Ionizable cationic lipid for RNA delivery

Abstract

What is described is a compound of formula (1)

##STR00001##
wherein

• • R₁ and R₂ are the same or different, each a linear or branched alkyl with 1-9 carbons, or an alkenyl or alkynyl with 2 to 11 carbon atoms;
  • L₁ and L₂ are the same or different, each a linear alkyl having 5 to 18 carbon atoms, or form a heterocycle with N;
  • X₁ is a bond, or is —CO—O— whereby L₂-CO—O—R₂ is formed;
  • X₂ is S or O; L₃ is a bond or a lower alkyl, or form a heterocycle with N;
  • R₃ is a lower alkyl; and R₄ and R₅ are the same or different, each a lower alkyl;
    or a pharmaceutically acceptable salt thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of Provisional U.S. patent application No. 61/905,724, filed Nov. 18, 2013, the contents of which is incorporated herein by reference in its entirety.

BACKGROUND

A number of different types of nucleic acids are currently being developed as therapeutics for the treatment of a number of diseases. These nucleic acids include DNA in gene therapy, plasmids-based interfering nucleic acids, small interfering nucleic acids for use in RNA interference (RNAi), including siRNA, miRNA, antisense molecules, ribozymes and aptamers. As these molecules are being developed, there has been developed a need to produce them in a form that is stable and has a long shelf-life and that can be easily incorporated into an anhydrous organic or anhydrous polar aprotic solvent to enable encapsulations of the nucleic acids without the side-reactions that can occur in a polar aqueous solution or nonpolar solvents.

The present invention relates to novel lipid compositions that facilitate the intracellular delivery of biologically active and therapeutic molecules. The present invention relates also to pharmaceutical compositions that comprise such lipid compositions, and that are useful to deliver therapeutically effective amounts of biologically active molecules into the cells of patients.

The delivery of a therapeutic compound to a subject is important for its therapeutic effects and usually it can be impeded by limited ability of the compound to reach targeted cells and tissues. Improvement of such compounds to enter the targeted cells of tissues by a variety of the means of delivery is crucial. The present invention relates the novel lipids, incompositions and methods for preparation that facilitate the targeted intracellular delivery of biological active molecules.

Examples of biologically active molecules for which effective targeting to a patient's tissues is often not achieved include: (1) numerous proteins including immunoglobin proteins, (2) polynucleotides such as genomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and (4) many low molecular weight compounds, whether synthetic or naturally occurring, such as the peptide hormones and antibiotics.

One of the fundamental challenges now facing medical practitioners is that a number of different types of nucleic acids are currently being developed as therapeutics for the treatment of a number of diseases. These nucleic acids include DNA in gene therapy, plasmids small interfering nucleic acids (iNA) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, microRNA and aptamers. As these nucleic are being developed, there is a need to produce lipid formulations that are easy to make and can be readily delivered to a target tissue.

SUMMARY

What is described herein is a compound of Formula 1

##STR00002##
in which

R₁ and R₂ are the same or different, each a linear or branched alkyl, alkenyl, or alkynyl,

• • L₁ and L₂ are the same or different, each a linear alkyl having at least five carbon atoms, or form a heterocycle with the N,
  • X₁ is a bond, or is —CO—O— whereby L₂-CO—O—R₂ is formed
  • X₂ is S or O,
  • L₃ is a bond or a lower alkyl,
  • R₃ is a lower alkyl,
  • R₄ and R₅ are the same or different, each a lower alkyl.

What is also described herein is the compound of formula 1, in which L₃ is absent, R₁ and R₂ each consists of at least seven carbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are methyl or ethyl, and L₁ and L₂ each consists of a linear alkyl having at least five carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is absent, R₁ and R₂ each consists of at least seven carbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are methyl or ethyl, and L₁ and L₂ each consists of a linear alkyl having at least five carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is absent, R₁ and R₂ each consists of an alkenyl of at least nine carbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are methyl or ethyl, and L₁ and L₂ each consists of a linear alkyl having at least five carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is methylene, R₁ and R₂ each consists of at least seven carbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are methyl or ethyl, and L₁and L₂ each consists of a linear alkyl having at least five carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is methylene, R₁ and R₂ each consists of at least nine carbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are each methyl, L₁ and L₂ each consists of a linear alkyl having at least seven carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is methylene, R₁ consists of an alkenyl having at least nine carbon atoms and R₂ consists of an alkenyl having at least seven carbon atoms, R₃ is n-propylene, R₄ and R₅ are each methyl, L₁ and L₂ each consists of a linear alkyl having at least seven carbon atoms.

What is also described herein is the compound of formula 1, in which L₃ is methylene, R₁ and R₂ each consists of an alkenyl having at least nine carbon atoms, R₃ is ethylene, R₄ and R₅ are each methyl, L₁ and L₂ each consists of a linear alkyl having at least seven carbon atoms.

What is also described herein is a compound having the structure

##STR00003##

What is also described herein is a compound having the structure

##STR00004##

What is also described herein is a compound having the structure

##STR00005##

What is also described herein is a compound having the structure

##STR00006##

What is also described herein is a compound having the structure

##STR00007##

What is also described herein is a compound having the structure

##STR00008##

What is also described herein is a compound having the structure

##STR00009##

What is also described herein is a compound having the structure

##STR00010##

What is also described herein is a compound having structure

##STR00011##

What is also described herein is a compound having the structure

##STR00012##

What is also described herein is a compound having the structure

##STR00013##

What is also described herein is a compound having the structure

##STR00014##

What are also described herein are any of the compounds listed in ATX-001 to ATX-032 listed in Table 1, below or a pharmaceutically acceptable salt thereof, in a lipid composition, comprising a nanoparticle or a bilayer of lipid molecules. The lipid bilayer preferably further comprises a neutral lipid or a polymer. The lipid composition preferably comprise a liquid medium. The composition preferably further encapsulates a nucleic acid. The nucleic acid preferably has an activity of suppressing the expression of the target gene by utilizing RNA interference (RNAi). The lipid composition preferably further comprises a nucleic acid and a neutral lipid or a polymer. The lipid composition preferably encapsulates the nucleic acid.

The nucleic acid preferably has an activity of suppressing the expression of a target gene. The target gene preferably is a gene associated with inflammation.

What is also described herein is a method for introducing a nucleic acid into a cell of a mammal by using any of the compositions, above. The cell may be in a liver, lung, kidney, brain, blood, spleen, or bone. The composition preferably is administered intravenously, subcutaneously, intraperitoneally, or intrathecally. Preferably, the compositions described herein are used in a method for treating cancer or inflammatory disease. The disease may be one selected from the group consisting of immune disorder, cancer, renal disease, fibrotic disease, genetic abnormality, inflammation, and cardiovascular disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the knockdown activity of siRNA encapsulated by different cationic lipids. The lipids include MC3 (0.3 mg/kg), NC1 (0.3 mg/kg), ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0 mg/kg), and ATX-003 (0.3 and 1.0 mg/kg). The amount of Factor VII knockdown in mouse plasma is shown following administration of the siRNA formulation to C57BL6 mice, compared to injection of vehicle alone. The amount of Factor VII in abnormal and normal human plasma is included as a control. Statistically significant decreases in Factor VII levels (p<0.01) is shown by an asterix (*).

FIG. 2 shows an evaluation of the effect of siRNA of Factor VII activity based on the results shown in FIG. 2, and normalized to percentage knockdown compared to the vehicle alone.

FIG. 3 shows the knockdown activity of siRNA encapsulated by different cationic lipids. The lipids include MC3 (0.3 and 1.5 mg/kg), or listed at Table 1 of formula (1) are provided in Table 1.

TABLE 1
				KD @
Lipid				0.3
ID	Novel Lipid	MW	pKa	mg/kg
ATX- 001	##STR00015##	695.1	8.9	~0
ATX- 002	##STR00016##	681	8.7	98
ATX- 003	##STR00017##	695.1	9.3	~0
ATX- 004	##STR00018##	709.13	9.4	~0
ATX- 005	##STR00019##	709.13	9.0	~0
ATX- 006	##STR00020##	723.15	9.8	~0
ATX- 007	##STR00021##	723.15	9.5	n/a
ATX- 008	##STR00022##	737.18	10.3	n/a
ATX- 009	##STR00023##	695.1	8.8	~0
ATX- 010	##STR00024##	709.13	9.6	30
ATX- 011	##STR00025##	709.13	9.4	n/a
ATX- 012	##STR00026##	723.15	10.2	~0
ATX- 013	##STR00027##	681.01		n/a
ATX- 014	##STR00028##	695.1		n/a
ATX- 015	##STR00029##	695.1		n/a
ATX- 016	##STR00030##	709.13		15
ATX- 017	##STR00031##	695.1		n/a
ATX- 018	##STR00032##	554.92		40 (@ .05 mpk)
ATX- 019	##STR00033##	611.03		30 (@ .05 mpk)
ATX- 020	##STR00034##	667.13		40 (@ .05 mpk)
ATX- 021	##STR00035##	679.04		n/a
ATX- 022	##STR00036##	665.01		n/a
ATX- 023	##STR00037##	695.1		n/a
ATX- 024	##STR00038##	925.5		0
ATX- 025	##STR00039##	869.39		15
ATX- 026	##STR00040##	681.07		n/a
ATX- 027	##STR00041##	695.1		n/a
ATX- 028	##STR00042##	681.07		n/a
ATX- 029	##STR00043##	681.1		n/a
ATX- 030	##STR00044##	695.1		n/a
ATX- 031	##STR00045##	663.1		n/a
ATX- 032	##STR00046##	645.13		n/a

Table 1 shows the name and structure of each compound, its molecular weight, its pKa, and its knockdown bioactivity (KD) in an assay described below in Example 19.

Example 2

Synthesis of Methyl 8-Bromooctanoate

##STR00047##

S.	Chemicals/Reagents
No.	and solvents	M.Wt.	Moles	Eq.	Wt.
1	8-Bromooctanoic acid	223	269.05	1	60	gm.
2	Dry MeOH				400	ml
3	Con H2SO4				10	drop

Under N2 atmosphere, 8-bromooctanoic acid was dissolved in dry methanol. Concentrated H₂SO₄ was added drop-wise and the reaction mixture was stirred under reflux for three hours.

The reaction was monitored by thin layer chromatography until completed. Solvent was completely removed under vacuum. The reaction mixture was diluted with ethyl acetate and washed with water. The water layer was re-extracted with ethyl acetate. The total organic layer was washed with a saturated NaHCO₃ solution. The organic layer was washed again with water and finally washed with brine. The product was dried over anhydrous Na₂SO₄ and concentrated.

Example 3

Synthesis of dimethyl 8,8′-(benzanediyl)dioctanoate

##STR00048##

S.	Chemicals/Reagents
No.	and solvents	M.Wt.	Moles	Eq.	Wt.
1	Benzyl amine	107	126.54	1	13.54
2	Methyl 8-bromooctanoate	237	253.08	2	60	g
3	Dry K2CO3	138	759.25	6	104.7
4	Dry DMF				500	ml

Dry K₂CO₃ was taken and added to dry dimethylformawide under N₂. Benzyl amine in dimethylformamide was slowly added. Methyl 8-bromooctanoate dissolved in dimethylformamide was then added at room temperature. The reaction mixture was heated to 80° C. and the reaction was maintained for 36 hours with stirring.

The reaction was monitored by thin layer chromatography until completed. The reaction product was cooled to room temperature and water was added. The compound was extracted with ethyl acetate. The water layer was re-extracted with ethyl acetate. The total organic layer was washed with water and finally with brine solution. The product was dried over anhydrous Na₂SO₄ and concentrated.

The reaction product was purified by silica gel column chromatography in 3% methanol in chloroform. 44 gm of pure product was recovered.

Using TLC system of 10% methanol in chloroform, the product migrated with a Rf: 0.8, visualizing by charring in ninhydrine. The overall yield was 82%. The compound was a light brown liquid. The structure was confirmed by 1H-NMR.

Example 4

Synthesis of Dimethyl 8,8′-Azanediyldioctanoate

##STR00049##

S.	Chemicals/
No.	Reagents and solvents	M.Wt.	mmoles	Eq.	Wt.
1	Dimethyl 8,8′-	419.60	8.34	1	3.5	gm
	(benzanediyl) dioctanoate
2	10% Pd/C		20% wt		700	mg
3	Ethanol				90	ml

Dimethyl 8,8′-(benzanediyl)dioctanoate was transferred to hydrogenation glass vessel, and ethanol was added followed by 10% Pd/C. The reaction mixture was shaken in a Parr-shaker apparatus under 50 pounds per square inch [psi] H₂ atmosphere pressure for two hours at room temperature.

The reaction product was filtered through celite and washed with hot ethyl acetate. The filtrate was concentrated under vacuum.

Example 5

Synthesis of Dimethyl 8,8′-((Tertbutoxycarbonyl)Azanedil) Dioctanoate

##STR00050##

S.	Chemicals/
No	reagents/solvents	Mw	Mole's	Eq	wt
1	Dimethyl 8,8′-	329	0.0972	1	32	gm
	azanediyl-dioctanoate
2	Boc anhydride	218	0.145	1.5	31.3	gm
3	Et3N (Dry)	101	0.389	4	9	gm
4	DCM(Dry)				700	ml

Dimethyl 8,8′-azanediyldioctanoate was transferred to DCM and Et₃N to the reaction mass and cooled to 0° C. Boc anhydride diluted in DCM was added drop to the above reaction. After the addition was completed, the reaction mixture was stirred at room temperature for three hours.

The reaction was quenched with water and the DCM layer was separated. The water phase was re-extracted with DCM and the combined DCM layers were washed with brine solution and dried with Na₂SO₄. After concentration, 40 gm of crude compound was collected.

Crude reaction product was purified by column chromatography using 0-12% ethyl acetate in hexane. The yield recovered was 48%. A single product migrated by thin layer chromatography in 20% ethyl acetate in hexane with an Rf of 0.5, charring with ninhydrine.

Example 6

Synthesis of 8,8′-((Tertbutoxycarbonyl)Azanediyl) Dioctanoic Acid

##STR00051##

S.
No	Chemicals/reagents/solvents	Mw	Mole's	Eq	wt
1	Dimethyl 8,8′ ((tertbutoxy-	429	0.0489	1	21	gm
	carbonyl)azanediyl)
	dioctanoate
2	6N NaOH (aq.)				175	ml
3	Dry THF				200	ml

Dimethyl 8,8′-((tertbutoxycarbonyl)azanedil) dioctanoate was transferred to THF. A 6N sodium hydroxide solution was added at room temperature. The reaction was maintained with stirring overnight at room temperature.

Reaction mass was evaporated under vacuum at 25° C. to remove THF. The reaction product was acidified with 5N HCl. Ethyl acetate was added to the aqueous layer. The separated organic layer was washed with water and the water layer was re-extracted with ethyl acetate. The combined organic layers were washed with brine solution and dried over anhydrous Na₂SO₄. Concentration of the solution gave 18 gm of crude mass.

Example 7

Synthesis of di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl)

##STR00052##

S.	Chemicals/reagents/
No	solvents	Mw	Mole's	Eq	wt
1	8,8′-((tertbutoxycarbonyl)	549.5	0.03275	1	18	gm
	azanediyl) dioctanoic
	acid
2	Cis-2-nonene-1-ol	142.24	0.065514	2	9.31	gm
3	HATU	380.23	0.06878	2.1	26.15	gm
4	Di-Isopropyl ethyl amine	129.25	0.1146	3.5	14.81	gm
5	DMAP	122.17	0.003275	0.1	400	mg
6	Dry-DCM				150	ml

8,8′-((tertbutoxycarbonyl)azanediyl) dioctanoic acid was dissolved in dry DCM. HATU was added to this solution. Di-isopropyl ethyl amine was added slowly to the reaction mixture at room temperature. The internal temp rose to 40° C. and a pale yellow color solution was formed. DMAP was added to the reaction mixture followed by cis-2-nonene-1-ol solution in dry DCM. The reaction changed to brown color. The reaction was stirred for five hours at room temperature.

The reaction was checked by thin layer chromatography under completion. Water was added to the reaction product, which was extracted with DCM. The DCM layer was washed with water followed by brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to obtain 35 gm of crude compound.

Example 8

Synthesis of ATX-001

##STR00053##

Di((Z)-non-2-en-1 -yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (0.023 mol, 15 g) was dissolved in dry dichloromethane (DCM) (200 ml). Trifluoroacetic acid (TFA) was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. The collected crude product (12 grams) was dissolved in dry DCM (85 ml) under nitrogen gas. Triphosgene were added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. DCM solvent was removed from the reaction mass by distillation under N₂.The reaction product was cooled to 0° C., diluted with DCM (50 ml), and 2-((2-(dimethylamino)ethyl)thio) acetic acid (0.039 mol, 6.4 g) and carbodiimide (EDC HCl) (0.054 mol, 10.4 g). The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was diluted with 0.3M HCl solution (75 ml), and the organic layer was separated. The aqueous layer was reextracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution (75 ml) and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10 gram. The crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 10.5 g (68%).

Example 9

Synthesis of ATX-002

##STR00054##

Di((Z)-non-2-en-1 -yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (13.85 mmol, 9 grams) was dissolved in dry DCM (150 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. The collected crude product was dissolved in dry DCM (85 ml) under nitrogen gas. Triphosgene were added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. DCM solvent was removed from the reaction mass by distillation under N₂. The reaction product was cooled to 0° C., diluted with DCM (50 ml), and 2-(dimethylamino) ethanethiol HCl (0.063 mol, 8.3 g) was added, followed by Et₃N (dry). The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was diluted with 0.3M HCl solution (75 ml), and the organic layer was separated. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution (75 ml) and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10 gram. The crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 3.1 gram.

Example 10

Synthesis of ATX-003

##STR00055##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (0.00337 mol, 2.2 g) was dissolved in dry DCM (20 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated under reduced pressure. The collected crude product was dissolved in dry DCM (10 ml) under nitrogen gas. Triphosgene (0.0182 mol, 5.4 g) was added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. DCM solvent was removed from the reaction mass by distillation under N₂. The reaction product was cooled to 0° C., diluted with DCM (15 ml), and 2-(dimethylamino)propanethiol HCl (0.0182 mol, 2.82 g) was added, followed by Et₃N (dry). The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was diluted with 0.3 M HCl aqueous solution (20 ml), and the organic layer was separated. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution (50 ml) and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 5 gram. The crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 0.9 gram.

Example 11

Synthesis of ATX-004

##STR00056##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (0.023 mol, 15 g) was dissolved in DCM (200 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. The collected crude product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (5.853 mmol, 3.2 g) was dissolved in dry dimethyl formamide (DMF) under nitrogen, and 2-((3-(dimethylamino)propyl) thio)acetic acid (10.48 mmol, 1.85 g) and EDC HCl (14.56 mmol, 2.78 g) was added. The reaction mixture was stirred for overnight at room temperature. The reaction was quenched with water (30 ml) and diluted with DCM (30 ml), and the organic layer was separated. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution and dried over anhydrous Na₂SO₄. The crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1 gram (24.2%).

Example 12

Synthesis of ATX-005

##STR00057##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (0.023 mol, 15 g) was dissolved in dry DCM (200 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. Crude reaction product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (5.853 mmol, 3.2 g) was dissolved in DMF under nitrogen gas. 2-((3-(dimethylamino)propyl)thio)acetic acid (10.48 mmol, 1.85 g) and EDC HCl (14.56 mmol, 2.78 g) were added and the reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was quenched with water (30 ml) and diluted with DCM (30 ml). The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution (75 ml) and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 5 gram. Crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1 gram (24.2%).

Example 13

Synthesis of ATX-006

##STR00058##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate was dissolved in dry DCM (150 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. The collected crude product was dissolved in dry DCM (85 ml) under nitrogen gas. Triphosgene were added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The crude reaction produce was dissolved in dry DMF under nitrogen atmosphere, and 2-((2-(diethylamino)ethyl)thio)acetic acid (3.93 mmol, 751 mg) and EDC HCl (5.45 mmol, 1.0 g) were added. The reaction mixture was stirred for overnight at room temperature. The reaction was quenched with water (3 ml) and excess DMF was removed under vacuum at 25° C. The reaction product was diluted with water and aqueous layer was extracted thrice with DCM (20 ml). The combined organic layers were washed with brine solution and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 2 gram. After purification by silica gel column (100-200 mesh) using 3% MeOH/DCM., the yield was 1.2 grams (76%).

Example 14

Synthesis of ATX-009

##STR00059##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (13.85 mmol, 9 grams) was dissolved in dry DCM (20 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. Di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (0.909 mmol, 500 mg) was dissolved in dry DCM (20 ml) under nitrogen atmosphere. Triphosgene were added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. DCM solvent was removed from the reaction mass by distillation under nitrogen atmosphere. 2-(ethyl(methyl)amino)ethane-1-thiol hydrochloride (4.575 mmol, 715 mg) was dissolved in DMF (7 ml) and tetrahydrofuran (THF) (5 ml), and was added drop wise to the sodium hydride suspension in THF at 0° C. The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was diluted with ethyl acetate and cold water. The reaction was neutralized with 5% HCl (9 ml), and the organic layer was separated. The aqueous layer was re-extracted with ethyl acetate (EtOAc) (20 ml), washed in cold water and brine, and the combined organic layers were washed dried over anhydrous Na₂SO₄. Concentration of the solvent gave 1 gram or crude product. The compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM to yield 100 mg.

Example 15

Synthesis of ATX-010

##STR00060##

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl) azanediyl) dioctanoate (3.079 mmol, 2 g) was dissolved in dry DCM (20 ml). TFA was added at 0° C. to initiate a reaction. The reaction temperature was slowly allowed to warm to room temperature over for 30 minutes with stirring. Thin layer chromatography showed that the reaction was completed. The reaction product was concentrated under vacuum at 40° C. and the crude residue was diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated. The collected crude product was dissolved in dry DCM (20 ml) under nitrogen gas. Triphosgene (14.55 mmol, 4.32 g) was added and the reaction mixture was cooled to 0° C., and Et₃N was added drop wise. The reaction mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. DCM solvent was removed from the reaction mass by distillation under N₂. The reaction product was cooled to 0° C., diluted with DCM (20 ml), and 2-(dimethylamino)ethanethiol HCl (0.063 mol, 8.3 g) was added, followed by Et₃N (dry). The reaction mixture was then stirred overnight at room temperature. Thin layer chromatography showed that the reaction was completed. The reaction product was diluted with 0.3 M HCl solution (20 ml), and the organic layer was separated. The aqueous layer was re-extracted with DCM, and the combined organic layers were washed with 10% K₂CO₃ aqueous solution 20 ml) and dried over anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10 gram. The crude compound was purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1.4 g (75%)

Example 16

Synthesis of ATX-011 to ATX-030 and ATX-32

The synthesis of ATX-011 to ATX-30 follows the synthesis of Examples 1-15, by substituting appropriate starting ingredients for synthetic reactions described therein.

Example 17

Synthesis of ATX-031

##STR00061## ##STR00062##

Example 19

In Vivo Mouse Factor VII Silencing

Using a liver-directed in vivo screen of the liposome libraries, a series of compounds were tested that facilitate high levels of siRNA mediated gene silencing in hepatocytes, the cells comprising the liver parenchyma. Factor VII, a blood clotting factor, is a suitable target gene for assaying functional siRNA delivery to liver. Because this factor is produced specifically in hepatocytes, gene silencing indicates successful delivery to parenchyma, as opposed to delivery to the cells of the reticulo-endothelial system (e.g., Kupffer cells). Furthermore, Factor VII is a secreted protein that can be readily measured in serum, obviating the need to euthanize animals. Silencing at the mRNA level can be readily determined by measuring levels of protein. This is because the protein's short half-life (2-5 hour). C57BL/6 mice (Charles River Labs) received either saline or siRNA in liposome formulations via tail vein injection at a volume of 0.006 ml/g. At 48 h after administration, animals were anesthetized by isofluorane inhalation and blood was collected into serum separator tubes by retroorbital bleed. Serum levels of Factor VII protein were determined in samples using a chromogenic assay (Biophen FVII, Aniara Corporation) according to manufacturers' protocols. A standard curve was generated using serum collected from saline-treated animals.

Compositions with siRNA directed to Factor VIII VII were formulated with ATX-001,ATX-002, ATX-003, and ATX-547, and comparator samples NC1 and MC3 (Alnylam). These were injected into animals at 0.3 mg/kg and at 1 mg/kg. The siRNA encapsulated by MC3 (0.3 mg/kg), NC1 (0.3 mg/kg), ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0 mg/kg), and ATX-003 (0.3 and 1.0 mg/kg) was measured for the ability to knockdown Factor VII in mouse plasma following administration of the siRNA formulation to C57BL6 mice. The results showed that ATX-001 and ATX-002 were was most effective at 0.3 mg/kg, compared to controls (FIGS. 1 and 2).

The siRNA encapsulated MC3 (0.3 and 1.5 mg/kg), NC1 (0.3 mg/kg), ATX-547 (0.1 and 0.3 mg/kg), ATX-004 (0.3 mg/kg), ATX-006 (0.3 and 1.0 mg/kg), ATX-010 (0.3 mg/kg), and ATX-001 (0.3 and 1.5 mg/kg), was measured for Factor VII knockdown in mouse plasma following administration of the siRNA formulation to C57BL6 mice. The results showed that ATX-001 ATX-002 and ATX-010 were most effective (FIGS. 3 and 4). The knockdown activity of the exemplary compounds is shown for 0.3 mg/kg or at 0.05 mg/kg for ATX-018, ATX-019, and ATX-020 (Table 1).

Claims

1. A compound of formula:

2. The compound of claim 1, wherein the X₂ is S.

3. The compound of claim 1, wherein the R₃ is ethylene.

4. The compound of claim 1, wherein the R₃ is n-propylene or isobutylene.

5. The compound of claim 1, wherein the R₄ and R₅ are separately methyl, ethyl, or isopropyl.

6. The compound of claim 1, wherein L₁ and L₂ are the same.

7. The compound of claim 1, wherein L₁ and L₂ differ.

8. The compound of claim 1, wherein L₁ or L₂ consists of a linear alkylene having seven carbons.

9. The compound of claim 1, wherein L₁ or L₂ consists of a linear alkylene having nine carbons.

10. The compound of claim 1, wherein R₁ and R₂ are the same.

11. The compound of claim 1, wherein R₁ and R₂ differ.

12. The compound of claim 10, wherein R₁ and R₂ each consists of an alkenyl.

13. The compound of claim 10, wherein R₁ and R₂ each consists of an alkyl.

14. The compound of claim 12 1, wherein the alkenyl of R₁and R₂ consists of a single double bond.

15. The compound of claim 10 1, wherein R1 R₁ or R2 R₂ consists of nine carbons.

16. The compound of claim 10 1, wherein R1 R₁ or R2 R₂ consists of eleven carbons.

17. The compound of claim 10 1, wherein R1 R₁ or R2 R₂ consists of seven carbons.

18. The compound of claim 1, wherein L₃ is a bond, R₃ is ethylene, X₂ is S, and R₄ and R₅ are each methyl.

19. The compound of claim 1, wherein L₃ is a bond, R₃ is n-propylene, X₂ is S, R₄ and R₅ are each methyl.

20. The compound of claim 1, wherein L₃ is a bond, R₃ is ethylene, X₂ is S, and R₄ and R₅ are each ethyl.

21. The A compound of claim 1, selected from the group consisting of the compounds of formula ATX-001 to ATX-028, and ATX-031

22. A compound of formula I

23. The compound of claim 22 consisting of the compound of formula ATX-029

24. The compound of claim 21, wherein the compound is

25. The compound of claim 21, wherein the compound is