# US 5,705,693 A · Cationic lipids

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- Number: US 5,705,693 A
- Document ID: US5705693A
- Status: Granted
- Kind: A
- Application number: 08/499,699
- Filed: 1995-07-07
- Granted: 1998-01-06
- Earliest claimed: 1994-07-08
- Assignees: Gilead Sciences, Inc.
- Inventors: Randolph B. DePrince; Kevin L. Facchine; George S. Lewis; Jason G. Lewis; Kuei-Ying Lin; Mark D. Matteucci; Robert A. Mook, Jr.; Richard W. Wagner
- Web page: https://patentgrep.com/patent/US5705693A

## CPC

- C07K 5/06095: {Arg-amino acid}
- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- C07C 237/06: having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C 237/20: the carbon skeleton containing six-membered aromatic rings
- C07C 237/22: having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated (peptides C07K)
- C07C 271/22: to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
- C07C 279/14: being further substituted by carboxyl groups
- C07C 279/22: Y being a hydrogen or a carbon atom, e.g. benzoylguanidines
- C07C 279/24: Y being a hetero atom
- C07J 41/0055: {the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives}
- C07K 5/06086: {with the first amino acid being basic}
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07J: STEROIDS (seco-steroids C07C)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to new cationic lipids and intermediates in their synthesis that are useful for transfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. The lipids comprise one or two substituted arginine, lysine or ornithine residues, or derivatives thereof, linked to a lipophilic moiety. The lipids form a complex when mixed with polyanions such as nucleic acids or peptides. The complexes permit efficient transfer of polyanions into cells, usually without significant toxicity to the cells.

## Claims

### Claim 1

A compound having the structure ##STR24## wherein each R.sup.1 is independently alkyl (10-22 C) or a mono unsaturated alkenyl (10-22 C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
R.sup.15 is alkyl; alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; alkenyl; alkenyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; aryl; aryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; heteroaryl; heteroaryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; N(H)alkyl; N(alkyl).sub.2 wherein each alkyl group is independently chosen; O-alkyl; O-aryl; alkylaryl; O-alkylaryl; alkylheteroaryl; or O-alkylheteroaryl;
L is an integer having a value of 2, 3, 4, 5, 6 or 7;
m is an integer having a value of 1;
n is an integer having a value of 0 or 1;
provided that when L is 3, n is 1, each R.sup.1 is independently alkyl (12-22C) or alkenyl (12-22C), and R.sup.15 together with the C(O) group to which it is linked, is a protecting group, then R.sup.4 is hydrogen;
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 2 (depends on 1)

The compound of claim 1 wherein L is 3 or 4, n is 1, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H and R.sup.15 is alkyl (1-6C).

### Claim 3 (depends on 1)

The compound of claim 1 wherein R.sup.15 is alkyl (1-4C).

### Claim 4 (depends on 3)

The compound of claim 3 wherein L is 3 or 4, n is 1, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H and R.sup.15 is --CH.sub.3 or --C(CH.sub.3).sub.3, --O--CH(CH.sub.3).sub.2 or --O--C(CH.sub.3).sub.3.

### Claim 5 (depends on 4)

The compound of claim 4 wherein each R.sup.1 is n-C.sub.14 H.sub.29 and L is 3.

### Claim 6 (depends on 1)

The compound of claim 1 wherein L is 3 or 4, n is 1, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H and R.sup.15 is alkyl (3-6C).

### Claim 7 (depends on 6)

The compound of claim 6 wherein each R.sup.1 is n-C.sub.14 H.sub.29.

### Claim 8 (depends on 1)

The compound of claim 1 wherein L is 3 or 4, n is 0, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H, R.sup.15 is --CH.sub.3, --CH.sub.2 CH.sub.3, --CH.sub.2 CH.sub.2 CH.sub.3, --CH(CH.sub.3).sub.2 or --C(CH.sub.3).sub.3.

### Claim 9 (depends on 8)

The compound of claim 8 wherein each R.sup.1 is n-C.sub.14 H.sub.29.

### Claim 10

A compound having the structure ##STR25## wherein each R.sup.1 is independently alkyl (10-11C) or a mono unsaturated alkenyl (10-11C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
R.sup.15 is alkyl; alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; alkenyl; alkenyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; aryl; aryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; heteroaryl; heteroaryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; N(H)alkyl; N(alkyl).sub.2 wherein each alkyl group is independently chosen; O-alkyl; O-aryl; alkylaryl; O-alkylaryl; alkylheteroaryl; or O-alkylheteroaryl;
L is an integer having a value of 2, 3, 4, 5, 6 or 7;
m is an integer having a value of 0 or 1;
n is an integer having a value of 0 or 1;
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 11

A compound having the structure ##STR26## wherein each R.sup.1 is independently alkyl (10-22C) or a mono unsaturated alkenyl (10-22C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
R.sup.15 is alkyl; alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; alkenyl; alkenyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; aryl; aryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; heteroaryl; heteroaryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; N(H)alkyl; N(alkyl).sub.2 wherein each alkyl group is independently chosen; O-alkyl; O-aryl; alkylaryl; O-alkylaryl; alkylheteroaryl; or O-alkylheteroaryl;
L is an integer having a value of 5, 6 or 7;
m is an integer having a value of 0 or 1;
n is an integer having a value of 0 or 1;
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 12

A compound having the structure ##STR27## wherein each R.sup.1 is independently alkyl (10-22C) or a mono unsaturated alkenyl (10-22C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
m is an integer having a value of 0 or 1;
L is an integer having a value of 5, 6 or 7;
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 13 (depends on 12)

The compound of claim 12 wherein m is 1.

### Claim 14 (depends on 12)

The compound of claim 12 wherein L is 5 or 6.

### Claim 15

A compound having the structure ##STR28## wherein each R.sup.1 is independently alkyl (10-22C) or a mono unsaturated alkenyl (10-22C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
L is an integer having a value of 2, 5, 6 or 7;
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 16

A compound of structure 35 ##STR29## wherein R1 is n-C.sub.14 C.sub.29 and the salts and solvates thereof.

### Claim 17

A composition comprising a polyanion and a lipid having the structure F ##STR30## wherein each R.sup.1 is independently alkyl (10-22 C) or a mono unsaturated alkenyl (10-22 C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.5 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
R.sup.15 is alkyl; alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; alkenyl; alkenyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; aryl; aryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; heteroaryl; heteroaryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; N(H)alkyl; N(alkyl).sub.2 wherein each alkyl group is independently chosen; O-alkyl; O-aryl; alkylaryl; O-alkylaryl; alkylheteroaryl; or O-alkylheteroaryl;
L is an integer having a value of 2, 3, 4, 5, 6 or 7;
m is an integer having a value of 1;
n is an integer having a value of 0 or 1;
provided that when L is 3, n is 1, each R.sup.1 is independently alkyl (12-22C) or alkenyl (12-22C), and R.sup.15 together with the C(O) group to which it is linked, is a protecting group, then R.sup.4 is hydrogen; and
and the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 18 (depends on 17)

The composition of claim 17 wherein
L is 3 or 4, n is 1, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H and R.sup.15 is alkyl (1-4C); or
L is 3 or 4, n is 1, each R.sup.1 is independently alkyl (12-16C), R.sup.4 is hydrogen or t-BOC, R.sup.5 is H and R.sup.15 is --CH.sub.3, --O--CH(CH.sub.3).sub.2, or --C(CH.sub.3).sub.3.

### Claim 19 (depends on 17)

The composition of claim 17 wherein the polyanion is a nucleopolymer.

### Claim 20 (depends on 19)

The composition of claim 19 wherein the nucleopolymer is an oligonucleotide or an expression vector wherein the expression vector is capable of expressing a polypeptide in a cell.

### Claim 21

A composition comprising a polyanion and a lipid having the structure ##STR31## wherein each R.sup.1 is independently alkyl (10-22 C) or a mono unsaturated alkenyl (10-22 C) group;
each R.sup.4 is independently hydrogen or is a protecting group;
R.sup.15 is H, CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2 CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2 CH.sub.3, CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 C.sub.6 H.sub.4 -p-OH, CH.sub.2 OH, CH(OH)CH.sub.3, (CH.sub.2).sub.4 NHR.sup.4, (CH.sub.2).sub.3 NHR.sup.4 or (CH.sub.2).sub.3 NR.sup.4 C(NH)NHR.sup.4 ;
R.sup.15 is alkyl; alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; alkenyl; alkenyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; aryl; aryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; heteroaryl; heteroaryl substituted with halogen, hydroxyl, alkyl, or alkyl substituted with halogen, hydroxyl, S(O).sub.2, O-alkyl or O-aryl; N(H)alkyl; N(alkyl).sub.2 wherein each alkyl group is independently chosen; O-alkyl; O-aryl; alkylaryl; O-alkylaryl; alkylheteroaryl; or O-alkylheteroaryl;
L is an integer having a value of 5, 6 or 7;
m is an integer having a value of 0 or 1;
n is an integer having a value of 0 or 1; and
the salts, solvates, resolved and unresolved enantiomers and diastereomers thereof.

### Claim 22 (depends on 21)

The composition of claim 21 wherein the polyanion is a nucleopolymer.

### Claim 23 (depends on 22)

The composition of claim 22 wherein the nucleopolymer is an oligonucleotide or an expression vector wherein the expression vector is capable of expressing a polypeptide in a cell.