U.S. patent application number 14/643845 was filed with the patent office on 2016-01-14 for amine-containing lipids and uses thereof.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Daniel Griffith Anderson, Michael Solomon Goldberg, Robert S. Langer, Elizaveta S. Leshchiner, Andreas Zumbuehl.
Application Number | 20160009657 14/643845 |
Document ID | / |
Family ID | 37571105 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160009657 |
Kind Code |
A1 |
Anderson; Daniel Griffith ;
et al. |
January 14, 2016 |
AMINE-CONTAINING LIPIDS AND USES THEREOF
Abstract
Nitrogen-containing lipids prepared from the conjugate addition
of amines to acrylates, acrylamides, or other carbon-carbon double
bonds conjugated to electron-withdrawing groups are described.
Methods of preparing these lipids from commercially available
starting materials are also provided. These amine-containing lipids
or salts forms of these lipids are preferably biodegradable and
biocompatible and may be used in a variety of drug delivery
systems. Given the amino moiety of these lipids, they are
particularly suited for the delivery of polynucleotides. Complexes
or nanoparticles containing the inventive lipid and polynucleotide
have been prepared. The inventive lipids may also be used to in
preparing microparticle for drug delivery. They are particularly
useful in delivering labile agents given their ability to buffer
the pH of their surroundings.
Inventors: |
Anderson; Daniel Griffith;
(Framingham, MA) ; Zumbuehl; Andreas; (Cambridge,
MA) ; Leshchiner; Elizaveta S.; (Belmont, MA)
; Langer; Robert S.; (Newton, MA) ; Goldberg;
Michael Solomon; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
37571105 |
Appl. No.: |
14/643845 |
Filed: |
March 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11453222 |
Jun 14, 2006 |
9006487 |
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14643845 |
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60785176 |
Mar 23, 2006 |
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60690608 |
Jun 15, 2005 |
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Current U.S.
Class: |
424/450 ;
514/785; 514/788; 548/340.1; 560/169; 560/170; 564/160 |
Current CPC
Class: |
A61K 48/0033 20130101;
C07C 229/16 20130101; C07C 231/12 20130101; C07D 233/61 20130101;
A61K 47/22 20130101; A61K 9/1617 20130101; A61P 43/00 20180101;
C07D 317/28 20130101; C07C 229/14 20130101; C07D 243/08 20130101;
C07C 229/12 20130101; A61K 47/18 20130101; C07C 227/10 20130101;
C07D 211/26 20130101; C07C 237/06 20130101; C07D 307/14 20130101;
C07D 295/12 20130101; C07C 229/18 20130101; C12N 15/88 20130101;
A61K 48/00 20130101 |
International
Class: |
C07D 233/61 20060101
C07D233/61; C07C 229/12 20060101 C07C229/12; A61K 47/22 20060101
A61K047/22; C07C 231/12 20060101 C07C231/12; C07C 227/10 20060101
C07C227/10; A61K 47/18 20060101 A61K047/18; C07C 229/16 20060101
C07C229/16; C07C 237/06 20060101 C07C237/06 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No. R01-EB000244 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A compound of the formula: ##STR00259## wherein V is selected
from the group consisting of C.dbd.O, C.dbd.S, S.dbd.O, and
SO.sub.2; R.sub.1 is selected from the group consisting of
hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; or --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; R.sub.2 is
selected from the group consisting of hydrogen; halogen; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched heteroaliphatic; substituted or
unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.B;
--C(.dbd.O)R.sub.B; --CO.sub.2R.sub.B; --CN; --SCN; --SR.sub.B;
--SOR.sub.B; --SO.sub.2R.sub.B; --NO.sub.2; --N.sub.3;
--N(R.sub.B).sub.2; --NHC(.dbd.O)R.sub.B;
--NR.sub.BC(.dbd.O)N(R.sub.B).sub.2; --OC(.dbd.O)OR.sub.B;
--OC(.dbd.O)R.sub.B; --OC(.dbd.O)N(R.sub.B).sub.2;
--NR.sub.BC(.dbd.O)OR.sub.B; or --C(R.sub.B).sub.3; wherein each
occurrence of R.sub.B is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; wherein
R.sub.1 and R.sub.2 may be taken together to form a cyclic
structure; R.sub.3 is selected from the group consisting of
hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; each
occurrence of R.sub.5 is independently selected from the group
consisting of hydrogen and C.sub.1-C.sub.6 alkyl; each occurrence
of R.sub.6 is independently selected from the group consisting of
hydrogen and C.sub.1-C.sub.6 alkyl; with the proviso that when all
occurrences of R.sub.5 and R.sub.6 are hydrogen, V is C.dbd.O,
R.sub.1 is --OR.sub.A, R.sub.2 is --OR.sub.B, and R.sub.1 and
R.sub.2 are the same, then R.sub.3 is not
--CH.sub.2CH.sub.2OR.sub.C', wherein R.sub.C' is methyl, ethyl,
propyl, isopropyl, butyl, s-butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, hexyl, cyclohexyl, decyl, methoxymethyl,
2-methoxyethyl, 1-ethoxyethyl, 2-ethoxyethyl,
(2-methoxyethoxy)methyl, 2-tetrahydrofuranyl, 2-tetrahydropyranyl,
tetrahydrofurfuryl, formyl, acetyl, propionyl, butyryl, isobutyryl,
pivaloyl, valeryl, methoxyacetyl, ethoxyacetyl, acetoxyacetyl,
2-formyloxyethyl, 2-acetoxyethyl, 2-oxopropyl, 2-oxobutyl,
2-oxocyclopentyl, 2-oxo-3-tetrahydrofuranyl,
2-oxo-3-tetrahydropyranyl, methoxycarbonyl, ethoxycarbonyl, and
t-butoxycarbonyl; and with the proviso that when all occurrences of
R.sub.5 and R.sub.6 are hydrogen, V is C.dbd.O, R.sub.1 is
--OR.sub.A, wherein R.sub.A is a straight-chain, branched or cyclic
alkyl group of 1 to 20 carbon atoms which may contain an ether,
carbonyl, or carbonyloxy group; R.sub.2 is --OR.sub.B, wherein
R.sub.B is a straight-chain, branched or cyclic alkyl group of 1 to
20 carbon atoms which may contain an ether, carbonyl, or
carbonyloxy group; and R.sub.1 and R.sub.2 are the same, the
R.sub.3 is not --CH.sub.2CH.sub.2OR.sub.C'', wherein R.sub.C'' is a
straight-chain, branched or cyclic alkyl group of 1 to 20 carbon
atoms which may contain an ether, carbonyl, or carbonyloxy group;
and salts thereof.
2. A compound of the formula: ##STR00260## wherein V is selected
from the group consisting of C.dbd.O, C.dbd.S, S.dbd.O, and
SO.sub.2; R.sub.1 is selected from the group consisting of
hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; or --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; R.sub.2 is
selected from the group consisting of hydrogen; halogen; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched heteroaliphatic; substituted or
unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.B;
--C(.dbd.O)R.sub.B; --CO.sub.2R.sub.B; --CN; --SCN; --SR.sub.B;
--SOR.sub.B; --SO.sub.2R.sub.B; --NO.sub.2; --N.sub.3;
--N(R.sub.B).sub.2; --NHC(.dbd.O)R.sub.B;
--NR.sub.BC(.dbd.O)N(R.sub.B).sub.2; --OC(.dbd.O)OR.sub.B;
--OC(.dbd.O)R.sub.B; --OC(.dbd.O)N(R.sub.B).sub.2;
--NR.sub.BC(.dbd.O)OR.sub.B; or --C(R.sub.B).sub.3; wherein each
occurrence of R.sub.B is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; wherein
R.sub.1 and R.sub.2 may be taken together to form a cyclic
structure; R.sub.3 is selected from the group consisting of
hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; each
occurrence of R.sub.5 is independently selected from the group
consisting of hydrogen and C.sub.1-C.sub.6 alkyl; each occurrence
of R.sub.6 is independently selected from the group consisting of
hydrogen and C.sub.1-C.sub.6 alkyl; R.sub.7 is hydrogen or
C.sub.1-C.sub.6 aliphatic; and X is an anion; with the proviso that
when all occurrences of R.sub.5 and R.sub.6 are hydrogen, V is
C.dbd.O, R.sub.1 is --OR.sub.A, R.sub.2 is --OR.sub.B, and R.sub.1
and R.sub.2 are the same, then R.sub.3 is not
--CH.sub.2CH.sub.2OR.sub.C', wherein R.sub.C' is methyl, ethyl,
propyl, isopropyl, butyl, s-butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, hexyl, cyclohexyl, decyl, methoxymethyl,
2-methoxyethyl, 1-ethoxyethyl, 2-ethoxyethyl,
(2-methoxyethoxy)methyl, 2-tetrahydrofuranyl, 2-tetrahydropyranyl,
tetrahydrofurfuryl, formyl, acetyl, propionyl, butyryl, isobutyryl,
pivaloyl, valeryl, methoxyacetyl, ethoxyacetyl, acetoxyacetyl,
2-formyloxyethyl, 2-acetoxyethyl, 2-oxopropyl, 2-oxobutyl,
2-oxocyclopentyl, 2-oxo-3-tetrahydrofuranyl,
2-oxo-3-tetrahydropyranyl, methoxycarbonyl, ethoxycarbonyl, and
t-butoxycarbonyl; and with the proviso that when all occurrences of
R.sub.5 and R.sub.6 are hydrogen, V is C.dbd.O, R.sub.1 is
--OR.sub.A, wherein R.sub.A is a straight-chain, branched or cyclic
alkyl group of 1 to 20 carbon atoms which may contain an ether,
carbonyl, or carbonyloxy group; R.sub.2 is --OR.sub.B, wherein
R.sub.B is a straight-chain, branched or cyclic alkyl group of 1 to
20 carbon atoms which may contain an ether, carbonyl, or
carbonyloxy group; and R.sub.1 and R.sub.2 are the same, the
R.sub.3 is not --CH.sub.2CH.sub.2OR.sub.C'', wherein R.sub.C'' is a
straight-chain, branched or cyclic alkyl group of 1 to 20 carbon
atoms which may contain an ether, carbonyl, or carbonyloxy group;
and salts thereof.
3-6. (canceled)
7. The compound of claim 1, wherein each occurrence of R.sub.5 and
R.sub.6 is hydrogen.
8. The compound of claim 1, wherein ##STR00261## are the same.
9. The compound of claim 1, wherein ##STR00262## are the same and
are different than R.sub.3.
10. (canceled)
11. The compound of claim 1, wherein ##STR00263## are independently
selected from the group consisting of: ##STR00264##
12-13. (canceled)
14. The compound of claim 1, wherein ##STR00265## are independently
selected from the group consisting of: ##STR00266##
15-16. (canceled)
17. The compound of claim 1, wherein ##STR00267## is selected from
the group consisting of: ##STR00268## ##STR00269##
18-19. (canceled)
20. The compound of claim 1 of formula: ##STR00270## wherein
R.sub.3' is C.sub.1-6alkyl, hydroxyl, thiol; C.sub.1-6alkoxy;
amino, C.sub.1-6alkylamino, diC.sub.1-6alkylamino; carbocyclic
moiety; heterocyclic moiety; aryl; or heteroaryl moiety; n is an
integer between 5 and 20, inclusive; and m is an integer between 1
and 10, inclusive; and pharmaceutically acceptable salts
thereof.
21-25. (canceled)
26. The compound of claim 1 selected from the group consisting of:
##STR00271## ##STR00272##
27. The compound of claim 1 of the formula: ##STR00273##
28. The compound of claim 1 of the formula: ##STR00274## wherein V
is C.dbd.O.
29. The compound of claim 28 of the formula: ##STR00275## wherein
R.sub.1 is --OR.sub.A; and R.sub.2 is --OR.sub.B.
30. (canceled)
31. The compound of claim 29, wherein R.sub.A and R.sub.B are
C.sub.6-C.sub.30 straight chain alkyl groups.
32-50. (canceled)
51. The compound of claim 28 of the formula: ##STR00276## wherein
R.sub.1 is --N(R.sub.A).sub.2; and R.sub.2 is
--N(R.sub.B).sub.2.
52. The compound of claim 28 of the formula: ##STR00277## wherein
R.sub.1 is --NHR.sub.A; and R.sub.2 is --NHR.sub.B.
53-82. (canceled)
83. A composition prepared by the method of reacting an amine of
one of the formula (1-117): ##STR00278## ##STR00279## with an
acrylate or acrylamide of formula: ##STR00280##
84-95. (canceled)
96. A microparticle, liposome, or micelle comprising a compound of
claim 1 and an agent to be delivered.
97-108. (canceled)
109. A pharmaceutical composition comprising a compound of claim 1
and pharmaceutical agent.
110-111. (canceled)
112. A method of preparing a compound of claim 1, the method
comprising steps of: reacting one or more equivalents of a compound
of formula: ##STR00281## wherein R.sub.1 is selected from the group
consisting of hydrogen; halogen; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic; cyclic or acyclic,
substituted or unsubstituted, branched or unbranched
heteroaliphatic; substituted or unsubstituted, branched or
unbranched acyl; substituted or unsubstitued, branched or
unbranched aryl; substituted or unsubstituted, branched or
unbranched heteroaryl; --OR.sub.A; --C(.dbd.O)R.sub.A;
--CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A; --SOR.sub.A;
--SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3; --N(R.sub.A).sub.2;
--NHC(.dbd.O)R.sub.A; --NR.sub.AC(.dbd.O)N(R.sub.A).sub.2;
--OC(.dbd.O)OR.sub.A; --OC(.dbd.O)R.sub.A;
--OC(.dbd.O)N(R.sub.A).sub.2; --NR.sub.AC(.dbd.O)OR.sub.A; or
--C(R.sub.A).sub.3; wherein each occurrence of R.sub.A is
independently a hydrogen; a protecting group; halogen; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched heteroaliphatic; substituted or
unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; and R.sub.5
is independently selected from the group consisting of hydrogen and
C.sub.1-C.sub.6 alkyl; with an amine of formula: ##STR00282##
wherein R.sub.3 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstitued, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; to form a
compound of formula: ##STR00283##
113-131. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of and claims
priority under 35 U.S.C. .sctn.120 to U.S. application Ser. No.
11/453,222, filed Jun. 14, 2006, now U.S. Pat. No. 9,006,487, which
claims priority under 35 U.S.C. .sctn.119(e) to U.S. provisional
patent applications, U.S. Ser. No. 60/690,608, filed Jun. 15, 2005,
and U.S. Ser. No. 60/785,176, filed Mar. 23, 2006, each of which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The treatment of human diseases through the application of
nucleotide-based drugs such as DNA and RNA has the potential to
revolutionize the medical field (Anderson Nature 392(Suppl.):25-30,
1996; Friedman Nature Med. 2:144-147, 1996; Crystal Science
270:404-410, 1995; Mulligan Science 260:926-932, 1993; each of
which is incorporated herein by reference). Thus far, the use of
modified viruses as gene transfer vectors has generally represented
the most clinically successful approach to gene therapy. While
viral vectors are currently the most efficient gene transfer
agents, concerns surrounding the overall safety of viral vectors,
which include the potential for unsolicited immune responses, have
resulted in parallel efforts to develop non-viral alternatives (for
leading references, see: Luo et al. Nat. Biotechnol. 18:33-37,
2000; Behr Acc. Chem. Res. 26:274-278, 1993; each of which is
incorporated herein by reference). Current alternatives to viral
vectors include polymeric delivery systems (Zauner et al. Adv. Drug
Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.
6:7-20, 1995; each of which is incorporated herein by reference),
liposomal formulations (Miller Angew. Chem. Int. Ed. 37:1768-1785,
1998; Hope et al. Molecular Membrane Technology 15:1-14, 1998;
Deshmukh et al. New J. Chem. 21:113-124, 1997; each of which is
incorporated herein by reference), and "naked" DNA injection
protocols (Sanford Trends Biotechnol. 6:288-302, 1988; incorporated
herein by reference). While these strategies have yet to achieve
the clinical effectiveness of viral vectors, the potential safety,
processing, and economic benefits offered by these methods
(Anderson Nature 392(Suppl.):25-30, 1996; incorporated herein by
reference) have ignited interest in the continued development of
non-viral approaches to gene therapy (Boussif et al. Proc. Natl.
Acad. Sci. USA 92:7297-7301, 1995; Putnam et al. Macromolecules
32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639,
1999; Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999;
Kukowska-Latallo et al. Proc. Natl. Acad. Sci. USA 93:4897-4902,
1996; Tang et al. Bioconjugate Chem. 7:703-714, 1996; Haensler et
al. Bioconjugate Chem. 4:372-379, 1993; each of which is
incorporated herein by reference).
[0004] There exists a continuing need for non-toxic, biodegradable,
biocompatible lipids that can be used to transfect nucleic acids
and that are easily prepared efficiently and economically. Such
lipids would have several uses, including the delivery of nucleic
acids in gene therapy as well as in the packaging and/or delivery
of diagnostic, therapeutic, and prophylactic agents.
SUMMARY OF THE INVENTION
[0005] The present invention provides novel lipids of the formula
(I):
##STR00001##
These lipids may be prepared by the addition of a primary amine to
a double bond conjugated with an electron withdrawing groups such
as a carbonyl moiety. Two equivalents of an
.alpha.,.beta.-unsaturated ketone such as an acrylate are reacted
with one equivalent of a primary amine to prepare the inventive
lipids as shown in the scheme below:
##STR00002##
These lipids typically have a hydrophobic half and a hydrophilic
half. The hydrophobic portion is typically provided by fatty acid
moieties attached to the acrylate, and the hydrophilic portion is
provided by the esters, amines, and side chain of the amine. The
fatty acid groups may be straight chain alkyl groups
(C.sub.1-C.sub.30) with no substitutions. In certain embodiments,
the fatty acid groups are substituted and/or branched. The amine
may be protonated or alkylated thereby forming a positively charged
amine. These lipids may be used in the delivery of therapeutic
agents to a subject. The inventive lipids are particularly useful
in delivering negatively charged agents given the tertiary amine
available for protonation thus forming a positive charge. For
example, these lipids may be used to delivery DNA, RNA, or other
polynucleotides to a subject or to a cell. As would be appreciated
by one of skill in the art, the above reaction may result in a
mixture with some lipids have one acrylate tail and other having
two acrylate tails. Also, two different acrylates may be used in
the reaction mixture to prepare a lipid with two different acrylate
tails.
[0006] In another aspect, the invention provides lipids of the
formula (II):
##STR00003##
Lipids of the formula (II) are prepared by the addition of a
primary or secondary diamine to a double bond conjugated to an
electron-withdrawing group such as a carbonyl. The lipids of
formula (II) have two amines per lipid molecule as compared to the
one amine per lipid molecule in the lipids of formula (I). These
amines may be protonated or alkylated to form positively charged
amino groups. These lipids may also be used to deliver DNA, RNA, or
other polynucleotides. As with the primary amine, the acrylate
tails may be the same or different. Also, the lipid may include any
where from one acrylate tail to as many acrylate tails as is
chemically possible.
[0007] In another aspect, the invention provides lipis of the
formulae (III) or (IV):
##STR00004##
Lipids of the formulae (III) or (IV) are prepared by the addition
of primary or secondary amino groups to a double bond conjugated to
an electron-withdrawing groups as a carbonyl. The lipids of
formulae (III) and (IV) have multiple amino groups per lipid
molecule. In certain embodiments, the number of amino groups per
lipid molecule is 3, 4, 5, 6, 7, 8, 9, or 10. These amines may be
protonated or alkylated to form positively charged amino groups.
The acrylate tails may all be the same or they may be different.
Any number of acrylate tails may be present on the molecule. The
lipids may be used to delivery DNA, RNA, or other
polynucleotides.
[0008] In one aspect of the invention, the inventive lipids are
combined with an agent to form microparticles, liposomes, or
micelles. The agent to be delivered by the microparticles,
liposomes, or micelles may be in the form of a gas, liquid, or
solid, and the agent may be a polynucleotide, protein, peptide, or
small molecule. The inventive lipids may be combined with other
lipids, polymers, surfactants, cholesterol, carbohydrates,
proteins, etc. to form the particles. These particles may be
combined with a pharmaceutically excipient to form pharmaceutical
compositions.
[0009] The invention also provides methods of making the inventive
lipids. One or more equivalents of an acrylate are allowed to react
with one equivalent of a primary amine, diamine, or polyamine under
suitable conditions to form a lipid of the formula (I), (II),
(III), or (IV). In certain embodiments, all the amino groups of the
amine are fully reacted with acrylates to form tertiary amines. In
other embodiments, all the amino groups of the amine are not fully
reacted with acrylate to form tertiary amines thereby resulting in
primary or secondary amines in the lipid molecule. These primary or
secondary amines are left as is or may be reacted with another
electrophile such as a different acrylate. As will be appreciated
by one of skill in this art, reacting an amine with less than an
excess of acrylate will result in a plurality of different lipid
amines. Certain molecules may include a full complement of acrylate
moieties while other molecules will not include a full complement
of acrylates. For example, a diamine or polyamine may include only
one, two, three, four, five, or six acrylate moieties off the
various amino moieties of the molecule resulting in primary,
secondary, and tertiary amines. In certain embodiments, it is
preferred that all the amino groups not be fully functionalized. In
certain embodiments, the two of the same type of acrylate are used.
In other embodiments, two or more different acrylates are used. The
synthesis of the lipid may be performed with or without solvent,
and the synthesis may be performed at temperatures ranging from
25.degree. C. to 100.degree. C., preferably approximately
95.degree. C. The prepared lipids may be optionally purified. For
example, the mixture of lipids may be purified to yield a lipid
with a certain number of acrylate moieties. The lipids may also be
alkylated using an alkyl halide (e.g., methyl iodide) or other
alkylating agent.
[0010] The invention also provides libraries of lipids prepared by
the inventive methods. These lipids may be prepared and/or screened
using high-throughput techniques involving liquid handlers, robots,
microtiter plates, computers, etc. In certain embodiments, the
lipids are screened for their ability to transfect DNA, RNA, or
other polynucleotides into the cell.
DEFINITIONS
[0011] Definitions of specific functional groups and chemical terms
are described in more detail below. For purposes of this invention,
the chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 75.sup.th Ed., inside cover, and specific functional
groups are generally defined as described therein. Additionally,
general principles of organic chemistry, as well as specific
functional moieties and reactivity, are described in "Organic
Chemistry", Thomas Sorrell, University Science Books, Sausalito:
1999, the entire contents of which are incorporated herein by
reference.
[0012] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0013] Isomeric mixtures containing any of a variety of isomer
ratios may be utilized in accordance with the present invention.
For example, where only two isomers are combined, mixtures
containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3,
98:2, 99:1, or 100:0 isomer ratios are all contemplated by the
present invention. Those of ordinary skill in the art will readily
appreciate that analogous ratios are contemplated for more complex
isomer mixtures.
[0014] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0015] One of ordinary skill in the art will appreciate that the
synthetic methods, as described herein, utilize a variety of
protecting groups. By the term "protecting group", as used herein,
it is meant that a particular functional moiety, e.g., O, S, or N,
is temporarily blocked so that a reaction can be carried out
selectively at another reactive site in a multifunctional compound.
In preferred embodiments, a protecting group reacts selectively in
good yield to give a protected substrate that is stable to the
projected reactions; the protecting group should be selectively
removable in good yield by readily available, preferably non-toxic
reagents that do not attack the other functional groups; the
protecting group forms an easily separable derivative (more
preferably without the generation of new stereogenic centers); and
the protecting group has a minimum of additional functionality to
avoid further sites of reaction. As detailed herein, oxygen,
sulfur, nitrogen, and carbon protecting groups may be utilized.
Hydroxyl protecting groups include methyl, methoxylmethyl (MOM),
methylthiomethyl (MTM), t-butylthiomethyl,
(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),
p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),
guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),
siloxymethyl, 2-methoxyethoxymethyl (MEM),
2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,
2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),
3-bromotetrahydropyranyl, tetrahydrothiopyranyl,
1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP),
4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl
S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl
(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(mmethoxyphenyl)phenylmethyl, tri(mmethoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec),
2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl
carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl
p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl
p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate,
alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl
S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl
dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,
4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,
2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,
4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxycarbonyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts). For protecting 1,2- or 1,3-diols, the protecting groups
include methylene acetal, ethylidene acetal, 1-t-butylethylidene
ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene
acetal, 2,2,2-trichloroethylidene acetal, acetonide,
cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene
ketal, benzylidene acetal, p-methoxybenzylidene acetal,
2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,
2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene
acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho
ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene
ortho ester, .alpha.-methoxybenzylidene ortho ester,
1-(N,N-dimethylamino)ethylidene derivative,
.alpha.-(N,N'-dimethylamino)benzylidene derivative,
2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),
1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),
tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic
carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.
Amino-protecting groups include methyl carbamate, ethyl carbamante,
9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl
carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),
1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl
carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl
carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl
carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate,
benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl
derivative, N'-p-toluenesulfonylaminocarbonyl derivative,
N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide,
chloroacetamide, trichloroacetamide, trifluoroacetamide,
phenylacetamide, 3-phenylpropanamide, picolinamide,
3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,
p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,
acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,
4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide
(Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,
N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),
5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one,
5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one,
1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine,
N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine,
amine N-oxide, diphenylphosphinamide (Dpp),
dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt),
dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl
phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide
(Nps), 2,4-dinitrobenzenesulfenamide,
pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,
triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), O-trimethylsilylethanesulfonamide (SES),
9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and
phenacylsulfonamide. Exemplary protecting groups are detailed
herein, however, it will be appreciated that the present invention
is not intended to be limited to these protecting groups; rather, a
variety of additional equivalent protecting groups can be readily
identified using the above criteria and utilized in the method of
the present invention. Additionally, a variety of protecting groups
are described in Protective Groups in Organic Synthesis, Third Ed.
Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New
York: 1999, the entire contents of which are hereby incorporated by
reference.
[0016] It will be appreciated that the compounds, as described
herein, may be substituted with any number of substituents or
functional moieties. In general, the term "substituted" whether
preceded by the term "optionally" or not, and substituents
contained in formulas of this invention, refer to the replacement
of hydrogen radicals in a given structure with the radical of a
specified substituent. When more than one position in any given
structure may be substituted with more than one substituent
selected from a specified group, the substituent may be either the
same or different at every position. As used herein, the term
"substituted" is contemplated to include all permissible
substituents of organic compounds. In a broad aspect, the
permissible substituents include acyclic and cyclic, branched and
unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic
substituents of organic compounds. For purposes of this invention,
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
which satisfy the valencies of the heteroatoms. Furthermore, this
invention is not intended to be limited in any manner by the
permissible substituents of organic compounds. Combinations of
substituents and variables envisioned by this invention are
preferably those that result in the formation of stable compounds
useful in the treatment, for example, of infectious diseases or
proliferative disorders. The term "stable", as used herein,
preferably refers to compounds which possess stability sufficient
to allow manufacture and which maintain the integrity of the
compound for a sufficient period of time to be detected and
preferably for a sufficient period of time to be useful for the
purposes detailed herein.
[0017] The term "aliphatic", as used herein, includes both
saturated and unsaturated, straight chain (i.e., unbranched),
branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons,
which are optionally substituted with one or more functional
groups. As will be appreciated by one of ordinary skill in the art,
"aliphatic" is intended herein to include, but is not limited to,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl
moieties. Thus, as used herein, the term "alkyl" includes straight,
branched and cyclic alkyl groups. An analogous convention applies
to other generic terms such as "alkenyl", "alkynyl", and the like.
Furthermore, as used herein, the terms "alkyl", "alkenyl",
"alkynyl", and the like encompass both substituted and
unsubstituted groups. In certain embodiments, as used herein,
"lower alkyl" is used to indicate those alkyl groups (cyclic,
acyclic, substituted, unsubstituted, branched or unbranched) having
1-6 carbon atoms.
[0018] In certain embodiments, the alkyl, alkenyl, and alkynyl
groups employed in the invention contain 1-20 aliphatic carbon
atoms. In certain other embodiments, the alkyl, alkenyl, and
alkynyl groups employed in the invention contain 1-10 aliphatic
carbon atoms. In yet other embodiments, the alkyl, alkenyl, and
alkynyl groups employed in the invention contain 1-8 aliphatic
carbon atoms. In still other embodiments, the alkyl, alkenyl, and
alkynyl groups employed in the invention contain 1-6 aliphatic
carbon atoms. In yet other embodiments, the alkyl, alkenyl, and
alkynyl groups employed in the invention contain 1-4 carbon atoms.
Illustrative aliphatic groups thus include, but are not limited to,
for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,
--CH.sub.2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, cyclobutyl, --CH.sub.2-cyclobutyl, n-pentyl,
sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,
--CH.sub.2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,
--CH.sub.2-cyclohexyl moieties and the like, which again, may bear
one or more substituents. Alkenyl groups include, but are not
limited to, for example, ethenyl, propenyl, butenyl,
1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups
include, but are not limited to, ethynyl, 2-propynyl (propargyl),
1-propynyl, and the like.
[0019] The term "alkyl" as used herein refers to saturated,
straight- or branched-chain hydrocarbon radicals derived from a
hydrocarbon moiety containing between one and twenty carbon atoms
by removal of a single hydrogen atom. Examples of alkyl radicals
include, but are not limited to, methyl, ethyl, propyl, isopropyl,
n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl,
n-octyl, n-decyl, n-undecyl, and dodecyl.
[0020] The term "alkenyl" denotes a monovalent group derived from a
hydrocarbon moiety having at least one carbon-carbon double bond by
the removal of a single hydrogen atom. Alkenyl groups include, for
example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the
like.
[0021] The term "alkynyl" as used herein refers to a monovalent
group derived form a hydrocarbon having at least one carbon-carbon
triple bond by the removal of a single hydrogen atom.
Representative alkynyl groups include ethynyl, 2-propynyl
(propargyl), 1-propynyl, and the like.
[0022] The term "alkoxy", or "thioalkyl" as used herein refers to
an alkyl group, as previously defined, attached to the parent
molecule through an oxygen atom or through a sulfur atom. In
certain embodiments, the alkyl, alkenyl, and alkynyl groups contain
1-20 alipahtic carbon atoms. In certain other embodiments, the
alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon
atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl
groups employed in the invention contain 1-8 aliphatic carbon
atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl
groups contain 1-6 aliphatic carbon atoms. In yet other
embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4
aliphatic carbon atoms. Examples of alkoxy, include but are not
limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,
tert-butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyl
include, but are not limited to, methylthio, ethylthio, propylthio,
isopropylthio, n-butylthio, and the like.
[0023] The term "alkylamino" refers to a group having the structure
--NHR', wherein R' is aliphatic, as defined herein. In certain
embodiments, the aliphatic group contains 1-20 aliphatic carbon
atoms. In certain other embodiments, the aliphatic group contains
1-10 aliphatic carbon atoms. In yet other embodiments, the
aliphatic group employed in the invention contain 1-8 aliphatic
carbon atoms. In still other embodiments, the aliphatic group
contains 1-6 aliphatic carbon atoms. In yet other embodiments, the
aliphatic group contains 1-4 aliphatic carbon atoms. Examples of
alkylamino groups include, but are not limited to, methylamino,
ethylamino, n-propylamino, iso-propylamino, cyclopropylamino,
n-butylamino, tert-butylamino, neopentylamino, n-pentylamino,
hexylamino, cyclohexylamino, and the like.
[0024] The term "carboxylic acid" as used herein refers to a group
of formula --CO.sub.2H.
[0025] The term "dialkylamino" refers to a group having the
structure --NRR', wherein R and R' are each an aliphatic group, as
defined herein. R and R' may be the same or different in an
dialkyamino moiety. In certain embodiments, the aliphatic groups
contains 1-20 aliphatic carbon atoms. In certain other embodiments,
the aliphatic groups contains 1-10 aliphatic carbon atoms. In yet
other embodiments, the aliphatic groups employed in the invention
contain 1-8 aliphatic carbon atoms. In still other embodiments, the
aliphatic groups contains 1-6 aliphatic carbon atoms. In yet other
embodiments, the aliphatic groups contains 1-4 aliphatic carbon
atoms. Examples of dialkylamino groups include, but are not limited
to, dimethylamino, methyl ethylamino, diethylamino,
methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino,
di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino,
di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino,
di(cyclohexyl)amino, and the like. In certain embodiments, R and R'
are linked to form a cyclic structure. The resulting cyclic
structure may be aromatic or non-aromatic. Examples of cyclic
diaminoalkyl groups include, but are not limited to, aziridinyl,
pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl,
1,3,4-trianolyl, and tetrazolyl.
[0026] Some examples of substituents of the above-described
aliphatic (and other) moieties of compounds of the invention
include, but are not limited to aliphatic; heteroaliphatic; aryl;
heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy;
heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;
heteroarylthio; F; Cl; Br; I; --OH; --NO.sub.2; --CN; --CF.sub.3;
--CH.sub.2CF.sub.3; --CHCl.sub.2; --CH.sub.2OH;
--CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--CH.sub.2SO.sub.2CH.sub.3; --C(O)R.sub.x; --CO.sub.2(R.sub.x);
--CON(R.sub.x).sub.2; --OC(O)R.sub.x; --OCO.sub.2R.sub.x;
--OCON(R.sub.x).sub.2; --N(R.sub.x).sub.2; --S(O).sub.2R.sub.x;
--NR.sub.x(CO)R.sub.x wherein each occurrence of R.sub.x
independently includes, but is not limited to, aliphatic,
heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
wherein any of the aliphatic, heteroaliphatic, arylalkyl, or
heteroarylalkyl substituents described above and herein may be
substituted or unsubstituted, branched or unbranched, cyclic or
acyclic, and wherein any of the aryl or heteroaryl substituents
described above and herein may be substituted or unsubstituted.
Additional examples of generally applicable substituents are
illustrated by the specific embodiments shown in the Examples that
are described herein.
[0027] In general, the terms "aryl" and "heteroaryl", as used
herein, refer to stable mono- or polycyclic, heterocyclic,
polycyclic, and polyheterocyclic unsaturated moieties having
preferably 3-14 carbon atoms, each of which may be substituted or
unsubstituted. Substituents include, but are not limited to, any of
the previously mentioned substitutents, i.e., the substituents
recited for aliphatic moieties, or for other moieties as disclosed
herein, resulting in the formation of a stable compound. In certain
embodiments of the present invention, "aryl" refers to a mono- or
bicyclic carbocyclic ring system having one or two aromatic rings
including, but not limited to, phenyl, naphthyl,
tetrahydronaphthyl, indanyl, indenyl, and the like. In certain
embodiments of the present invention, the term "heteroaryl", as
used herein, refers to a cyclic aromatic radical having from five
to ten ring atoms of which one ring atom is selected from S, O, and
N; zero, one, or two ring atoms are additional heteroatoms
independently selected from S, O, and N; and the remaining ring
atoms are carbon, the radical being joined to the rest of the
molecule via any of the ring atoms, such as, for example, pyridyl,
pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,
oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,
furanyl, quinolinyl, isoquinolinyl, and the like.
[0028] It will be appreciated that aryl and heteroaryl groups can
be unsubstituted or substituted, wherein substitution includes
replacement of one, two, three, or more of the hydrogen atoms
thereon independently with any one or more of the following
moieties including, but not limited to: aliphatic; heteroaliphatic;
aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy;
heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;
heteroarylthio; --F; --Cl; --Br; --I; --OH; --NO.sub.2; --CN;
--CF.sub.3; --CH.sub.2CF.sub.3; --CHCl.sub.2; --CH.sub.2OH;
--CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--CH.sub.2SO.sub.2CH.sub.3; --C(O)R.sub.x; --CO.sub.2(R.sub.x);
--CON(R.sub.x).sub.2; --OC(O)R.sub.x; --OCO.sub.2R.sub.x;
--OCON(R.sub.x).sub.2; --N(R.sub.x).sub.2; --S(O).sub.2R.sub.x;
--NR.sub.x(CO)R.sub.x, wherein each occurrence of R.sub.x
independently includes, but is not limited to, aliphatic,
heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
wherein any of the aliphatic, heteroaliphatic, arylalkyl, or
heteroarylalkyl substituents described above and herein may be
substituted or unsubstituted, branched or unbranched, cyclic or
acyclic, and wherein any of the aryl or heteroaryl substituents
described above and herein may be substituted or unsubstituted.
Additional examples of generally applicable substitutents are
illustrated by the specific embodiments shown in the Examples that
are described herein.
[0029] The term "cycloalkyl", as used herein, refers specifically
to groups having three to seven, preferably three to ten carbon
atoms. Suitable cycloalkyls include, but are not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
the like, which, as in the case of other aliphatic,
heteroaliphatic, or hetercyclic moieties, may optionally be
substituted with substituents including, but not limited to
aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;
heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;
alkylthio; arylthio; heteroalkylthio; heteroarylthio; --F; --Cl;
--Br; --I; --OH; --NO.sub.2; --CN; --CF.sub.3; --CH.sub.2CF.sub.3;
--CHCl.sub.2; --CH.sub.2OH; --CH.sub.2CH.sub.2OH;
--CH.sub.2NH.sub.2; --CH.sub.2SO.sub.2CH.sub.3; --C(O)R.sub.x;
--CO.sub.2(R.sub.x); --CON(R.sub.x).sub.2; --OC(O)R.sub.x;
--OCO.sub.2R.sub.x; --OCON(R.sub.x).sub.2; --N(R.sub.x).sub.2;
--S(O).sub.2R.sub.x; --NR.sub.x(CO)R.sub.x, wherein each occurrence
of R.sub.x independently includes, but is not limited to,
aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,
arylalkyl, or heteroarylalkyl substituents described above and
herein may be substituted or unsubstituted, branched or unbranched,
cyclic or acyclic, and wherein any of the aryl or heteroaryl
substituents described above and herein may be substituted or
unsubstituted. Additional examples of generally applicable
substitutents are illustrated by the specific embodiments shown in
the Examples that are described herein.
[0030] The term "heteroaliphatic", as used herein, refers to
aliphatic moieties that contain one or more oxygen, sulfur,
nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon
atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic
or acyclic and include saturated and unsaturated heterocycles such
as morpholino, pyrrolidinyl, etc. In certain embodiments,
heteroaliphatic moieties are substituted by independent replacement
of one or more of the hydrogen atoms thereon with one or more
moieties including, but not limited to aliphatic; heteroaliphatic;
aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy;
heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;
heteroarylthio; --F; --Cl; --Br; --I; --OH; --NO.sub.2; --CN;
--CF.sub.3; --CH.sub.2CF.sub.3; --CHCl.sub.2; --CH.sub.2OH;
--CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--CH.sub.2SO.sub.2CH.sub.3; --C(O)R.sub.x; --CO.sub.2(R.sub.x);
--CON(R.sub.x).sub.2; --OC(O)R.sub.x; --OCO.sub.2R.sub.x;
--OCON(R.sub.x).sub.2; --N(R.sub.x).sub.2; --S(O).sub.2R.sub.x;
--NR.sub.x(CO)R.sub.x, wherein each occurrence of R.sub.x
independently includes, but is not limited to, aliphatic,
heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
wherein any of the aliphatic, heteroaliphatic, arylalkyl, or
heteroarylalkyl substituents described above and herein may be
substituted or unsubstituted, branched or unbranched, cyclic or
acyclic, and wherein any of the aryl or heteroaryl substituents
described above and herein may be substituted or unsubstituted.
Additional examples of generally applicable substitutents are
illustrated by the specific embodiments shown in the Examples that
are described herein.
[0031] The terms "halo" and "halogen" as used herein refer to an
atom selected from fluorine, chlorine, bromine, and iodine.
[0032] The term "haloalkyl" denotes an alkyl group, as defined
above, having one, two, or three halogen atoms attached thereto and
is exemplified by such groups as chloromethyl, bromoethyl,
trifluoromethyl, and the like.
[0033] The term "heterocycloalkyl" or "heterocycle", as used
herein, refers to a non-aromatic 5-, 6-, or 7-membered ring or a
polycyclic group, including, but not limited to a bi- or tri-cyclic
group comprising fused six-membered rings having between one and
three heteroatoms independently selected from oxygen, sulfur and
nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds
and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen
and sulfur heteroatoms may be optionally be oxidized, (iii) the
nitrogen heteroatom may optionally be quaternized, and (iv) any of
the above heterocyclic rings may be fused to a benzene ring.
Representative heterocycles include, but are not limited to,
pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,
imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl,
isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and
tetrahydrofuryl. In certain embodiments, a "substituted
heterocycloalkyl or heterocycle" group is utilized and as used
herein, refers to a heterocycloalkyl or heterocycle group, as
defined above, substituted by the independent replacement of one,
two or three of the hydrogen atoms thereon with but are not limited
to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;
heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;
alkylthio; arylthio; heteroalkylthio; heteroarylthio; --F; --Cl;
--Br; --I; --OH; --NO.sub.2; --CN; --CF.sub.3; --CH.sub.2CF.sub.3;
--CHCl.sub.2; --CH.sub.2OH; --CH.sub.2CH.sub.2OH;
--CH.sub.2NH.sub.2; --CH.sub.2SO.sub.2CH.sub.3; --C(O)R.sub.x;
--CO.sub.2(R.sub.x); --CON(R.sub.x).sub.2; --OC(O)R.sub.x;
--OCO.sub.2R.sub.x; --OCON(R.sub.x).sub.2; --N(R.sub.x).sub.2;
--S(O).sub.2R.sub.x; --NR.sub.x(CO)R.sub.x, wherein each occurrence
of R.sub.x independently includes, but is not limited to,
aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,
arylalkyl, or heteroarylalkyl substituents described above and
herein may be substituted or unsubstituted, branched or unbranched,
cyclic or acyclic, and wherein any of the aryl or heteroaryl
substituents described above and herein may be substituted or
unsubstituted. Additional examples of generally applicable
substitutents are illustrated by the specific embodiments shown in
the Examples which are described herein.
[0034] "Carbocycle": The term "carbocycle", as used herein, refers
to an aromatic or non-aromatic ring in which each atom of the ring
is a carbon atom.
[0035] "Independently selected": The term "independently selected"
is used herein to indicate that the R groups can be identical or
different.
[0036] "Labeled": As used herein, the term "labeled" is intended to
mean that a compound has at least one element, isotope, or chemical
compound attached to enable the detection of the compound. In
general, labels typically fall into three classes: a) isotopic
labels, which may be radioactive or heavy isotopes, including, but
not limited to, .sup.2H, .sup.3H, .sup.32P, .sup.35S, .sup.67Ga,
.sup.99mTc (Tc-99m), .sup.111In, .sup.123I, .sup.125I, .sup.169Yb
and .sup.186Re; b) immune labels, which may be antibodies or
antigens, which may be bound to enzymes (such as horseradish
peroxidase) that produce detectable agents; and c) colored,
luminescent, phosphorescent, or fluorescent dyes. It will be
appreciated that the labels may be incorporated into the compound
at any position that does not interfere with the biological
activity or characteristic of the compound that is being detected.
In certain embodiments of the invention, photoaffinity labeling is
utilized for the direct elucidation of intermolecular interactions
in biological systems. A variety of known photophores can be
employed, most relying on photoconversion of diazo compounds,
azides, or diazirines to nitrenes or carbenes (See, Bayley, H.,
Photogenerated Reagents in Biochemistry and Molecular Biology
(1983), Elsevier, Amsterdam.), the entire contents of which are
hereby incorporated by reference. In certain embodiments of the
invention, the photoaffinity labels employed are o-, m- and
p-azidobenzoyls, substituted with one or more halogen moieties,
including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic
acid.
[0037] The terms halo and halogen as used herein refer to an atom
selected from fluorine, chlorine, bromine, and iodine.
[0038] The term "heterocyclic", as used herein, refers to a
non-aromatic partially unsaturated or fully saturated 3- to
10-membered ring system, which includes single rings of 3 to 8
atoms in size and bi- and tri-cyclic ring systems which may include
aromatic six-membered aryl or aromatic heterocyclic groups fused to
a non-aromatic ring. These heterocyclic rings include those having
from one to three heteroatoms independently selected from oxygen,
sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms
may optionally be oxidized and the nitrogen heteroatom may
optionally be quaternized.
[0039] The term "heteroaryl", as used herein, refers to a cyclic
aromatic radical having from five to ten ring atoms of which one
ring atom is selected from sulfur, oxygen, and nitrogen; zero, one,
or two ring atoms are additional heteroatoms independently selected
from sulfur, oxygen, and nitrogen; and the remaining ring atoms are
carbon, the radical being joined to the rest of the molecule via
any of the ring atoms, such as, for example, pyridyl, pyrazinyl,
pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,
isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,
quinolinyl, isoquinolinyl, and the like.
[0040] Specific heterocyclic and aromatic heterocyclic groups that
may be included in the compounds of the invention include:
3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,
4-(bis-(4-fluorophenyl)methyl)piperazine,
4-(diphenylmethyl)piperazine, 4-(ethoxycarbonyl)piperazine,
4-(ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine,
4-(1-phenylethyl)piperazine,
4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)
amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine,
4-(2-chlorophenyl)piperazine, 4-(2-cyanophenyl)piperazine,
4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine,
4-(2-fluorophenyl)piperazine, 4-(2-hydroxyethyl)piperazine,
4-(2-methoxyethyl)piperazine, 4-(2-methoxyphenyl)piperazine,
4-(2-methylphenyl)piperazine, 4-(2-methylthiophenyl) piperazine,
4-(2-nitrophenyl)piperazine, 4-(2-nitrophenyl)piperazine,
4-(2-phenylethyl)piperazine, 4-(2-pyridyl)piperazine,
4-(2-pyrimidinyl)piperazine, 4-(2,3-dimethylphenyl)piperazine,
4-(2,4-difluorophenyl) piperazine,
4-(2,4-dimethoxyphenyl)piperazine,
4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine,
4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine,
4-(3-methylphenyl)piperazine,
4-(3-trifluoromethylphenyl)piperazine,
4-(3,4-dichlorophenyl)piperazine, 4-3,4-dimethoxyphenyl)piperazine,
4-(3,4-dimethylphenyl)piperazine,
4-(3,4-methylenedioxyphenyl)piperazine,
4-(3,4,5-trimethoxyphenyl)piperazine,
4-(3,5-dichlorophenyl)piperazine,
4-(3,5-dimethoxyphenyl)piperazine,
4-(4-(phenylmethoxy)phenyl)piperazine,
4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,
4-(4-chloro-3-trifluoromethylphenyl)piperazine,
4-(4-chlorophenyl)-3-methylpiperazine,
4-(4-chlorophenyl)piperazine, 4-(4-chlorophenyl)piperazine,
4-(4-chlorophenylmethyl)piperazine, 4-(4-fluorophenyl)piperazine,
4-(4-methoxyphenyl)piperazine, 4-(4-methylphenyl)piperazine,
4-(4-nitrophenyl)piperazine, 4-(4-trifluoromethylphenyl)piperazine,
4-cyclohexylpiperazine, 4-ethylpiperazine,
4-hydroxy-4-(4-chlorophenyl)methylpiperidine,
4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine,
4-methylpiperazine, 4-phenylpiperazine, 4-piperidinylpiperazine,
4-(2-furanyl)carbonyl)piperazine,
4-((1,3-dioxolan-5-yl)methyl)piperazine,
6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline,
1,4-diazacylcloheptane, 2,3-dihydroindolyl, 3,3-dimethylpiperidine,
4,4-ethylenedioxypiperidine, 1,2,3,4-tetrahydroisoquinoline,
1,2,3,4-tetrahydroquinoline, azacyclooctane, decahydroquinoline,
piperazine, piperidine, pyrrolidine, thiomorpholine, and
triazole.
[0041] The terms "substituted," whether preceded by the term
"optionally" or not, and substituent, as used herein, refer to the
ability, as appreciated by one skilled in this art, to change one
functional group for another functional group provided that the
valency of all atoms is maintained. When more than one position in
any given structure may be substituted with more than one
substituent selected from a specified group, the substituent may be
either the same or different at every position. The substituents
may also be further substituted (e.g., an aryl group substituent
may have another substituent off it, such as another aryl group,
which is further substituted with fluorine at one or more
positions).
[0042] The following are more general terms used throughout the
present application:
[0043] "Animal": The term animal, as used herein, refers to humans
as well as non-human animals, including, for example, mammals,
birds, reptiles, amphibians, and fish. Preferably, the non-human
animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a
monkey, a dog, a cat, a primate, or a pig). An animal may be a
transgenic animal.
[0044] "Associated with": When two entities are "associated with"
one another as described herein, they are linked by a direct or
indirect covalent or non-covalent interaction. Preferably, the
association is covalent. Desirable non-covalent interactions
include hydrogen bonding, van der Waals interactions, hydrophobic
interactions, magnetic interactions, electrostatic interactions,
etc.
[0045] "Biocompatible": The term "biocompatible", as used herein is
intended to describe compounds that are not toxic to cells.
Compounds are "biocompatible" if their addition to cells in vitro
results in less than or equal to 20% cell death, and their
administration in vivo does not induce inflammation or other such
adverse effects.
[0046] "Biodegradable": As used herein, "biodegradable" compounds
are those that, when introduced into cells, are broken down by the
cellular machinery or by hydrolysis into components that the cells
can either reuse or dispose of without significant toxic effect on
the cells (i.e., fewer than about 20% of the cells are killed when
the components are added to cells in vitro). The components
preferably do not induce inflammation or other adverse effects in
vivo. In certain preferred embodiments, the chemical reactions
relied upon to break down the biodegradable compounds are
uncatalyzed.
[0047] "Effective amount": In general, the "effective amount" of an
active agent or drug delivery device refers to the amount necessary
to elicit the desired biological response. As will be appreciated
by those of ordinary skill in this art, the effective amount of an
agent or device may vary depending on such factors as the desired
biological endpoint, the agent to be delivered, the composition of
the encapsulating matrix, the target tissue, etc. For example, the
effective amount of microparticles containing an antigen to be
delivered to immunize an individual is the amount that results in
an immune response sufficient to prevent infection with an organism
having the administered antigen.
[0048] "Peptide" or "protein": According to the present invention,
a "peptide" or "protein" comprises a string of at least three amino
acids linked together by peptide bonds. The terms "protein" and
"peptide" may be used interchangeably. Peptide may refer to an
individual peptide or a collection of peptides. Inventive peptides
preferably contain only natural amino acids, although non-natural
amino acids (i.e., compounds that do not occur in nature but that
can be incorporated into a polypeptide chain) and/or amino acid
analogs as are known in the art may alternatively be employed.
Also, one or more of the amino acids in an inventive peptide may be
modified, for example, by the addition of a chemical entity such as
a carbohydrate group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. In a preferred
embodiment, the modifications of the peptide lead to a more stable
peptide (e.g., greater half-life in vivo). These modifications may
include cyclization of the peptide, the incorporation of D-amino
acids, etc. None of the modifications should substantially
interfere with the desired biological activity of the peptide.
[0049] "Polynucleotide" or "oligonucleotide": Polynucleotide or
oligonucleotide refers to a polymer of nucleotides. Typically, a
polynucleotide comprises at least three nucleotides. The polymer
may include natural nucleosides (i.e., adenosine, thymidine, guano
sine, cytidine, uridine, deoxyadenosine, deoxythymidine,
deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and
2-thiocytidine), chemically modified bases, biologically modified
bases (e.g., methylated bases), intercalated bases, modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose), or modified phosphate groups (e.g., phosphorothioates and
5'-N-phosphoramidite linkages).
[0050] "Small molecule": As used herein, the term "small molecule"
refers to organic compounds, whether naturally-occurring or
artificially created (e.g., via chemical synthesis) that have
relatively low molecular weight and that are not proteins,
polypeptides, or nucleic acids. Typically, small molecules have a
molecular weight of less than about 1500 g/mol. Also, small
molecules typically have multiple carbon-carbon bonds. Known
naturally-occurring small molecules include, but are not limited
to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin.
Known synthetic small molecules include, but are not limited to,
ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
BRIEF DESCRIPTION OF THE DRAWING
[0051] FIGS. 1A to 1B show acrylates and amines used in the
synthesis of exemplary amine-containing lipids.
[0052] FIGS. 2A to 2F show .sup.1H NMR spectra of lipids LD28 (FIG.
2A), LD86 (FIG. 2B), LD87 (FIG. 2C), ND32 (FIG. 2D), ND86 (FIG.
2E), and ND87 (FIG. 2F).
[0053] FIG. 3 shows the DNA transfection efficiency of several of
the inventive amine-containing lipids.
[0054] FIG. 4 shows the percentage of luciferase knockdown for
several of the inventive lipids.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE
INVENTION
[0055] The present invention provides novel lipids and delivery
systems based on the use of amino lipids. The system may be used in
the pharmaceutical/drug delivery arts to delivery polynucleotides,
proteins, small molecules, peptides, antigen, drugs, etc. to a
patient, tissue, organ, cell, etc.
[0056] The amino lipids of the present invention provide for
several different uses in the drug delivery art. The lipids with
their amine-containing hydrophilic portion may be used to complex
polynucleotides and thereby enhance the delivery of polynucleotide
and prevent their degradation. The lipids may also be used in the
formation of nanoparticles, microparticles, liposomes, and micelles
containing the agent to be delivered. Preferably, the lipids are
biocompatible and biodegradable, and the formed particles are also
biodegradable and biocompatible and may be used to provide
controlled, sustained release of the agent. These lipids and their
corresponding particles may also be responsive to pH changes given
that these lipids are protonated at lower pH.
Lipids
[0057] The lipids of the present invention are lipids containing
primary, secondary, or tertiary amines and salts thereof. In a
particularly preferred embodiment, the inventive lipids are
relatively non-cytotoxic. In another particularly preferred
embodiment, the inventive lipids are biocompatible and
biodegradable. In a particularly preferred embodiment, the lipids
of the present invention have pK.sub.as in the range of 5.5 to 7.5,
more preferably between 6.0 and 7.0. In another particularly
preferred embodiment, the lipid may be designed to have a desired
pK.sub.a between 3.0 and 9.0, more preferably between 5.0 and 8.0.
The inventive lipids are particularly attractive for drug delivery
for several reasons: 1) they contain amino groups for interacting
with DNA, RNA, other polynucleotides, and other negatively charged
agents, for buffering the pH, for causing endosomolysis, etc.; 2)
they can be synthesized from commercially available starting
materials; and 3) they are pH responsive and can be engineered with
a desired pK.sub.a.
[0058] In certain embodiments, the lipids of the present invention
are of the formula (I):
##STR00005##
wherein each occurrence of V is independently selected from the
group consisting of C.dbd.O, C.dbd.S, S.dbd.O, and SO.sub.2;
[0059] R.sub.1 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; or --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0060] R.sub.2 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.B;
--C(.dbd.O)R.sub.B; --CO.sub.2R.sub.B; --CN; --SCN; --SR.sub.B;
--SOR.sub.B; --SO.sub.2R.sub.B; --NO.sub.2; --N.sub.3;
--N(R.sub.B).sub.2; --NHC(.dbd.O)R.sub.B;
--NR.sub.BC(.dbd.O)N(R.sub.B).sub.2; --OC(.dbd.O)OR.sub.B;
--OC(.dbd.O)R.sub.B; --OC(.dbd.O)N(R.sub.B).sub.2;
--NR.sub.BC(.dbd.O)OR.sub.B; or --C(R.sub.B).sub.3; wherein each
occurrence of R.sub.B is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0061] wherein R.sub.1 and R.sub.2 may be taken together to form a
cyclic structure;
[0062] R.sub.3 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; [0063] each
occurrence of R.sub.5 is independently selected from the group
consisting of hydrogen and C.sub.1-C.sub.6 alkyl;
[0064] each occurrence of R.sub.6 is independently selected from
the group consisting of hydrogen and C.sub.1-C.sub.6 alkyl; and
salts thereof.
[0065] In certain embodiments, the tertiary amine of formula (I) is
protonated or alkylated to form a compound of formula (Ia):
##STR00006##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.6, and V are
defined above;
[0066] R.sub.7 is hydrogen or C.sub.1-C.sub.6 aliphatic, preferably
C.sub.1-C.sub.6 alkyl, more preferably hydrogen or methyl; and
[0067] X is any anion. Possible anions include fluoride, chloride,
bromide, iodide, sulfate, bisulfate, phosphate, nitrate, acetate,
fumarate, oleate, citrate, valerate, maleate, oxalate,
isonicotinate, lactate, salicylate, tartrate, tannate,
pantothenate, bitartrate, ascorbate, succinate, gentisinate,
gluconate, glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate).
[0068] In certain embodiments, V is C.dbd.O. In other embodiments,
V is C.dbd.S. In yet other embodiments, V is S.dbd.O. In still
other embodiments, V is SO.sub.2.
[0069] In certain embodiments, R.sub.1 is hydrogen. In other
embodiments, R.sub.1 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.1 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocyclic 5- or 6-membered ring system. In
certain embodiments, R.sub.1 is --OR.sub.A, --SR.sub.A,
--N(R.sub.A).sub.2, or --NHR.sub.A. In certain embodiments, R.sub.1
is --OR.sub.A. In other embodiments, R.sub.1 is --N(R.sub.A).sub.2
or --NHR.sub.A. In certain embodiments, R.sub.A is hydrogen. In
other embodiments, R.sub.A is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.A is an acyclic, substituted
or unsubstituted aliphatic moiety. In certain other embodiments,
R.sub.A is an acyclic, unsubstituted, unbranched aliphatic moiety,
preferably C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In
certain embodiments, R.sub.A is an unsubstituted, straight chain
alkyl group with at least 5 carbons. In certain embodiments,
R.sub.A is an unsubstituted, straight chain alkyl group, preferably
C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.9 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.10 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.11 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.A is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0070] In certain embodiments, R.sub.2 is hydrogen. In other
embodiments, R.sub.2 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.2 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocylic 5- or 6-membered ring system. In
certain embodiments, R.sub.2 is --OR.sub.B, --SR.sub.B,
--N(R.sub.B).sub.2, or --NHR.sub.B. In certain embodiments, R.sub.2
is --OR.sub.B. In other embodiments, R.sub.2 is --N(R.sub.B).sub.2
or --NHR.sub.B. In certain embodiments, R.sub.B is hydrogen. In
other embodiments, R.sub.B is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.B is an acyclic, substituted
or unsubstituted aliphatic moiety. In certain embodiments, R.sub.B
is an unsubstituted, straight chain alkyl group with at least 5
carbons. In certain other embodiments, R.sub.B is an acyclic,
unsubstituted, unbranched aliphatic moiety, preferably
C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In certain
embodiments, R.sub.B is an unsubstituted, straight chain alkyl
group, preferably C.sub.6-C.sub.30, more preferably
C.sub.10-C.sub.20. In certain embodiments, R.sub.2 is --OR.sub.B,
wherein R.sub.B is an unsubstituted, unbranched C.sub.9 alkyl
chain. In certain embodiments, R.sub.2 is --OR.sub.B, wherein
R.sub.B is an unsubstituted, unbranched C.sub.10 alkyl chain. In
certain embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.11 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.B is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0071] In certain embodiments, R.sub.3 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched aliphatic
moiety. In other embodiments, R.sub.3 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched
heteroaliphatic moiety. In certain embodiments, R.sub.3 is a
polyethylene glycol moiety. In certain embodiments, R.sub.3 is an
aliphatic moiety substituted with one or more hydroxyl groups. In
other embodiments, R.sub.3 is an aliphatic moiety substituted with
one or more amino, alkylamino, or dialkylamino groups. In certain
embodiments, R.sub.3 is a heteroaliphatic moiety. In certain
embodiments, R.sub.3 is cyclic aliphatic, preferably a monocylic
ring system with a 5- or 6-membered ring. In other embodiments,
R.sub.3 is aryl or heteroaryl, preferably a monocyclic ring system
with a 5- or 6-membered ring. In certain embodiments, the lipids
are prepared from the primary amines 1, 11, 20, 24, 25, 28, 31, 32,
36, 76, 77, 80, 86, 87, 93, 94, 95, 96, 99, or 100 shown in FIGS.
1A and 1B. In certain other embodiments the lipids are prepared
from the primary amines 31, 93, or 94 as shown in FIG. 1A.
[0072] In certain embodiments, each occurrence of R.sub.5 is
hydrogen. In certain embodiments, at least one occurrence of
R.sub.5 is methyl and the other occurrences are hydrogen. In
certain embodiments, at least two occurrences of R.sub.5 are
methyl, and the other occurrences are hydrogen. In other
embodiments, at least two occurrences of R.sub.5 are hydrogen.
[0073] In certain embodiments, each occurrence of R.sub.6 is
hydrogen. In certain other embodiments, at least two occurrences of
R.sub.6 are hydrogen. In certain embodiments, at least one
occurrence of R.sub.6 is methyl, and the other occurrences are
hydrogen. In certain embodiments, at least two occurrences of
R.sub.6 are methyl, and the other occurrences are hydrogen.
[0074] In certain embodiments,
##STR00007##
which are attached to N, are the same. In other embodiments,
##STR00008##
which are attached to N are the same and are different than
R.sub.3. In yet other embodiments,
##STR00009##
and R.sub.3 are all different.
[0075] In certain subclasses of lipids, the lipids are of the
formula:
##STR00010##
wherein V, R.sub.1, R.sub.2, and R.sub.3 are defined as above; and
all occurrences of R.sub.5 and R.sub.6 are hydrogen. In certain
embodiments, R.sub.1 and R.sub.2 are the same. In other
embodiments, R.sub.1 and R.sub.2 are different. In certain
embodiments, V is C.dbd.O as shown in the formula:
##STR00011##
In certain embodiments, R.sub.1 and R.sub.2 are the same. In other
embodiments, R.sub.1 and R.sub.2 are different. In certain
embodiments, R.sub.1 is --OR.sub.A and R.sub.2 is --OR.sub.B, as
shown in the formula below:
##STR00012##
In certain embodiments, R.sub.A and R.sub.B are the same. In other
embodiments, R.sub.A and R.sub.B are different. In certain
embodiments, at least one of R.sub.A and R.sub.B is an
unsubstituted, straight chain alkyl group with at least 5 carbons.
In certain embodiments, both of R.sub.A and R.sub.B are an
unsubstituted, straight chain alkyl group with at least 5 carbons.
In certain embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30
straight chain alkyl groups, or C.sub.21-C.sub.30 straight chain
alkyl groups, preferably C.sub.9-C.sub.20 straight chain alkyl
groups. In certain embodiments, R.sub.A and R.sub.B are
C.sub.6-C.sub.30 straight chain alkenyl groups, or
C.sub.21-C.sub.30 straight chain alkenyl groups, preferably
C.sub.9-C.sub.20 straight chain alkenyl groups. In certain
embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30 straight
chain alkynyl groups, or C.sub.21-C.sub.30 straight chain alkynyl
groups, preferably C.sub.9-C.sub.20 straight chain alkynyl groups.
In certain embodiments, when R.sub.A and R.sub.B are the same,
R.sub.A and R.sub.B are not methyl, ethyl, n-propyl,
##STR00013##
In other embodiments, when R.sub.A and R.sub.B are the same,
R.sub.A and R.sub.B each comprise at least 4 carbon atoms. In other
embodiments, when R.sub.A and R.sub.B are the same, R.sub.A and
R.sub.B each comprise at least 5 carbon atoms. In other
embodiments, when R.sub.A and R.sub.B are the same, R.sub.A and
R.sub.B each comprise at least 6 carbon atoms. In other
embodiments, R.sub.A and R.sub.B each comprise at least 4 carbon
atoms. In other embodiments, R.sub.A and R.sub.B each comprise at
least 5 carbon atoms. In other embodiments, R.sub.A and R.sub.B
each comprise at least 6 carbon atoms. Exemplary classes of the
above formula include:
##STR00014##
In certain embodiments, the acrylate used in the synthesis of the
lipid is acrylate LD, LF, or LG in FIG. 1A. In certain embodiments
the acrylate is acrylate LF in FIG. 1A. In certain embodiments the
acrylate is acrylate LG in FIG. 1A. In certain embodiments, R.sub.3
is not
##STR00015##
wherein R.sub.C is defined as above. In certain embodiments,
R.sub.3 is not --CH.sub.2CH.sub.2OR.sub.C', wherein R.sub.C' is
methyl, ethyl, propyl, isopropyl, butyl, s-butyl, isobutyl,
t-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, decyl,
methoxymethyl, 2-methoxyethyl, 1-ethoxyethyl, 2-ethoxyethyl,
(2-methoxyethoxy)methyl, 2-tetrahydrofuranyl, 2-tetrahydropyranyl,
tetrahydrofurfuryl, formyl, acetyl, propionyl, butyryl, isobutyryl,
pivaloyl, valeryl, methoxyacetyl, ethoxyacetyl, acetoxyacetyl,
2-formyloxyethyl, 2-acetoxyethyl, 2-oxopropyl, 2-oxobutyl,
2-oxocyclopentyl, 2-oxo-3-tetrahydrofuranyl,
2-oxo-3-tetrahydropyranyl, methoxycarbonyl, ethoxycarbonyl, and
t-butoxycarbonyl. In yet other embodiments, R.sub.3 is not
CH.sub.2CH.sub.2OR.sub.C'', wherein R.sub.C'' is a straight chain,
branched or cyclic alkyl group of 1 to 20 carbons atoms, which may
contain an ether, carbonyl, or carbonyloxy group. In yet other
embodiments, R.sub.3 is not --CH.sub.2CH.sub.2OR.sub.C'', wherein
R.sub.C'' is a straight chain, branched or cyclic alkyl group of 1
to 10 carbons atoms, which may contain an ether, carbonyl, or
carbonyloxy group. In certain particular embodiments, R.sub.3 is
not --CH.sub.2CH.sub.2OR.sub.C'', wherein R.sub.C'' is formyl;
acetyl; or methyl group.
[0076] In other embodiments, R.sub.1 is --NR.sub.A and R.sub.2 is
--NR.sub.B, as shown in the formula below:
##STR00016##
In certain embodiments, R.sub.A and R.sub.B are the same. In other
embodiments, R.sub.A and R.sub.B are different. In certain
embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30 straight
chain alkyl groups, or C.sub.21-C.sub.30 straight chain alkyl
groups, preferably C.sub.9-C.sub.20 straight chain alkyl groups. In
certain embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30
straight chain alkenyl groups, or C.sub.21-C.sub.30 straight chain
alkenyl groups, preferably C.sub.9-C.sub.20 straight chain alkenyl
groups. In certain embodiments, R.sub.A and R.sub.B are
C.sub.6-C.sub.30 straight chain alkynyl groups, or
C.sub.21-C.sub.30 straight chain alkynyl groups, preferably
C.sub.9-C.sub.20 straight chain alkynyl groups. In certain
embodiments, when R.sub.A and R.sub.B are the same, R.sub.A and
R.sub.B are not methyl, ethyl, n-propyl,
##STR00017##
In other embodiments, when R.sub.A and R.sub.B are the same,
R.sub.A and R.sub.B each comprise at least 4 carbon atoms. In other
embodiments, when R.sub.A and R.sub.B are the same, R.sub.A and
R.sub.B each comprise at least 5 carbon atoms. In other
embodiments, when R.sub.A and R.sub.B are the same, R.sub.A and
R.sub.B each comprise at least 6 carbon atoms. In other
embodiments, R.sub.A and R.sub.B each comprise at least 4 carbon
atoms. In other embodiments, R.sub.A and R.sub.B each comprise at
least 5 carbon atoms. In other embodiments, R.sub.A and R.sub.B
each comprise at least 6 carbon atoms. Exemplary classes of the
above formula include:
##STR00018##
In certain embodiments, the acrylate used in the synthesis of the
lipid is acrylate ND, NF, NG, or NP in FIG. 1A. In certain
embodiments the acrylate is acrylate ND in FIG. 1A. In certain
embodiments the acrylate is acrylate NF in FIG. 1A. In certain
embodiments the acrylate is acrylate NP in FIG. 1A.
[0077] Particular exemplary compounds include:
##STR00019## ##STR00020## ##STR00021##
[0078] In other subclasses of lipids, the lipids are of the
formula:
##STR00022##
wherein V, R.sub.1, and R.sub.3 are defined as above; all
occurrences of R.sub.6 are hydrogen; and R.sub.5 is defined as in
the formula. In certain embodiments, R.sub.1 and R.sub.2 are the
same. In certain embodiments, V is C.dbd.O as shown in the
formula:
##STR00023##
preferably R.sub.1 and R.sub.2 are the same. In certain
embodiments, R.sub.1 is --OR.sub.A and R.sub.2 is --OR.sub.B, as
shown in the formula below:
##STR00024##
preferably R.sub.A and R.sub.B are the same. In certain
embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30 straight
chain alkyl groups, preferably C.sub.9-C.sub.20 straight chain
alkyl groups. In other embodiments, R.sub.1 is --NR.sub.A and
R.sub.2 is --NR.sub.B, as shown in the formula below:
##STR00025##
preferably R.sub.A and R.sub.B are the same. In certain
embodiments, R.sub.A and R.sub.B are C.sub.6-C.sub.30 straight
chain alkyl groups, preferably Co-Coo straight chain alkyl
groups.
[0079] In certain embodiments,
##STR00026##
in formulae (I) and (Ia) are selected from the group consisting
of:
##STR00027##
In certain embodiments, the lipids are prepared using acrylates LC,
LD, LE, LF, and LG in FIG. 1A.
[0080] In certain embodiments,
##STR00028##
in formulae (I) and (Ia) are selected from the group consisting
of:
##STR00029##
In certain embodiments, the lipids are prepared using acrylates NC,
ND, NF, NG, or NP in FIG. 1A. In certain embodiments, the lipids
are prepared using acrylate ND. In other embodiments, the lipids
are prepared using acrylate NF.
[0081] In certain embodiments,
##STR00030##
is selected from the group consisting of:
##STR00031## ##STR00032##
[0082] In certain embodiments,
##STR00033##
is selected from the group consisting of:
##STR00034##
[0083] In certain embodiments,
##STR00035##
wherein n is an interger between 0 and 10, inclusive; and R.sub.3'
is hydrogen, aliphatic, heteroaliphatic, carbocyclic, heterocyclic,
aryl, acyl, or heteroaryl. In certain embodiments, R.sub.3' is
hydrogen, In other embodiments, R.sub.3' is C.sub.1-C.sub.6 alkyl.
In yet other embodiments, R.sub.3' is acyl (e.g., acetyl).
[0084] In certain embodiments, the inventive lipid is of
formula:
##STR00036##
wherein
[0085] n is an integer between 5 and 20, inclusive; and
[0086] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
n is 11. In other embodiments, n is 12. In yet other embodiments, n
is 13. In still other embodiments, n is 14. In certain embodiments,
m is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0087] In certain embodiments, the inventive lipid is of
formula:
##STR00037##
wherein
[0088] n is an integer between 5 and 20, inclusive; and
[0089] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
n is 11. In other embodiments, n is 12. In yet other embodiments, n
is 13. In still other embodiments, n is 14. In certain embodiments,
m is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0090] In certain embodiments, the inventive lipid is of
formula:
##STR00038##
wherein
[0091] R.sub.3' is C.sub.1-6alkyl;
[0092] n is an integer between 5 and 20, inclusive; and
[0093] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
R.sub.3' is methyl. In other embodiments, R.sub.3' is ethyl. In
other embodiments, R.sub.3' is n-propyl. In still other
embodiments, R.sub.3' is iso-propyl. In certain embodiments, n is
11. In other embodiments, n is 12. In yet other embodiments, n is
13. In still other embodiments, n is 14. In certain embodiments, m
is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0094] In certain embodiments, the inventive lipid is of
formula:
##STR00039##
wherein
[0095] R.sub.3' is C.sub.1-6alkyl;
[0096] n is an integer between 5 and 20, inclusive; and
[0097] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
R.sub.3' is methyl. In other embodiments, R.sub.3' is ethyl. In
other embodiments, R.sub.3' is n-propyl. In still other
embodiments, R.sub.3' is iso-propyl. In certain embodiments, n is
11. In other embodiments, n is 12. In yet other embodiments, n is
13. In still other embodiments, n is 14. In certain embodiments, m
is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0098] In certain embodiments, the inventive lipid is of
formula:
##STR00040##
wherein
[0099] R.sub.3' is carbocyclic; heterocyclic; aryl or
heteroaryl;
[0100] n is an integer between 5 and 20, inclusive; and
[0101] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
R.sub.3' is phenyl. In other embodiments, R.sub.3' is heteroaryl.
In other embodiments, R.sub.3' is aryl. In still other embodiments,
R.sub.3' is histidine. In certain embodiments, n is 11. In other
embodiments, n is 12. In yet other embodiments, n is 13. In still
other embodiments, n is 14. In certain embodiments, m is 1. In
other embodiments, m is 2. In other embodiments, m is 3. In other
embodiments, m is 4. In other embodiments, m is 5. In other
embodiments, m is 6.
[0102] In certain embodiments, the inventive lipid is of
formula:
##STR00041##
wherein
[0103] R.sub.3' is carbocyclic; heterocyclic; aryl or
heteroaryl;
[0104] n is an integer between 5 and 20, inclusive; and
[0105] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
R.sub.3' is phenyl. In other embodiments, R.sub.3' is heteroaryl.
In other embodiments, R.sub.3' is aryl. In still other embodiments,
R.sub.3' is histidine. In certain embodiments, n is 11. In other
embodiments, n is 12. In yet other embodiments, n is 13. In still
other embodiments, n is 14. In certain embodiments, m is 1. In
other embodiments, m is 2. In other embodiments, m is 3. In other
embodiments, m is 4. In other embodiments, m is 5. In other
embodiments, m is 6.
[0106] In certain embodiments, the inventive lipid is of
formula:
##STR00042##
wherein
[0107] n is an integer between 5 and 20, inclusive; and
[0108] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
n is 11. In other embodiments, n is 12. In yet other embodiments, n
is 13. In still other embodiments, n is 14. In certain embodiments,
m is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0109] In certain embodiments, the inventive lipid is of
formula:
##STR00043##
wherein
[0110] n is an integer between 5 and 20, inclusive; and
[0111] m is an integer between 1 and 10, inclusive; and
pharmaceutically acceptable salts thereof. In certain embodiments,
n is 11. In other embodiments, n is 12. In yet other embodiments, n
is 13. In still other embodiments, n is 14. In certain embodiments,
m is 1. In other embodiments, m is 2. In other embodiments, m is 3.
In other embodiments, m is 4. In other embodiments, m is 5. In
other embodiments, m is 6.
[0112] The present invention also provides amino lipids prepared
from reacting acrylates with diamines, triamines, or polyamines.
The amino moieties are completely or partially reacted with
acrylate or acrylamides. Also, as would be appreciated by one of
skill in this art, amino lipids with different number of acrylate
or acrylamide tails will result in various isomers. These various
forms of the linventive lipids are prepared individually, or the
lipid is prepared as a mixture and then purified from the other
forms. A single form mya be used in a composition, or a mixture of
forms may be used.
[0113] The tails of the inventive amino lipids may also be the same
or different. Non-exhaustively reacted amino groups may be reacted
with a second acrylate, second acrylamide, or other electrophiles
to created a mixed amino lipid. Again, various isomeric forms may
be prepared and may optionally be purified.
[0114] In certain embodiments, the lipids of the present invention
are of the formula (II):
##STR00044##
wherein A is selected from the group consisting of cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched heteroaliphatic; substituted or
unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; and substituted or
unsubstituted, branched or unbranched heteroaryl;
[0115] V is selected from the group consisting of C.dbd.O, C.dbd.S,
S.dbd.O, and SO.sub.2;
[0116] R.sub.1 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; and --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0117] R.sub.2 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.B;
--C(.dbd.O)R.sub.B; --CO.sub.2R.sub.B; --CN; --SCN; --SR.sub.B;
--SOR.sub.B; --SO.sub.2R.sub.B; --NO.sub.2; --N.sub.3;
--N(R.sub.B).sub.2; --NHC(.dbd.O)R.sub.B;
--NR.sub.BC(.dbd.O)N(R.sub.B).sub.2; --OC(.dbd.O)OR.sub.B;
--OC(.dbd.O)R.sub.B; --OC(.dbd.O)N(R.sub.B).sub.2;
--NR.sub.BC(.dbd.O)OR.sub.B; or --C(R.sub.B).sub.3; wherein each
occurrence of R.sub.B is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0118] wherein R.sub.1 and R.sub.2 may be taken together to form a
cyclic structure;
[0119] R.sub.3 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0120] R.sub.4 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.D;
--C(.dbd.O)R.sub.D; --CO.sub.2R.sub.D; --CN; --SCN; --SR.sub.D;
--SOR.sub.D; --SO.sub.2R.sub.D; --NO.sub.2; --N.sub.3;
--N(R.sub.D).sub.2; --NHC(.dbd.O)R.sub.D;
--NR.sub.CC(.dbd.O)N(R.sub.D).sub.2; --OC(.dbd.O)OR.sub.D;
--OC(.dbd.O)R.sub.D; --OC(.dbd.O)N(R.sub.D).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.D; or --C(R.sub.D).sub.3; wherein each
occurrence of R.sub.D is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0121] wherein R.sub.3 and R.sub.4 may be taken together to form a
cyclic structure; [0122] each occurrence of R.sub.5 is
independently selected from the group consisting of hydrogen and
C.sub.1-C.sub.6 alkyl;
[0123] each occurrence of R.sub.6 is independently selected from
the group consisting of hydrogen and C.sub.1-C.sub.6 alkyl; and
salts thereof. In certain embodiments, the lipid is prepared using
amine 95, 96, 99, 100, 103, and 109 in FIG. 1A. In certain
embodiments, the lipid is prepared using amine 99 in FIG. 1A. In
certain embodiments, the lipid is prepared using amine 100 in FIG.
1A. In certain embodiments, the lipid is prepared using acrylate
ND, NF, NP, LF, and LG in FIG. 1A. In certain embodiments, the
lipid is prepared using acrylate ND in FIG. 1A. In certain
embodiments, the lipid is prepared using acrylate NF in FIG. 1A. In
certain embodiments, the lipid is prepared using acrylate NP in
FIG. 1A.
[0124] In certain embodiments, the tertiary amine of formula (II)
is protonated or alkylated to form a compound of formula (IIa):
##STR00045##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and V
are defined above;
[0125] each occurrence of R.sub.7 is hydrogen or C.sub.1-C.sub.6
aliphatic, preferably C.sub.1-C.sub.6 alkyl, more preferably
hydrogen or methyl; [0126] each dashed line represents a bond or
the absence of a bond, wherein when the dashed line represents a
bond, the attached nitrogen is positively charged; and
[0127] X is any anion. Possible anions include fluoride, chloride,
bromide, iodide, sulfate, bisulfate, phosphate, nitrate, acetate,
fumarate, oleate, citrate, valerate, maleate, oxalate,
isonicotinate, lactate, salicylate, tartrate, tannate,
pantothenate, bitartrate, ascorbate, succinate, gentisinate,
gluconate, glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate). In certain
embodiments, both dashed lines presents bonds, and both nitrogen
atoms are positively charged.
[0128] In certain embodiments, A is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched aliphatic or
heteroaliphatic group. In certain embodiments, A is a substituted
or unsubstituted, branched or unbranched aliphatic group. In
certain particular embodiments, A is a substituted or
unsubstituted, branched or unbranched alkyl group. In certain
embodiments, A is an unsubstituted, C.sub.1-C.sub.6 straight chain
alkyl group. In other embodiments, A is a polyethylene group. In
yet other embodiments, A is a polyethylene glycol moiety. In
certain embodiments, A, the two nitrogen atoms attached to A,
R.sub.3 and R.sub.4 form a heterocyclic ring. In certain
embodiments, the ring is aromatic. In other embodiments, the ring
is non-aromatic. In certain embodiments,
##STR00046##
is selected from the group consisting of:
##STR00047##
In certain particular embodiments,
##STR00048##
In certain embodiments,
##STR00049##
In certain embodiments, A is
##STR00050##
wherein n is an interger between 0 and 10, inclusive.
[0129] In certain embodiments, V is C.dbd.O. In other embodiments,
V is C.dbd.S. In yet other embodiments, V is S.dbd.O. In still
other embodiments, V is SO.sub.2.
[0130] In certain embodiments, R.sub.1 is hydrogen. In other
embodiments, R.sub.1 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.1 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocylic 5- or 6-membered ring system. In
certain embodiments, R.sub.1 is --OR.sub.A, --SR.sub.A,
--N(R.sub.A).sub.2, or --NHR.sub.A. In certain embodiments, R.sub.1
is --OR.sub.A. In other embodiments, R.sub.1 is --N(R.sub.A).sub.2
or --NHR.sub.A. In certain embodiments, R.sub.A is hydrogen. In
certain embodiments, R.sub.A is not hydrogen. In other embodiments,
R.sub.A is a cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic or heteroaliphatic moiety. In
certain embodiments, R.sub.A is an acyclic, substituted or
unsubstituted aliphatic moiety. In certain embodiments, R.sub.A is
an unsubstituted, straight chain alkyl group with at least 5
carbons. In certain other embodiments, R.sub.A is an acyclic,
unsubstituted, unbranched aliphatic moiety, preferably
C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In certain
embodiments, R.sub.A is an unsubstituted, straight chain alkyl
group, preferably C.sub.6-C.sub.30, more preferably
C.sub.10-C.sub.20. In certain embodiments, R.sub.1 is --OR.sub.A,
wherein R.sub.A is an unsubstituted, unbranched C.sub.9 alkyl
chain. In certain embodiments, R.sub.1 is --OR.sub.A, wherein
R.sub.A is an unsubstituted, unbranched C.sub.10 alkyl chain. In
certain embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.11 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.A is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0131] In certain embodiments, R.sub.2 is hydrogen. In other
embodiments, R.sub.2 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.2 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocylic 5- or 6-membered ring system. In
certain embodiments, R.sub.2 is --OR.sub.B, --SR.sub.B,
--N(R.sub.B).sub.2, or --NHR.sub.B. In certain embodiments, R.sub.2
is --OR.sub.B. In other embodiments, R.sub.2 is --N(R.sub.B).sub.2
or --NHR.sub.B. In certain embodiments, R.sub.B is hydrogen. In
certain embodiments, R.sub.B is not hydrogen. In other embodiments,
R.sub.B is a cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic or heteroaliphatic moiety. In
certain embodiments, R.sub.B is an acyclic, substituted or
unsubstituted aliphatic moiety. In certain embodiments, R.sub.B is
an unsubstituted, straight chain alkyl group with at least 5
carbons. In certain other embodiments, R.sub.B is an acyclic,
unsubstituted, unbranched aliphatic moiety, preferably
C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In certain
embodiments, R.sub.B is an unsubstituted, straight chain alkyl
group, preferably C.sub.6-C.sub.30, more preferably
C.sub.10-C.sub.20. In certain embodiments, R.sub.2 is --OR.sub.B,
wherein R.sub.B is an unsubstituted, unbranched C.sub.9 alkyl
chain. In certain embodiments, R.sub.2 is --OR.sub.B, wherein
R.sub.B is an unsubstituted, unbranched C.sub.10 alkyl chain. In
certain embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.11 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.B is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0132] In certain embodiments, R.sub.3 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched aliphatic
moiety. In other embodiments, R.sub.3 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched
heteroaliphatic moiety. In certain embodiments, R.sub.3 is an
aliphatic moiety substituted with one or more hydroxyl groups. In
other embodiments, R.sub.3 is an aliphatic moiety substituted with
one or more amino, alkylamino, or dialkylamino groups. In certain
embodiments, R.sub.3 is C.sub.1-C.sub.6 alkyl. In certain
embodiments, R.sub.3 is methyl. In certain embodiments, R.sub.3 is
ethyl. In other embodiments, R.sub.3 is n-propyl. In other
embodiments, R.sub.3 is iso-propyl. In certain embodiments, R.sub.3
is hydrogen. In certain embodiments, R.sub.3 is a heteroaliphatic
moiety. In certain embodiments, R.sub.3 is cyclic aliphatic,
preferably a monocylic ring system with a 5- or 6-membered ring. In
other embodiments, R.sub.3 is aryl or heteroaryl, preferably a
monocyclic ring system with a 5- or 6-membered ring. In certain
embodiments, R.sub.3 is
##STR00051##
wherein n is an interger between 0 and 10, inclusive; and R.sub.3'
is hydrogen, aliphatic, heteroaliphatic, carbocyclic, heterocyclic,
aryl, acyl, or heteroaryl. In certain embodiments, R.sub.3' is
hydrogen, In other embodiments, R.sub.3' is C.sub.1-C.sub.6 alkyl.
In yet other embodiments, R.sub.3' is acyl (e.g., acetyl). In
certain embodiments. R.sub.3 is
##STR00052##
In other embodiments, R.sub.3 is
##STR00053##
[0133] In certain embodiments, R.sub.4 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched aliphatic
moiety. In other embodiments, R.sub.4 is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched
heteroaliphatic moiety. In certain embodiments, R.sub.4 is an
aliphatic moiety substituted with one or more hydroxyl groups. In
other embodiments, R.sub.4 is an aliphatic moiety substituted with
one or more amino, alkylamino, or dialkylamino groups. In certain
embodiments, R.sub.4 is C.sub.1-C.sub.6 alkyl. In certain
embodiments, R.sub.3 is methyl. In certain embodiments, R.sub.3 is
ethyl. In other embodiments, R.sub.3 is n-propyl. In other
embodiments, R.sub.3 is iso-propyl. In certain embodiments, R.sub.4
is hydrogen. In certain embodiments, R.sub.4 is a heteroaliphatic
moiety. In certain embodiments, R.sub.3 is cyclic aliphatic,
preferably a monocylic ring system with a 5- or 6-membered ring. In
other embodiments, R.sub.4 is aryl or heteroaryl, preferably a
monocyclic ring system with a 5- or 6-membered ring. In certain
embodiments, R.sub.4 is
##STR00054##
wherein n is an interger between 0 and 10, inclusive; and R.sub.4'
is hydrogen, aliphatic, heteroaliphatic, carbocyclic, heterocyclic,
aryl, acyl, or heteroaryl. In certain embodiments, R.sub.4' is
hydrogen, In other embodiments, R.sub.4' is C.sub.1-C.sub.6 alkyl.
In yet other embodiments, R.sub.4' is acyl (e.g., acetyl). In
certain embodiments. R.sub.4 is
##STR00055##
In other embodiments, R.sub.4 is
##STR00056##
[0134] In certain embodiments, R.sub.3 and R.sub.4 are the same. In
other embodiments, R.sub.3 and R.sub.4 are different.
[0135] In certain embodiments, each occurrence of R.sub.5 is
hydrogen. In certain embodiments, at least one occurrence of
R.sub.5 is methyl and the other occurrences are hydrogen. In
certain embodiments, at least two occurrences of R.sub.5 are
methyl, and the other occurrences are hydrogen. In other
embodiments, at least two occurrences of R.sub.5 are hydrogen.
[0136] In certain embodiments, each occurrence of R.sub.6 is
hydrogen. In certain other embodiments, at least two occurrences of
R.sub.6 are hydrogen. In certain embodiments, at least one
occurrence of R.sub.6 is methyl, and the other occurrences are
hydrogen. In certain embodiments, at least two occurrences of
R.sub.6 are methyl, and the other occurrences are hydrogen.
[0137] In certain embodiments,
##STR00057##
which are attached to N are the same. In other embodiments,
##STR00058##
which are attached to N are the same and are different than
R.sub.3. In yet other embodiments,
##STR00059##
and R.sub.3 are all different.
[0138] In certain embodiments,
##STR00060##
in formulae (II) and (IIa) are selected from the group consisting
of:
##STR00061##
In certain embodiments, the lipids are prepared using acrylates LC,
LD, LE, LF, and LG in FIG. 1A.
[0139] In certain embodiments,
##STR00062##
in formulae (II) and (IIa) are selected from the group consisting
of:
##STR00063##
In certain embodiments, the lipids are prepared using acrylates NC,
ND, NF, NG, or NP in FIG. 1A. In certain embodiments, the lipids
are prepared using acrylate ND. In other embodiments, the lipids
are prepared using acrylate NF. In other embodiments, the lipids
are prepared using acrylate NP.
[0140] In certain embodiments,
##STR00064##
in formulae (II) and (IIa) are the same. In other embodiments,
##STR00065##
formulae (II) and (IIa) are different.
[0141] In certain embodiments,
##STR00066##
is selected from the group consisting of:
##STR00067##
[0142] In certain embodiments,
##STR00068##
is selected from the group consisting of:
##STR00069##
wherein R.sub.3 and R.sub.4 form a cyclic structure.
[0143] In other embodiments, the lipids of the present invention
are of the formula (III):
##STR00070##
wherein
[0144] A is selected from the group consisting of cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched heteroaliphatic; substituted or
unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; and substituted or
unsubstituted, branched or unbranched heteroaryl;
[0145] V is selected from the group consisting of C.dbd.O, C.dbd.S,
S.dbd.O, and SO.sub.2;
[0146] n is an integer between 0 and 10, inclusive;
[0147] R.sub.1 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; and --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0148] R.sub.2 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.B;
--C(.dbd.O)R.sub.B; --CO.sub.2R.sub.B; --CN; --SCN; --SR.sub.B;
--SOR.sub.B; --SO.sub.2R.sub.B; --NO.sub.2; --N.sub.3;
--N(R.sub.B).sub.2; --NHC(.dbd.O)R.sub.B;
--NR.sub.BC(.dbd.O)N(R.sub.B).sub.2; --OC(.dbd.O)OR.sub.B;
--OC(.dbd.O)R.sub.B; --OC(.dbd.O)N(R.sub.B).sub.2;
--NR.sub.BC(.dbd.O)OR.sub.B; or --C(R.sub.B).sub.3; wherein each
occurrence of R.sub.B is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0149] wherein R.sub.1 and R.sub.2 may be taken together to form a
cyclic structure;
[0150] R.sub.3 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.C;
--C(.dbd.O)R.sub.C; --CO.sub.2R.sub.C; --CN; --SCN; --SR.sub.C;
--SOR.sub.C; --SO.sub.2R.sub.C; --NO.sub.2; --N.sub.3;
--N(R.sub.C).sub.2; --NHC(.dbd.O)R.sub.C;
--NR.sub.CC(.dbd.O)N(R.sub.C).sub.2; --OC(.dbd.O)OR.sub.C;
--OC(.dbd.O)R.sub.C; --OC(.dbd.O)N(R.sub.C).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.C; or --C(R.sub.C).sub.3; wherein each
occurrence of R.sub.C is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0151] R.sub.4 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.D;
--C(.dbd.O)R.sub.D; --CO.sub.2R.sub.D; --CN; --SCN; --SR.sub.D;
--SOR.sub.D; --SO.sub.2R.sub.D; --NO.sub.2; --N.sub.3;
--N(R.sub.D).sub.2; --NHC(.dbd.O)R.sub.D;
--NR.sub.CC(.dbd.O)N(R.sub.D).sub.2; --OC(.dbd.O)OR.sub.D;
--OC(.dbd.O)R.sub.D; --OC(.dbd.O)N(R.sub.D).sub.2;
--NR.sub.CC(.dbd.O)OR.sub.D; or --C(R.sub.D).sub.3; wherein each
occurrence of R.sub.D is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0152] wherein R.sub.3 and R.sub.4 may be taken together to form a
cyclic structure;
[0153] each occurrence of R.sub.5 is independently selected from
the group consisting of hydrogen and C.sub.1-C.sub.6 alkyl;
[0154] each occurrence of R.sub.6 is independently selected from
the group consisting of hydrogen and C.sub.1-C.sub.6 alkyl;
[0155] R.sub.7 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.G;
--C(.dbd.O)R.sub.G; --CO.sub.2R.sub.G; --CN; --SCN; --SR.sub.G;
--SOR.sub.G; --SO.sub.2R.sub.G; --NO.sub.2; --N.sub.3;
--N(R.sub.G).sub.2; --NHC(.dbd.O)R.sub.G;
--NR.sub.GC(.dbd.O)N(R.sub.G).sub.2; --OC(.dbd.O)OR.sub.G;
--OC(.dbd.O)R.sub.G; --OC(.dbd.O)N(R.sub.G).sub.2;
--NR.sub.GC(.dbd.O)OR.sub.G; and --C(R.sub.G).sub.3; wherein each
occurrence of R.sub.G is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety and salts
thereof. In certain embodiments, n is 0. In other embodiments, n is
1. In still other embodiments, n is 2. In other embodiments, n is
3. In yet other embodiments, n is 4. In other embodiments, n is 5.
In other embodiments, n is 6. In certain embodiments, the lipid is
prepared using amine 98. In other embodiments, the lipid is
prepared using amine 100.
[0156] In certain embodiments, the tertiary amine of formula (III)
is protonated or alkylated to form a compound of formula
(IIIa):
##STR00071##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, n, and V are defined above;
[0157] each occurrence of R.sub.8 is hydrogen or C.sub.1-C.sub.6
aliphatic, preferably C.sub.1-C.sub.6 alkyl, more preferably
hydrogen or methyl;
[0158] each dashed line represents a bond or the absence of a bond,
wherein when the dashed line represents a bond, the attached
nitrogen is positively charged; and
[0159] X is any anion. Possible anions include fluoride, chloride,
bromide, iodide, sulfate, bisulfate, phosphate, nitrate, acetate,
fumarate, oleate, citrate, valerate, maleate, oxalate,
isonicotinate, lactate, salicylate, tartrate, tannate,
pantothenate, bitartrate, ascorbate, succinate, gentisinate,
gluconate, glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate). In certain
embodiments, both dashed lines presents bonds, and both nitrogen
atoms are positively charged.
[0160] In certain embodiments, A is a cyclic or acyclic,
substituted or unsubstituted, branched or unbranched aliphatic or
heteroaliphatic group. In certain embodiments, A is a substituted
or unsubstituted, branched or unbranched aliphatic group. In
certain particular embodiments, A is a substituted or
unsubstituted, branched or unbranched alkyl group. In certain
embodiments, A is an unsubstituted, C.sub.1-C.sub.6 straight chain
alkyl group. In other embodiments, A is a polyethylene group. In
yet other embodiments, A is a polyethylene glycol moiety. In
certain embodiments, A, the two nitrogen atoms attached to A,
R.sub.3 and R.sub.4 form a heterocyclic ring. In certain
embodiments, the ring is aromatic. In other embodiments, the ring
is non-aromatic.
[0161] In certain embodiments, V is C.dbd.O. In other embodiments,
V is C.dbd.S. In yet other embodiments, V is S.dbd.O. In still
other embodiments, V is SO.sub.2.
[0162] In certain embodiments, R.sub.1 is hydrogen. In other
embodiments, R.sub.1 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.1 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocylic 5- or 6-membered ring system. In
certain embodiments, R.sub.1 is --OR.sub.A, --SR.sub.A,
--N(R.sub.A).sub.2, or --NHR.sub.A. In certain embodiments, R.sub.1
is --OR.sub.A. In other embodiments, R.sub.1 is --N(R.sub.A).sub.2
or --NHR.sub.A. In certain embodiments, R.sub.A is hydrogen. In
other embodiments, R.sub.A is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.A is an acyclic, substituted
or unsubstituted aliphatic moiety. In certain other embodiments,
R.sub.A is an acyclic, unsubstituted, unbranched aliphatic moiety,
preferably C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In
certain embodiments, R.sub.A is an unsubstituted, straight chain
alkyl group, preferably C.sub.6-C.sub.30, more preferably
C.sub.10-C.sub.20. In certain embodiments, R.sub.1 is --OR.sub.A,
wherein R.sub.A is an unsubstituted, unbranched C.sub.9 alkyl
chain. In certain embodiments, R.sub.1 is --OR.sub.A, wherein
R.sub.A is an unsubstituted, unbranched C.sub.10 alkyl chain. In
certain embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.ii alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.1 is --OR.sub.A, wherein R.sub.A is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.A is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0163] In certain embodiments, R.sub.2 is hydrogen. In other
embodiments, R.sub.2 is a cyclic or acyclic, substituted or
unsubstituted, branched or un branched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.2 is a substituted or
unsubstituted aryl or heteroaryl moiety. Preferably, the aryl or
heteroaryl moiety is a monocylic 5- or 6-membered ring system. In
certain embodiments, R.sub.2 is --OR.sub.B, --SR.sub.B,
--N(R.sub.B).sub.2, or --NHR.sub.B. In certain embodiments, R.sub.2
is --OR.sub.B. In other embodiments, R.sub.2 is --N(R.sub.B).sub.2
or --NHR.sub.B. In certain embodiments, R.sub.B is hydrogen. In
other embodiments, R.sub.B is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic or heteroaliphatic
moiety. In certain embodiments, R.sub.B is an acyclic, substituted
or unsubstituted aliphatic moiety. In certain other embodiments,
R.sub.B is an acyclic, unsubstituted, unbranched aliphatic moiety,
preferably C.sub.6-C.sub.30, more preferably C.sub.10-C.sub.20. In
certain embodiments, R.sub.B is an unsubstituted, straight chain
alkyl group, preferably C.sub.6-C.sub.30, more preferably
C.sub.10-C.sub.20. In certain embodiments, R.sub.2 is --OR.sub.B,
wherein R.sub.B is an unsubstituted, unbranched C.sub.9 alkyl
chain. In certain embodiments, R.sub.2 is --OR.sub.B, wherein
R.sub.B is an unsubstituted, unbranched C.sub.10 alkyl chain. In
certain embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.11 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.12 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.13 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.14 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.15 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.16 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.17 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.A, wherein R.sub.B is an
unsubstituted, unbranched C.sub.18 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.19 alkyl chain. In certain
embodiments, R.sub.2 is --OR.sub.B, wherein R.sub.B is an
unsubstituted, unbranched C.sub.20 alkyl chain. In yet other
embodiments, R.sub.B is a substituted or unsubstituted aryl or
heteroaryl moiety.
[0164] In certain embodiments, R.sub.3 is hydrogen. In certain
embodiments, R.sub.3 is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic moiety. In other
embodiments, R.sub.3 is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic moiety. In
certain embodiments, R.sub.3 is an aliphatic moiety substituted
with one or more hydroxyl groups. In other embodiments, R.sub.3 is
an aliphatic moiety substituted with one or more amino, alkylamino,
or dialkylamino groups. In certain embodiments, R.sub.3 is
C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.3 is hydrogen.
In certain embodiments, R.sub.3 is a heteroaliphatic moiety. In
certain embodiments, R.sub.3 is cyclic aliphatic, preferably a
monocylic ring system with a 5- or 6-membered ring. In other
embodiments, R.sub.3 is aryl or heteroaryl, preferably a monocyclic
ring system with a 5- or 6-membered ring. In certain embodiments,
R.sub.3 is
##STR00072##
wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6, and V are defined as
above.
[0165] In other embodiments, R.sub.4 is hydrogen. In certain
embodiments, R.sub.4 is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched aliphatic moiety. In other
embodiments, R.sub.4 is a cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic moiety. In
certain embodiments, R.sub.4 is an aliphatic moiety substituted
with one or more hydroxyl groups. In other embodiments, R.sub.4 is
an aliphatic moiety substituted with one or more amino, alkylamino,
or dialkylamino groups. In certain embodiments, R.sub.4 is
C.sub.1-C.sub.6 alkyl. In certain embodiments, R.sub.4 is hydrogen.
In certain embodiments, R.sub.4 is a heteroaliphatic moiety. In
certain embodiments, R.sub.3 is cyclic aliphatic, preferably a
monocylic ring system with a 5- or 6-membered ring. In other
embodiments, R.sub.4 is aryl or heteroaryl, preferably a monocyclic
ring system with a 5- or 6-membered ring. In certain embodiments,
R.sub.4 is
##STR00073##
wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6, and V are defined as
above.
[0166] In certain embodiments, R.sub.3 and R.sub.4 are the same. In
other embodiments, R.sub.3 and R.sub.4 are different. In certain
embodiments, both R.sub.3 and R.sub.4 are hydrogen. In certain
embodiments, only one of R.sub.3 and R.sub.4 is hydrogen. In
certain embodiments, both R.sub.3 and R.sub.4 are
##STR00074##
wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6, and V are defined as
above. In certain embodiments, one of R.sub.3 and R.sub.4 is
##STR00075##
wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6, and V are defined as
above; and the other is hydrogen. In certain embodiments, both
R.sub.3 and R.sub.4 are
##STR00076##
wherein R.sub.1 is as defined above. In certain embodiments, one of
R.sub.3 and R.sub.4 is
##STR00077##
wherein R.sub.1 is defined as above; and the other is hydrogen.
[0167] In certain embodiments, each occurrence of R.sub.5 is
hydrogen. In certain embodiments, at least one occurrence of
R.sub.5 is methyl and the other occurrences are hydrogen. In
certain embodiments, at least two occurrences of R.sub.5 are
methyl, and the other occurrences are hydrogen. In other
embodiments, at least two occurrences of R.sub.5 are hydrogen.
[0168] In certain embodiments, each occurrence of R.sub.6 is
hydrogen. In certain other embodiments, at least two occurrences of
R.sub.6 are hydrogen. In certain embodiments, at least one
occurrence of R.sub.6 is methyl, and the other occurrences are
hydrogen. In certain embodiments, at least two occurrences of
R.sub.6 are methyl, and the other occurrences are hydrogen.
[0169] In certain embodiments, R.sub.7 is
##STR00078##
wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6, and V are defined as
above. In certain embodiments, R.sub.7,
##STR00079##
are the same. In other embodiments, R.sub.7,
##STR00080##
are different. In certain embodiments, R.sub.7 and
##STR00081##
are the same. In other embodiments, R.sub.7 and
##STR00082##
are the same. In certain embodiments, all R.sub.7 are the same.
[0170] In certain embodiments,
##STR00083##
which are attached to N are the same. In other embodiments,
##STR00084##
which are attached to N are the same and are different than R.sub.3
or R.sub.4. In yet other embodiments,
##STR00085##
R.sub.3, and R.sub.4 are all different. In certain embodiments,
R.sub.3 and R.sub.4 are the same. In other embodiments, R.sub.3 and
R.sub.4 are different
[0171] In certain embodiments,
##STR00086##
in formulae (III) and (IIIa) are selected from the group consisting
of:
##STR00087##
In certain embodiments, the lipids are prepared using acrylates LC,
LD, LE, LF, and LG in FIG. 1A.
[0172] In certain embodiments,
##STR00088##
in formulae (III) and (IIIa) are selected from the group consisting
of:
##STR00089##
In certain embodiments, the lipids are prepared using acrylates NC,
ND, NF, NG, and NP in FIG. 1A. In certain embodiments, the lipids
are prepared using acrylate ND. In other embodiments, the lipids
are prepared using acrylate NF.
[0173] In certain embodiments,
##STR00090##
is selected from the group consisting of:
##STR00091##
In certain particular embodiments,
##STR00092##
In certain particular embodiments,
##STR00093##
and n is 0, 1, 2, 3, 4, 5, or 6. In certain particular
embodiments,
##STR00094##
and n is 2. In certain embodiments,
##STR00095##
In certain embodiments,
##STR00096##
and n is 0, 1, 2, 3, 4, 5, or 6. In certain embodiments,
##STR00097##
and n is 2.
[0174] In certain embodiments, the lipid is of the formula
(IV):
##STR00098##
wherein
[0175] each occurrence of x is an integer between 1 and 10,
inclusive; preferably, between 1 and 6, inclusive;
[0176] y is an integer between 0 and 10, inclusive; preferably,
between 0 and 6, inclusive;
[0177] each occurrence of R.sub.7 is hydrogen; substituted or
unsubstituted, branched or unbranched aliphatic; substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted aryl; substituted or unsubstituted heteroaryl;
or
##STR00099##
[0178] R.sub.1 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; and --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety; and salts
thereof. In certain embodiments, x is 1, 2, 3, 4, or 5. In certain
particular embodiments, x is 1. In other particular embodiments, x
is 2. In certain embodiments, y is 0. In certain embodiments, y is
1. In other embodiments, y is 2. In yet other embodiments, y is 3.
In still other embodiments, y is 4. In certain embodiments, R.sub.1
is --OR.sub.A. In other embodiments, R.sub.1 is --NHR.sub.A. In
certain embodiments, at least one R.sub.1 is C.sub.1-C.sub.20
alkyl. In certain embodiments, all R.sub.7 are of the formula
##STR00100##
In certain embodiments, at least one R.sub.7 is branched or
unbranched, substituted or unsubstituted aliphatic. In certain
embodiments, at least one R.sub.7 is C.sub.1-C.sub.20 alkyl. In
certain embodiments, at least one R.sub.7 is C.sub.1-C.sub.12
alkyl. In certain embodiments, at least one R.sub.7 is branched or
unbranched, substituted or unsubstituted heteroaliphatic. In
certain embodiments, at least one R.sub.7 is
##STR00101##
wherein k is an interger between 0 and 10, inclusive, and R.sub.7'
is hydrogen or C.sub.1-6alkyl. In certain embodiments, at least one
R.sub.7 is
##STR00102##
In other embodiments, at least one R.sub.7 is
##STR00103##
In other embodiments, at least one R.sub.7 is a hydrogen. In other
embodiments, at least two R.sub.7 are each hydrogen. In still other
embodiments, at least three R.sub.7 are each hydrogen. In still
further embodiments, at least four R.sub.7 are each hydrogen.
[0179] In certain embodiments, each R.sub.7 in formulae (IV) is
independently selected from the group consisting of hydrogen
and
##STR00104##
[0180] In certain embodiments, each R.sub.7 in formulae (IV) is
independently selected from the group consisting of hydrogen
and
##STR00105##
[0181] In certain embodiments, the lipid is of the formula (V),
(VI), or (VII):
##STR00106##
wherein
[0182] x is an integer between 1 and 10, inclusive; preferably,
between 1 and 6, inclusive; more preferably, between 1 and 3,
inclusive;
[0183] each occurrence of R.sub.7 is hydrogen or
##STR00107##
[0184] R.sub.1 is selected from the group consisting of hydrogen;
halogen; cyclic or acyclic, substituted or unsubstituted, branched
or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; --OR.sub.A;
--C(.dbd.O)R.sub.A; --CO.sub.2R.sub.A; --CN; --SCN; --SR.sub.A;
--SOR.sub.A; --SO.sub.2R.sub.A; --NO.sub.2; --N.sub.3;
--N(R.sub.A).sub.2; --NHC(.dbd.O)R.sub.A;
--NR.sub.AC(.dbd.O)N(R.sub.A).sub.2; --OC(.dbd.O)OR.sub.A;
--OC(.dbd.O)R.sub.A; --OC(.dbd.O)N(R.sub.A).sub.2;
--NR.sub.AC(.dbd.O)OR.sub.A; and --C(R.sub.A).sub.3; wherein each
occurrence of R.sub.A is independently a hydrogen; a protecting
group; halogen; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched aliphatic; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched heteroaliphatic; substituted
or unsubstituted, branched or unbranched acyl; substituted or
unsubstituted, branched or unbranched aryl; substituted or
unsubstituted, branched or unbranched heteroaryl; an acyl moiety;
alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,
dialkylamino, heteroaryloxy; or heteroarylthio moiety;
[0185] each occurrence of R.sub.8 is independently hydrogen,
C.sub.1-C.sub.6 alkyl, hydroxy-C.sub.1-C.sub.6-alkyl; or
##STR00108##
wherein k is an interger between 0 and 10, inclusive, and R.sub.8'
is hydrogen or C.sub.1-6alkyl; and salts thereof. In certain
embodiments, x is 1, 2, 3, 4, or 5. In certain particular
embodiments, x is 1. In other particular embodiments, x is 2. In
other embodiments, x is 3. In certain embodiments, R.sub.1 is
--OR.sub.A. In other embodiments, R.sub.1 is --NHR.sub.A. In
certain embodiments, all R.sub.7 are of the formula
##STR00109##
In other embodiments, at least one R.sub.7 is a hydrogen. In other
embodiments, at least two R.sub.7 are each hydrogen. In still other
embodiments, at least three R.sub.7 are each hydrogen. In still
further embodiments, at least four R.sub.7 are each hydrogen. In
certain embodiments, all R.sub.8 are the same. In certain
particular embodiments, R.sub.8 is hydrogen. In certain
embodiments, R.sub.8 is methyl. In other embodiments, R.sub.8 is
ethyl. In yet other embodiments, R.sub.8 is hydroxymethyl. In still
other embodiments, R.sub.8 is hydroxyethyl.
[0186] In certain embodiments, each R.sub.7 in formula (V), (VI),
or (VII) is independently selected from the group consisting of
hydrogen and
##STR00110##
[0187] In certain embodiments, each R.sub.7 in formula (V), (VI),
or (VII) is independently selected from the group consisting of
hydrogen and
##STR00111##
[0188] Exemplary compounds of the formulae (V), (VI), and (VII) are
of the formula:
##STR00112##
In certain embodiments, the lipid is one of the formulae:
##STR00113##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0189] In other embodiments, the compound is of one of the
formulae:
##STR00114##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0190] In certain embodiments, the lipid is of one of the
formulae:
##STR00115##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0191] In certain embodiments, the lipid is of one of the
formulae:
##STR00116##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0192] In certain embodiments, the lipid is of one of the
formulae:
##STR00117## ##STR00118## ##STR00119## ##STR00120##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0193] In certain embodiments, the lipid is of one of the
formulae:
##STR00121## ##STR00122## ##STR00123## ##STR00124##
wherein n is an integer ranging from 1 to 15, inclusive;
preferably, n is an integer ranging from 6 to 12, inclusive, or 1
to 6, inclusive. In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15. In certain particular embodiments,
n is 10, 11, or 12. In certain embodiments, n is 11. In other
embodiments, n is 10. In certain embodiments, each n is
independently an integer ranging from 1 to 15, inclusive. In other
embodiments, all n are the same integer. In certain embodiments,
one n is different from the other n in the compound.
[0194] In another aspect of the invention, the lipid or composition
of lipids of the invention is lipid or composition prepared by
reacting an amine of one of the formula (1-117):
##STR00125## ##STR00126## ##STR00127## ##STR00128##
##STR00129##
with an acrylate of formula:
##STR00130## ##STR00131##
In certain embodiments, one equivalent of amine is reacted with one
equivalent of acrylate. In certain embodiments, one equivalent of
amine is reacted with one, two, three, four, five, six, or more
equivalents of acrylate. In certain embodiments, the amount of
acrylate is limiting to prevent the functionalization of all amino
groups. The resulting lipid or lipid composition in these instances
contain secondary amino groups or primary amino groups. Lipids
having secondary amines are particular useful in certain instances.
In certain embodiments, amine-containing lipids that have not been
fully functionalize are further reacted with another electrophile
(e.g., an acrylate, acrylamide, alkylating agent, acylating agent,
etc.). Such further functionalization of the amines of the lipid
results in lipids with different tails. One, two, three, four,
five, or more tails may be different from the other tails of the
lipid.
[0195] In certain embodiments, the amine and acrylate are reacted
together neat. In other embodiments, the reaction is done in a
solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH, EtOH, CHCl.sub.3,
hexanes, toluene, benzene, CCl.sub.4, glyme, diethyl ether, etc.).
In certain embodiments, the reaction mixture is heated. In a
particularly preferred embodiment, the reaction mixture is heated
to temperature ranging from 50-150.degree. C. In another
particularly preferred embodiment, the reaction mixture is heated
to approximately 95.degree. C. The reaction may also be catalyzed.
For example, the reaction may be catalyzed by the addition of an
acid, base, or metal. The reaction may be allowed to proceed for
hours, days, or weeks. In certain embodiments, the reaction is
allowed to proceed for 1-7 days, preferably 7 days. The resulting
composition may be used with or without purification. In certain
embodiments, the lipids are subsequently subjected to an alkylation
step (e.g., reaction with methyl iodide) to form quanternary amine
salts. Optionally, various salt forms of the lipids may be
prepared. In certain embodiments, the salts are pharmaceutically
acceptable salts.
[0196] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate NC to form lipid NC98. In certain
embodiments, the lipid NC98 is of one of the formulae
##STR00132## ##STR00133## ##STR00134## ##STR00135##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0197] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate NC to form lipid NC99. In certain
embodiments, the lipid NC99 is of one of the formulae below:
##STR00136## ##STR00137##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0198] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate NC to form lipid NC100. In certain
embodiments, the lipid NC100 is of one of the formulae below:
##STR00138##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0199] In certain embodiments, the lipid is prepared by reacting
amine 20 with acrylate ND to form lipid ND20. In certain
embodiments, the lipid ND20 is of one of the formulae below:
##STR00139##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0200] In certain embodiments, the lipid is prepared by reacting
amine 24 with acrylate ND to form lipid ND24. In certain
embodiments, the lipid ND24 is of one of the formulae
##STR00140##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0201] In certain embodiments, the lipid is prepared by reacting
amine 25 with acrylate ND to form lipid ND25. In certain
embodiments, the lipid ND25 is of one of the formulae below:
##STR00141##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0202] In certain embodiments, the lipid is prepared by reacting
amine 28 with acrylate ND to form lipid ND28. In certain
embodiments, the lipid ND28 is of one of the formulae
##STR00142##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0203] In certain embodiments, the lipid is prepared by reacting
amine 32 with acrylate ND to form lipid ND32. In certain
embodiments, the lipid ND32 is of one of the formulae below:
##STR00143##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0204] In certain embodiments, the lipid is prepared by reacting
amine 36 with acrylate ND to form lipid ND36. In certain
embodiments, the lipid ND36 is of one of the formulae
##STR00144##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0205] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate ND to form lipid ND98. In certain
embodiments, the lipid ND98 is of one of the formulae below:
##STR00145## ##STR00146## ##STR00147## ##STR00148##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0206] In certain embodiments, the lipid is prepared by reacting
amine 94 with acrylate ND to form lipid ND94. In certain
embodiments, the lipid ND94 is of one of the formulae below:
##STR00149##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0207] In certain embodiments, the lipid is prepared by reacting
amine 95 with acrylate ND to form lipid ND95. In certain
embodiments, the lipid ND95 is of one of the formulae below:
##STR00150##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0208] In certain embodiments, the lipid is prepared by reacting
amine 96 with acrylate ND to form lipid ND96. In certain
embodiments, the lipid ND96 is of one of the formulae below:
##STR00151##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0209] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate ND to form lipid ND99. In certain
embodiments, the lipid ND99 is of one of the formulae below:
##STR00152## ##STR00153##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, ND99 is treated with MeI
or another alkylating agent to form lipids of the formulae:
##STR00154## ##STR00155##
[0210] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate ND to form lipid ND100. In certain
embodiments, the lipid ND100 is of one of the formulae below:
##STR00156##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0211] In certain embodiments, the lipid is prepared by reacting
amine 103 with acrylate ND to form lipid ND103. In certain
embodiments, the lipid ND103 is of one of the formulae below:
##STR00157##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0212] In certain embodiments, the lipid is prepared by reacting
amine 109 with acrylate ND to form lipid ND109. In certain
embodiments, the lipid ND109 is of one of the formulae below:
##STR00158##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0213] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate NE to form lipid NE98. In certain
embodiments, the lipid NE98 is of one of the formulae below:
##STR00159## ##STR00160## ##STR00161## ##STR00162##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0214] In certain embodiments, the lipid is prepared by reacting
amine 94 with acrylate NE to form lipid NE94. In certain
embodiments, the lipid NE94 is of one of the formulae below:
##STR00163##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0215] In certain embodiments, the lipid is prepared by reacting
amine 95 with acrylate NE to form lipid NE95. In certain
embodiments, the lipid NE95 is of one of the formulae below:
##STR00164##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0216] In certain embodiments, the lipid is prepared by reacting
amine 96 with acrylate NE to form lipid NE96. In certain
embodiments, the lipid NE96 is of one of the formulae below:
##STR00165##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0217] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate NE to form lipid NE99. In certain
embodiments, the lipid NE99 is of one of the formulae below:
##STR00166## ##STR00167##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, NE99 is treated with MeI
or another alkylating agent to form lipids of the formulae:
##STR00168## ##STR00169##
[0218] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate NE to form lipid NE100. In certain
embodiments, the lipid NE100 is of one of the formulae below:
##STR00170##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0219] In certain embodiments, the lipid is prepared by reacting
amine 103 with acrylate NE to form lipid NE103. In certain
embodiments, the lipid NE103 is of one of the formulae below:
##STR00171##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0220] In certain embodiments, the lipid is prepared by reacting
amine 109 with acrylate NE to form lipid NE109. In certain
embodiments, the lipid NE109 is of one of the formulae below:
##STR00172##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0221] In certain embodiments, the lipid is prepared by reacting
amine 1 with acrylate NF to form lipid NF1. In certain embodiments,
the lipid NF1 is of one of the formulae below:
##STR00173##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0222] In certain embodiments, the lipid is prepared by reacting
amine 10 with acrylate NF to form lipid NF10. In certain
embodiments, the lipid NF10 is of one of the formulae below:
##STR00174##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0223] In certain embodiments, the lipid is prepared by reacting
amine 11 with acrylate NF to form lipid NF11. In certain
embodiments, the lipid NF10 is of one of the formulae below:
##STR00175##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0224] In certain embodiments, the lipid is prepared by reacting
amine 20 with acrylate NF to form lipid NF20. In certain
embodiments, the lipid NF20 is of one of the formulae below:
##STR00176##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0225] In certain embodiments, the lipid is prepared by reacting
amine 25 with acrylate NF to form lipid NF25. In certain
embodiments, the lipid NF25 is of one of the formulae below:
##STR00177##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0226] In certain embodiments, the lipid is prepared by reacting
amine 28 with acrylate NF to form lipid NF28. In certain
embodiments, the lipid NF28 is of one of the formulae below:
##STR00178##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0227] In certain embodiments, the lipid is prepared by reacting
amine 32 with acrylate NF to form lipid NF32. In certain
embodiments, the lipid NF32 is of one of the formulae below:
##STR00179##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0228] In certain embodiments, the lipid is prepared by reacting
amine 36 with acrylate NF to form lipid NF36. In certain
embodiments, the lipid NF36 is of one of the formulae below:
##STR00180##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0229] In certain embodiments, the lipid is prepared by reacting
amine 60 with acrylate NF to form lipid NF60. In certain
embodiments, the lipid NF60 is of one of the formulae below:
##STR00181##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0230] In certain embodiments, the lipid is prepared by reacting
amine 61 with acrylate NF to form lipid NF61. In certain
embodiments, the lipid NF61 is of one of the formulae below:
##STR00182##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0231] In certain embodiments, the lipid is prepared by reacting
amine 63 with acrylate NF to form lipid NF63. In certain
embodiments, the lipid NF63 is of one of the formulae below:
##STR00183##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0232] In certain embodiments, the lipid is prepared by reacting
amine 64 with acrylate NF to form lipid NF64. In certain
embodiments, the lipid NF64 is of one of the formulae below:
##STR00184##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0233] In certain embodiments, the lipid is prepared by reacting
amine 61 with acrylate NF to form lipid NF70. In certain
embodiments, the lipid NF70 is of one of the formulae below:
##STR00185##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0234] In certain embodiments, the lipid is prepared by reacting
amine 86 with acrylate NF to form lipid NF86. In certain
embodiments, the lipid NF86 is of one of the formulae below:
##STR00186##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0235] In certain embodiments, the lipid is prepared by reacting
amine 87 with acrylate NF to form lipid NF87. In certain
embodiments, the lipid NF87 is of one of the formulae below:
##STR00187##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0236] In certain embodiments, the lipid is prepared by reacting
amine 91 with acrylate NF to form lipid NF91. In certain
embodiments, the lipid NF91 is of one of the formulae below:
##STR00188##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0237] In certain embodiments, the lipid is prepared by reacting
amine 95 with acrylate NF to form lipid NF95. In certain
embodiments, the lipid NF95 is of one of the formulae below:
##STR00189##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0238] In certain embodiments, the lipid is prepared by reacting
amine 96 with acrylate NF to form lipid NF96. In certain
embodiments, the lipid NF96 is of one of the formulae below:
##STR00190##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0239] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate NF to form lipid NF98. In certain
embodiments, the lipid NF98 is of one of the formulae below:
##STR00191## ##STR00192## ##STR00193## ##STR00194##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0240] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate NF to form lipid NF99. In certain
embodiments, the lipid NF99 is of one of the formulae below:
##STR00195## ##STR00196##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, NF99 is treated with MeI
or another alkylating agent to form lipids of the formula:
##STR00197## ##STR00198##
[0241] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate NF to form lipid NF100. In certain
embodiments, the lipid NF100 is of one of the formulae below.
##STR00199##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0242] In certain embodiments, the lipid is prepared by reacting
amine 103 with acrylate NF to form lipid NF103. In certain
embodiments, the lipid NE103 is of one of the formulae below:
##STR00200##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0243] In certain embodiments, the lipid is prepared by reacting
amine 109 with acrylate NF to form lipid NF109. In certain
embodiments, the lipid NF109 is of one of the formulae below:
##STR00201##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0244] In certain embodiments, the lipid is prepared by reacting
amine 61 with acrylate NG to form lipid NG61. In certain
embodiments, the lipid NG61 is of one of the formulae below:
##STR00202##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0245] In certain embodiments, the lipid is prepared by reacting
amine 64 with acrylate NG to form lipid NG64. In certain
embodiments, the lipid NG64 is of one of the formulae below:
##STR00203##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0246] In certain embodiments, the lipid is prepared by reacting
amine 77 with acrylate NG to form lipid NG77. In certain
embodiments, the lipid NG77 is of one of the formulae
##STR00204##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0247] In certain embodiments, the lipid is prepared by reacting
amine 86 with acrylate NG to form lipid NG86. In certain
embodiments, the lipid NG86 is of one of the formulae below:
##STR00205##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0248] In certain embodiments, the lipid is prepared by reacting
amine 87 with acrylate NG to form lipid NG87. In certain
embodiments, the lipid NG87 is of one of the formulae
##STR00206##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0249] In certain embodiments, the lipid is prepared by reacting
amine 95 with acrylate NG to form lipid NG95. In certain
embodiments, the lipid NG95 is of one of the formulae below:
##STR00207##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0250] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate NG to form lipid NG100. In certain
embodiments, the lipid NG100 is of one of the formulae below:
##STR00208##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, NG100 is alkylated with
methyl iodide or another alkylating agent.
[0251] In certain embodiments, the lipid is prepared by reacting
amine 62 with acrylate NP to form lipid NP62. In certain
embodiments, the lipid NP62 is of one of the formulae below:
##STR00209##
[0252] In other embodiments, the lipid is a composition of one or
more of the above lipids.
In certain embodiments, the lipid is prepared by reacting amine 63
with acrylate NP to form lipid NP63. In certain embodiments, the
lipid NP63 is of one of the formulae below:
##STR00210##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0253] In certain embodiments, the lipid is prepared by reacting
amine 86 with acrylate NP to form lipid NP86. In certain
embodiments, the lipid NP86 is of one of the formulae below:
##STR00211##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0254] In certain embodiments, the lipid is prepared by reacting
amine 87 with acrylate NP to form lipid NP87. In certain
embodiments, the lipid NP87 is of one of the formulae below:
##STR00212##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0255] In certain embodiments, the lipid is prepared by reacting
amine 96 with acrylate NP to form lipid NP96. In certain
embodiments, the lipid NP96 is of one of the formulae below:
##STR00213##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0256] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate NP to form lipid NP98. In certain
embodiments, the lipid NP98 is of one of the formulae below:
##STR00214## ##STR00215## ##STR00216## ##STR00217##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0257] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate NP to form lipid NP99. In certain
embodiments, the lipid NP99 is of one of the formulae below:
##STR00218## ##STR00219##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, NF99 is treated with MeI
or another alkylating agent to form lipids of the formula:
##STR00220## ##STR00221##
[0258] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate NP to form lipid NP100. In certain
embodiments, the lipid NP100 is of one of the formulae below:
##STR00222##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0259] In certain embodiments, the lipid is prepared by reacting
amine 103 with acrylate NP to form lipid NP103. In certain
embodiments, the lipid NP103 is of one of the formulae below:
##STR00223##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0260] In certain embodiments, the lipid is prepared by reacting
amine 31 with acrylate LD to form lipid LD31. In certain
embodiments, the lipid LD31 is of one of the formulae below:
##STR00224##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0261] In certain embodiments, the lipid is prepared by reacting
amine 98 with acrylate LD to form lipid LD98. In certain
embodiments, the lipid LD98 is of one of the formulae below:
##STR00225## ##STR00226## ##STR00227## ##STR00228##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0262] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate LD to form lipid LD99. In certain
embodiments, the lipid LD99 is of one of the formulae below:
##STR00229##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, LD99 is treated with MeI
or another alkylating agent to form lipids (QD99) of the
formula:
##STR00230##
[0263] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate LD to form lipid LD100. In certain
embodiments, the lipid LD100 is of one of the formulae below:
##STR00231##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, LD100 is treated with MeI
or another alkylating agent to form lipids (QD100) of the
formula:
##STR00232##
[0264] In certain embodiments, the lipid is prepared by reacting
amine 87 with acrylate LE to form lipid LE87. In certain
embodiments, the lipid LE87 is of one of the formulae below:
##STR00233##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0265] In certain embodiments, the lipid is prepared by reacting
amine 94 with acrylate LE to form lipid LE94. In certain
embodiments, the lipid LE94 is of one of the formulae below:
##STR00234##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0266] In certain embodiments, the lipid is prepared by reacting
amine 31 with acrylate LF to form lipid LF31. In certain
embodiments, the lipid LF31 is of one of the formulae below:
##STR00235##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0267] In certain embodiments, the lipis is prepared by reacting
amine 94 with acrylate LF to form lipid LF94. In certain
embodiments, the lipid LF94 is of one of the formulae below:
##STR00236##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0268] In certain embodiments, the lipid is prepared by reacting
amine 95 with acrylate LF to form lipid LF95. In certain
embodiments, the lipid LF95 is of one of the formulae below:
##STR00237##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0269] In certain embodiments, the lipid is prepared by reacting
amine 99 with acrylate LF to form lipid LF99. In certain
embodiments, the lipid LF99 is of one of the formulae below:
##STR00238##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, LF99 is treated with MeI
or another alkylating agent to form lipid (QF99) of the
formula:
##STR00239##
[0270] In certain embodiments, the lipid is prepared by reacting
amine 32 with acrylate LG to form lipid LG32. In certain
embodiments, the lipid LG32 is of one of the formulae below:
##STR00240##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0271] In certain embodiments, the lipid is prepared by reacting
amine 77 with acrylate LG to form lipid LG77. In certain
embodiments, the lipid LG77 is of one of the formulae below:
##STR00241##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0272] In certain embodiments, the lipid is prepared by reacting
amine 80 with acrylate LG to form lipid LG80. In certain
embodiments, the lipid LG80 is of one of the formulae below:
##STR00242##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0273] In certain embodiments, the lipid is prepared by reacting
amine 96 with acrylate LG to form lipid LG96. In certain
embodiments, the lipid NG96 is of one of the formulae below:
##STR00243##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0274] In certain embodiments, the lipid is prepared by reacting
amine 100 with acrylate LG to form lipid LG100. In certain
embodiments, the lipid LG100 is of one of the formulae below:
##STR00244##
In other embodiments, the lipid is a composition of one or more of
the above lipids. In certain embodiments, LG100 is treated with MeI
or another alkylating agent to form lipids (QG100) of the
formula:
##STR00245##
[0275] In certain embodiments, the lipid is prepared by reacting
amine 109 with acrylate LG to form lipid LG109. In certain
embodiments, the lipid NG109 is of one of the formulae below:
##STR00246##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0276] In certain embodiments, the lipid is prepared by reacting
amine 64 with acrylate LG to form lipid LG64. In certain
embodiments, the lipid LG64 is of one of the formulae below:
##STR00247##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0277] In certain embodiments, the lipid is prepared by reacting
amine 31 with acrylate LG to form lipid LG31. In certain
embodiments, the lipid LG31 is of one of the formulae below:
##STR00248##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
[0278] In certain embodiments, the lipid is prepared by reacting
amine 32 with acrylate LG to form lipid LG32. In certain
embodiments, the lipid NG32 is of one of the formulae below:
##STR00249##
In other embodiments, the lipid is a composition of one or more of
the above lipids.
Synthesis of Lipids
[0279] The inventive lipids may be prepared by any method known in
the art. Preferably the lipids are prepared from commercially
available starting materials, such acrylates or acrylamides, and
amines. In another preferred embodiment, the lipids are prepared
from easily and/or inexpensively prepared starting materials. As
would be appreciated by one of skill in the art, the inventive
lipids can be prepared by total synthesis starting from
commercially available starting materials A particular lipid may be
the desired final product of the synthesis, or a mixture of lipids
may be the desired final product.
[0280] In a particularly preferred embodiment, the inventive lipid
is prepared via the conjugate addition of primary amines to
acrylates or acrylamides. An exemplary reaction scheme is shown
below:
##STR00250##
Any primary amine is useful in preparing inventive lipids. Primary
amines useful in this invention include, but are not limited to,
methylamine, ethylamine, isopropylamine, aniline, substituted
anilines, and ethanolamine. The primary amine may be a bis(primary
amine). Preferably, the amine is commercially available. In certain
embodiments, the amine used in the synthesis of the lipid is of the
formula:
##STR00251## ##STR00252## ##STR00253## ##STR00254##
##STR00255##
[0281] Acrylate or acrylamide monomers that are useful in the
present invention include any acrylates and acrylamides In certain
embodiments, the acrylates or acrylamides are acrylates or
acrylamides of straight chain alkyl groups. In certain embodiments,
the acrylate or acrylamide is of the formula:
##STR00256##
In other embodiments, the acrylate or acrylamide may include
branched, substituted, or cyclic aliphatic or heteroaliphatic
groups. In certain embodiments, the acrylate or acrylamide is
substituted with C1-C6 alkyl group, halogens, amino groups,
hydroxyl groups, alkoxy groups, etc.
[0282] In certain embodiments, the reaction is performed neat
without the use of a solvent. In other embodiments, a solvent is
used for the reaction. Both or one of the monomers is dissolved in
an organic solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH, EtOH,
CHCl.sub.3, hexanes, toluene, benzene, CCl.sub.4, glyme, diethyl
ether, etc.). The resulting solutions are combined, and the
reaction mixture is heated to yield the desired lipid. In a
particularly preferred embodiment, the reaction mixture is heated
to temperature ranging from 50-150.degree. C. In another
particularly preferred embodiment, the reaction mixture is heated
to approximately 95.degree. C. The reaction may also be catalyzed.
For example, the reaction may be catalyzed by the addition of an
acid, base, or metal. The reagents may be allowed to react for
fours, days, or weeks. Preferably, the reaction is allowed to
proceed from overnight (e.g., 8-2 hours) to 7 days.
[0283] In another particularly preferred embodiment, the inventive
lipids are prepared by the conjugate addition of a bis(amine) to an
acrylate. The bis(amine) may be a bis(secondary amine) or a
bis(primary amine). En exemplary reaction scheme using bis(amines)
is shown below:
##STR00257##
In certain embodiments, the reaction is performed neat without a
solvent. In other embodiments, the reaction is performed in a
solvent. One or both of the monomers are dissolved in an organic
solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH, EtOH, CHCl.sub.3,
hexanes, CCl.sub.4, glyme, diethyl ether, etc.). Organic solvents
are preferred due to the susceptibility of polyesters to
hydrolysis. The resulting solutions are combined, and the reaction
mixture is heated to yield the desired lipid. In a particularly
preferred embodiment, the reaction mixture is maintained at a
temperature ranging from 50-150.degree. C. In another particularly
preferred embodiment, the reaction mixture is heated to
approximately 95.degree. C. The reaction may also be catalyzed. For
example, the reaction may be catalyzed by the addition of an acid,
base, or metal.
[0284] In yet another particularly preferred embodiment, the
inventive lipids are prepared by the conjugate addition of a
poly(amine) to an acrylate or acrylamide. The poly(amine) may
include primary, secondary, tertiary, or quaternary amines. In
certain embodiments, the poly(amine) contains only primary and
secondary amines. An exemplary reaction scheme using poly(amines)
is shown below:
##STR00258##
In certain embodiments, the reaction is performed with an excess of
acrylate or acrylamide to fully funcationlize all amino groups of
the poly(amine). In other embodiments, the equivalents of acrylate
are limiting; therefore, all amino groups of the poly(amine) are
not functionalized. In certain embodiments, the reaction is
performed neat without a solvent. In other embodiments, the
reaction is performed in a solvent. One or both of the monomers are
dissolved in an organic solvent (e.g., THF, CH.sub.2Cl.sub.2, MeOH,
EtOH, CHCl.sub.3, hexanes, CCl.sub.4, glyme, diethyl ether, etc.).
Organic solvents are preferred due to the susceptibility of
polyesters to hydrolysis. The resulting solutions are combined, and
the reaction mixture is heated to yield the desired lipid. In a
particularly preferred embodiment, the reaction mixture is
maintained at a temperature ranging from 50-150.degree. C. In
another particularly preferred embodiment, the reaction mixture is
heated to approximately 95.degree. C. The reaction may also be
catalyzed. For example, the reaction may be catalyzed by the
addition of an acid, base, or metal.
[0285] The synthesized lipid may be purified by any technique known
in the art including, but not limited to, precipitation,
crystallization, chromatography, distillation, etc. In a
particularly preferred embodiment, the lipid is purified through
repeated precipitations in organic solvent (e.g., diethyl ether,
hexane, etc.). In a particularly preferred embodiment, the lipid is
isolated as a salt. The lipid is reacted with an acid (e.g., an
organic acid or inorganic acid) to form the corresponding salt. In
certain embodiments, the tertiary amine is alkylated to form a
quaternary ammonium salt of the lipid. The tertiary amines may be
alkylated with any alkylating agent, for example, alkyl halides
such as methyl iodide may be used to from the quaternary amino
groups. The anion associated with the quaternary amine may be any
organic or inorganic anion. Preferably, the anion is a
pharmaceutically acceptable anion.
[0286] In certain embodiments, the reaction mixture results in a
mixture of isomers with varying numbers and positions of acrylate
tails. Such mixtures of products may be used as is, or a single
isomer may be purified from the reaction mixture. When an amine is
not exhaustively alkylated, the resulting primary, secondary, or
tertiary amines may be further reacted with another acrylate,
acrylamide, or other electrophile. The resulting lipid may then be
optionally purified.
[0287] In certain embodiments, a desired lipid is prepared by
traditional total synthesis. In certain embodiments, a commercially
available amine is the starting material. One or more amino groups
of the amine are optionally protected. The unprotected amino groups
are reacted with a acrylate or acrylamide. The product is
optionally purified. Protecting groups are removed, and the free
amino groups are optionally reacted with another acrylate,
acrylamide, or other electrophile. Such a sequence may be repeated
depending on the desired complexity of the inventive product being
prepared. The final product may then be optionally purified.
[0288] In one embodiment, a library of different lipids is prepared
in parallel. A different amine and/or acrylate is added to each
vial in a set of vials or to each well of a multi-well plate used
to prepare the library. The array of reaction mixtures is incubated
at a temperature and length of time sufficient to allow formation
of the lipids to occur. In one embodiment, the vials are incubated
at approximately 95.degree. C. overnight. In other embodiments, the
vials are incubated from 1 to 7 days at approximately 95.degree. C.
The lipids may then be isolated and purified using techniques known
in the art. The lipids may then be screened using high-throughput
techniques to identify lipids with a desired characteristic (e.g.,
solubility in water, solubility at different pH, ability to bind
polynucleotides, ability to bind heparin, ability to bind small
molecules, ability to form microparticles, ability to increase
tranfection efficiency, etc.). In certain embodiments the lipids
may be screened for properties or characteristics useful in gene
therapy (e.g., ability to bind polynucleotides, increase in
transfection efficiency).
Polynucleotide Complexes
[0289] The ability of cationic compounds to interact with
negatively charged polynucleotides through electrostatic
interactions is well known. Cationic lipids such as Lipofectamine
have been prepared and studied for their ability to complex and
transfect polynucleotides. The interaction of the lipid with the
polynucleotide is thought to at least partially prevent the
degradation of the polynucleotide. By neutralizing the charge on
the backbone of the polynucleotide, the neutral or
slightly-positively-charged complex is also able to more easily
pass through the hydrophobic membranes (e.g., cytoplasmic,
lysosomal, endosomal, nuclear) of the cell. In a particularly
preferred embodiment, the complex is slightly positively charged.
In another particularly preferred embodiment, the complex has a
positive .zeta.-potential, more preferably the .zeta.-potential is
between +1 and +30.
[0290] The lipids of the present invention possess tertiary amines.
Although these amines are hindered, they are available to interact
with a polynucleotide (e.g., DNA, RNA, synthetic analogs of DNA
and/or RNA, DNA/RNA hydrids, etc.). Polynucleotides or derivatives
thereof are contacted with the inventive lipids under conditions
suitable to form polynucleotide/lipid complexes. The lipid is
preferably at least partially protonated so as to form a complex
with the negatively charged polynucleotide. In a preferred
embodiment, the polynucleotide/lipid complexes form nanoparticles
that are useful in the delivery of polynucleotides to cells. In
certain embodiments, multiple lipid molecules may be associated
with a polynucleotide molecule. The complex may include 1-100 lipid
molecules, 1-1000 lipid molecules, 10-1000 lipid molecules, or
100-10,000 lipid molecules. In certain embodiments, the complex may
form a nanoparticle. In a particularly preferred embodiment, the
diameter of the nanoparticles ranges from 10-500 nm, more
preferably the diameter of the nanoparticles ranges from 10-1200
nm, and most preferably from 50-150 nm. The nanoparticles may be
associated with a targeting agent as described below.
Polynucleotide
[0291] The polynucleotide to be complexed, encapsulated by the
inventive lipids, or included in a composition with the inventive
lipds may be any nucleic acid including but not limited to RNA and
DNA. In certain embodiments, the polynucleotide is DNA. In other
embodiments, the polynucleotide is RNA. In other embodiments, the
polynucleotide is an siRNA. In other embodiments, the
polynucleotide is an shRNA. The polynucleotides may be of any size
or sequence, and they may be single- or double-stranded. In certain
preferred embodiments, the polynucleotide is greater than 100 base
pairs long. In certain other preferred embodiments, the
polynucleotide is greater than 1000 base pairs long and may be
greater than 10,000 base pairs long. The polynucleotide is
preferably purified and substantially pure. Preferably, the
polynucleotide is greater than 50% pure, more preferably greater
than 75% pure, and most preferably greater than 95% pure. The
polynucleotide may be provided by any means known in the art. In
certain preferred embodiments, the polynucleotide has been
engineered using recombinant techniques (for a more detailed
description of these techniques, please see Ausubel et al. Current
Protocols in Molecular Biology (John Wiley & Sons, Inc., New
York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); each of which is incorporated herein by reference).
The polynucleotide may also be obtained from natural sources and
purified from contaminating components found normally in nature.
The polynucleotide may also be chemically synthesized in a
laboratory. In a preferred embodiment, the polynucleotide is
synthesized using standard solid phase chemistry.
[0292] The polynucleotide may be modified by chemical or biological
means. In certain preferred embodiments, these modifications lead
to increased stability of the polynucleotide. Modifications include
methylation, phosphorylation, end-capping, etc.
[0293] Derivatives of polynucleotides may also be used in the
present invention. These derivatives include modifications in the
bases, sugars, and/or phosphate linkages of the polynucleotide.
Modified bases include, but are not limited to, those found in the
following nucleoside analogs: 2-aminoadenosine, 2-thiothymidine,
inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, and 2-thiocytidine. Modified sugars include,
but are not limited to, 2'-fluororibose, ribose, 2'-deoxyribose,
3'-azido-2',3'-dideoxyribose, 2',3'-dideoxyribose, arabinose (the
2'-epimer of ribose), acyclic sugars, and hexoses. The nucleosides
may be strung together by linkages other than the phosphodiester
linkage found in naturally occurring DNA and RNA. Modified linkages
include, but are not limited to, phosphorothioate and
5'-N-phosphoramidite linkages. Combinations of the various
modifications may be used in a single polynucleotide. These
modified polynucleotides may be provided by any means known in the
art; however, as will be appreciated by those of skill in this art,
the modified polynucleotides are preferably prepared using
synthetic chemistry in vitro.
[0294] The polynucleotides to be delivered may be in any form. For
example, the polynucleotide may be a circular plasmid, a linearized
plasmid, a cosmid, a viral genome, a modified viral genome, an
artificial chromosome, etc.
[0295] The polynucleotide may be of any sequence. In certain
preferred embodiments, the polynucleotide encodes a protein or
peptide. The encoded proteins may be enzymes, structural proteins,
receptors, soluble receptors, ion channels, pharmaceutically active
proteins, cytokines, interleukins, antibodies, antibody fragments,
antigens, coagulation factors, albumin, growth factors, hormones,
insulin, etc. The polynucleotide may also comprise regulatory
regions to control the expression of a gene. These regulatory
regions may include, but are not limited to, promoters, enhancer
elements, repressor elements, TATA box, ribosomal binding sites,
stop site for transcription, etc. In other particularly preferred
embodiments, the polynucleotide is not intended to encode a
protein. For example, the polynucleotide may be used to fix an
error in the genome of the cell being transfected.
[0296] The polynucleotide may also be provided as an antisense
agent or RNA interference (RNAi) (Fire et al. Nature 391:806-811,
1998; incorporated herein by reference). Antisense therapy is meant
to include, e.g., administration or in situ provision of single- or
double-stranded oligonucleotides or their derivatives which
specifically hybridize, e.g., bind, under cellular conditions, with
cellular mRNA and/or genomic DNA, or mutants thereof, so as to
inhibit expression of the encoded protein, e.g., by inhibiting
transcription and/or translation (Crooke "Molecular mechanisms of
action of antisense drugs" Biochim. Biophys. Acta 1489(1):31-44,
1999; Crooke "Evaluating the mechanism of action of
antiproliferative antisense drugs" Antisense Nucleic Acid Drug Dev.
10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes
313-314, 1999; each of which is incorporated herein by reference).
The binding may be by conventional base pair complementarity, or,
for example, in the case of binding to DNA duplexes, through
specific interactions in the major groove of the double helix
(i.e., triple helix formation) (Chan et al. J. Mol. Med.
75(4):267-282, 1997; incorporated herein by reference).
[0297] In a particularly preferred embodiment, the polynucleotide
to be delivered comprises a sequence encoding an antigenic peptide
or protein. Nanoparticles containing these polynucleotides can be
delivered to an individual to induce an immunologic response
sufficient to decrease the chance of a subsequent infection and/or
lessen the symptoms associated with such an infection. The
polynucleotide of these vaccines may be combined with interleukins,
interferon, cytokines, and adjuvants such as cholera toxin, alum,
Freund's adjuvant, etc. A large number of adjuvant compounds are
known; a useful compendium of many such compounds is prepared by
the National Institutes of Health and can be found on the internet
(http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf,
incorporated herein by reference; see also Allison Dev. Biol.
Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol.
6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, 1992, each
of which is incorporated herein by reference).
[0298] The antigenic protein or peptides encoded by the
polynucleotide may be derived from such bacterial organisms as
Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus
aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae,
Listeria monocytogenes, Bacillus anthracis, Clostridium tetani,
Clostridium botulinum, Clostridium perfringens, Neisseria
meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,
Pseudomonas aeruginosa, Salmonella typhi, Haemophilus
parainfluenzae, Bordetella pertussis, Francisella tularensis,
Yersinia pestis, Vibrio cholerae, Legionella pneumophila,
Mycobacterium tuberculosis, Mycobacterium leprae, Treponema
pallidum, Leptospirosis interrogans, Borrelia burgdorferi,
Camphylobacter jejuni, and the like; from such viruses as smallpox,
influenza A and B, respiratory syncytial virus, parainfluenza,
measles, HIV, varicella-zoster, herpes simplex 1 and 2,
cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,
adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,
coxsackieviruses, equine encephalitis, Japanese encephalitis,
yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus,
and the like; and from such fungal, protozoan, and parasitic
organisms such as Cryptococcus neoformans, Histoplasma capsulatum,
Candida albicans, Candida tropicalis, Nocardia asteroides,
Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,
Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,
Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,
Trichomonas vaginalis, Schistosoma mansoni, and the like.
Microparticles
[0299] The lipids of the present invention may also be used to form
drug delivery devices. The inventive lipids may be used to
encapsulate agents including polynucleotides, small molecules,
proteins, peptides, metals, organometallic compounds, etc. The
inventive lipids have several properties that make them
particularly suitable in the preparation of drug delivery devices.
These include 1) the ability of the lipid to complex and "protect"
labile agents; 2) the ability to buffer the pH in the endosome; 3)
the ability to act as a "proton sponge" and cause endosomolysis;
and 4) the ability to neutralize the charge on negatively charged
agents. In a preferred embodiment, the lipids are used to form
microparticles containing the agent to be delivered. These
microparticles may include other materials such as proteins,
carbohydrates, synthetic polymers (e.g., PEG, PLGA), and natural
polymers. In a particularly preferred embodiment, the diameter of
the microparticles ranges from between 500 nm to 50 micrometers,
more preferably from 1 micrometer to 20 micrometers, and most
preferably from 1 micrometer to 10 micrometers. In another
particularly preferred embodiment, the microparticles range from
1-5 micrometers.
Methods of Preparing Microparticles
[0300] The inventive microparticles may be prepared using any
method known in this art. These include, but are not limited to,
spray drying, single and double emulsion solvent evaporation,
solvent extraction, phase separation, simple and complex
coacervation, and other methods well known to those of ordinary
skill in the art. Particularly preferred methods of preparing the
particles are the double emulsion process and spray drying. The
conditions used in preparing the microparticles may be altered to
yield particles of a desired size or property (e.g.,
hydrophobicity, hydrophilicity, external morphology, "stickiness",
shape, etc.). The method of preparing the particle and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may also depend on the agent being encapsulated
and/or the composition of the matrix.
[0301] Methods developed for making microparticles for delivery of
encapsulated agents are described in the literature (for example,
please see Doubrow, M., Ed., "Microcapsules and Nanoparticles in
Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz and
Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et al.
Reactive Polymers 6:275-283, 1987; Mathiowitz et al. J. Appl.
Polymer Sci. 35:755-774, 1988; each of which is incorporated herein
by reference).
[0302] If the particles prepared by any of the above methods have a
size range outside of the desired range, the particles can be
sized, for example, using a sieve. The particle may also be coated.
In certain embodiments, the particles are coated with a targeting
agent. In other embodiments, the particles are coated to achieve
deisirable surface properties (e.g., a particular charge).
Micelles and Liposomes
[0303] The lipids of the invention may be used to prepare micelles
or liposomes. Many techniques for preparing micelles and liposomes
are known in the art, and any method may be used with the inventive
lipids to make micelles and liposomes. In addition, any agent
including polynucleotides, small molecules, proteins, peptides,
metals, organometallic compounds, etc. may be included in a micelle
or liposome. Micelles and liposomes are particularly useful in
delivering hydrophobic agents such as hydrophobic small
molecules.
[0304] In certain embodiments, liposomes (lipid vesicles) are
formed through spontaneous assembly. In other embodiments,
liposomes are formed when thin lipid films or lipid cakes are
hydrated and stacks of lipid crystalline bilayers become fluid and
swell. The hydrated lipid sheets detach during agitation and
self-close to form large, multilamellar vesicles (LMV). This
prevents interaction of water with the hydrocarbon core of the
bilayers at the edges. Once these particles have formed, reducing
the size of the particle can be modified through input of sonic
energy (sonication) or mechanical energy (extrusion). See Walde, P.
"Preparation of Vesicles (Liposomes)" In Encylopedia of Nanoscience
and Nanotechnology; Nalwa, H. S. Ed. American Scientific
Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka et al.
"Comparative Properties and Methods of Preparation of Lipid
Vesicles (Liposomes)" Ann. Rev. Biophys. Bioeng. 9:467-508, 1980;
each of which is incorporated herein. The preparation of lipsomes
involves preparing the lipid for hydration, hydrating the lipid
with agitation, and sizing the vesicles to achieve a homogenous
distribution of liposomes. Lipids are first dissolved in an organic
solvent to assure a homogeneous mixture of lipids. The solvent is
then removed to form a lipid film. This film is thoroughly dried to
remove residual organic solvent by placing the vial or flask on a
vaccuum pump overnight. Hydration of the lipid film/cake is
accomplished by adding an aqueous medium to the container of dry
lipid and agitating the mixture. Disruption of LMV suspensions
using sonic energy typically produces small unilamellar vesicles
(SUV) with diameters in the range of 15-50 nm. Lipid extrusion is a
technique in which a lipid suspension is forced through a
polycarbonate filter with a defined pore size to yield particles
having a diameter near the pore size of the filter used. Extrusion
through filters with 100 nm pores typically yields large,
unilamellar vesicles (LUV) with a mean diameter of 120-140 nm.
[0305] In certain embodiments of the invention, liposomes are
formed comprising an inventive lipid, PEG-ceramide, cholesterol,
and a polynucleotide. In certain embodiments, the polynucleotide is
an RNA molecule (e.g., an RNAi molecule). In other embodiments, the
polynucleotide is a DNA molecule. In certain embodiments, the lipid
is ND98. In other embodiments, the lipid is ND28, ND32, LF94, ND99,
ND95, NP103, NP98, ND25, ND20, ND100, NF96, NF103, NF109, NF11,
ND24, NF86, NP96, ND36, NF61, NF87, NF95, QG100, NF60, NP100, NF1,
NP99, QD99, NF63, LG109, ND103, LF95, QF99, LG100, LF31, LG32,
NF109, NF64, LE87, LG77, LG96, ND96, LD31, NG64, ND109, or LG80. In
certain embodiments, the amount of lipid in the liposome ranges
from 30-80 mol %, preferably 40-70 mol %, more preferably 60-70 mol
%. In certain embodiments, the amount of PEG-ceramide in the
liposomes ranges from 5-20 mol %, preferably 10-15 mol %, more
preferably approximately 10 mol %. In certain embodiments, the
amount of cholesterol in the liposome ranges from 5-25 mol %,
preferably 10-20 mol %, more preferably approximately 15 mol %. In
certain embodiments, the amount of cholesterol in the liposome is
approximately 20 mol %. These liposomes may be prepared using any
method known in the art. In certain embodiments (e.g., liposomes
containing RNAi molecules), the liposomes are prepared by lipid
extrusion.
[0306] Certain lipids can spontaneously self assemble around
certain molecules, such as DNA and RNA, to form liposomes. For some
applications such as the delivery of polynucleotides, these are
preferred. Use of these lipids allows for simple assembly of
liposomes without the need for additional steps or devices such as
an extruder.
[0307] The following scientific papers described other methods for
preparing liposomes and micelles: Narang et al. "Cationic Lipids
with Increased DNA Binding Affinity for Nonviral Gene Transfer in
Dividing and Nondividing Cells" Bioconjugate Chem. 16:156-68, 2005;
Hofland et al. "Formation of stable cationic lipid/DNA complexes
for gene transfer" Proc. Natl. Acad. Sci. USA 93:7305-7309, July
1996; Byk et al. "Synthesis, Activity, and Structure--Activity
Relationship Studies of Novel Cationic Lipids for DNA Transfer" J.
Med. Chem. 41(2):224-235, 1998; Wu et al. "Cationic Lipid
Polymerization as a Novel Approach for Constructing New DNA
Delivery Agents" Bioconjugate Chem. 12:251-57, 2001; Lukyanov et
al. "Micelles from lipid derivatives of water-soluble polymers as
delivery systems for poorly soluble drugs" Advanced Drug Delivery
Reviews 56:1273-1289, 2004; Tranchant et al. "Physicochemical
optimisation of plasmid delivery by cationic lipids" J. Gene Med.
6:S24-S35, 2004; van Balen et al. "Liposome/Water Lipophilicity:
Methods, Information Content, and Pharmaceutical Applications"
Medicinal Research Rev. 24(3):299-324, 2004; each of which is
incorporated herein by reference.
Agent
[0308] The agents to be delivered by the system of the present
invention may be therapeutic, diagnostic, or prophylactic agents.
Any chemical compound to be administered to an individual may be
delivered using the inventive comlexes, nanoparticles, or
microparticles. The agent may be a small molecule, organometallic
compound, nucleic acid, protein, peptide, polynucleotide, metal, an
isotopically labeled chemical compound, drug, vaccine,
immunological agent, etc.
[0309] In a preferred embodiment, the agents are organic compounds
with pharmaceutical activity. In another embodiment of the
invention, the agent is a clinically used drug. In a particularly
preferred embodiment, the drug is an antibiotic, anti-viral agent,
anesthetic, steroidal agent, anti-inflammatory agent,
anti-neoplastic agent, antigen, vaccine, antibody, decongestant,
antihypertensive, sedative, birth control agent, progestational
agent, anti-cholinergic, analgesic, anti-depressant,
anti-psychotic, .beta.-adrenergic blocking agent, diuretic,
cardiovascular active agent, vasoactive agent, non-steroidal
anti-inflammatory agent, nutritional agent, etc.
[0310] In a preferred embodiment of the present invention, the
agent to be delivered may be a mixture of agents.
[0311] Diagnostic agents include gases; metals; commercially
available imaging agents used in positron emissions tomography
(PET), computer assisted tomography (CAT), single photon emission
computerized tomography, x-ray, fluoroscopy, and magnetic resonance
imaging (MRI); and contrast agents. Examples of suitable materials
for use as contrast agents in MRI include gadolinium chelates, as
well as iron, magnesium, manganese, copper, and chromium. Examples
of materials useful for CAT and x-ray imaging include iodine-based
materials.
[0312] Prophylactic agents include, but are not limited to,
antibiotics, nutritional supplements, and vaccines. Vaccines may
comprise isolated proteins or peptides, inactivated organisms and
viruses, dead organisms and viruses, genetically altered organisms
or viruses, and cell extracts. Prophylactic agents may be combined
with interleukins, interferon, cytokines, and adjuvants such as
cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents
include antigens of such bacterial organisms as Streptococccus
pneumoniae, Haemophilus influenzae, Staphylococcus aureus,
Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria
monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium
botulinum, Clostridium perfringens, Neisseria meningitidis,
Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas
aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,
Bordetella pertussis, Francisella tularensis, Yersinia pestis,
Vibrio cholerae, Legionella pneumophila, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum,
Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter
jejuni, and the like; antigens of such viruses as smallpox,
influenza A and B, respiratory syncytial virus, parainfluenza,
measles, HIV, varicella-zoster, herpes simplex 1 and 2,
cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,
adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,
coxsackieviruses, equine encephalitis, Japanese encephalitis,
yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus,
and the like; antigens of fungal, protozoan, and parasitic
organisms such as Cryptococcus neoformans, Histoplasma capsulatum,
Candida albicans, Candida tropicalis, Nocardia asteroides,
Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,
Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,
Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,
Trichomonas vaginalis, Schistosoma mansoni, and the like. These
antigens may be in the form of whole killed organisms, peptides,
proteins, glycoproteins, carbohydrates, or combinations
thereof.
Targeting Agents
[0313] The inventive complexes, liposomes, micelles,
microparticles, and nanoparticles may be modified to include
targeting agents since it is often desirable to target a particular
cell, collection of cells, or tissue. A variety of targeting agents
that direct pharmaceutical compositions to particular cells are
known in the art (see, for example, Cotten et al. Methods Enzym.
217:618, 1993; incorporated herein by reference). The targeting
agents may be included throughout the particle or may be only on
the surface. The targeting agent may be a protein, peptide,
carbohydrate, glycoprotein, lipid, small molecule, etc. The
targeting agent may be used to target specific cells or tissues or
may be used to promote endocytosis or phagocytosis of the particle.
Examples of targeting agents include, but are not limited to,
antibodies, fragments of antibodies, low-density lipoproteins
(LDLs), transferrin, asialycoproteins, gp120 envelope protein of
the human immunodeficiency virus (HIV), carbohydrates, receptor
ligands, sialic acid, etc. If the targeting agent is included
throughout the particle, the targeting agent may be included in the
mixture that is used to form the particles. If the targeting agent
is only on the surface, the targeting agent may be associated with
(i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals,
or other interactions) the formed particles using standard chemical
techniques.
Pharmaceutical Compositions
[0314] Once the complexes, micelles, liposomes, microparticles, or
nanoparticles have been prepared, they may be combined with one or
more pharmaceutical excipients to form a pharmaceutical composition
that is suitable to administer to animals including humans. As
would be appreciated by one of skill in this art, the excipients
may be chosen based on the route of administration as described
below, the agent being delivered, time course of delivery of the
agent, etc.
[0315] Pharmaceutical compositions of the present invention and for
use in accordance with the present invention may include a
pharmaceutically acceptable excipient or carrier. As used herein,
the term "pharmaceutically acceptable carrier" means a non-toxic,
inert solid, semi-solid or liquid filler, diluent, encapsulating
material or formulation auxiliary of any type. Some examples of
materials which can serve as pharmaceutically acceptable carriers
are sugars such as lactose, glucose, and sucrose; starches such as
corn starch and potato starch; cellulose and its derivatives such
as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose
acetate; powdered tragacanth; malt; gelatin; talc; excipients such
as cocoa butter and suppository waxes; oils such as peanut oil,
cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and
soybean oil; glycols such as propylene glycol; esters such as ethyl
oleate and ethyl laurate; agar; detergents such as Tween 80;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline;
Ringer's solution; ethyl alcohol; and phosphate buffer solutions,
as well as other non-toxic compatible lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator. The pharmaceutical compositions of this invention can
be administered to humans and/or to animals, orally, rectally,
parenterally, intracisternally, intravaginally, intranasally,
intraperitoneally, topically (as by powders, creams, ointments, or
drops), bucally, or as an oral or nasal spray.
[0316] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups, and elixirs. In addition to the active
ingredients (i.e., microparticles, nanoparticles, liposomes,
micelles, polynucleotide/lipid complexes), the liquid dosage forms
may contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0317] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In a
particularly preferred embodiment, the particles are suspended in a
carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose
and 0.1% (v/v) Tween 80.
[0318] The injectable formulations can be sterilized, for example,
by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0319] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
particles with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol, or a suppository wax which
are solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
microparticles.
[0320] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the particles are mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or a) fillers or extenders such as starches, lactose,
sucrose, glucose, mannitol, and silicic acid, b) binders such as,
for example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets,
and pills, the dosage form may also comprise buffering agents.
[0321] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0322] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes.
[0323] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0324] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The particles are admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, and
eye drops are also contemplated as being within the scope of this
invention.
[0325] The ointments, pastes, creams, and gels may contain, in
addition to the particles of this invention, excipients such as
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc, and zinc oxide, or mixtures
thereof.
[0326] Powders and sprays can contain, in addition to the particles
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates, and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0327] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the microparticles or
nanoparticles in a proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
can be controlled by either providing a rate controlling membrane
or by dispersing the particles in a polymer matrix or gel.
[0328] These and other aspects of the present invention will be
further appreciated upon consideration of the following Examples,
which are intended to illustrate certain particular embodiments of
the invention but are not intended to limit its scope, as defined
by the claims.
Examples
Example 1
Preparation and Testing of Amine-Containing Lipids
[0329] Lipid Synthesis.
[0330] Monomers were purchased from Aldrich (Milwaukee, Wis.), TCI
(Portland, Oreg.), Pfaltz & Bauer (Waterbury, Conn.), Matrix
Scientific (Columbia, S.C.), Acros-Fisher (Pittsburgh, Pa.),
Scientific Polymer (Ontario, N.Y.), Polysciences (Warrington, Pa.),
and Dajac monomer-polymer (Feasterville, Pa.). The acrylate and
amine monomers were used neat to prepare the lipids. All possible
pair wise combinations of amine and acrylate monomers shown in
FIGS. 1A and 1B were prepared in sealed vials. The vials were then
incubated overnight at approximately 95.degree. C. with shaking.
The synthesized lipids were used without further purification.
[0331] The molecular weights of the synthesized lipids were
determined by mass spectroscopy and compared to predicted molecular
weights to confirm synthesis of the lipid. Mass spectrometric data
are shown in the table below.
TABLE-US-00001 TABLE 1 Mass-spectrometry data of amine-containing
lipids. Lipid Formula Predicted MW Actual MW LF1
C3.sub.8H.sub.75NO.sub.5 626.572 626.651 LF6
C.sub.39H.sub.75NO.sub.6 654.5667 654.6604 LF7
C.sub.40H7.sub.7NO.sub.6 668.5824 668.6972 LF10
C.sub.37H.sub.73NO.sub.5 612.5562 612.5917 LF11
C.sub.38H.sub.75NO.sub.5 626.5718 626.6789 LF15
C.sub.39H.sub.76NO.sub.5 638.5723 638.6649 LF17
C.sub.44H.sub.79NO.sub.6 718.598 718.6921 LF20
C.sub.37H.sub.73NO.sub.5 612.5562 612.5946 LF21
C.sub.37H.sub.73NO.sub.5 612.556 612.5959 LG1
C.sub.40H.sub.79NO.sub.5 654.6036 654.8644 LG6
C.sub.41H.sub.79NO.sub.6 682.5985 682.8408 LG7
C.sub.42H.sub.81NO.sub.6 696.6142 696.9988 LG10
C.sub.39H.sub.78NO.sub.5 640.588 640.9817 LG13
C.sub.40H.sub.80NO.sub.6 670.5985 670.9156 LG15
C.sub.41H.sub.79NO.sub.5 666.6036 666.9696 LG17
C.sub.46H.sub.83NO.sub.5 746.6298 746.9586 LG20
C.sub.39H.sub.77NO.sub.5 640.588 640.9586 LG21
C.sub.39H.sub.77NO.sub.5 640.588 640.9292 LG22 C.sub.39H.sub.77NO5
640.588 640.8809 LG24 C.sub.39H.sub.77NO.sub.6 656.5829 656.9402
QF1 C.sub.39H.sub.78NO.sub.5 640.588 640.6866 QF6
C.sub.40H.sub.78NO.sub.6 668.5829 668.7032 QF7
C.sub.41H.sub.80NO.sub.6 682.5985 682.7867 QF10
C.sub.38H.sub.76NO.sub.5 626.5723 626.6509 QF11
C.sub.39H.sub.78NO.sub.5 640.588 640.6297 ND25
C.sub.33H.sub.67N.sub.3O.sub.4 570.5204 570.6493 ND36
C.sub.36H.sub.73N.sub.3O.sub.3 596.5725 596.6654 ND75
C.sub.36H.sub.74N.sub.4O.sub.2 595.5885 595.6977 ND87
C.sub.37H.sub.76N.sub.4O.sub.4 641.5939 641.7349 NH32
C4.sub.7H.sub.96N.sub.3O.sub.3 750.7451 750.8913 NH36
C.sub.48H.sub.98N.sub.3O.sub.3 764.7608 764.8723 NH60
C.sub.45H.sub.93N.sub.4O.sub.2 735.7455 735.8695 NH86
C.sub.48H.sub.99N4O.sub.4 795.7666 795.804 NH87
C.sub.49H.sub.100N.sub.4O.sub.4 809.7822 809.8638 Q in the lipid
name indicates that the ammo groups of the lipid were quaternized
using methyl iodide. L indicates lipids prepared from the indicated
acrylates and amines. N indicates that the ester functional group
of the acrylate has been replaced with an amide group.
[0332] Transfection Experiments.
[0333] 14,000 cos-7 cells (ATCC, Manassas, Va.) were seeded into
each well of a solid white or clear 96 well plate (Corning-Costar,
Kennebunk, Me.) and allowed to attached overnight in growth medium,
composed of: 500 ml phenol red minus DMEM, 50 ml heat inactivated
FBS, 5 ml penicillin/streptomycin (Invitrogen, Carlsbad,
Calif.).
[0334] A small liquot of lipid was tranferred to an Eppendorf tube.
Based on the mass of the lipid in the tube, sterile 25 mM sodium
acetate buffer was added to each tube to yield a concentration of
60 mg/ml. The resulting mixture was vortexed from approximately 20
minutes until the lipid was fully dissolved. DNA was prepared based
on 300 ng DNA per well of a 96-well plate. 291 .mu.g of Lc DNA was
dissolved in 9210 .mu.l of 25 mM sodium acetate buffer. Aliquots of
30 .mu.l of DNA solution were added to each well expect for the
last column which was reserved for the Lipo2000 standard. For the
last column of the plate, 61 .mu.g of DNA was added to 1940 .mu.l
Optimem. 150 .mu.l of media/Optimem was added to each well of
plates. 50 .mu.l of lipid solution was aliquoted into wells of
robot plate. The following amounts were aliquoted to obtain the
correct ratios of DNA to lipid:
For 300 ng DNA well:
TABLE-US-00002 w/w ratio .mu.l of lipid from robot plate .mu.l of
NaOAc buffer 2.5 5 195 5 10 190 10 20 180 15 30 170 20 40 160 25 50
150
In quadruplicate, 30 .mu.l of lipid was aliquoted onto DNA in four
rows for each ratio. For the Lipo2000 control (2.5 w/w ratio to
DNA), 152.5 .mu.g of Lipo sample was aliquoted into 1847.5 .mu.l of
Optimem. 30 .mu.l of this solution was aliquoted onto DNA in the
Optimem in the last columns of each plate. The plates were
incubated for 15-20 minutes, and then 36.5 .mu.l of lipid+DNA
complexes was transferred into 150 .mu.l of media/Optimem, then add
to cells. The media was aspirated off the cells, and 105 .mu.l of
the lipid/DNA/media/Optimem solution was added to the cells. The
luciferase assay was performed after 48 hours.
[0335] Luminescence was analyzed using bright-glo assay kits
(Promega). Briefly, 100 .mu.l of bright-glo solution was added to
each well of the microtiter plate containing media and cells.
Luminescence was measured using a Mithras Luminometer (Berthold,
Oak Ridge, Tenn.). In some cases, a neutral density filter (Chroma,
Brattleboro, Vt.) was used to prevent saturation of the
luminometer. A standard curve for Luciferase was generated by
titration of Luciferase enzyme (Promega) into growth media in white
microtiter plates. Luciferase in ng per well are calculated for
each of the lipids at 2.5 w/w, 5 w/w, 10 w/w, 15 w/w, 20 w/w, and
25 w/w lipid to DNA based on the standard curve. These data are
shown in the table below. eGFP expression was examined using a
Zeiss Aciovert 200 inverted microscope.
TABLE-US-00003 TABLE 2 Luciferase expression (measured in relative
light units) as a percentage of that achieved using Lipofectamine
.TM. 2000 (ng per well) for lipids at specific lipid/DNA (w/w)
ratios using 300 ng Luciferase DNA per well 2.5 w/w 5 w/w 10 w/w 15
w/w 20 w/w 25 w/w QG7 0.4 0.3 0.5 0.3 0.4 0.3 QB1 0.4 0.3 0.4 0.3
0.4 0.4 QF1 0.4 0.3 0.4 0.3 0.3 0.3 QG1 0.4 0.5 0.4 0.4 0.3 0.3
QB77 0.4 0.4 0.4 0.3 0.3 0.3 QF77 0.4 0.4 0.7 1.3 2.7 9.9 QG77 0.5
0.6 1.5 5.6 18.8 29.5 LD90 0.5 0.4 0.4 0.6 0.4 0.4 LE90 0.5 0.3 0.4
0.4 0.4 0.4 LF90 0.5 0.4 0.7 0.4 0.4 0.4 LG90 0.6 0.6 0.4 0.4 0.4
0.4 LB64 0.1 0.1 0.2 0.1 0.1 0.2 LD64 0.1 0.1 0.1 0.1 0.1 0.1 LE64
0.1 0.1 0.1 0.0 0.1 0.1 LF64 0.1 0.1 0.4 0.1 0.2 0.1 LG64 0.2 0.1
0.1 0.1 0.1 0.2 LB31 1.2 0.8 8.3 1.9 1.3 4.2 LD31 44.2 38.7 18.8
11.3 42.8 174.1 LE31 1.0 1.0 0.9 2.5 2.9 9.5 LF31 64.1 78.7 13.4
69.5 97.3 266.8 LG31 19.6 27.6 34.0 8.5 14.3 94.0 LB63 0.1 0.2 0.1
1.0 0.1 0.2 ND28 124.8 116.0 28.9 0.0 0.0 0.0 ND86 0.5 0.2 0.0 0.0
0.0 0.0 ND87 0.0 0.0 0.0 0.0 0.0 0.0 QB6 0.0 0.0 0.0 0.0 0.0 0.0
QF6 0.0 0.0 0.0 0.0 0.0 0.0 QG6 0.0 0.0 0.0 0.0 0.0 0.0 QB7 0.0 0.0
0.0 0.0 0.0 0.0 QF7 0.0 0.0 0.0 0.0 0.0 0.0 LB1 0.1 0.1 0.1 0.1 0.1
0.2 LB6 0.1 0.1 0.1 0.1 0.1 0.1 LB7 0.1 0.1 0.1 0.1 0.1 0.2 LB10
0.1 0.1 0.1 0.1 0.1 0.1 LB11 0.1 0.1 0.1 0.1 0.1 0.1 LB13 0.1 0.1
0.1 0.1 0.1 0.1 LB15 0.1 0.1 0.1 0.1 0.1 0.1 LB17 0.1 0.1 0.1 0.1
0.1 0.1 LB20 0.1 0.1 0.4 0.2 0.3 0.7 LB21 0.7 0.8 0.7 0.7 0.7 0.8
LB22 0.3 0.3 0.4 0.4 0.4 0.4 LB24 0.1 0.2 0.4 0.1 0.1 0.1 LB25 0.8
2.4 2.6 3.3 2.3 1.8 LB26 0.1 0.1 0.1 0.1 0.1 0.1 LB28 0.1 0.1 0.1
0.1 0.1 0.1 LB31 0.1 0.1 0.1 0.1 0.1 0.1 LB32 0.1 0.1 0.1 0.1 0.1
0.1 LB33 0.1 0.1 0.1 0.1 0.1 0.1 LB34 0.1 0.1 0.2 0.4 0.1 0.1 LB36
0.1 0.1 0.1 0.1 0.1 0.1 LB38 0.7 0.8 0.7 0.8 0.7 0.8 LB60 0.4 0.4
0.4 0.4 0.4 0.4 LB61 0.1 0.1 0.1 0.1 0.1 0.1 LB62 0.1 0.1 0.1 0.1
0.1 0.1 LB63 0.1 0.1 0.1 0.1 0.1 0.1 LB64 0.1 0.1 0.1 0.1 0.1 0.1
LB70 0.1 0.1 0.1 0.1 0.1 0.1 LB75 0.1 0.1 0.1 0.1 0.1 0.1 LB76 0.1
0.1 0.1 0.1 0.1 0.1 LB77 0.1 0.1 0.1 0.2 0.1 0.3 LB79 0.1 0.1 0.1
0.1 0.1 0.1 LB80 0.7 0.8 0.1 0.2 1.0 1.5 LB81 0.4 0.4 0.2 0.3 0.4
0.5 LF1 0.1 0.1 0.0 0.0 0.1 0.1 LF64 0.1 0.1 0.0 0.0 0.1 0.1 LF7
0.1 0.1 0.0 0.0 0.1 0.1 LF10 0.1 0.1 0.0 0.0 0.1 0.1 LF11 0.1 0.1
0.0 0.0 0.1 0.1 LF13 0.1 0.1 0.0 0.0 0.1 0.1 LF15 0.1 0.1 0.0 0.0
0.1 0.1 LF17 0.1 0.1 0.1 0.1 0.1 0.1 LF20 0.2 0.4 0.3 0.2 0.1 0.1
LF21 0.4 0.4 0.4 0.4 0.4 0.4 LD28 1.7 8.5 LD86 29.6 16.0 LD87 53.9
43.3 LG34 1.4 0.8 LG77 43.5 34.0 LH28 0.2 0.2 QD28 0.1 0.1 QD86 2.0
2.0 QD87 0.5 0.7 LF22 0.1 0.1 0.1 0.1 0.1 0.1 LF24 0.1 0.1 0.1 0.2
0.3 0.3 LF25 0.1 0.2 0.3 0.4 0.9 1.5 LF26 0.1 0.2 0.7 0.1 0.1 0.3
LF28 0.1 0.1 0.1 0.2 0.3 0.4 LF32 0.1 0.1 0.2 0.1 0.4 0.4 LF33 0.1
0.1 0.1 0.1 0.1 0.1 LF34 0.1 0.3 1.1 0.5 0.2 0.3 LF36 0.1 0.1 0.1
0.1 0.1 0.1 LF38 0.8 0.9 0.9 0.9 0.8 1.0 LF60 0.4 0.4 0.5 0.5 0.5
0.5 LF61 0.1 0.1 0.1 0.1 0.1 0.1 LF62 0.1 0.1 0.2 0.1 0.4 0.2 LF63
0.1 0.1 0.1 0.3 0.3 0.4 LF64 0.1 0.1 0.1 0.1 0.1 0.1 LF70 0.1 0.1
0.2 0.3 0.2 0.3 LF75 0.7 1.0 0.7 1.9 1.7 1.7 LF76 1.7 5.6 9.8 24.3
22.2 19.5 LF77 3.7 25.1 28.2 24.1 17.0 22.1 LF79 0.1 0.3 0.3 0.3
0.3 0.3 LF80 2.5 35.1 35.5 34.3 19.9 14.8 LF81 0.5 1.6 1.5 4.9 4.6
4.0 LF82 0.4 0.3 0.7 0.5 0.6 0.8 LF86 21.5 17.9 19.6 21.2 10.5 10.4
LF87 19.4 11.3 30.2 13.3 11.0 10.0 LF90 0.4 0.5 0.3 1.3 1.1 1.5
LF91 0.5 0.5 0.8 1.4 1.3 1.6 LF93 32.0 50.4 15.0 150.7 143.2 171.3
LF94 41.8 37.7 96.3 114.7 99.0 98.6 LF95 15.3 51.3 44.3 71.8 64.6
75.1 LF96 52.4 62.8 79.3 47.7 64.4 36.0 LF98 2.5 7.9 17.8 17.2 9.5
9.9 LF99 32.2 49.8 26.5 10.7 6.0 6.2 LF100 17.6 70.0 69.0 85.9 44.2
50.9 LF103 43.9 11.6 65.4 91.8 61.6 61.4 LF109 16.0 28.3 16.9 21.9
28.7 49.5 LG1 0.0 0.0 0.0 0.0 0.0 0.0 LG64 0.0 0.0 0.0 0.0 0.0 0.0
LG77 0.0 0.0 0.0 0.0 0.0 0.0 LG10 0.0 0.0 0.0 0.0 0.0 0.0 LG11 0.0
0.0 0.0 0.0 0.0 0.0 LG13 0.0 0.0 0.0 0.0 0.0 0.0 LG15 0.7 0.8 0.5
0.6 0.7 0.8 LG17 0.4 0.3 0.1 0.0 0.3 0.3 LG20 2.0 2.5 0.4 0.2 0.1
0.1 LG21 0.1 0.1 0.1 0.2 0.2 0.4 LG22 0.2 0.3 0.4 0.5 0.4 0.7 LG24
2.0 3.3 4.8 11.2 16.4 32.5 LG25 15.9 32.0 43.2 56.9 42.1 63.9 LG26
0.6 13.0 1.9 0.5 0.3 0.4 LG28 0.2 0.5 0.2 0.2 0.1 0.2 LG32 0.2 10.2
1.0 0.7 0.4 0.7 LG33 0.1 0.1 0.1 0.1 0.1 0.1 LG60 0.9 1.0 0.8 0.9
0.8 1.0 LG61 0.4 0.5 0.4 0.4 0.4 0.5 LG63* 0.5 0.5 0.5 0.5 0.5 0.7
LG64* 0.3 0.3 1.8 1.8 1.2 1.6 LG75* 0.9 1.1 3.1 4.7 3.1 5.3 LG76*
6.2 14.1 21.4 48.6 54.1 92.5 LG79* 0.6 0.4 1.5 1.6 1.4 1.4 LG93*
45.0 43.8 310.5 281.8 185.9 183.8 160A* 0.4 0.4 1.7 1.5 1.1 1.1
160B* 0.8 0.8 1.1 1.1 0.9 0.9 160C* 0.5 0.5 0.7 0.7 0.6 0.8 160D*
0.5 0.3 0.4 0.4 0.3 0.3 160E* 0.6 0.5 0.6 0.6 0.6 0.3 LD109 9.3
18.6 31.1 20.7 7.0 2.0 LD103 11.6 17.2 24.4 27.1 12.3 6.6 LD100 3.9
1.5 12.0 15.3 6.9 1.3 LD99 4.4 12.8 44.6 27.0 6.2 0.8 LD98 0.2 0.7
0.8 1.0 0.8 0.6 LD96 2.3 0.3 1.3 3.6 1.2 0.4 LD95 1.2 19.5 3.6 9.1
9.4 5.9 LD94 1.5 5.9 2.0 8.5 9.2 7.3 LD93 1.8 4.2 3.9 24.6 15.8
10.8 LD91 0.2 0.2 0.2 0.2 0.2 0.2 LD90 0.4 0.4 0.4 0.4 0.5 0.5 LD82
0.1 0.1 0.2 0.3 0.4 0.7 LD81 0.1 0.1 15.2 7.6 5.4 2.3 LD80 0.1 0.1
3.7 6.5 7.1 2.1 LD79 0.1 0.1 0.1 0.2 0.3 0.2 LD77 0.1 0.1 6.9 11.1
6.4 2.9 LD76 0.1 0.1 0.2 0.3 0.2 0.4 LD75 0.1 0.1 0.1 0.2 0.2 0.2
LD70 0.1 0.1 0.6 0.6 0.6 0.6 LD64 0.1 0.1 0.3 0.3 0.3 0.4 LD63 0.7
0.8 0.3 0.3 0.4 0.6 LD62 0.4 0.4 0.4 0.4 0.4 0.5 LG109 16.3 36.0
23.4 37.9 25.7 34.5 LG100 21.5 32.7 11.8 18.0 8.6 8.1 LG98 0.7 2.3
13.2 9.8 6.8 7.8 LG96 46.7 82.9 37.3 32.6 14.1 18.5 LG93 3.6 9.2
19.8 37.3 24.0 24.6 LG91 0.2 0.2 0.1 0.2 0.1 0.1 LG90 0.1 0.1 0.0
0.0 0.1 0.0 LG87 14.5 7.5 9.2 10.4 5.9 4.7 LG82 0.2 0.2 0.6 0.8 0.5
1.2 LG81 0.7 0.8 1.2 4.1 14.8 19.9 LG80 2.2 11.9 19.8 31.3 21.9
17.4 LH1 0.0 0.0 0.0 0.0 0.0 0.0 LH6 0.0 0.0 0.0 0.0 0.0 0.0 LH7
0.0 0.0 0.0 0.0 0.0 0.0 LH10 0.0 0.0 0.0 0.0 0.0 0.0 LH11 0.0 0.0
0.0 0.0 0.0 0.0 LH13 0.0 0.0 0.0 0.0 0.0 0.0 LH15 0.0 0.0 0.0 0.0
0.0 0.0 LH17 0.0 0.0 0.0 0.0 0.0 0.0 LH20 0.0 0.0 0.0 0.0 0.0 0.0
LH21 0.0 0.0 0.0 0.0 0.0 0.0 LH22 0.0 0.0 0.0 0.0 0.0 0.0 Q in the
lipid name indicates that the tertiary amines of the lipid were
quaternized using methyl iodide. L indicates lipids prepared from
the indicated acrylates and amines. N indicates that the ester
functional group of the acrylate has been replaced with an amide
group. *indicates 72 hours incubation before bright-glo.
[0336] The table below summarizes the data as a % of the luciferase
activity obtained from the use of Lipofectamine 2000. The table
indicates the best lipids for transfection.
TABLE-US-00004 TABLE 3 2.5 w/w 5 w/w 10 w/w 15 w/w 20 w/w 25 w/w
LD28 1.7 8.5 LD31 44.2 38.7 18.8 11.3 42.8 174.1 LD77 0.1 0.1 6.9
11.1 6.4 2.9 LD81 0.1 0.1 15.2 7.6 5.4 2.3 LD86 29.6 16.0 LD87 53.9
43.3 LD93 1.8 4.2 3.9 24.6 15.8 10.8 LD94 1.5 5.9 2.0 8.5 9.2 7.3
LD95 1.2 19.5 3.6 9.1 9.4 5.9 LD99 4.4 12.8 44.6 27.0 6.2 0.8 LD100
3.9 1.5 12.0 15.3 6.9 1.3 LD103 11.6 17.2 24.4 27.1 12.3 6.6 LD109
9.3 18.6 31.1 20.7 7.0 2.0 LE86 31.1 22.7 22.6 2.5 0.0 0.0 LE87
10.5 9.0 38.4 4.3 0.0 0.0 LE96 13.5 0.5 0.0 0.0 0.0 0.0 LE99 9.9
5.4 13.7 2.7 0.0 0.0 LE103 20.4 22.1 11.8 2.8 0.0 0.0 LE109 1.5 5.3
28.7 18.0 1.8 0.6 LF31 64.1 78.7 13.4 69.5 97.3 266.8 LF76 1.7 5.6
9.8 24.3 22.2 19.5 LF77 3.7 25.1 28.2 24.1 17.0 22.1 LF80 2.5 35.1
35.5 34.3 19.9 14.8 LF86 21.5 17.9 19.6 21.2 10.5 10.4 LF87 19.4
11.3 30.2 13.3 11.0 10.0 LF93 32.0 50.4 15.0 150.7 143.2 171.3 LF94
41.8 37.7 96.3 114.7 99.0 98.6 LF95 15.3 51.3 44.3 71.8 64.6 75.1
LF96 52.4 62.8 79.3 47.7 64.4 36.0 LF98 2.5 7.9 17.8 17.2 9.5 9.9
LF99 32.2 49.8 26.5 10.7 6.0 6.2 LF100 17.6 70.0 69.0 85.9 44.2
50.9 LF103 43.9 11.6 65.4 91.8 61.6 61.4 LF109 16.0 28.3 16.9 21.9
28.7 49.5 LG25 15.9 32.0 43.2 56.9 42.1 63.9 LG31 19.6 27.6 34.0
8.5 14.3 94.0 LG32 0.2 10.2 1.0 0.7 0.4 0.7 LG76* 6.2 14.1 21.4
48.6 54.1 92.5 LG77 43.5 34.0 LG80 2.2 11.9 19.8 31.3 21.9 17.4
LG81 0.7 0.8 1.2 4.1 14.8 19.9 LG87 14.5 7.5 9.2 10.4 5.9 4.7 LG93*
3.6 9.2 19.8 37.3 24.0 24.6 LG96 46.7 82.9 37.3 32.6 14.1 18.5 LG98
0.7 2.3 13.2 9.8 6.8 7.8 LG100 21.5 32.7 11.8 18.0 8.6 8.1 LG109
16.3 36.0 23.4 37.9 25.7 34.5 LG93 45.0 43.8 310.5 281.8 185.9
183.8 ND28 124.8 116.0 28.9 0.0 0.0 0.0 QG75 10.0 20.6 34.0 2.7 1.0
0.8 QG76 5.6 16.9 20.7 2.5 0.0 0.0 QG80 1.3 4.9 32.2 68.4 36.2 24.5
QG81 0.7 3.6 41.1 15.5 2.4 1.4 QG82 25.0 24.6 32.6 8.6 2.3 1.4 QG87
89.0 94.0 42.4 64.7 62.1 44.1 QG90 1.1 4.3 7.7 17.4 7.3 5.4 QG91
0.4 3.3 25.9 45.2 18.5 2.8 QG98 2.1 10.3 22.3 14.9 9.3 4.5 QG100
11.2 32.1 57.1 102.6 93.0 94.5 QG109 29.9 40.6 31.3 52.6 51.3 46.6
Q in the lipid name indicates that the tertiary amines of the lipid
were quaternized using methyl iodide. L indicates lipids prepared
from the indicated acrylates and amines. N indicates that the ester
functional group of the acrylate has been replaced with an amide
group. *indicates 72 hour incubuation before bright-glo.
Example 2
Testing of Lipids for RNA Delivery
[0337] Reporter-protein knockdown achieved by the top transfecting
lipids relative to Lipofectamine.TM. 2000 (where negative values
indicate improved knockdown). The assay accounts for toxicity,
monitoring expression of both renilla and firefly luciferases,
where the latter serves as viability control. For each lipid, 50 ng
of siRNA was added per well at specific lipid/RNA w/w ratios (from
top to bottom: 2.5, 5, 10, 15).
TABLE-US-00005 ND95 ND98 ND99 ND100 NF96 LD31 LE87 LF31 LF95 LG32
19.8058 -4.00473 -10.1961 10.88263 -6.262 15.3558 5.504325 50.09235
36.29344 58.10496 8.318854 -4.48242 -9.86007 -7.54577 -2.03466
-7.89703 -6.92331 11.94445 28.86141 41.51079 -5.20544 -4.63444
-18.2728 -21.4945 63.19863 -15.5821 -8.53319 -0.82516 -2.22361
44.71105 -7.16345 -13.9514 -20.0273 -8.3303 90.8142 -16.8831
-15.2929 -10.5366 -8.09928 -13.6483 LG77 LG80 LG96 LG100 LG109 QD99
QF99 NG64 QF100 LG31 71.00772 2.006687 27.952 33.5374 55.7409
11.53579 9.026287 -11.7697 24.82954 41.00232 -0.05414 -9.08975
-20.9722 15.36957 43.90063 5.050401 -1.83976 -34.6712 1.751482
21.06244 -7.72114 -15.9409 -38.0741 -21.8017 -5.23097 -27.9145
-8.99917 3.073073 -0.8059 -2.71354 -21.3897 -21.0263 -31.1828
-20.7012 -11.1255 -22.3471 -34.0996 1.439032 0.212713 -5.76419
Example 3
Testing of Lipds for DNA Delivery
[0338] Raw values for luciferase expression (relative lights units)
of best-transfecting lipids at lipid/DNA (w/w) ratios listed. 300
ng of DNA was added per well.
TABLE-US-00006 2.5 w/w 5 w/w 10 w/w 15 w/w ND20 6868960 4880532
ND21 1170534 1213862 ND22 721270 1083569 LIPO2000 2467624 1754800
ND24 5256861 1231973 ND25 7548491 5088120 ND28 20925873 1299887
ND32 17713202 2745231 ND31 1154404 936815 ND36 4517622 2945357 ND36
2043526 658985 LIPO2000 3334772 4043316 LIPO2000 2424297 2086728
ND66 835433 1291599 LIPO2000 30344816 28204810 ND94 774425 1382667
ND95 2875811 4619654 ND95 7977064 10279185 ND96 2910700 42837 ND98
5649897 791660 LIPO2000 25489210 14945620 LIPO2000 21192523
19239706 ND99 10252260 9886021 ND100 2713327 294518 ND109 2197484
2087995 NF1 778473 227587 NF10 1192178 838283 LIPO2000 13040527
10774995 NF20 1814886 1801431 NF25 2473702 1366194 LIPO2000
19020395 15427061 NF61 4332331 3689556 NF60 2401570 3739668 NF63
1141469 865897 NF63 3031718 1138429 NF64 384317 2283250 NF70
1605749 887500 LIPO2000 6776111 5451663 LIPO2000 8309497 7617169
NF86 4798383 4087285 NF87 3904993 3436007 NF91 1993690 1670695
LIPO2000 8463204 7664209 NF95 1839462 2082965 NF95 1906488 918612
NF96 3095896 650770 NF100 1061441 8015 NF103 2559344 347240 NF109
2867865 678968 LIPO2000 1397825 1469076 LIPO2000 1897360 2712535
NG61 1452027 765795 NG64 2585784 1972838 LIPO2000 7992593 9351046
NG77 972002 1184771 NG86 1357355 1521687 NG86 1513703 1223360 NG87
823764 1005875 LIPO2000 6188050 5383340 LIPO2000 4484218 6094431
NG95 834849 1208120 LIPO2000 2308433 3627875 NP62 1443678 33188
NP63 1180418 1543275 NP63 1388102 798342 LIPO2000 15535841 14019561
LIPO2000 15019226 14524819 NP86 595340 1323915 NP87 1207376 1192838
LIPO2000 8746668 10523338 NP96 4598787 288439 NP98 7860614 24812
NP99 3202948 9767 NP103 8798627 308844 NP109 37952 19829 NF95 12051
3775212 NF103 19086 7008052 NF103 2886271 772518 NF109 85471
3352713 NF109 1151621 108854 LIPO2000 10602791 11196771 LIPO2000
11152535 11535693 ND20 7521540 435731 ND98 6947385 5165088 ND98
1824773 643862 ND99 11757295 7656193 ND100 5544902 2936058 NF61
458095 3053999 NF96 7037710 3202668 NF96 2048655 167043 NF103
5865613 4028907 NF109 6605163 1767496 LIPO2000 8861025 7852498
LIPO2000 7017261 6964853 2.5 w/w 5 w/w 10 w/w 15 w/w 20 w/w 25 w/w
LD31 44.2 38.7 18.8 11.3 42.8 174.1 LD87 53.9 43.3 LF31 64.1 78.7
13.4 69.5 97.3 266.8 LF93 32.0 50.4 15.0 150.7 143.2 171.3 LF94
41.8 37.7 96.3 114.7 99.0 98.6 LF95 15.3 51.3 44.3 71.8 64.6 75.1
LF96 52.4 62.8 79.3 47.7 64.4 36.0 LF99 32.2 49.8 26.5 10.7 6.0 6.2
LF100 17.6 70.0 69.0 85.9 44.2 50.9 LF103 43.9 11.6 65.4 91.8 61.6
61.4 LF109 16.0 28.3 16.9 21.9 28.7 49.5 LG25 15.9 32.0 43.2 56.9
42.1 63.9 LG31 19.6 27.6 34.0 8.5 14.3 94.0 LG76 6.2 14.1 21.4 48.6
54.1 92.5 * 72 hours incubation before bright-glo LG96 46.7 82.9
37.3 32.6 14.1 18.5 LG93 45.0 43.8 310.5 281.8 185.9 183.8 * 72
hours incubation before bright-glo ND28 124.8 116.0 28.9 0.0 0.0
0.0 QF80 0.4 1.4 10.9 52.7 35.1 20.8 QF86 61.1 58.1 53.2 27.8 22.2
19.6 QF87 11.7 41.8 57.2 64.0 53.5 45.4 QF91 0.3 2.1 36.4 51.2 23.0
12.1 QF94 34.2 37.6 42.5 53.6 47.7 35.6 QD99 5.1 22.9 16.9 83.1
91.7 96.9 QD109 40.3 65.7 71.0 60.5 44.0 32.9 LB100 0.0 0.9 13.2
39.9 46.6 29.0 LB109 0.0 0.9 1.9 13.5 47.1 61.6 QG80 1.3 4.9 32.2
68.4 36.2 24.5 QG87 89.0 94.0 42.4 64.7 62.1 44.1 QG100 11.2 32.1
57.1 102.6 93.0 94.5 QG109 29.9 40.6 31.3 52.6 51.3 46.6 ND20 278.4
278.1 0.2 0.1 0.0 0.0 ND20 84.9 5.5 0.1 0.0 ND21 47.4 69.2 18.7 2.6
0.1 0.4 ND22 29.2 61.7 48.0 4.4 0.4 0.2 ND24 157.6 30.5 2.4 0.2 0.0
0.0 ND25 226.4 125.8 19.5 0.5 0.0 0.0 ND26 13.4 2.3 0.1 0.0 0.0 0.0
ND28 627.5 32.1 0.1 0.1 0.0 0.0 ND31 7.7 5.6 47.6 44.9 7.6 19.7
ND32 531.2 67.9 1.1 0.1 0.0 0.0 ND36 135.5 72.8 84.3 31.6 0.8 0.0
ND95 11.3 30.9 37.6 53.4 37.8 54.0 ND98 22.2 5.3 0.5 0.4 0.4 0.3
ND98 78.4 65.8 26.0 9.2 ND99 16.8 36.5 78.6 91.7 ND99 132.7 97.5
6.8 2.2 ND100 45.7 48.0 20.8 2.7 ND100 62.6 37.4 2.5 0.6 NF60 6.8
21.9 28.9 49.1 NF61 5.2 38.9 8.6 1.1 NF61 63.9 67.7 7.0 2.9 NF64
5.7 41.9 0.3 0.1 NF86 56.7 53.3 4.5 2.3 NF87 46.1 44.8 0.3 0.0 NF95
131.6 141.8 100.5 33.9 NF95 6.0 9.4 4.7 12.1 NF96 221.5 44.3 0.2
0.1 NF96 79.4 40.8 29.2 2.4 NF100 75.9 0.5 0.0 0.0 NF103 183.1 23.6
0.0 0.0 NF103 66.2 51.3 0.6 0.0 NF109 205.2 46.2 1.7 0.0 NF109 74.5
22.5 3.9 1.6 NP96 43.4 2.6 7.1 8.2 NP98 74.1 0.2 0.3 0.4 NP98 0.4
0.3 0.3 0.2 NP103 83.0 2.8 0.0 1.5 NP103 2.1 2.3 3.8 1.4 The data
in the second set of columns represents is a replicate set of data
for certain experiments.
Other Embodiments
[0339] The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those of ordinary skill in
the art will appreciate that various changes and modifications to
this description may be made without departing from the spirit or
scope of the present invention, as defined in the following
claims.
* * * * *
References