SYNTHESIS AND USE OF AMINO LIPIDS

Abstract

The present invention provides novel amino lipids and a method for synthesizing these compounds. According to the invention, the amino lipids have a structure of the following formula:

##STR00001##

wherein Z is hydrogen or X.sup.1R.sup.1,
R.sup.1 and R.sup.2 are the same or different and independently C.sub.6-C.sub.24 alkyl, C.sub.6-C.sub.24 alkenyl, C.sub.6-C.sub.24 alkynyl, or C.sub.6-C.sub.24 acyl,
X.sup.1 and X.sup.2 are the same or different, S, SO or S(O).sub.2,
Y is

##STR00002##

or heterocycles of the formula

##STR00003##

wherein k and l are integers from 0 to 2,
R.sup.3 and R.sup.4 are either the same or different and independently C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, or C.sub.1-C.sub.12 alkynyl, wherein alkyl, alkenyl or alkynyl may be optionally substituted with a C.sub.1-C.sub.6 hydrocarbyl group, or R.sup.3 and R.sup.4 optionally join to form, together with the nitrogen atom to which they are bound, an optionally substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are the same or different alkyl amines;
R.sup.5 and R.sup.6 are absent or are hydrogen or C.sub.1-C.sub.12 alkyl,
R.sup.7 is hydrogen or C.sub.1-C.sub.12 alkyl,
m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3, wherein YN for p=2 or 3.

Claims

1-18. (canceled)

19. An amino lipid comprising a structure of the following formula: ##STR00158## wherein Z is selected from the group consisting of hydrogen and X.sup.1R.sup.1; wherein R.sup.1 and R.sup.2 are one of the same and different, and independently selected from the group consisting of C.sub.6-C.sub.24 alkyl, C.sub.6-C.sub.24 alkenyl, C.sub.6-C.sub.24 alkynyl, and C.sub.6-C.sub.24 acyl; wherein X.sup.1 and X.sup.2 are one of the same and different, and independently selected from the group consisting of S, SO and S(O).sub.2; wherein Y is selected from the group consisting of ##STR00159## NH, NR.sup.7, ##STR00160## and heterocycles of the formula ##STR00161## wherein k and l are integers from 0 to 2; wherein R.sup.3 and R.sup.4 are one of the same and different, and independently selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, and C.sub.1-C.sub.12 alkynyl, or R.sup.3 and R.sup.4 join to form, together with a nitrogen atom to which R.sup.3 and R.sup.4 are bound, a substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are one of the same and different alkyl amines; wherein R.sup.5 and R.sup.6 are absent or are selected from the group consisting of hydrogen and C.sub.1-C.sub.12alkyl; wherein R.sup.7 is selected from the group consisting of hydrogen and C.sub.1-C.sub.12 alkyl; wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; wherein YN for p=2 or for p=3, or Y is ##STR00162## for p=2.

20. The amino lipid of claim 19, wherein at least one of R.sup.1 and R.sup.2 can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

21. The amino lipid of claim 19, wherein, in connection with R.sup.3 and R.sup.4, one or more of the C.sub.1-C.sub.12 alkyl, the C.sub.1-C.sub.12 alkenyl, and the C.sub.1-C.sub.12 alkynyl can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

22. The amino lipid of claim 19, comprising: the structure of ##STR00163## or the structure of ##STR00164##

23. The amino lipid of claim 22, wherein at least one of R.sup.1 and R.sup.2 can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

24. The amino lipid of claim 22, wherein, in connection with R.sup.3 and R.sup.4, one or more of the C.sub.1-C.sub.12 alkyl, the C.sub.1-C.sub.12 alkenyl, and the C.sub.1-C.sub.12 alkynyl can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

25. The amino lipid of claim 19, having the structure of formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIn), (IIIo), (IIIp), (IIIr), (IIIs), (IIIt), (IIIu), (IIIv), or (IIIw): ##STR00165## ##STR00166## ##STR00167## wherein R.sup.1 and R.sup.2 are the same C.sub.11-C.sub.12 alkyl; wherein R.sup.3 and R.sup.4 are the same C.sub.1-C.sub.2 alkyl; and wherein m is an integer from 1 to 2, and n is an integer from 1 to 3.

26. An amino lipid comprising the structure of the following formula (Ic): ##STR00168## wherein R.sup.1 and R.sup.2 are one of the same and different, and independently selected from the group consisting of C.sub.6-C.sub.24 alkyl, C.sub.6-C.sub.24 alkenyl, C.sub.6-C.sub.24 alkynyl, and C.sub.6-C.sub.24 acyl; X.sup.1 and X.sup.2 are one of the same and different, and independently selected from the group consisting of S, SO and S(O).sub.2; wherein Y is selected from the group consisting of ##STR00169## wherein Z is selected from the group consisting of ##STR00170## k and l are integers from 0 to 2, m is an integer from 1 to 12 and n is an integer from 1 to 12.

27. The amino lipid of claim 26, wherein at least one of R.sup.1 and R.sup.2 can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

28. An amino lipid comprising: the structure of ##STR00171## or the structure of ##STR00172## wherein R.sup.1 and R.sup.2 are the same C.sub.6-C.sub.18 alkyl; Y is selected from the group consisting of ##STR00173## NH, NR.sup.7, ##STR00174## and heterocycles of the formula ##STR00175## wherein R.sup.3 and R.sup.4 are one of the same and different and each a C.sub.1-C.sub.12 alkyl, or R.sup.3 and R.sup.4 join to form, together with the nitrogen atom to which they are bound, a substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are one of the same and different alkyl amines; wherein R.sup.5 and R.sup.6 are absent or selected from the group consisting of hydrogen and a C.sub.1-C.sub.12 alkyl; wherein R.sup.7 is selected from the group consisting of hydrogen and a C.sub.1-C.sub.12 alkyl; wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; wherein YN for p=2 or for p=3, or Y is ##STR00176## for p=2.

29. The amino lipid of claim 28, wherein, in connection with R.sup.3 and R.sup.4, the C.sub.1-C.sub.12 alkyl is substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

30. A lipid particle containing an amino lipid comprising a structure of the following formula: ##STR00177## wherein Z is selected from the group consisting of hydrogen and X.sup.1R.sup.1; wherein R.sup.1 and R.sup.2 are one of the same and different, and independently selected from the group consisting of C.sub.6-C.sub.24 alkyl, C.sub.6-C.sub.24 alkenyl, C.sub.6-C.sub.24 alkynyl, and C.sub.6-C.sub.24 acyl; wherein X.sup.1 and X.sup.2 are one of the same and different, and independently selected from the group consisting of S, SO and S(O).sub.2; wherein Y is selected from the group consisting of ##STR00178## NH, NR.sup.7, ##STR00179## and heterocycles of the formula ##STR00180## wherein k and l are integers from 0 to 2; wherein R.sup.3 and R.sup.4 are one of the same and different, and independently selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, and C.sub.1-C.sub.12 alkynyl, or R.sup.3 and R.sup.4 join to form, together with a nitrogen atom to which R.sup.3 and R.sup.4 are bound, a substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are one of the same and different alkyl amines; wherein R.sup.5 and R.sup.6 are absent or are selected from the group consisting of hydrogen and C.sub.1-C.sub.12 alkyl; wherein R.sup.7 is selected from the group consisting of hydrogen and C.sub.1-C.sub.12 alkyl; wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; wherein YN for p=2 or for p=3, or Y is ##STR00181## for p=2.

31. The lipid particle of claim 30, wherein the lipid particle is a liposome.

32. The lipid particle of claim 30, further containing at least one of a non-cationic lipid, a sterol and a bioactive agent, wherein the bioactive agent is selected from the group consisting of a nucleic acid, an antineoplastic agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, an agent acting on a central nervous system, a polypeptide and a polypeptoid.

33. The lipid particle of claim 30, wherein the lipid particle is used for delivering a bioactive agent into a cell.

34. The lipid particle of claim 30, wherein the lipid particle is used as a medicament in treatment of at least one of a viral infection, a liver disease, a liver disorder, and a cancer.

35. A method for synthesizing an amino lipid comprising: performing a reaction of alkynes or alkenes of formula (VI): ##STR00182## wherein E is one of CHCH.sub.2 and CCH; with compounds of the formula HSR.sup.1 and HSR.sup.2 to yield a compound of formula (VIIa) or (VIIb): ##STR00183## wherein R.sup.1 and R.sup.2 are one of the same and different, and independently selected from the group consisting of C.sub.6-C.sub.24 alkyl, C.sub.6-C.sub.24 alkenyl, C.sub.6-C.sub.24 alkynyl, or C.sub.6-C.sub.24 acyl; wherein Y is selected from the group consisting of ##STR00184## NH, NR.sup.7, ##STR00185## and a heterocycle of the formula ##STR00186## wherein k and l are integers from 0, or wherein k and l are integers from 0 to 2; wherein R.sup.3 and R.sup.4 are one of the same and different and independently selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, and C.sub.1-C.sub.12 alkynyl, or R.sup.3 and R.sup.4 join to form, together with the nitrogen atom to which they are bound, an substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are one of the same and different alkyl amines; wherein R.sup.5 and R.sup.6 are absent or are selected from the group consisting of hydrogen and C.sub.1-C.sub.12alkyl; wherein R.sup.7 is one of hydrogen and C.sub.1-C.sub.12 alkyl; wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; wherein YN for p=2 or p=3, or Y is ##STR00187## for p=2.

36. The method of claim 35, wherein at least one of R.sup.1 and R.sup.2 can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

37. The method of claim 35, wherein, in connection with R.sup.3 and R.sup.4, one or more of the C.sub.1-C.sub.12 alkyl, the C.sub.1-C.sub.12 alkenyl, and the C.sub.1-C.sub.12 alkynyl can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

38. The method of claim 35, wherein the reaction of the alkynes or the alkenes of the formula (VI) with the compounds of the formula HSR.sup.1 and HSR.sup.2 is performed under UV-irradiation or using a radical initiator.

39. The method of claim 35, wherein the product of the reaction of the alkynes or the alkenes of the formula (VI) with the compounds of the formula HSR.sup.1 and HSR.sup.2 is a compound according to formula (VIIb).

39. The method of claim 35, further comprising oxidizing thioethers of the formula (VIIa) or (VIIb) into at least one of sulfoxide (SO) and sulfone (S(O).sub.2) using an oxidation agent to synthesize a sulfoxide or sulfone group containing an amino lipid.

40. The method of claim 35, further comprising: performing a reaction of alkynes or alkenes of the formula (IVa), (IVb), (IVc), or (IVd): ##STR00188## wherein n is an integer from 1 to 12, with the compound ##STR00189## to yield a compound of the formula (Va), (Vb), (Vc), or (Vd) ##STR00190##

41. The method of claim 40, further comprising: performing a reaction of alkynes or alkenes of the formula (Va), (Vb), (Vc), or (Vd): ##STR00191## with an amine of the formula (R.sup.3R.sup.4R.sup.5N)(CH.sub.2).sub.mZ, wherein m is an integer from 1 to 12, Z is selected from the group consisting of OH, NH.sub.2, NH, and a secondary heterocyclic amine of the formula ##STR00192## wherein k and l are integers from 0 to 2, to yield a compound of the formula (VIa) or (VIb): ##STR00193## wherein Y is selected from the group consisting of ##STR00194## and heterocycles of the formula ##STR00195## wherein k and l are integers from 0 to 2, wherein R.sup.3 and R.sup.4 are one of the same or different and independently C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, or C.sub.1-C.sub.12 alkynyl, wherein alkyl, alkenyl or alkynyl is optionally substituted with a C.sub.1-C.sub.6 hydrocarbyl group, or R.sup.3 and R.sup.4 optionally join to form, together with the nitrogen atom to which they are bound, an optionally substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, or R.sup.3 and R.sup.4 are one of the same and different alkyl amines; wherein R.sup.5 is absent or is selected from the group consisting of hydrogen and C.sub.1-C.sub.12 alkyl, wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; and wherein YN for p=2 or p=3.

42. The method of claim 35, further comprising: performing a reaction of alkynes or alkenes of the formula (IVe), (IVf), (IVg), or (IVh): ##STR00196## wherein n is an integer from 1 to 12, with an amine of the formula (R.sup.3R.sup.4R.sup.5N)(CH.sub.2).sub.mZ, wherein m is an integer from 1 to 12, and Z is selected from the group consisting of NH.sub.2, NH, N and a secondary heterocyclic amine of the formula ##STR00197## wherein k and l are integers from 0 to 2, to yield a compound of the formula (VIa) or (VIb): ##STR00198## wherein Y is selected from the group consisting of NH, NR.sup.7, ##STR00199## and heterocycles of the formula ##STR00200## wherein k and l are integers from 0 to 2, R.sup.3 and R.sup.4 are one of the same or different and independently selected from the group consisting of C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkenyl, or C.sub.1-C.sub.12 alkynyl, or R.sup.3 and R.sup.4 join to form, together with the nitrogen atom to which they are bound, a substituted N-heterocyclic ring of 3 to 10 atoms comprising 1 to 7 nitrogen atoms, wherein R.sup.5 and R.sup.6 are absent or are selected from the group consisting of hydrogen and C.sub.1-C.sub.12alkyls; wherein R.sup.7 is selected from the group consisting of hydrogen and C.sub.1-C.sub.12 alkyl; wherein m is an integer from 1 to 12, n is an integer from 1 to 12, and p is an integer from 1 to 3; and wherein YN for p=2 or p=3, or Y is ##STR00201## for p=2.

43. The method of claim 42, wherein, in connection with R.sup.3 and R.sup.4, one or more of the C.sub.1-C.sub.12 alkyl, the C.sub.1-C.sub.12 alkenyl, and the C.sub.1-C.sub.12 alkynyl can be substituted with a C.sub.1-C.sub.6 hydrocarbyl group.

Description

FIGURES

[0086] The following figures are presented to provide a better understanding of the description of procedures and conceptual aspects of the invention.

[0087] FIG. 1: Comparative microscope pictures showing results of lipofection by use of amino liposomal reagent #30 and one commercially available transfection reagent.

[0088] FIG. 2: Comparative microscope pictures showing results of lipofection by use of an amino liposomal reagent #60 and one commercially available transfection reagent.

[0089] FIG. 3: Graphical overview of the transfection efficiency of a library of transfection reagents of example compounds 1 to 44 compared to a commercially available reagent.

[0090] FIG. 4: Graphical overview of the transfection efficiency of a library of transfection reagents of example compounds 47 to 76 compared to a commercially available reagent.

[0091] FIG. 5: siRNA gene silencing in MEF cells using lipid reagent #24.

[0092] FIG. 6: siRNA gene silencing in MEF cells using lipid reagent #64.

EXAMPLES

[0093] The following 81 examples are presented to provide a better understanding of the description of procedures and conceptual aspects of the invention.

Example 1: Synthesis and Characterization of 2,3-bis(dodecylthio)propyl (2-(dimethylamino)ethyl) carbamate

[0094] ##STR00078##

[0095] The amino lipid was synthesized by two steps. The first step is to synthesize prop-2-yn-1-yl (2-(dimethylamino)ethyl) carbamate. 0.1 mmol carbonyiimidazole and 0.1 mmol propargyl alcohol were dissolved in 2 ml DCM and added into a 20 ml glass vial covered with aluminium foil. The solution was shaked for 1 h, then 0.1 mmol dimethylethane-1,2-diamine and 0.01 mmol DMAP were added, and the solution was shaken overnight.

[0096] The second step is the photo addition of dodecanethiol on to prop-2-yn-1-yl (2-(dimethylamino)ethyl) carbamate. To do this, 0.2 mmol dodecanthiol and 3 mg DMPA were added to the solution from first step and the vial was degassed by argon and irradiated under 365 nm UV for 2 h. The vial was then dried and stored under 4 C.

[0097] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 574.46 W/z.

[0098] Synthesis of the compounds of the examples 2-22 were carried out similarly to example 1. Step 1 differs in the used amine and the stoichiometric ratios differ for examples 17-22. Step 2 differs in the educts, whereas the stoichiometric ratios were maintained. The resulting compounds and the corresponding MW/z values are summarized in Table 1:

TABLE-US-00001 TABLE 1 Examples of synthesized compounds 2-22 and the corresponding MW/z values. Ex- am- ple Compound MW/z 2 2,3-bis(undecylthio)propyl (2-(dimethylamino)ethyl)carbamate 546.43 [00079] 3 2,3-bis(dodecylthio)propyl (2-(pyrrolidin-1-yl)ethyl)carbamate 600.47 [00080] 4 2,3-bis(undecylthio)propyl (2-(pyrrolidin-1-yl)carbamate 572.44 [00081] 5 2,3-bis(dodecylthio)propyl (2-(diethylamino)ethyl)carbamate 602.49 [00082] 6 2,3-bis(undecylthio)propyl (2-(diethylamino)ethyl)carbamate 574.46 [00083] 7 2,3-bis(dodecylthio)propyl (3-(diethylamino)propyl)carbamate 616.5 [00084] 8 2,3-bis(undecylthio)propyl (3-(diethylamino)propyl)carbamate 588.47 [00085] 9 2,3-bis(dodecylthio)propyl (3-(dimethylamino)propyl)carbamate 588.47 [00086] 10 2,3-bis(undecylthio)propyl (3-(dimethylamino)propyl)carbamate 560.44 [00087] 11 3-(dodecylthio)propyl (2-(dimethylamino)ethyl)carbamate 374.3 [00088] 12 3-(undecylthio)propyl (2-(dimethylamino)ethyl)carbamate 360.28 [00089] 13 3-(dodecylthio)propyl (2-(diethylamino)ethyl)carbamate 402.33 [00090] 14 3-(undecylthio)propyl (2-(diethylamino)ethyl)carbamate 388.31 [00091] 15 3-(dodecylthio)propyl (3-(diethylamino)propyl)carbamate 416.34 [00092] 16 3-(undecylthio)propyl (3-(diethylamino)propyl)carbamate 402.33 [00093] 17 bis(3-(dodecylthio)propyl) (piperazine-1,4-diylbis(propane-3,1-diyl))dicarbamate 772.59 [00094] 18 bis(3-(undecylthio)propyl) (piperazine-1,4-diylbis(propane-3,1-diyl))dicarbamate 744.56 [00095] 19 bis(3-(dodecylthio)propyl) ((methylazanediyl)bis(propane-3,1-diyl))dicarbamate 717.55 [00096] 20 bis(3-(undecylthio)propyl) ((methylazanediyl)bis(propane-3,1-diyl))dicarbamate 689.52 [00097] 21 3-(dodecylthio)propyl 4-(2-(((3-(dodecylthio)propoxy)carbonyl)amino)ethyl)piperazine-1-carboxylate 701.52 [00098] 22 3-(undecylthio)propyl 4-(2-(((3-(undecylthio)propoxy)carbonyl)amino)ethyl)piperazine-1-carboxylate 673.49 [00099]

Example 23: Synthesis and Characterization of 1-(2,3-bis(dodecylthio)propyl)-3-(2-(dimethylamino)ethyl)urea

[0099] ##STR00100##

[0100] The synthesis procedure of 1-(2,3-bis(dodecylthio)propyl)-3-(2-imethylamino)ethyl)urea is similar to the previous examples.

[0101] The first step is to synthesize 1-(2-(dimethylamino)ethyl)-3-(prop-2-yn-1-yl)urea. 0.1 mmol carbonyiimidazole and 0.1 mmol propargyl amine were dissolved in 2 ml DCM and added into a 20 ml glass vial covered with aluminium foil. The solution was shaked for 1 h, then 0.1 mmol dimethylethane-1,2-diamine and 0.01 mmol DMAP were added, the solution was shaked for overnight.

[0102] The second step is the photo addition of thiol onto 1-(2-(dimethylamino)ethyl)-3-(prop-2-yn-1-yl)urea. To do this, 0.2 mmol dodecanthiol and 3 mg DMPA were added to the solution from first step and the vial was degassed by argon and irradiated under 365 nm UV for 2 h. The vial was then dried and stored under 4 C.

[0103] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 573.47 MW/z.

[0104] Syntheses according to the examples 24-44 were carried out similarly to example 23. Step 1 differs in the used amine as well as the stoichiometric ratio for the examples 39-44. Step 2 differs in the educts, whereas the stoichiometric ratios were maintained. The resulting compounds and the corresponding molecular ion are resumed in table 2:

TABLE-US-00002 TABLE 2 Examples of synthesized compounds 24-44 and the corresponding MW/z values. Ex- am- ple Compound MW/z 24 1-(2,3-bis(undecylthio)propyl)-3-(2-(dimethylamino)ethyl)urea 545.44 [00101] 25 1-(2,3-bis(dodecylthio)propyl)-3-(2-(pyrrolidin-1-yl)ethyl)urea 599.49 [00102] 26 1-(2,3-bis(undecylthio)propyl)-3-(2-(pyrrolidin-1-yl)ethyl)urea 571.46 [00103] 27 1-(2,3-bis(dodecylthio)propyl)-3-(2-(diethylamino)ethyl)urea 601.50 [00104] 28 1-(2,3-bis(undecylthio)propyl)-3-(2-(diethylamino)ethyl)urea 573.47 [00105] 29 1-(2,3-bis(dodecylthio)propyl)-3-(3-(diethylamino)propyl)urea 615.52 [00106] 30 1-(2,3-bis(undecylthio)propyl)-3-(3-(diethylamino)propyl)urea 587.49 [00107] 31 1-(2,3-bis(dodecylthio)propyl)-3-(3-(dimethylamino)propyl)urea 587.49 [00108] 32 1-(2,3-bis(undecylthio)propyl)-3-(3-(dimethylamino)propyl)urea 559.46 [00109] 33 1-(2-(dimethylamino)ethyl)-3-(3-(dodecylthio)propyl)urea 373.31 [00110] 34 1-(2-(dimethylamino)ethyl)-3-(2-(undecylthio)propyl)urea 359.30 [00111] 35 1-(2-(diethylamino)ethyl)-3-(2-(dodecylthio)ethyl)urea 401.34 [00112] 36 1-(2-(diethylamino)ethyl)-3-(2-(undecylthio)propyl)urea 387.33 [00113] 37 1-(3-(diethylamino)propyl)-3-(3-(dodecylthio)propyl)urea 415.36 [00114] 38 1-(3-(diethylamino)propyl)-3-(3-(undecylthio)propyl)urea 401.34 [00115] 39 1,1-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(3-(3-(dodecylthio)propyl)urea) 770.63 [00116] 40 1,1-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(3-(3-(undecylthio)propyl)urea) 742.59 [00117] 41 1,1-((methylazanediyl)bis(propane-3,1-diyl))bis(3-(3-(dodecylthio)propyl)urea) 715.58 [00118] 42 1,1-((methylazanediyl)bis(propane-3,1-diyl))bis(3-(3-(undecylthio)propyl)urea) 687.55 [00119] 43 N-(3-(dodecylthio)propyl)-4-(2-(3-(3-(dodecylthio)propyl)ureido)ethyl)piperazine-1-carboxamide 699.55 [00120] 44 N-(3-(undecylthio)propyl)-4-(2-(3-(3-(undecylthio)propyl)ureido)ethyl)piperazine-1-carboxamide 671.52 [00121]

Example 45: Synthesis and Characterization of 3-(dodecylsulfinyl)-2-(dodecylthio)propyl (2-(dimethylamino)ethyl)carbamate

[0105] ##STR00122##

[0106] The amino lipid is synthesized in one step. 1 mmol 2,3-bis(dodecylthio)propyl (2-(dimethylamino)ethyl)carbamate (product of example 1) was mixed with 10 mmol aqueous hydrogen peroxide (30%) in 10 ml methanol, and stirred at room temperature for 1 h. Then the mixture was transferred to a 50 ml flask and evaporated.

[0107] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 590.5 MW/z.

Example 46: Synthesis and Characterization of 2,3-bis(dodecylsulfonyl)propyl (2-(dimethylamino)ethyl)carbamate

[0108] ##STR00123##

[0109] The amino lipid is synthesized in one step. 1 mmol 2,3-bis(dodecylthio)propyl (2-(dimethylamino)ethyl)carbamate (product of example 1) was mixed with 10 mmol aqueous hydrogen peroxide (30%) in 10 ml methanol, and stirred at room temperature for 2 d. Then the mixture was transferred to a 50 ml flask and evaporated.

[0110] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 638.4 MW/z.

Example 47: Synthesis and Characterization of N.SUP.1.,N.SUP.1.-bis(3-(dodecylthio)propyl)-N.SUP.2.,N.SUP.2.-dimethylethane-1,2-diamine

[0111] ##STR00124##

[0112] The synthesis procedure of N.sup.1,N.sup.1-bis(3-(dodecylthio)propyl)-N.sup.2,N.sup.2-dimethylethane-1,2-diamine is similar to the previous examples.

[0113] 1 mmol of N.sup.1,N.sup.1-dimethylethane-1,2-diamine, and 2 mmol 3-bromoprop-1-ene were added to a 20 ml glass vial containing 2 ml THF and 1 wt % Cs.sub.2CO.sub.3. The solution was shaked for overnight at 40 C. The solution vial was then removed from the shaker, centrifuged and the supernatant was transferred into a new 20 ml vial. 2 mmol dodecane-1-thiol and 5-7 wt % 2,2-Dimethoxy-2-phenylacetonphenone (DMPA) solution in THF were added to the above 20 ml glass vial. The mixture was irradiated under UV 365 nm for 2 h and then the THF was evaporated out.

[0114] This step was carried out similar to the previous examples. The stoichiometric ratios were maintained.

[0115] To verify the identity of the molecules, crude product was tested by mass spectrometry (m/z 572.51).

[0116] Syntheses according to the examples 48-59 were carried out similarly to example 47. Step 1 differs in the used amine, whereas the stoichiometric ratios were maintained. The resulting compounds and the corresponding molecular ion are resumed in table 3.

TABLE-US-00003 TABLE 3 Examples of synthesized compounds 48-56 and the corresponding MW/z values. Example Compound MW/z 48 3-(dodecylthio)-N-(3-(dodecylthio)propyl)-N-(2-(pyrrolidin-1-yl)ethyl)propan-1-amine 598.52 [00125] 49 N.sup.1,N.sup.1-bis(3-(dodecylthio)propyl)-N.sup.2,N.sup.2-diethylethane-1,2-diamine 600.54 [00126] 50 N.sup.1,N.sup.1-bis(3-(dodecylthio)propyl)-N.sup.3,N.sup.3-diethylpropane-1,3-diamine 614.56 [00127] 51 N.sub.1,N.sub.1-bis(3-(dodecylthio)propyl)-N.sub.3,N.sub.3-dimethylpropane-1,3-diamine 586.52 [00128] 52 N.sup.1,N.sup.1-dimethyl-N.sup.2,N.sup.2-bis(3-(undecylthio)propyl)ethane-1,2-diamine 544.48 [00129] 53 N-(2-(pyrrolidin-1-yl)ethyl)-3-(undecylthio)-N-(3-(undecylthio)propyl)propan-1-amine 570.50 [00130] 54 N.sup.1,N.sup.1-diethyl-N.sup.2,N.sup.2-bis(3-(undecylthio)propyl)ethane-1,2-diamine 572.51 [00131] 55 N.sup.1,N.sup.1-diethyl-N.sup.3,N.sup.3-bis(3-(undecylthio)propyl)propane-1,3-diamine 586.53 [00132] 56 N.sup.1,N.sup.1-dimethyl-N.sup.3,N.sup.3-bis(3-(undecylthio)propyl)propane-1,3-diamine 558.50 [00133]

Example 57: Synthesis and Characterization of 11-(dodecylthio)-2-methyl-9-oxa-13-thia-2,5-diazapentacosan-7-ol

[0117] ##STR00134##

[0118] The lipid was synthesized as follows, 1 mmol N.sup.1,N.sup.1-dimethylethane-1,2-diamine, and 1 mmol 2-((prop-2-yn-1-yloxy)methyl)oxirane were dissolved in 2 ml ethanol in a 20 ml glass vial covered with septum. The solution was shaken for overnight at 40 C. The solution vial was then removed from the shaker, ethanol was evaporated and 2 ml fresh THF was added and mixed well, the solution was then degassed for 5 min and filled with Argon (Ar) for 3 min, then covered with a cap. 2 mmol dodecane-1-thiol and 5-7 wt % 2,2-Dimethoxy-2-phenylacetonphenone (DMPA) solution in THF were added to the above 20 ml glass vial. The mixture was irradiated under UV 365 nm for 2 h and then the THF was evaporated out.

[0119] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 605.08 MW/z.

[0120] Synthesis of the compounds of the examples 58-66 were carried out similarly to example 57. The resulting compounds and the corresponding MW/z values are summarized in Table 4:

TABLE-US-00004 TABLE 4 Examples of synthesized compounds 58-66 and the corresponding MW/z values. Example Compound MW/z 58 1-(2,3-bis(dodecylthio)propoxy)-3-((2-(pyrrolidin-1-yl)ethyl)amino)propan-2-ol 631.11 [00135] 59 12-(dodecylthio)-3-ethyl-10-oxa-14-thia-3,6-diazahexacosan-8-ol 633.13 [00136] 60 13-(dodecylthio)-3-ethyl-11-oxa-15-thia-3,7-diazaheptacosan-9-ol 647.16 [00137] 61 12-(dodecylthio)-2-methyl-10-oxa-14-thia-2,6-diazahexacosan-8-ol 619.10 [00138] 62 2-methyl-11-(undecylthio)-9-oxa-13-thia-2,5-diazatetracosan-7-ol 577.02 [00139] 63 1-(2,3-bis(undecylthio)propoxy)-3-((2-(pyrrolidin-1-yl)ethyl)amino)propan-2-ol 603.06 [00140] 64 3-ethyl-12-(undecylthio)-10-oxa-14-thia-3,6-diazapentacosan-8-ol 605.08 [00141] 65 3-ethyl-13-(undecylthio)-11-oxa-15-thia-3,7-diazahexacosan-9-ol 619.10 [00142] 66 2-methyl-12-(undecylthio)-10-oxa-14-thia-2,6-diazapentacosan-8-ol 519.05 [00143]

Example 67: Synthesis and Characterization of 21-(2-(dimethylamino)ethyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol

[0121] ##STR00144##

[0122] The synthesis procedure of 21-(2-(dimethylamino)ethyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol is similar to the previous examples. 1 mmol N.sup.1,N.sup.1-dimethylethane-1,2-diamine, and 2 mmol 2-((allyloxy)methyl)oxirane were dissolved in 2 ml ethanol in a 20 ml glass vial covered with septum. The solution was shaken for overnight at 40 C. The solution vial was then removed from the shaker, ethanol was evaporated and 2 ml fresh THF was added and mixed well, the solution was then degassed for 5 min and filled with Argon (Ar) for 3 min, then covered with a cap. 2 mmol dodecane-1-thiol and 5-7 wt % 2,2-Dimethoxy-2-phenylacetonphenone (DMPA) solution in THF were added to the above 20 ml glass vial. The mixture was irradiated under UV 365 nm for 2 h and then the THF was evaporated out.

[0123] This step was carried out similar to the previous examples. The stoichiometric ratios were maintained.

[0124] To verify the identity of the molecules, crude product was tested by mass spectrometry (m/z 721.24).

[0125] Syntheses according to the examples 68-76 were carried out similarly to example 67. The resulting compounds and the corresponding molecular ion are resumed in table 5:

TABLE-US-00005 TABLE 5 Examples of synthesized compounds 68-76 and the corresponding MW/z values. Example Compound MW/z 68 21-(2-(pyrrolidin-1-yl)ethyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol 747.27 [00145] 69 21-(2-(diethylamino)ethyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol 749.29 [00146] 70 21-(4-(diethylamino)butyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol 777.34 [00147] 71 21-(4-(dimethylamino)butyl)-17,25-dioxa-13,29-dithia-21-azahentetracontane-19,23-diol 749.29 [00148] 72 20-(2-(dimethylamino)ethyl)-16,24-dioxa-12,28-dithia-20-azanonatriacontane-18,22-diol 693.18 [00149] 73 20-(2-(pyrrolidin-1-yl)ethyl)-16,24-dioxa-12,28-dithia-20-azanonatriacontane-18,22-diol 719.12 [00150] 74 20-(2-(diethylamino)ethyl)-16,24-dioxa-12,28-dithia-20-azanonatriacontane-18,22-diol 721.24 [00151] 75 20-(4-(diethylamino)butyl)-16,24-dioxa-12,28-dithia-20-azanonatriacontane-18,22-diol 749.29 [00152] 76 20-(4-(dimethylamino)butyl)-16,24-dioxa-12,28-dithia-20-azanonatriacontane-18,22-diol 721.24 [00153]

Example 77: Synthesis and Characterization of N.SUP.1.-(3-(dodecylsulfinyl)-2-(dodecylthio)propyl)-N.SUP.2.,N.SUP.2.-dimethylethane-1,2-diamine

[0126] ##STR00154##

[0127] The amino lipid is synthesized in one step. 1 mmol N.sup.1-(2,3-bis(dodecylthio)propyl)-N.sup.2,N.sup.2-dimethylethane-1,2-diamine (product of example 50) was mixed with 10 mmol aqueous hydrogen peroxide (30%) in 10 ml methanol, and stirred at room temperature for 1 h. Then the mixture was transferred to a 50 ml flask and evaporated.

[0128] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 546.5 MW/z.

Example 78: Synthesis and Characterization of N.SUP.1.-(2,3-bis(dodecylsulfonyl)propyl)-N.SUP.2.,N.SUP.2.-dimethylethane-1,2-diamine

[0129] ##STR00155##

[0130] The amino lipid is synthesized in one step. 1 mmol N.sup.1-(2,3-bis(dodecylthio)propyl)-N.sup.2,N.sup.2-dimethylethane-1,2-diamine (product of example 50) was mixed with 10 mmol aqueous hydrogen peroxide (30%) in 10 ml methanol, and stirred at room temperature for 2 d. Then the mixture was transferred to a 50 ml flask and evaporated.

[0131] To verify the identity of the molecules, crude product was analyzed by mass spectrometry. The molecular ion was clearly identified as 594.4 MW/z.

[0132] Screening of the Cationic Lipid for Cell Transfection

Example 79: Initial Determination of Optimal Lipid Ratios for Cell Transfection

[0133] The well documented HEK 293T cell line is used for examples 79 and 80.

[0134] The natural phospholipid dioleolylphosphatidylethanolamine (DOPEstructure shown below) was selected as the required co-lipid (also termed helper lipid). It is required not for the stability of liposomes per se, but rather the breakdown of the lipid membranes in the endocytic compartment (endosomes) of cells, allowing release of the bioactive agent to the cytosol and/or nucleus. Basically, it is required for the desired effect for stable liposome formation in combination with our cationic amino lipids (DPDEC). DOPE was mixed with a representative cationic amino lipid (structure shown below) in different ratios. Both lipids were dissolved in ethanol at 50 mg/ml and combined to a final volume of 30 l.

[0135] DOPE (Co-Lipid):

##STR00156##

[0136] 2,3-bis(dodecylthio)propyl (2-(dimethylamino)ethyl) carbamate (DPDEC) as representative novel cationic amino lipids:

[0137] DPDEC:

##STR00157##

TABLE-US-00006 TABLE 6 DPDEC ratios tested DPDEC DOPE 0 1 1 3 1 2 1 1 2 1 3 1 1 0

[0138] These 30 l ethanol mixtures were then added to 70 l of 0.2 mol/l Sodium acetate buffer (pH 5.0) with constant vortexing for 30 s, followed by sonication for 5 min to form liposomes. Final lipid content is 2 mg/ml. This final 2 mg/ml liposome sample is referred to as the lipid reagent.

[0139] 0.1 l, 0.2 l, 0.3 l, 0.4 l and 0.5 l of the above lipid reagents were combined with either 50 ng or 100 ng plasmid DNA (comprising the pCS-LacZ and pH2B-YFP plasmids at a ratio of 5:1, respectively) and mixed with cells as described below:

(amounts shown are for one well of a 96-well culture plate)
1. 0.1 l-0.5 l lipid reagent diluted in 10 l of 50 mmol/l sodium acetate buffer, pH 5.0.
2. After 2-5 min incubation, added diluted lipid reagent from (1) to either 50 ng or 100 ng plasmid DNA (DNA dissolved in 10 l of 50 mmol/l sodium acetate buffer, pH 5.0).
3. Samples were left at RT for 30 min to form Lipid/DNA transfection complexes. As DNA is negatively charged, it associates non-specifically with the positively charged head groups of the cationic lipids in the liposomes.
4. After 30 min, 50 l of freshly suspended HEK 293 cells (approximately 50,000 cells, in DMEM culture medium supplemented with 10% fetal calf serum) were added to the lipid/DNA complexes, mixed with pipette action and 65 l of the cells+lipid/DNA complexes added to a single 96-well.

[0140] To assess the ability of the lipid mixtures to deliver the plasmid DNA into cells, microscopy was used to visualize fluorescence emitted by the yellow fluorescent protein (YFP) 20-24 hours after initial transfection. The YFP protein is encoded by the pH2B-YFP plasmid and is efficiently synthesized and located within the nucleus of successfully transfected cells.

[0141] Results:

[0142] A very clear optimal ratio of lipid:DOPE was identified as 2:1 and the optimal lipid reagent:DNA ratio was 0.4 l lipid reagent per 75 ng DNA. These conditions were therefore used for the primary screen to identify the lipid reagents with highest cell transfection efficiency and lowest cell toxicity, as described in example 80 below.

Example 80: Primary Screen Using Novel Lipids Reagents

[0143] Cell line: HEK 293 cells
Screen format: 96-well
Detection (read-out): YFP fluorescence relative to total cell number (total cell number assessed using the nuclear dye, Hoechstsee FIGS. 1 and 2)

[0144] A commercially available liposomal transfection reagent was used as a reference (reference reagent) according to manufacturers' instructions, see FIGS. 1 and 2.

[0145] Method:

[0146] All steps performed in 96 tube/plate format using 8- or 12-channel multi-pipettes. Amounts shown are for duplicate (2) wells of a 96-well plate.

1. 0.8 l lipid reagents diluted in 20 l 50 mmol/l NaOAc buffer, pH 5.0.
2. Diluted lipid reagents from (1) were added to 150 ng DNA (25 ng pH2B-YFP+125 ng pCS-LacZ plasmids) in 20 l NaOAc buffer, pH 5.0 and mixed with pipette action.
3. After 30 min incubation at RT, added 100 l freshly resuspended cells (510.sup.4 cells/well DMEM culture medium supplemented with 10% fetal calf serum) and mixed with pipette.
4. Duplicate 65 l aliquots of the cells+lipid/DNA complexes were immediately transferred to separate wells of a 96-well culture plate and placed in 37 C. incubator with 5% CO.sub.2.
5. 20 to 24 hours after initial transfection of cells, Hoechst 33258 dye was added to cells at a final concentration of 0.2 g/ml and cells incubated for a further 30 min at 37 C. Cells were then placed on an inverted microscope and 2 independent sets of images of the cells captured from each well as shown in FIGS. 1 and 2.

[0147] For each sample, 3 images were captured: bright field image of cells (FIGS. 1 and 2 upper panels), Hoechst dye stained image of total cell nuclei (FIGS. 1 and 2 middle panels) and YFP images showing cells successfully transfected with plasmid DNA and expressing YFP protein (FIGS. 1 and 2 lower panels). Microscope images of transfected HEK 293 cells, showing both the transfection efficiency and toxicity level of two of our lipid molecules (#30 and #60) compared to a commonly used commercial transfection reagent (reference reagent). Lipid reagents #30 and #60 have a transfection efficiency of approximately 90% and have low cell toxicity (very few brightly stained apoptotic nuclei).

[0148] According to the protocol given in Example 80, a library of newly synthesized compounds according to claim 1 have been tested for their ability to transfect HEK 293 cells. The graphs in FIGS. 3 and 4 show the transfection efficiency of these lipid compounds compared to a commercially available transfection agent reference. 1 of the lipid molecules is efficient at delivering plasmid DNA (YFP gene) to HEK 293 cells when compared to a widely used commercial transfection reagent, indicated by the solid line. Two reagents, in particular have been identified as possessing very low toxicity and have the ability to very efficiently deliver siRNA molecules to cells (#24, see FIGS. 5 and #64; see FIG. 6).

Example 81: Screen of Library Hits for Ability to Transfect siRNA

[0149] One of the key technologies for manipulation of gene function, both in cells and whole organisms, is gene silencing through RNA interference (RNAi). Delivery of small interfering RNA (siRNA) molecules into mammalian cells is crucial for this technology and has significant clinical/therapeutic implications.

[0150] Thus, in addition to screening our lipids for delivery of plasmid DNA (the genes) we have also screened amino lipids according to the present invention for their ability to efficiently deliver siRNA molecules (the gene silencers).

[0151] In order to screen for this two different types of cells were used to test the ability of our lipid reagents to deliver siRNA targeting Low density lipoprotein receptor related protein 6 (LRP6). This is a 200 kD single-pass transmembrane receptor for Wnt ligands and activates the Wnt/b-catenin signalling pathway. It is expressed at relatively high levels in MEF cells.

[0152] Assay: Transfection of siRNA in mouse embryonic fibroblast (MEF) cells

[0153] Method:

[0154] All steps performed in 0.2 ml PCR tubes and 96 well plate format. Amounts shown are for one well of a 96-well plate.

1. 0.25 l lipid reagents diluted in 8 l 50 mmol/l NaOAc buffer, pH 5.0.
2. Diluted lipid reagents from (1) added to 1 pmol (0.2 l of 5 mol/1) siRNA molecules in 8 l NaOAc buffer, pH 5.0 and mixed with pipette action. The siRNA molecules used had either a scrambled sequence not specific for any known gene (Con siRNA), or a sequence specifically targeting the endogenous mRNA from the LRP6 gene (LRP6 siRNA).
3. After 30 min incubation at RT, added 50 l freshly resuspended cells (210.sup.4 cells/well DMEM culture medium supplemented with 10% fetal calf serum) in one well of a 96-well plate and placed in 37 C. incubator with 5% CO.sub.2.
4. 24 hours after initial transfection, cells, were lysed in 25 l detergent buffer (50 mmol/l Tris, 1% Triton X-100, 0.15 mol/l NaCl, pH 7.0, containing protease and phosphatase inhibitors), spun to remove insoluble cell debris and 30 l clarified lysates added to 10 l of 4 SDS loading buffer (250 mmol/l Tris HCl, 40% Glycerol, 8% SDS, 0.01% Bromophenol Blue, 5% 2-Mercaptoethanol, pH 6.8).
5. Samples were denatures by heating at 96 C. for 2 min and 12 l loaded on a 9% SDS-PAGE gels for separation of proteins according to molecular weight. Separated proteins were transferred from the SDS-PAGE gel to nitrocellulose membrane for Western Blot (WB) analysis.
6. WB was performed using an automated BioLane HTI instrument using antibodies against LRP6 and -tubublin proteins. HRP linked secondary antibodies and chemiluminescence were used to detect the proteins on the membrane.

[0155] Results

[0156] FIGS. 5 and 6 show the Western Blot (WB) analysis of LRP6 protein from total lysates of MEF (mouse embryonic fibroblast) cells transfected with the indicated siRNA molecules and cultured for 24 hours. Lipid reagents #24 and #64 are quite effective compared with the commercial reagent A at siRNA mediated gene silencing. This demonstrates the striking difference in properties of related transfection reagents and highlights the importance of using our novel method to easily synthesize hundreds of related lipids that can be screened to identify the ones having optimal properties (such as highly efficient DNA and siRNA delivery as well as low cellular toxicity).

LIST OF ABBREVIATIONS

[0157] Ar Argon [0158] DCM Dichloromethane [0159] DEDPA N-(2-(Dimethylamino)ethyl)-4,5-bis(dodecylthio)pentanamide [0160] DIC N, N-Diisopropylcarbodiimide [0161] DMEM Culture medium [0162] DMF Dimethylformamide [0163] DMPA 2,2-Dimethoxy-2-phenylacetonphenone [0164] DNA Desoxyribonucleic acid [0165] DOPE Dioleolylphosphatidylethanolamine [0166] YFP Yellow Fluorescent Protein [0167] HOBt Hydroxylbenzotriazole [0168] HRP Horseradish Peroxidase [0169] kD kilo Dalton [0170] LRP6 Low density lipoprotein receptor related protein 6 [0171] MEF Mouse embryonic fibroblast [0172] PEG Polyethylene glycol [0173] RNA Ribonucleic acid [0174] RNAi RNA interference [0175] siRNA small interfering RNA [0176] SDS Sodium dodecyl sulfate [0177] THF Tetrahydrofuran [0178] WB Western Blot [0179] Wnt Signalling proteins in cell differentiation