and derivatives thereof); phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine; diquaternary ammonium salts such as N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,2-ethanediamine (TmedEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,3-propanediamine (PropEce), N,N′-dioleyl-N,N,N′,N′-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N,N′-dicetyl saturated analogues (TmedAce, PropAce and HexAce), diphosphatidylglycerols; fatty acid esters; monocationic transfection lipids such as 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol; 1-deoxy-1-[methyl(ditetradecyl)ammonio]-Darabinitol; 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol; 1-deoxy-1-[methyl(dioctadecyl)ammonio]-Darabinitol, glycerol esters; sphingolipids; cardolipin; cerebrosides; and ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3βOH-sterols as well as derivatives thereof phosphatidyl choline or commercially available cationic lipid mixtures such as, for example, LIPOFECTIN® CELLFECTIN® (1:1.5 (M/M) formulation of N, NI,NII, NIII-tetramethyl-N, NI, NII, NIII-tetrapalmitylspermine (TMTPS) and dioleoyl phosphatidylethanolamine (DOPE), LIPOFECTACE®, GS 2888 CYTOFECTIN®, FUGENE 6®, EFFECTENE®, and LIPOFECTAMINE®, LIPOFECTAMINE 2000®, LIPOFECTAMINE PLUS®, LIPOTAXI®, POLYECT®, SUPERFECT®, TFXN™, TRANSFAST™, TRANSFECTAM®, TRANSMESSENGER®, vectamidine (3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine (diC14-amidine), OLIGOFECTAMINE®. 
     
     
         38 . The transfection complex of  claim 23 , further comprising at least one pegylated lipid. 
     
     
         39 . The transfection complex of  claim 23 , further comprising at least one bioactive agent. 
     
     
         40 . The transfection complex of  claim 39 , wherein the bioactive agent is a DNA molecule, and RNA molecule, a protein of a drug. 
     
     
         41 . The transfection complex of  claim 40 , wherein the RNA molecule is an siRNA, an shRNA, an miRNA and stRNA or an mRNA. 
     
     
         42 . The transfection complex of  claim 39 , wherein the bioactive agent is an siRNA molecule. 
     
     
         43 . The transfection complex of  claim 39 , wherein the bioactive agent is an mRNA molecule. 
     
     
         44 . The transfection complex of  claim 39 , wherein the bioactive agent is an DNA molecule. 
     
     
         45 . The transfection complex of  claim 23 , further comprising a targeting moiety. 
     
     
         46 . The transfection complex of  claim 23 , further comprising a targeting moiety selected from the list consisting of peptide, a modified peptide, an antibody, a modified antibody, a receptor molecule, a modified receptor molecule, a single or a double stranded nucleic acid molecule, a modified single or double stranded nucleic acid molecule, a peptide or nucleic acid aptamer, a modified peptide or nucleic acid aptamer, an organic molecule, a polysaccharide.