LIPIDS, LIPID COMPOSITIONS, LIPOSOMES, AND LIPOPLEXES RELATED APPLICATION This application is related to (and where permitted by law, claims priority to) United Kingdom patent application number GB 0106041.7 filed 12 March 2001, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD This invention pertains generally to the field of lipids and liposomes, and more particularly to certain lipids and lipid compositions, liposomes and lipoplexes formed therefrom, methods for their synthesis and preparation, compositions and medicaments comprising such liposomes and lipoplexes, and methods of cellular delivery, transfection, and medical treatment employing such liposomes and lipoplexes.

BACKGROUND Gene Therapy Gene therapy may be defined as the genetic modification of cells for therapeutic benefit (see, e. g., reference 1). The aim is to deliver genes to correct genetic defects or to alter the physiopathology of diseased cells in view of their cure or elimination (see, e. g., reference 2). In cancer gene therapy, therapeutic benefit may be achieved by targeting either non-malignant or malignant cells (see, e. g., reference 3). Examples of the former include cytokine gene therapy, where the aim is to elicit an immune response against tumour cells (see, e. g., reference 1). The genes encoding cytokines such as the interleukins have been used in combination with suicide gene therapy (see, e. g., reference 4). Gene therapy may also be directed at the malignant cell itself and encompasses techniques for rendering the malignant cells more susceptible to chemotherapy. This may involve either suppressing the expression of drug resistance genes (such as multidrug resistance, MDR), or the introduction of suicide genes to the tumour cells, which increase sensitivity to chemotherapeutic agents (see, e. g., reference 1). Suicide gene therapy can be subdivided into two categories; toxin gene therapy, in which the genes introduced to the cell encode a toxic product directly, and GDEPT.

GDEPT Conventional GDEPT requires; (1) a gene for an exogenous enzyme; (2) a vector to transport the gene into cells and (3) a relatively non-cytotoxic prodrug that is converted by the enzyme into a cytotoxic drug.

In the first step, the DNA encoding the enzyme is transported into the target tumour cells by means of a vehicle or"vector."Transfected cells will then produce the exogenous enzyme. Once this has occurred the prodrug is administered. When the prodrug reaches the enzyme, it is converted to the cytotoxic drug, killing the cell. Preferably, the surrounding cells have a"Bystander Effect"whereby cells not expressing the exogenous enzyme are killed by passage of the cytotoxic drug. Examples of prodrug/drug combinations along with their relevant activating enzyme include ganciclovir and HSV-tk; nitrogen mustard glutamic acid conjugates and carboxypeptidase G2 (CPG2); CB 1954 and nitroreductasse (NR) (see, e. g., reference 5).

Vectors A vector is usually required to transport exogenous DNA into a target cell, and this process is of paramount importance to GDEPT. Most of the current vectors can be conveniently divided into two groups, viral and non-viral. Both have their intrinsic advantages and disadvantages.

Viral Vectors Many different types of virus have been researched as vectors in GDEPT. The two most widely used are retroviruses (a type of RNA virus) and adenovirus (a DNA virus) (see, e. g., reference 1). The general principle for constructing these vectors is the excision of 2 or 3 genes encoding vital proteins from the viral genome, and replacement with the therapeutic gene to be used. A packaging cell line is then produced, engineered to express the genes deleted in the viral genome (see, e. g., reference 11). Infectious virions (the vector) can then be produced, with the packaging cell line supplying the necessary proteins that have been deleted from the virus. The vectors will still be lacking the genes encoding these proteins, and so once a target cell is infected, further replication is not possible.

Non-Viral Vectors Complications associated with viral vectors have led to much effort in the development of synthetic non-viral vectors. The main drawback in their development has been that they are not as efficient as viruses, and need to be presented to the target cells in high concentration (see, e. g., reference 12). There is also a problem with non-viral vectors being rapidly removed from circulation by white blood cells and the reticulo-endothelial system (RES), and a significant proportion of lipid dose being deposited in the large capillary bed of the lungs (see, e. g., reference 14). However, there are fewer safety and immunogenicity concerns inherent in viral systems, and non-viral vectors, particularly cationic lipids and polymers, are easier and cheaper to produce, and can be obtained on a much larger scale. A greater degree of control can be exercised over the structure on a molecular level, and the products can be highly purified (see, e. g., reference 14). Most non-viral vectors may conveniently be classified as cationic polymers, peptides, and cationic lipids (e. g., in cationic liposomes).

Cationic polymers rely on the electrostatic interaction with the anionic phosphate groups of DNA to form a complex (see, e. g., reference 15). The ratio of mixing is such that the complex retains a net positive charge, and thus binds to negatively charged cell surfaces where they are taken up by endocytosis.

As well as polycationic peptides such as polylysine, other peptides have also been used in many ways, including DNA compaction, targeting, endosomal escape, and nuclear localising signals (see, e. g., references 23-30).

Cationic Liposomes Cationic liposomes are the largest and most extensively studied group of non-viral vectors. Liposomes are large, spherical structures that form spontaneously when their constituent lipids are exposed to an aqueous environment. The basic structure of a typical cationic lipid comprises a cationic head group joined by a linker group to a large hydrophobic region.

The cationic head group often consists of primary, secondary or tertiary amines, but quaternary ammonium salts, guanidino and imidazole groups have also been investigated

(see, e. g., references 31-37). The most common linkers used are ethers and esters, although amides and carbamates have also been employed (see, e. g., references 38-41).

The hydrophobic domain is typically either a double chain hydrocarbon or a cholesterol derivative. The double chain hydrocarbons are typically 14-18 carbon atoms in length, and are either completely saturated or partially unsaturated (e. g., oleyl). Cationic lipids are sometimes formulated into liposomes alone, but more often they are mixed with a neutral co-lipid such as cholesterol or dioleoyl phosphatidylethanolamine (DOPE) (see, e. g., references 31,33-35,37-40,42-46).

Liposome formation and structure Cationic lipids are amphiphiles, which means they contain both a hydrophobic and a hydrophilic region. When exposed to an aqueous environment they organise into layers, with two back-to-back layers of lipids. The polar, hydrophilic head groups face outwards, shielding the hydrophobic domain from the aqueous solution. A flat 2- dimensional sheet would still have the hydrophobic entities around its edges exposed to water (see, e. g., reference 47). Instead, the edges join up to form spherical structures, called liposomes, which present no hydrophobic domains to the aqueous environment.

Cationic liposomes may be composed of a single bilayer (called unilamellar liposomes), or many concentric bilayer (called multilamellar liposomes). Inside, liposomes contain an aqueous void. This void has been used to encapsulate various molecules for delivery, including DNA, chemotherapeutic drugs, and dyes (see, e. g., references 48,49).

Liposomes typically have an overall positive charge, derived from the polar head groups of the individual cationic lipids.

Known cationic lipids A range of cationic lipids, and liposomes formed therefrom, are known.

N- [1- (2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA) (see, e. g., reference 31) is commercially available as Lipofectin0 (a 3: 2 molar ratio of DOTMA and the neutral lipid DOPE).

Analogues of DOTMA include 1,2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP) (see, e. g., reference 32), and variants with an additional hydroxyalkyl group of varying

length as well as hydrophobic domains of differing sizes (see, e. g., reference 42); and (+/- )-N- (3-aminopropyl)-N, N-dimethyl-2, 3-bis (dodecyloxy)-1-propanaminium bromide (GAP- DLRIE) (see, e. g., reference 33).

Cholesterol-based lipids employ the cholesterol ring system as their hydrophobic domain.

Probably the most widely used cholesterol based lipid is the first reported example, 3p- [N-(N', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) (see, e. g., reference 38).

Various other cholesterol lipids have been described, including several homologous series of polyamines based on the cholesterol structure, e. g., N'S-cholesteryloxycarbonyl- 3,7,12-triazapentadecane-1,15-diamine (CTAP) (see, e. g., reference 39); lipids with guanidine head groups, such as bis-guanidinium-tris (2-aminoethyl) amine-cholesterol (BGTC) (see, e. g., reference 40); and cholesteryl spermidine, the active ingredient in the commercially available Transfectall Reagent (see, e. g., reference 41).

Perhaps the most well known and widely used polyamine is 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) (see, e. g., reference 35). This lipid is commercially available as Lipofectamine, which contains 25% by weight of the helper lipid DOPE.

Another well-known polyamine is dioctodecylamidoglycyl-spermine (DOGS) which is marketed commercially as the Transfectam0 Reagent (see, e. g., reference 36).

Other lipids include YKS-220 (see, e. g., references 37,52,53) which, as well as containing ester linkers for the hydrophobic chains, also contains one in the head group, to aid biodegradation further.

Lipids containing disulphide bonds are also known, such as 1,2-dioleoyl-sn-glycero-3- succinyl-2-hydroxyethyl disulphide ornithine conjugate (DOGSDSO), its carbon-carbon bond analogue 1,2-dioleoyl-sn-glycero-3-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), and cholesteryl hemidithiodiglycolyl tris (aminoethyl) amine (CHDTAEA), which possesses a disulphide bond as part of a dithioglycolic acid subunit (see, e. g., reference 43). Other disulfide lipids have also been reported (see, e. g., reference 54).

Cationic lipids which bear the positive charge on an arsenic or phosphorus atom, instead of nitrogen are also known (see, e. g., reference 55).

Single-tailed cationic lipids, such as 6-lauroxyhexyl ornithinate (LHON), are also known (see, e. g., reference 56).

Mechanism of Lipofection The process of cationic liposome-mediated gene transfer may conveniently be broken down into four discrete steps: (i) lipoplex formation; (ii) binding and uptake of lipoplex ; (iii) endosomal escape; and (iv) entry to nucleus and expression (see, e. g., reference 57).

Lipoplex Formation Lipoplexes are complexes formed between liposomes and DNA.

It is commonly believed that lipid fusion occurs between liposomes when they form a complex with DNA (see, e. g., references 58,59); results imply that fusion of liposomes occurs at the same point as condensation of the plasmid, and that liposomal complexation is directly responsible for DNA structure collapse. When this, and lipid membrane fusion, occurs, DNA is effectively surrounded by a continuous lipid bilayer.

Various lipoplex structures have been reported (see, e. g., references 60-65). At present, the exact mechanics of lipoplex formation are still unknown, but in general terms, one would predict that DNA induces aggregation of liposomes which consequently fuse, with the DNA becoming entrapped in the aggregate in the process (see, e. g., reference 66).

As well as forming electrostatic complexes with DNA, liposomes can be used to encapsulate DNA inside them (see, e. g., references 48,49). Related stabilised plasmid- lipid particles (SPLP) have also been reported (see, e. g., references 67-70).

Cell Binding and Uptake Initial uptake of untargeted cationic lipoplexes into cells is indiscriminate since it proceeds as a consequence of strong electrostatic forces being generated between the positively charged lipoplex and the negatively charged cell surface.

Proposed mechanisms for cell binding and uptake include lipid fusion (see, e. g., reference 31), in which cationic lipids within the lipoplex fuse with the lipids of the cells'

external membrane, releasing the DNA into the cytoplasm; endocytosis (see, e. g., reference 36), in which lipoplexes bind to the cell surface, enter the cell via endocytosis, and are finally contained in an endosome; and, membrane-directed movement (see, e. g., reference 73), in which lipoplexes incubated in polyanionic medium enlarge and form granules on the surface of cells, and then enter the cell by membrane-directed movement.

Endosomal Escape to the Cytoplasm For successful gene expression, the DNA must escape the endosome before it is degraded, and eventually dissociate itself from the lipoplex. Failure to do so usually precludes successful transfection.

Membrane disrupting peptides (see, e. g., reference 28), lipids that are designed to degrade in the endosome (see, e. g., references 43,44,54), and vectors/additives containing intrinsic buffering capacities (see, e. g., references 19-21,33,40), are just some of the strategies that have been employed to achieve this result.

The neutral co-lipid DOPE is often used in liposome formulations, since it often improves transfection. A common hypothesis is that DOPE aids endosomal escape (see, e. g., references 42,71,75) as a result of its tendency to fuse with lipid membrane via the formation of the unstable, inverted hexagonal Hn phase. ! t has been suggested that once inside the endosome, decreasing pH levels compel the DOPE to convert the lipidic structure from an La to an Hn arrangement. This triggers fusion of the cationic bilayer with the anionic bilayer of endosomal membrane, releasing the remaining lipoplex into the cytoplasm.

Nuclear Entry and Expression Another barrier encountered by the DNA before transfection is entry into the nucleus.

Cationic-mediated transfection depends on mitotic activity of the cell (see, e. g., reference 78). Only during the M phase, when the nuclear membrane is dismantled and then reformed, is there an opportunity for cytoplasmic DNA to gain access to the nucleus. The problem is compounded by the fact that DNA is also unstable in the cytoplasm (see, e. g., references 79,80). Transport into the nucleus and subsequent expression is typically very inefficient.

Despite this, it is true that several million protein and RNA molecules are transported between the cytoplasm and the nucleus of a eukaryotic cell every minute (see, e. g., reference 81), travelling through a Nuclear Pore Complex (NPC). The NPC provides an aqueous channel of approximately 9 nm in diameter that allows diffusion of macromolecules of up to 40-60 kDa in size. Molecules larger than this (up to 26-28 nm) may be actively transported through the NPC, but must possess an appropriate transport signal, termed a Nuclear Localisation Signal (NLS) (see, e. g., reference 81).

NLS peptides have been successfully coupled to plasmids resulting in increased expression (see, e. g., references 29,30).

Another strategy that has been proposed, which bypasses the need to traverse the nuclear membrane entirely, is the use of cytoplasmic expression systems (see, e. g., references 82-84).

Improving Transfection Rates Various methods have been employed in an effort to improve transfection rates.

Chloroquine has been reported to improve transfection, as reported by Felgner et al in the case of DMRIE/DOPE liposomes (see, e. g., reference 86). Lam and Cullis have reported that Ca2+ ions increase the potency of lipoplexes, in some cases by as much as 20-fold, when used in in vitro assays on a variety of cell lines (see, e. g., reference 143). Also, inclusion of small, non-toxic concentrations (1 %) of DMSO have been reported to increase gene expression by 10-or 14-fold, depending on the plasmid involved (see, e. g., reference 144).

Variation in the hydrophobic group has also been examined. However, there are few extensive reports in the literature focusing on variation of alkyl chain length, and still fewer which address asymmetric lipids. Byk et al. synthesised a series of four lipids with double alkyl chain lengths of 12,13,14 and 18 carbon atoms (see, e. g., reference 85).

They reported that the lipid with two C18 chains was the most efficient transfecting agent, when assayed in vitro on both HeLa and NIH3T3 cell lines. Balasubramaniam et al synthesised several series that included some asymmetric lipids (see, e. g., reference 86).

The in vitro activities of asymmetric lipids were usually superior to the best symmetric analogues, but the degree of improvement depended on the cell line. Other groups have

also reported homologous series with different hydrophobic domains that result in enhancement/depreciation of transfection activity (see, e. g., references 42,133).

Other groups however, have found that shorter alkyl chain length leads to better transfection efficiency and hypothesised that this was due to the shorter chain facilitating more efficient inter-membrane mixing (see, e. g., references 42,86). Floch et al found that the C14 lipid worked best in a series of symmetric dialkyl lipids ranging in length from C, 2 to C, 8 (including oleyl) (see, e. g., reference 133).

Felgner et al pioneered cationic lipid-mediated gene transfer with the DOTMA-containing formulation now available as Lipofectin0 (see, e. g., reference 31). DOTMA is a dialkyl lipid with ether linkages. Shortly after its publication, Silvius et a/synthesised an analogue with diester linkages, DOTAP. They reasoned that the diester bond, being more easily metabolised, would prove less cytotoxic, although they could not provide evidence of this (see, e. g., reference 50). Liu et al found the ester (DOTAP) to be up to 10-fold less active than the ether (DOTMA) in vivo (see, e. g., reference 51). In a more recent report, Liu et al synthesised and studied eleven structural analogues of DOTMA and DOTAP, and reported ethers and esters to be equivalent in vitro, but ethers to be superior in vivo (see, e. g., reference 34). Ghosh et al found ether-linked lipids to perform better than their ester analogues (see, e. g., reference 135) in experiments in vitro.

Some researchers have reported that the addition of certain compounds to cells along with lipoplexes increases transfection.

One form of targeted cationic lipoplex employs a protein or peptide fragment which binds (preferably selectively) to a cell surface receptor. The RGD tripeptide motif (arginine- glycine-aspartic acid), elucidated by Pierschbacher and Ruoslahti (see, e. g., reference 89), is known to have a high affinity for many of the integrin family of cell surface receptors. Most cell lines express several of the integrin receptors, binding their ligands with relatively low affinity, but present at about 10-to 100-fold the concentration of other receptors on the cell surface (see, e. g., reference 140). Work published by Kasono et al, demonstrated that incorporation of an RGD peptide into an adenoviral vector noticeably enhanced its gene transfer efficiency (see, e. g., reference 142) into these other cell lines.

Linear peptides could be susceptible to folding, which might obscure the binding motif from the target receptor. Cyclic analogues would be expected to hold the peptide in a

more rigid and restricted conformation, exposing certain preferred sequences (e. g., the RGD motif). Cyclisation could be introduced, for example, via a disulphide bridge between cystein residues, or by incorporation of a glutamic acid residue protected orthogonally to both the Aloc and Boc/t-butyl protecting groups, for example, using Dmab (see. e. g., reference 145).

Several of the more successful RGD peptides reported in the literature are cyclic, such as "RGD-4C,"elucidated from a phage displayed peptide library by Koivunen et al (see, e. g., reference 93). Binding studies showed this peptide (which is actually bicyclic) to be 200- fold more potent than its linear analogue. By incorporating RGD-4C it into an adenoviral vector, a 470-fold increase in gene transfer may be effected (see, e. g., reference 92).

Other studies comparing linear and cyclic peptides have also confirmed this observation (see, e. g., reference 96).

Another peptide sequence, the NGR tripeptide sequence (asparagine-glycine-arginine) has been shown to bind specifically to cells expressing the aminopeptidase N receptor (APN) on their surface (see, e. g., reference 26). First elucidated from a phage display library by Koivunen et al (see, e. g., reference 146), this motif has been coupled to doxorubicin and, along with an RGD-4C conjugate, used to treat nude mice bearing human breast xenografts with impressive results (see, e. g., reference 95).

Further Development Since the inception of cationic lipid mediated gene transfer, major steps have been taken towards its use as a clinically useful therapeutic tool. The ultimate goal is a"synthetic virus" : a transfecting agent synthesised in the laboratory which has all of the function and efficiency of a viral vector.

New strategies are required to overcome the major barriers of transfection: systemic delivery, uptake, endosomal escape, and nuclear entry. The cationic lipids of the present invention, and the associated liposomes, lipoplexes, and the like, address one or more of these barriers.

Liposomes prepared from several lipid formulations have been shown to transfect different cell lines with up to 10 times the efficacy of the commonly used DC-Chol/DOPE formulation. In addition, the cytotoxicity assay shows that the IC50 values are at least comparable to, and in the case the diether/diester lipids better than, the DC-Chol/DOPE controls.

Amine Diether Lipids

A number of symmetric (R1 = R2 = R) diether amines are known, including those with: R'and R2 as- (CH2) 5CH3 (6/6); R'and R2 as -(CH2)13CH3 (14/14); and R1 and R2 as -(CH2)15CH3(16/16).

A number of asymmetric (R'# R2) diether amines are known, including those with: R'as- (CH2) 17CH3 and R2 as- (CH2) 4CH3 (18/5); and R1 as -(CH2)17CH3 and R2 as -(CH2)11CH3 (18/12).

Amine Diester Lipids A number of symmetric (R'= R2 = R) diester amines are known, including those with: R1 and R2 as -(CH2)12CH3 ; R'and R2 as -(CH2)14CH3 ; R1 and R2 as -(CH2)16CH3 ; and, R'and R2 as cis-(CH2)7CH=CH(CH2)7CH3 (oleyl).

A number of asymmetric (R'# R2) diester amines are known, including those with: R1 as -(CH2)6CH3 and R2 as cis, cis-(CH2)7CH=CHCH2CH=CH(CH2)4CH3 ; and, R1 as -(CH2)14CH3 and R2 as cis, cis- (CH2) 7CH=CHCH2CH=CH (CH2) 4CH3.

Amino Acid Amides of Amine Diethers A few amino acid amides of amine diethers are known, including a Gly-amide (shown below) as well as a Gly-Gly-amide and a boc-Gly-Gly-amide :

European patent application EP 451763 A2 (1991) describes a number of short peptide amides of amine diethers, illustrated by the following formula, in which n is an integer from 0 to 5 (for example, 14/14, n=0, Asp; 14/14, n=1, Goy2 ; 14/14, n=2, GIy2Lys ; 14/14, n=3, Goy4) : Amino Acid Amides of Amine Diesters A number of amides of amine diesters are known, and have been used in lipoplex transfecion. See, for example : (a) Meyer et al., Gene Therapy, Vol. 7, No. 18, pp. 1606-1611; (b) Boussif et al., European Patent publication EP 1013772 A1 (2000); (c) Xiang Gao et al., published PCT application WO 00/30444 (2000); (d) Worth et al., Cancer Immunol. Immunother., Vol. 48, No. 6, pp. 312-320 (1999) ; (e) Nazih et al., Tetrahedron Lett., Vol. 40, No. 46, pp. 8089-8091 (1999); (f) Schughart et al., Gene Therapy, Vol. 6, No. 3, pp. 448-453 (1999); (g) Bischoff et al., published PCT application WO 98/34910 (1998); (h) Fast et al., Vaccine, Vol. 15, No. 16, pp. 1748-1752 (1997);

(i) Vosika et al., published PCT application WO 97/43308 (1997); (j) Fernholz et al., German patent publication DE 19521412 A1 (1996); (k) Vosika et al., U. S. Patent No. 5,416,070 (1995); (I) Vosika et al., published PCT application WO 91/16347 (1991).

Several cationic lipids of the present invention are described by Heyes et al., 2002.

Cationic Lipid Acronyms The following acronyms for common cationic lipids are used herein: dioleoylphosphatidylethanolamine (DOPE); 3p- [N- (N', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) ; dioleoylphosphatidylcholine (DOPC); dioctadecyldimethylammonium bromide (DODAB); N- [1- (2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA); 1,2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP); (+/-)-N-(3-aminopropyl)-N, N-dimethyl-2, 3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) ; 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); dioctodecylamidoglycylspermine (DOGS); 1,2-dilauryloxypropyl-3-dimethylhydroxyethylammonium bromide (DLRIE) ; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl-ammonium bromide (DMRIE) ; N'S-cholesteryloxycarbonyl-3, 7,12-triazapentadecane-1,15-diamine (CTAP); bis-guanidinium-tris (2-aminoethyl) amine-cholesterol (BGTC); 1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulphide ornithine conjugate (DOGSDSO); 1,2-dioleoyl-sn-glycero-3-succinyl-1,6-hexanediol ornithine conjugate (DOGSHDO); cholesteryl hemidithiodiglycolyl tris (aminoethyl) amine (CHDTAEA); and 6-lauroxyhexyl ornithinate (LHON).

SUMMARY OF THE INVENTION One aspect of the invention pertains to novel lipids, as described herein, including: amine diethers (class 1),

amine diesters (class 2), amino acid amides of amine diethers (class 3), amino acid amides of amine diesters (class 4), peptide amides of amine diethers (class 5), and peptide amides of amine diesters (class 6).

Another aspect of the present invention pertains to novel lipid compositions, as described herein.

Another aspect of the present invention pertains to liposomes and lipoplexes formed from the lipid compositions, as described herein.

Another aspect of the present invention pertains to pharmaceutical compositions comprising liposomes and lipoplexes formed from the lipid compositions, as described herein, and a pharmaceutical acceptable carrier.

Another aspect of the present invention pertains to medicaments comprising liposomes and lipoplexes formed from the lipid compositions as described herein.

Another aspect of the present invention pertains to methods of delivering one or more negatively charged (anionic) species into a cell, comprising contacting said cell with liposomes and/or lipoplexes formed from the lipid compositions as described herein.

Examples of anionic species include nucleic acids (e. g., DNA, RNA), oligonucleotides, mononucleotides, peptides, and proteins. Some preferred anionic species include nucleic acids (e. g., DNA, RNA) and oligonucleotides.

Another aspect of the present invention pertains to liposomes and lipoplexes formed from the lipid compositions, as described herein, for use in a method of treatment of the human or animal body by therapy, or for use in a diagnostic method practised on the human or animal body.

Another aspect of the present invention pertains to lipid compositions, as described herein, and/or liposomes and lipoplexes formed from the lipid compositions, as described herein, for use in the preparation of a medicament for the treatment of an undesired condition.

Another aspect of the present invention pertains to methods of treating a disease in a mammal via transfection, using the lipid compositions as described herein (e. g., using liposomes and lipoplexes formed therefrom), for example, by a method comprising administering to said mammal a liposome or lipoplex formed from the lipid compositions, as described herein.

Another aspect of the present invention pertains to kits having each of, or a combination of, the components of a lipid composition, liposome, plasmid, and/or lipoplex, as described herein, optionally including appropriate reagents (e. g., buffers, solvents) and devices (e. g., tubes, syringes) for assembly and use (e. g., administration).

Another aspect of the present invention pertains to novel methods for the chemical synthesis of lipids, as described herein, including, for example, methods for the synthesis of peptide amides of amine diethers (class 5).

As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of (3-Gal activity (V79 cells) versus per cent compound 43 of the total cationic lipid content of 43/DC-Chol/DOPE liposomes (not extruded).

Figure 2 is a graph of (3-Gal activity (V79 cells) versus per cent compound 43 of the total cationic lipid content of 43/DC-Chol/DOPE liposomes (extruded).

Figure 3 is a graph of ß-Gal activity (V79 cells) versus per cent compound 47 of the total cationic lipid content of 47/DC-Chol/DOPE liposomes (not extruded).

Figure 4 is a graph of ß-Gal activity (V79 cells) versus per cent compound 47 of the total cationic lipid content of 47/DC-Chol/DOPE liposomes (extruded).

Figure 5 is a graph of a-Gal activity (V79 cells) versus per cent compound 33 of the total cationic lipid content of 33/DC-Chol/DOPE liposomes (not extruded).

Figure 6 is a graph of P-Gal activity (V79 cells) versus per cent compound 33 of the total cationic lipid content of 33/DC-Chol/DOPE liposomes (extruded).

Figure 7 is a graph of (3-Gal activity (V79 cells) versus per cent compound 33 of the total cationic lipid content of 33/DC-Chol/DOPE liposomes, of (i) not extruded, (ii) 100 nm extruded, (iii), 200 nm extruded, and (iv) 400 nm extruded.

Figure 8 is a graph of (3-Gal activity (V79 cells) versus (+/-) charge ratio of liposomes with composition (i) DC-Chol/DOPE (3: 2), (ii) 33/DC-Chol/DOPE (1.5: 1.5: 2), and (iii) 43/DC-Chol/DOPE (1.5: 1.5: 2).

Figure 9 is a graph of ß-Gal activity (V79 cells) versus per cent of specified diether lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 33 (12/12); (ii) 42 (12/18); (iii) 45 (12/oleyl) ; (iv) 35 (12/16); (v) 41 (16/18); (vi) 38 (14/18); (vii) 43 (18/18); and, (viii) 47 (oleyl/oleyl).

Figure 10 is a graph of -Gal activity (V79 cells) versus per cent of specified diether lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 34 (12/14); (ii) 36 (14/14); (iii) 39 (14/oleyl) ; (iv) 46 (16/oleyl) ; (v) 40 (16/16); (vi) 37 (14/16); and, (vii) 44 (18/oleyl).

Figure 11 is a graph of ß-Gal activity (V79 cells) versus per cent of specified diester lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 52 (12/12); (ii) 53 (14/14); (iii) 54 (16/16); and, (iv) 55 (18/18).

Figure 12 is a graph of (3-Gal activity (V79 cells) versus per cent of specified lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 33 (12/12 diether); (ii) 59 (12/chol) ; and (iii) 60 (18/chol).

Figure 13 is a graph of ß-Gal activity (V79 cells) versus per cent of specified lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 33 (12/12 diether); (ii) 63 (12/12 Lys); (iii) 64 (12/18 Lys); (iv) 72 (12/12 His); (v) 73 (12/18 His); (vi) 78 (12/12 Arg); (vii) 79 (12/18 Arg); and, (viii) 67 (12/12 Trp).

Figure 14 is a graph of ß-Gal activity (V79 cells) versus per cent of specified lipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 33 (12/12 diether); (ii) 63 (12/12 Lys); and, (iii) 72 (12/12 His); and, (iv) 78 (12/12 Arg).

Figure 15 is a graph of ß-Gal activity (V79 cells) versus (+/-) charge ratio of liposomes with composition (i) 63/DOPE (3: 2), (ii) 72/DOPE (3: 2), and (iii) 78/DOPE (3: 2).

Figure 16 is a graph of (3-Gal activity (HT29 cells) versus (+/-) charge ratio of liposomes with composition (i) 33/DC-Chol/DOPE (1.5: 1.5: 2); (ii) DC-Chol/DOPE (3: 2), (iii) 63/DOPE (3: 2), and (iv) 78/DOPE (3: 2).

Figure 17 is a graph of (3-Gal activity (V79 cells) versus per cent of peptidolipid 85 of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes.

Figure 18 is a graph of (3-Gal activity versus per cent of peptidolipid of the total cationic lipid content of lipid/DC-Chol/DOPE liposomes, for: (i) 86/DC-Chol/DOPE ; (ii) 87/DC-Chol/DOPE ; and, (iii) 88/DC-Chol/DOPE.

Figure 19 is a graph of (3-Gal activity (V79 cells) versus per cent of peptidolipid (86,87, 88) of the total cationic lipid content of liposomes, for: (i) 33/DC-Chol/86/DOPE ; (ii) 33/DC-Chol/87/DOPE ; (iii) 33/DC-Chol/88/DOPE ; (iv) 63/86/DOPE; (v) 63/87/DOPE; (vi) 63/88/DOPE; (vii) 78/86/DOPE; (viii) 78/87/DOPE; and, (ix) 78/88/DOPE.

Figure 20 is a graph of (3-Gal activity (HT29 cells) versus per cent of peptidolipid (86,87, 88) of the total cationic lipid content of liposomes, for: (i) DC-Chol/86/DOPE ; (ii) DC-Chol/87/DOPE ; (iii) DC-Chol/88/DOPE ; (iv) 33/DC-Chol/86/DOPE ; (v) 33/DC- Chol/87/DOPE ; and, (vi) 33/DC-Chol/88/DOPE.

Figure 21 is a graph of ß-Gal activity (HT29 cells) versus per cent of peptidolipid (86,87, 88) of the total cationic lipid content of liposomes, for: (i) 63/86/DOPE; (ii) 63/87/DOPE; (iii) 63/88/DOPE; (iv) 78/86/DOPE; (v) 78/87/DOPE; and, (vi) 78/88/DOPE.

DETAILED DESCRIPTION OF THE INVENTION Lipids One aspect of the present invention pertains to certain cationic lipids.

Although usually depicted herein as neutral species, such lipids are conventionally referred to as"cationic"since, at physiological pH, they are cationic, for example, due to amino groups which are protonated.

For convenience, the cationic lipids of the present invention have been classified according to their lipid structure, as shown below.

Class 1-Amine Diethers Amine diethers of the present invention have the following formula, wherein R'and R2 are each independently a hydrophobic group: The carbon atom marked with an asterisk (*) may be in either R or S configuration. The compound may be in the form of (substantially) optically pure R or S isomer, or as a mixture (e. g., a racemic mixture) thereof.

In one embodiment, each of the hydrophobic groups, R'and R2, is a fatty group.

The term"fatty group,"as used herein, is defined in reference to fatty acids, which are compounds of the formula : where R is a"fatty group"and is a C130alkyl group. The alkyl group, R, may be linear or branched, saturated or unsaturated. Typically, the alkyl group, R, is saturated, or is unsaturated and has one, two, three, four, or five, or six ethylenic bonds (i. e., carbon- carbon double bonds), though more commonly one or two. Where one or more ethylenic

bonds are present, they may be in a cis-or trans-conformation, but preferably cis. The alkyl group is typically unsubstituted, but may be substituted with one or more substituents. If substituted, the alkyl group typically has from one to four substituents, for example, aryl groups.

Conventionally, fatty acids are denoted"X: Y" where X is the number of backbone carbon atoms, including the carboxylic acid carbon atom, and Y is the number of ethylenic bonds.

If ethylene bonds are present, their position and conformation (e. g., cis-and trans-) are usually indicated by a suitable prefix, c-, t-. If the conformation is not known, or is usually found as a mixture, it is usually denoted e-. If the unsaturation is acetylenic (triple bond) rather than ethylenic, it is usually denoted a-. If substituents are present, they too are usually indicated by a suitable prefix. Branched fatty acids are usually denoted as substituted linear fatty acids. For example, acetic acid (CH3COOH) is a"2: 0" fatty acid; butyric acid (CH3CH2CH2COOH) is a"4: 0" fatty acid; and oleic acid (cis-octadeca-9-enoic acid) is a"9c-18 : 1"fatty acid; isobutyric acid (CH3CH (CH3) COOH) is a"2-Me-3: 0" fatty acid. See, for example, Robinson (1982),"Common Names and Abbreviated Formula for Fatty Acids,"J. Lip. Res., Vol. 23, pp. 1251-1253.

Short-chain fatty acids, where R is C, 4alkyl, include acetic (ethanoic, 2: 0), propionic (propanoic, 3: 0), butyric (butanoic, 4: 0), and isobutyric (2-Me-3: 0).

Mid-chain fatty acids, where R is C5 7alkyl, include the following : valeric (5: 0); caproic (6: 0); isocaproic (4-Me-5 : 0); and enanthic (7: 0).

Long-chain fatty acids, where R is C8 30alkyl, include the following : caprylic (8: 0); pelargonic (9: 0); capric (10: 0); caproleic (9c-10: 1); isolauric (10-Me-11 : 0); lauric (12: 0); lauroleic (9c-12: 1); myristic (14: 0); myristoleic (9c-14: 1); palmitic (16: 0); palmitoleic (9c- 16: 1); stearic (18: 0); stearolic (9a-18: 0); oleic (9c-18: 1); linoleic (9c12c-18 : 2); linolenic (9c12c15c-18 : 3); elaidic (9t-18 : 1); arachidic (20: 0); behenic (22: 0); lignoceric (24: 0); cerotic (26: 0); montanic (28: 0); and, melissic (30: 0).

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C10 20alkyl group.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C, 0-20alkyl group, having from 0 to 2 carbon- carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C10-, C12-, C14-, C16-, C18-, or C2oalkyl group, having from 0 to 2 carbon-carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C12-, C14-, C16-, or C18alkyl group, having from 0 to 2 carbon-carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C1020alkyl group, as described above, but R' and R2 differ in chain length by at least 2 carbon atoms (e. g., C12 and C14), more preferably at least 4 carbon atoms (e. g., C12) and C16), more preferably at least 6 carbon atoms (e. g., C12 and C18).

Examples of such compounds include, but are not limited to, the following : 2,3-dilauryloxypropylamine; 2-myrisytyloxy-3-lauryloxypropylamine ; 2-palmityloxy-3-lauryloxypropylamine ; 2-stearyloxy-3-lauryloxypropylamine ; 2-oleyloxy-3-lauryloxypropylamine ; 2-lauryloxy-3-myristyloxypropylamine ; 2,3-dimyristyloxypropylamine; 2-palmityloxy-3-myristyloxypropylamine ; 2-stearyloxy-3-myristyloxypropylamine ; 2-oleyloxy-3-myristyloxypropylamine ; 2-lauryloxy-3-palmityloxypropylamine ; 2-myristyloxy-3-palmityloxypropylamine ; 2,3-dipalmityloxypropylamine; 2-stearyloxy-3-palmityloxypropylamine ;

2-oleyloxy-3-palmityloxypropylamine ; 2-lauryloxy-3-stearyloxypropylamine ; 2-myristyloxy-3-stearyloxypropylamine ; 2-palmityloxy-3-stearyloxypropylamine ; 2,3-distearyloxypropylamine; 2-oleyl-3-stearyloxypropylamine ; 2-lauryloxy-3-oleyloxypropylamine ; 2-myristyloxy-3-oleyloxypropylamine ; 2-palmityloxy-3-oleyloxypropylamine ; 2-stearyloxy-3-oleyloxypropylamine ; and, 2,3-dioleyloxypropylamine.

In one embodiment, one of the hydrophobic groups, R'and R2, is a fatty group, as described above, and the other is a steryl group (derived from a sterol), for example, a cholesteryl group (derived from cholesterol). In one preferred embodiment, R'is a steryl group, for example, a cholesteryl group, and R2 is a fatty group.

Examples of such compounds include, but are not limited to, the following : 2-lauryloxy-3-cholesteryloxypropylamine ; 2-myristyloxy-3-cholesteryloxypropylamine ; 2-palmityloxy-3-cholesteryloxypropylamine ; 2-stearyloxy-3-cholesteryloxypropylamine ; 2-oleyloxy-3-cholesteryloxypropylamine ; 2-cholesteryloxy-3-lauryloxy-propylamine ;

2-cholesteryloxy-3-myristyloxy-propylamine ; 2-cholesteryloxy-3-palmityloxy-propylamine ; 2-cholesteryloxy-3-stearyloxy-propylamine ; and, 2-cholesteryloxy-3-oleyloxy-propylamine.

Class 2-Amine Diesters Amine diesters of the present invention have the following formula, wherein R'and R2 are each independently a hydrophobic group: Again, the carbon atom marked with an asterisk (*) may be in either R or S configuration.

The compound may be in the form of (substantially) optically pure R or S isomer, or as a mixture (e. g., a racemic mixture) thereof.

In one embodiment, each of the hydrophobic groups, R'and R2, is a fatty group.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C9-19alkyl group.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C9-19alkyl group, having from 0 to 2 carbon- carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, Cg-, Cil-, C13-, C1s-, C17-, or C, 9alkyl group, having from 0 to 2 carbon-carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, C, 1-, C13-, C1s-or C17alkyl group, having from 0 to 2 carbon-carbon double bonds.

In one preferred embodiment, each of the hydrophobic groups, R'and R2, is a straight- chain, saturated or partially unsaturated, Cl 19alkyl group, as described above, but R'and R2 differ in chain length by at least 2 carbon atoms (e. g., Cil and C13), more preferably at least 4 carbon atoms (e. g., C11 and C15), more preferably at least 6 carbon atoms (e. g., C11 and C17).

Examples of such compounds include, but are not limited to, the following : 2,3-dilauroyloxypropylamine; 2-myrisytoyloxy-3-lauroyloxypropylamine ; 2-palmitoyloxy-3-lauroyloxypropylamine ; 2-stearoyloxy-3-lauroyloxypropylamine ; 2-oleoyloxy-3-lauroyloxypropylamine ; 2-lauroyloxy-3-myristoyloxypropylamine ; 2,3-dimyristoyloxypropylamine; 2-palmitoyloxy-3-myristoyloxypropylamine ; 2-stearoyloxy-3-myristoyloxypropylamine ; 2-oleoyloxy-3-myristoyloxypropylamine ; 2-lauroyloxy-3-palmitoyloxypropylamine ; 2-myristoyloxy-3-palmitoyloxypropylamine ; 2,3-dipalmitoyloxypropylamine; 2-stearoyloxy-3-palmitoyloxypropylamine ; 2-oleoyloxy-3-palmitoyloxypropylamine ; 2-lauroyloxy-3-stearoyloxypropylamine ; 2-myristoyloxy-3-stearoyloxypropylamine ; 2-palmitoyloxy-3-stearoyloxypropylamine ; 2,3-distearoyloxypropylamine; 2-oleoyl-3-stearoyloxypropylamine ; 2-lauroyloxy-3-oleoyloxypropylamine ; 2-myristoyloxy-3-oleoyloxypropylamine ; 2-palmitoyloxy-3-oleoyloxypropylamine ; 2-stearoyloxy-3-oleoyloxypropylamine ; and,

2,3-dioleoyloxypropylamine.

In one embodiment, one of the hydrophobic groups, R'and R2, is a fatty group, as described above, and the other is a steryl group (derived from a sterol), for example, a cholesteryl group (derived from cholesterol).

In one preferred embodiment, R'is a steryl group, for example, a cholesteryl group, and R2 is a fatty group.

Examples of such compounds include, but are not limited to, the following : 2-lauroyloxy-3-cholesteryloxypropylamine ; 2-myristoyloxy-3-cholesteryloxypropylamine ; 2-palmitoyloxy-3-cholesteryloxypropylamine ; 2-stearoyloxy-3-cholesteryloxypropylamine ; 2-oleoyloxy-3-cholesteryloxypropylamine ; 2-cholesteryloxy-3-lauroyloxy-propylamine ; 2-cholesteryloxy-3-myristoyloxy-propylamine ; 2-cholesteryloxy-3-palmitoyloxy-propylamine ; 2-cholesteryloxy-3-stearoyloxy-propylamine ; and, 2-cholesteryloxy-3-oleoyloxy-propylamine.

Class 3-Amino Acid Amides of Amine Diethers.

Amino acid amides of amine diethers of the present invention have the following formula, wherein R'and R2 are each independently a hydrophobic group, as defined herein, and "amino acid"denotes a single amino acid bound to the amine diether by an amide bond (- CO-NH-): amino acid- In embodiment, the amino acid is an a-amino acid, and is bound via its a-carboxy group, and has the following formula, wherein RASC denotes the amino acid side chain:

The carbon atom marked with an hash-sign (#) may be in either R or S configuration.

The compound may be in the form of (substantially) optically pure stereoisomer, or as a mixture (e. g., a racemic mixture) thereof. Preferably, the carbon has has an S configuration, as found in naturally occurring (L) a-amino acids.

In one embodiment, the amino acid is selected from those which have, at physiological pH, a cationic sidechain.

Thus, in one embodiment, the amino acid is selected from lysine, histidine, arginine, and tryptophan.

Class 4-Amino Acid Amides of Amine Diesters Amino acid amides of amine diesters of the present invention have the following formula, wherein R'and R2 are each independently a hydrophobic group, as defined herein, and "amino acid"denotes a single amino acid bound to the amine diester by an amide bond (- CO-NH-): 0 0 R2 LJ aminoacid-NO R' 3 3 0 In embodiment, the amino acid is an a-amino acid, and is bound via its a-carboxy group, and has the following formula, wherein R Asc denotes the amino acid side chain:

The carbon atom marked with an hash-sign (#) may be in either R or S configuration.

The compound may be in the form of (substantially) optically pure stereoisomer, or as a mixture (e. g., a racemic mixture) thereof. Preferably, the carbon has has an S configuration, as found in naturally occurring (L) a-amino acids.

In one embodiment, the amino acid is selected from those which have, at physiological pH, a cationic sidechain.

Thus, in one embodiment, the amino acid is selected from lysine, histidine, arginine, and tryptophan.

Class 5-Peptide Amides of Amine Diethers Peptide amides of amine diesters of the present invention (also referred to as peptido lipids) have the following formula, wherein R'and R2 are each independently a hydrophobic group, as defined herein, and"peptide"denotes a peptide bound to the amine diether by an amide bond (-CO-NH-): 0, R2 peptide-HO, peptide - N OR The term"peptide,"as used herein, relates to a polymer of two or more amino acids.

Preferably, the amino acids are linked via amide groups (-CO-NH-). The peptide may be linear, branched, or cyclic, or a combination thereof.

In one embodiment, the peptide comprises 5 or more amino acids (e. g., 5-30). In one embodiment, the peptide comprises 10 or more amino acids (e. g., 10-30). In one embodiment, the peptide comprises 15 or more amino acids (e. g., 15-30).

In one preferred embodiment, the amino acids are a-amino acids. In one preferred embodiment, the amino acids are selected from the naturally occurring L-a-amino acids.

When the amino acids are naturally occurring L-a-amino acids, they may be linked via their a-amino and/or a-carboxy groups, or via sidechain amino and/or carboxy groups (if present).

In one preferred embodiment, the peptide comprises a motif (or sequence) which enhances transfection. For example, the peptide may comprise one or more motifs which bind to one or more cell surface receptors (e. g., a targeting motif). For example, the peptide may be, or comprise, a fusogenic peptide, which facilitates fusion between a liposome and a cell membrane (see, e. g., international patent publication no.

WO 99/39742).

In one embodiment, the peptide comprises one more RGD tripeptide (arginine-glycine- aspartic acid) motifs.

In one embodiment, the peptide comprises one more NGR tripeptide (asparagine-glycine- arginine) motifs.

Examples of such compounds are described in the Examples below.

Class 6-Peptide Amides of Amine Diesters Peptide amides of amine diesters of the present invention (also referred to as peptido lipids) have the following formula: 0 oJ4R2 peptide-H 0 R 3 t ! 0 wherein R'and R2 are each independently a hydrophobic group, as defined herein, and "peptide"denotes a peptide bound to the amine diester by an amide bond (-CO-NH-), as described above.

Chemical Synthesis Methods Class 1: Amine Diethers The protected alkene was formed from the reaction between allyl bromide and potassium phthalimide, in DMF.

Scheme 1 The epoxide was synthesised in good yield (92%) via the Prilezhaev reaction, by treating with meta-chloroperoxybenzoic acid (mCPBA) (see, e. g., reference 102).

Scheme 2

Yamaguchi and Hirao selectively opened epoxide rings with alkynes using stoichiometric amounts of boron trifluoride etherate (BF3. Et2O) and BuLi (see, e. g., reference 101). Here, stoichiometric amounts of BuLi and BF3. Et2O appeared to deprotect the phthalimide group, the result being extensive polymerisation. Some of the protected monolipid was obtained, but in approximately 3% yield.

Guivisdalsky and Bittman reported using an alcohol stereoselectively to open an oxirane ring (see, e. g., reference 103), using only a catalytic amount of BF3. Et2O, and without a deprotonating agent (such as BuLi). Here, reaction with alcohol ROH, in the presence of a catalytic amount of BF3. Et2O (0.1 eq.), in dichloromethane, at 35-40°C, gave the protected monolipid in 72% yield, as a racemic mixture.

Scheme 3 Aoki and Poulter describe coupling to a secondary alcohol using sodium hydride (NaH) in tetrahydrofuran (THF), and then adding the triflate of the alkyl chain to be introduced (see, e. g., reference 104). Following this protocol, the triflates of five alkyl chains intended for addition to the monolipids were synthesised, by reaction of the alcohols with triflic anhydride and pyridine in dichloromethane.

Scheme 4

However, deprotonation of the monolipids with NaH followed by reaction with the triflates, in analogous fashion to Aoki and Poulter, gave products in low yield (-25%). By contrast, refluxing the monolipids and 1.8 equivalents of triflate together in the presence of Proton Sponge (PS) in dichloromethane for 3 days, as reported by Thompson et al (see, e. g., reference 105), gave the protected dilipids in much higher yield (-75%).

Scheme 5

The phthalimide head group was removed using the Ing-Manske procedure, whereby the phthalimide is refluxed in the presence of an large excess of hydrazine (see, e. g., reference 106). An exchange takes place, to give the free amine and the hydrazine adduct.

Scheme 6 A number of diether lipids were prepared, as described in the Examples below. Other diether lipids may be prepared by employing suitable reagents and by adapting and/or optimizing the above methods (or other known methods) as appropriate.

Class 2: Amine Diesters The cbz-protected diester lipids were prepared by reaction of 1-benzyloxycarbonylamino- propane-2,3-diol with the corresponding acyl chloride (2 eq.) in a condensation reaction.

A catalytic amount of the acyl transfer catalyst 4-dimethylaminopyridine (DMAP) (0.1 eq.) was used (see, e. g., reference 107), with triethylamine (TEA) (2 eq.) as a base, in dichloromethane.

Scheme 7

Attempts to remove the cbz-group via catalytic transfer hydrogenation (see, e. g., reference 108), specifically, refluxing in ethanol in the presence of palladium on charcoal (Pd-C) (0.1 eq.) and a hydrogen donor, cyclohexene (10 eq.), did not appear to give the desired product.

The cbz-groups were successfully removed by reaction with HBr, to give the acid addition salts as products.

Scheme 8 A number of diester lipids were prepared, as described in the Examples below. Other diester lipids may be prepared by employing suitable reagents and by adapting and/or optimizing the above methods (or other known methods) as appropriate.

Class 1 (Continued): Mixed Cholesteryl Diether Lipids The mixed cholestery diether lipids were synthesised in fashion similar to that for diether lipids described above.

The epoxide was refluxed in the presence of cholesterol (1.5 eq.) and BF3. Et2O (0. 1 eq) in dichloromethane to give the cholesteryl monolipid. In this case, the reaction mixture needed to be heated to the higher temperature of 70°C to achieve complete oxirane ring opening.

Scheme 9

The cholesterol monolipid was coupled to the relevant triflate to give the protected mixed di-lipids (using Proton Sponge (2 eq.), dichloromethane, reflux, 3 days).

Scheme 10

Removal of the phthalimide group, by reaction with hydrazine (25 eq.) in ethanol, at reflux for 18 hours, gave the deprotected mixed di-lipids.

Scheme 11

A number of mixed cholesterol lipids were prepared, as described in the Examples below. Other mixed cholesterol lipids may be prepared by employing suitable reagents and by adapting and/or optimizing the above methods (or other known methods) as appropriate.

Class 3: Amino Acid Amides of Diether Lipids Each of the four cationic amino acids (Arg, His, Lys and Trp) were coupled to diether lipids to give amino acid amides of diether lipids. The amino acids, with an activated C- terminus and a protected side chain and N-terminus, were synthesised or obtained from commercial sources.

Lysine Derivatised Diether Lipids An N-hydroxysuccinimide L-Lys ester with both amines protected by the t-butyloxycarbonyl (BOC) group was obtained from commercial sources, and coupled to an amine diether lipid (0.66 eq) in tetrahydrofuran (THF) at reflux.

Scheme 12

The BOC groups were removed with trifluoroacetic acid (TFA) that contained 5% H20 as a carbocation scavenger. The products were purified by column chromatography using a solvent gradient (CH2CI2 : MeOH : NH3, 96: 3.5: 0.5 to CHzCl2 : MeOH : NH3, 92: 7: 1). The ammonia content of this solution ensures that the products are in the form of the free base rather than the TFA salt.

Scheme 13

Tryptophan Derivatised Diether Lipids The pentafluorophenyl (PFP) ester of L-Trp was obtained from commercial sources, with the amine protected by the fluorenylmethoxycarbonyl (FMOC) group. Protection of the imidazole side chain is unnecessary since the nucleophilicity of the NH group is significantly reduced by delocalisation around the ring system.

The activated ester was coupled to the diether lipid (0.66 eq) by refluxing overnight in THF, to yield the protected, derivatised lipid.

Scheme 14

FMOC removal was then carried out in a 20% solution of piperidine in dimethylformamide (DMF) to afford the Trp-lipid.

Scheme 15

Histidine Derivatised Diether Lipids A suitable L-His-PFP ester was prepared from the trityl/BOC protected free acid (see, e. g., reference 113) and perfluorophenol (PFP) (1.25 eq.) in dichloromethane with 1- [3- (dimethylamino)-propyl]-3-ethylcarbodiimde hydrochloride (EDC) (1.25 eq.) as a coupling agent since the urea by-product is soluble in aqueous media and easily removed in the work-up (see, e. g., reference 114).

Scheme 16

The active ester (1.5 eq.) was then used to form the protected, His-derivatised lipid by refluxing overnight in tetrahydrofuran (THF) with the diether lipid.

Scheme 17

The protecting groups were removed using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water (95: 2.5: 2. 5). Highly staibilised cations such as those created by the trityl group are not irreversibly removed from the solution by water or scavengers such as thiols (see, e. g., reference 115), whereas trialkylsilanes are more effective. Deprotection afforded the deprotected His-derivatised lipid.

Scheme 18

Arqinine Derivatised Diether Lipids A suitable PFP ester of L-Arg was obtained from commercial sources, with the NH2 and side chain orthogonally protected by the fmoc and 4-methoxy-2,3,6-trimethyl benzene- sulphonyl (mtr) groups, respectively.

As with the previous compounds, the PFP ester was coupled to the diether lipid (0.66 eq.) by refluxing in tetrahydrofuran (THF) to give the protected, Arg-derivatised lipid.

Scheme 19 The fmoc group was removed first since cations developed during TFA cleavages could possibly modify the fmoc, altering its cleavage characteristics. The compounds were stirred in a 20% solution of piperidine in DMF, to give the partially deprotected lipids. The mtr group was then removed in acidic conditions using trifluoroacetic acid (TFA), phenol, water, 1,2-ethanedithiol (EDT), thioanisole, and triisopropylsilane (TIS) (81: 5: 5: 5: 2.5 : 1).

Due to the highly nucleophilic nature of the guanidino group, the cations generated during the cleavage must be removed particularly efficiently. For this reason, thiols such as 1,2- ethanedithiol (EDT) and thioanisole were included in deprotection mixture (see, e. g., reference 116). The mtr group is effectively a benzylsulphonyl group with additional electron donating groups to make it more acid labile. However, the developing cation is still less stable than t-butyl, hence the extended deprotection time of 8 hours.

Scheme 20

Class 4: Amino Acid Amides of Diester Lipids These compounds may be prepared using methods analogous to those described above for amino acid amides of diether lipids (Class 3), by employing the corresponding diester lipids.

Class 3 and Class 4 (Continued): Amino Acid Amides of Mixed Cholestervl Di-Lipids These compounds may be prepared using methods analogous to those described above for amino acid amides of diether lipids (Class 3), by employing the corresponding mixed cholesterol di-lipids.

Class 5 and Class 6: Peptido-Lipids In one method, the peptidolipid was made by initially coupling the lipid to an amino acid, and then subsequently coupling the lipo-amino acid to the N-terminus of a peptide on resin. This route predominantly relies on solution phase chemistry.

A suitable PFP ester was prepared from a commerically available L-Asp with orthogonally protected NH2 (BOC) and side-chain (Benzyl) and a free carboxylic acid. Initially this was achieved following the method of Kisfaludy and Schon (see, e. g., reference 113), using perfluorophenol (PFP) (1.25 eq.) in dichloromethane and dicyclohexyl-carbodiimide (DCC) (1.25 eq.) as the coupling agent, and purifying the product by recrystallisation.

However, when the method was modified by replacing the DCC with EDC (1.25 eq.), and the product purified by flash column chromatography, it was obtained in greater yield (98% compared to 69%).

Scheme 21

The activated Asp was then coupled to the diether lipid (0.66 eq.) by refluxing in tetrahydrofuran (THF) to yield the protected Asp derivative. Adding a catalytic amount of DMAP was found to reduce the reaction time.

The benzyl protecting group on the side-chain was then removed via hydrogenolysis, using Pd-C as a catalyst (0.1 eq.) in ethanol with a hydrogen atmosphere. This afforded the corresponding product with a free carboxyl side chain.

Scheme 22 In order to couple this product to a peptide, it was necessary to activate the carboxylic acid, and this was again achieved by formation of the PFP ester of Asp, using a slight excess of PFP (1.25 eq.) and EDC (1.25 eq.) in dichloromethane, with reflux.

The peptide was synthesised"on bead"using standard fmoc chemistry, on a 0.1 mmol scale, and the N-terminus was deprotected to leave the free amine.

The activated product was coupled to the N-terminus of a peptide on bead. The peptido- resin was suspended in anhydrous dimethylacetamide (DMA) and a 5-fold excess of both the activated ester and DMAP were added. After agitating overnight at 38°C, the

peptidolipid was cleaved from the resin with a mixture of trifluoroacetic acid (TFA), water, 1,2-ethanedithiol (EDT), and triisopropylsilane (TIS) (92.5: 2.5: 2.5: 2.5), and purified via FPLC.

Scheme 23 0 boc' HOR' H-r, 2 0-peptide-NH2 + 0 OR U O PfPO N boc' [yOR' O OR2 0 OR ---peptide H2N N//OR' - O OR NH peptide Class 5 and Class 6 (Continued): Peptido-Lipids Much of the chemistry in the method described above is carried out in the solution phase.

However, solid phase chemistry offers substantial advantages. For example, synthesis can be swift and simple. Large excesses of reagent can be used to drive a reaction to completion, and simply rinsed off, along with unwanted by-products after the reaction has finished. Purification is only necessary for the final compound, unlike solution phase chemistry where most intermediates require column chromatography after every step.

Also, the synthetic route might be shortened by direct coupling the lipid to the peptide, rather than coupling the lipid to a single amino acid first, and then the lipo-amino acid to a peptide.

This approach is illustrated by the following example.

A peptide was synthesized on a resin, with an N-terminus consisting of an L-Glu residue coupled to the peptide via the Glu side chain, with the Glu a carboxyl group orthogonally protected by an allyl ester group (denoted Glu). The corresponding Glu* monomer is commercially avialable. 0 0 (1-peptideN H ! NH2

After peptide synthesis, the N-terminus was re-protected with the Boc group. The peptido-resin was suspended in dimethylformamide (DMF) and shaken in the presence of an excess of di-t-butyl-dicarbonate ( (BOC) 20) (20 eq.) and triethylamine (TEA) (20 eq.).

Evidence of the reaction going to completion was provided by the Kaiser test.

Scheme 24 0 0 0-peptide-,, HNs HN. boc Next, allyl proptecting group was removed. While being stable to both piperidine and trifluoroacetic acid (TFA) treatment, the allyl ester is easily removed by Pd (0) catalysed allyl transfer, as described by Kates et al (see, e. g., reference 117) ((Ph3P) 4Pd (3 eq.) and chloroform, acetic acid, N-methylmorpholinne (37: 2: 1)).

Scheme 25 0 0 ---- peptide\ OH H i i H boc The resulting free carboxyl required activating before the lipid could be coupled. The carboxyl group was activated by forming the 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (dhbt) ester, using 3,4-dihydro-4-oxo-1,2,3-benzotriazine (9 eq.) and diisopropylcarbodiimde (DIC) (4 eq.), in tetrahydrofuran (THF).

Scheme 26 0 0 0-peptide-,, Dhbt HNs HN. boc

First reported by Konig and Geiger for their acylating properties (see, e. g., reference 118), their use in solid phase syntheses by Atherton et al has more recently been described (see, e. g., reference 119). Another useful property of these compounds is the development of an intense yellow colour when they react, due to the liberated Dhbt being ionised by the basic environment. It is therefore easy to see when these compounds are reacting without cleaving a sample from the resin. Unlike Atherton et al, here the Dhbt ester was being created on the solid phase, and coupled to an amine in solution, rather than vice versa. Therefore excesses of reagents could be used to ensure maximum conversion of the free acid the active ester.

The peptido-resin was suspended in anhydrous dimethylacetamide (DMA) and a 5-fold excess of the diether lipid (5 eq.) added. After agitating overnight at 50°C, the peptidolipid was cleaved from the resin with a mixture of trifluoroacetic acid (TFA) and water (95: 5), and purified by FPLC. The longer retention time of the product on the column indicated the addition of a hydrophobic group (i. e. the lipid). Mass spectrometry (MS) confirmed that the desired product had been formed.

Scheme 27 0 0 (1-peptide,dhbt + H N OR' J N O'f boc boc 0 0 0-peptide-,, OR 1 H ! H !,, HN OR boc O O ---- nu H Hz NU 2 OR DC-Chol DC-Chol (3ß-[N-(N', N'dimethylaminoethyl) carbamoyl] cholesterol) was synthesised following the method described by Gao and Huang (see, e. g., reference 38). Cholesteryl chloroformate was treated with an excess of N, N-dimethylethylenediamine in chloroform at 0°C, and the product purified by recrystallisation.

Scheme 28

Lipid Compositions Another aspect of the present invention pertains to lipid compositions which are, e. g., suitable for forming liposomes and/or lipoplexes.

The lipid compositions comprise: (a) a cationic lipid fraction, and (b) a non-ionic lipid fraction.

In one embodiment, said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis. In one embodiment, said components (a) and (b) account for at least 95% of said lipid composition, on a weight basis. In one embodiment, said components (a) and (b) account for substantially all (>99%) of said lipid composition, on weight basis.

The non-ionic lipid fraction comprises one or more of a number of known non-ionic lipids.

An especially preferred non-ionic lipid is dioleoyl phosphatidylethanolamine (DOPE). In one embodiment, the non-ionic lipid fraction comprises at least 50% DOPE, on a molar basis. In one embodiment, the non-ionic lipid fraction comprises at least 90% DOPE, on a molar basis. In one embodiment, the non-ionic lipid fraction comprises at least 95% DOPE, on a molar basis. In one embodiment, the non-ionic lipid fraction is substantially all (>99%) DOPE.

Preferably, the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2: 1.

In one preferred embodiment, the ratio is from about 1.25: 1 to about 1.75: 1. In one preferred embodiment, the ratio is from about 1.4: 1 to about 1.6: 1. In one preferred embodiment, the ratio is about 1.5: 1 (that is, about 3: 2).

Thus, in one preferred embodiment, the lipid composition comprises: (a) a cationic lipid fraction; and, (b) a non-ionic lipid fraction;

wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2: 1; and, wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

The cationic lipid fraction may comprise a single cationic lipid, or a mixture of different cationic lipids.

In one embodiment, the cationic lipid fraction comprises a mixture of different cationic lipids, which mixture includes 3ß-[N-(N', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol).

Thus, in one preferred embodiment, the lipid composition comprises : (a) a cationic lipid fraction, which comprises a mixture of different cationic lipids, and which mixture includes DC-Chol ; (b) a non-ionic lipid fraction; wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2: 1; and, wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

Amine Diether Lipids and Amine Diester Lipids In one embodiment, the cationic lipid fraction comprises a mixture of different cationic lipids, and which mixture includes: (i) DC-Chol ; and, (ii) an amine diether lipid, as described herein (class 1), or an amine diester lipid, as described herein (class 2), or a mixture thereof; In one embodiment, said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis. In one embodiment, said components (i) and (ii) account for substantially all (>99%) of said cationic lipid fraction.

In such embodiments, the molar ratio of (ii) to (i), in the cationic lipid fraction, is preferably from about 0.18: 1 (15%) to about 5.66 (85%).

In one preferred embodiment, the ratio is from about 0.25: 1 (20%) to about 4: 1 (80%).

In one preferred embodiment, the ratio is from about 0.25: 1 (20%) to about 2.33: 1 (70%).

In one preferred embodiment, the ratio is from about 0.43: 1 (30%) to about 2.33: 1 (70%).

In one preferred embodiment, the ratio is from about 0.66: 1 (40%) to about 2.33: 1 (70%).

In one preferred embodiment, the ratio is from about 0.25: 1 (20%) to about 1.5: 1 (60%).

In one preferred embodiment, the ratio is from about 0.43: 1 (30%) to about 1.5: 1 (60%).

In one preferred embodiment, the ratio is from about 0.66: 1 (40%) to about 1.5: 1 (60%).

Thus, in one preferred embodiment, the lipid composition comprises: (a) a cationic lipid fraction, which comprises a mixture of different cationic lipids, and which mixture includes: (i) DC-Chol ; and, (ii) an amine diether lipid, as described herein (class 1), or an amine diester lipid, as described herein (class 2), or a mixture thereof; and, (b) a non-ionic lipid fraction; wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2:1; wherein molar ratio of (ii) to (i), in the cationic lipid fraction, is from about 0.18: 1 (15%) to about 5.66 (85%); and, wherein said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis; and, wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

Amino Acid Amides of Amine Diether/Diester Lipids In one embodiment, the cationic lipid fraction comprises an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof.

In one embodiment, said amino acid amide accounts for at least 90% of said cationic lipid fraction, on a molar basis. In one embodiment, said amino acid amide accounts for substantially all (>99%) of said cationic lipid fraction.

Thus, in one preferred embodiment, the lipid composition comprises : (a) a cationic lipid fraction, which comprises a mixture of different cationic lipids, and which mixture includes an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof; and, (b) a non-ionic lipid fraction; wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2:1; wherein said amino acid amide accounts for at least 90% of said cationic lipid fraction, on a molar basis; and, wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

In one embodiment, the cationic lipid fraction comprises a mixture of different cationic lipids, and which mixture includes : (i) DC-Chol ; and, (ii) an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof.

In one embodiment, said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis. In one embodiment, said components (i) and (ii) account for substantially all (>99%) of said cationic lipid fraction.

In such embodiments, the molar ratio of (ii) to (i), in the cationic lipid fraction, is preferably from about 0.43: 1 (30%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 0.66: 1 (40%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 1: 1 (50%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 1.5: 1 (60%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 2.33: 1 (70%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 4: 1 (80%) to about 99: 1 (99%).

In one preferred embodiment, the ratio is from about 9: 1 (90%) to about 99: 1 (99%).

Thus, in one preferred embodiment, the lipid composition comprises:

(a) a cationic lipid fraction, which comprises a mixture of different cationic lipids, and which mixture includes: (i) DC-Chol ; and, (ii) an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof; and, (b) a non-ionic lipid fraction; wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2:1; wherein the molar ratio of (ii) to (i), in the cationic lipid fraction, is preferably from about 0.43: 1 (30%) to about 99: 1 (99%); wherein said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis; and, wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

Peptide Amides of Amine Diether/Diester Lipids In one embodiment, the cationic lipid fraction comprises a mixture of different cationic lipids, and which mixture includes: (i) one or more of: (i-a) DC-Chol ; (i-b) an amine diether lipid, as described herein (class 1), or an amine diester lipid, as described herein (class 2), or a mixture thereof; and, (i-c) an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof; and, (ii) a peptide amide of an amine diether lipid, as described herein (class 5), or a peptide amide of an amine diester lipid, as described herein (class 6), or a mixture thereof.

In one embodiment, (i) is (i-a).

In one embodiment, (i) is (i-b).

In one embodiment, (i) is (i-c).

In one embodiment, (i) is a mixture of (i-a) and (i-b).

In one embodiment, (i) is a mixture of (i-a) and (i-c).

In one embodiment, (i) is a mixture of (i-a), (i-b), and (i-c).

In one embodiment, said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis. In one embodiment, said components (i) and (ii) account for substantially all (>99%) of said cationic lipid fraction.

In such embodiments, the molar ratio of (ii) to (i), in the cationic lipid fraction, is preferably from about 0.01 (1%) to about 3: 1 (25%).

In one preferred embodiment, the ratio is from about 0.01 (1%) to about 0.25: 1 (20%).

In one preferred embodiment, the ratio is from about 0.01 (1%) to about 0.18: 1 (15%).

In one preferred embodiment, the ratio is from about 0.01 (1%) to about 0.1: 1 (10%).

In one preferred embodiment, the ratio is from about 0.01 (1%) to about 0.05: 1 (5%).

In one preferred embodiment, the ratio is from about 0.01 (1%) to about 0.03: 1 (3%).

Thus, in one preferred embodiment, the lipid composition comprises: (a) a cationic lipid fraction, which comprises a mixture of different cationic lipids, and which mixture includes: (i) one or more of: (i-a) DC-Chol ; (i-b) an amine diether lipid, as described herein (class 1), or an amine diester lipid, as described herein (class 2), or a mixture thereof; and, (i-c) an amino acid amide of an amine diether lipid, as described herein (class 3), or an amino acid amide of an amine diester lipid, as described herein (class 4), or a mixture thereof; and, (ii) a peptide amide of an amine diether lipid, as described herein (class 5), or a peptide amide of an amine diester lipid, as described herein (class 6), or a mixture thereof; and, (b) a non-ionic lipid fraction; wherein the molar ratio of cationic lipid to non-ionic lipid is from about 1: 1 to about 2:1; wherein the molar ratio of (ii) to (i), in the cationic lipid fraction, is preferably from about 0.01 (1%) to about 3: 1 (25%); and, wherein said components (i) and (ii) account for at least 90% of said cationic lipid fraction, on a molar basis; and,

wherein said components (a) and (b) account for at least 90% of said lipid composition, on a weight basis.

Liposomes and Lipoplexes Another aspect of the present invention pertains to liposomes and lipoplexes formed from the lipid compositions as described herein.

Liposomes and lipoplexes may be prepared using any of a variety of well known methods, including, for example, those described in the Examples below. These and/or other well known methods may be modified and/or adapted in known ways in order to facilitate the preparation of additional liposomes and lipoplexes within the scope of the present invention.

In one embodiment, the liposome or lipoplex further comprises an anionic species (e. g., an encapsulated and/or complexed anionic species).

In one embodiment, the anionic species is selected from: nucleic acids, oligonucleotides, mononucleotides, peptides, and proteins.

In one embodiment, the anionic species is selected from: nucleic acids and oligonucleotides.

In one embodiment, the (+/-) charge ratio (of cationic lipid to anionic species) is from about 0.3: 1 to about 12: 1.

In one embodiment, the (+/-) charge ratio is from about 1: 1 to about 5: 1.

In one embodiment, the (+/-) charge ratio is from about 3: 1 to about 5: 1.

In one embodiment, the (+/-) charge ratio is about 3: 1.

In one embodiment, the (+/-) charge ratio is about 5: 1.

Applications The cationic lipids and lipid compositions, as described herein, can be used alone, or with other known lipids, such as, for example, DOTMA or DOTAP, in any procedure comprising the use of liposomes or lipid vesicles to deliver substances intracellularly either in vitro or in vivo.

Thus, one aspect of the present invention pertains to pharmaceutical compositions comprising liposomes and lipoplexes formed from the lipid compositions as described herein, and a pharmaceutically acceptable carrier. Preferably, the composition additionally comprises one or more biologically active species (e. g., small molecules, peptides and proteins, nucleic acids).

The term"pharmaceutically acceptable"as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e. g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, adjuvant, excipient, etc. must also be"acceptable"in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Reminqton's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Another aspect of the present invention pertains to methods of delivering one or more negatively charged (anionic) species into a cell, comprising contacting said cell with liposomes and/or lipoplexes formed from the lipid compositions as described herein.

Examples of anionic species include nucleic acids (e. g., DNA, RNA), oligonucleotides, mononucleotides, peptides, and proteins.

For example, the cationic lipids and lipid compositions, as described herein, are useful as transfection agents. For example, they can be used in methods of liposomal delivery of DNA or mRNA sequences coding for therapeutically active polypeptides, as known in the art. Similarly, they can be used for liposomal delivery of the expressed gene product, the polypeptide or protein, itself. Thus, cationic lipid mediated delivery of DNA, mRNA, polypeptides, and/or proteins can provide therapy for genetic disease by supplying deficient or absent gene or gene product. These and other applications are described in the art, for example, in published international patent publication no. WO 00/30444.

Another aspect of the present invention pertains to lipid compositions, as described herein, and/or liposomes and lipoplexes formed from the lipid compositions, as described herein, for use in the preparation of a medicament for the treatment of an undesired condition.

The term"treatment,"as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e. g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition.

Treatment as a prophylactic measure (i. e., prophylaxis) is also included.

EXAMPLES The following are examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

Chemical Synthesis General Procedures All starting materials and reagents were purchased from Sigma-Aldrich Co. Ltd. unless otherwise stated. GPR solvents were purchased from BDH, apart from hexane (HPLC grade) which was purchased from Laserchrom. Thin layer chromatography (TLC) was carried out on aluminium sheets pre-coated with Kieselgel 60 F254 (Merck) and developed with either UV light or Mary's (0.4 g 4,4'-bis (dimethylamino) benzhydrol, 100 cm3 acetone), Molybdat (125 g ammonium molybdat tetrahydrate, 250 cm3 conc. H2SO4 2250 cm3 water), or Vanillin (3 grams vanillin, 100 cm3 ethanol, 100 cm3 water, 3 cm3 conc. H2SO4) dips as appropriate.

All compounds were characterised by'H NMR, high resolution accurate mass spectrometry, and elemental combustion analysis. Where elemental analysis could not confirm product to an accuracy of within 0.4%,'3C NMR was used to further substantiate characterisation.'H NMR and'3C NMR spectra were performed in CDC13 on either a Bruker AC250 or a Bruker Avance 250 spectrometer (both 250 MHz). Melting points were determined on a Kofler hot-stage (Reichert-Thermovar) and are uncorrected.

Elemental combustion analyses were carried out by C. H. N. Analysis Ltd., (Leicester, UK).

Numbering on chemical structures is for reference to NMR characterisation.

Example 1 N-benzyloxycarbonyl-1-aminopropan-2, 3-diol (1) A stirred solution ot 1-aminopropane-2, 3-diol (9.1 g, 100 mmol) in water (250 mi) was cooled to 0°C. Benzyl chloroformate (17.1 g, 14.3 ml, 100 mmol) and a solution of NaOH (4.0g, 100 mmol) in water (250 ml) were added separately and dropwise over a period of 30 minutes. The temperature of the mixture was not allowed to exceed 0°C. After a further 30 minutes, the cooling bath was removed and the reaction stirred for another 12 hours. At this point TLC (15% MeOH, 85% CH2CI2) indicated most of the starting material to have reacted, and the water was removed under reduced pressure. The resulting white crystalline solid was triturated repeatedly with CH2CI2 (4 x 250 ml) and the residue filtered off. The CH2CI2 extracts were then washed with water (2 x 100 ml) and brine (1 x 100 ml), dried over MgS04, and evaporated under reduced pressure. The product was purified by chromatography (1-10% MeOH/CH2CI2) to give N-benzyloxycarbonyl-1-aminopropan-2, 3- diol 44 as white crystals (16.1 g, 71.6%); Rf 0.5 (15% MeOH, 85% CH2CI2) ; mp 74-76°C ; 1H-NMR (250 MHz), 8H 7. 38-7.25 (m, 5H, H2'-6'), 5.66-5.56 (m, 1H, NH), 5.10-5.0 (s, 2H, H7), 3.78-3.61 (m, 3H, C, OH, C20H, H2), 3.61-3.40 (m, 2H, H3A/B) 3.35-3.07 (m, 2H, H1A/B) ; HRMS (FAB) found molecular ion (M+Na) @ 248.0899 (C11H15NO4Na requires (M+Na) @ 248.0892); CHN found C 58.38, H 6.66, N 6.23 (C"H, 5NO4 requires C 58.66, H 6.71, N 6. 22).

Example 2 Oleyl tosylate (2)

The THF was distilled over potassium prior to use. To a flask containing oleyl alcohol under argon (20.1 g, 75 mmol) was added THF (275 ml) and the solution cooled to-78°C.

A 1.6 M solution of BuLi in hexane (51.6 ml, 82.5 mmol, 1.1 eq.)) was then added dropwise over a period of 10 minutes and the mixture stirred for a further 30 minutes. In a separate flask THF (100 mi) was added to tosyl chloride (15.8 g, 82.5 mmol, 1.1 eq.) and this solution then transferred to the reaction mixture via canula and stirring continued for a 2 hours at room temperature. Water (10 ml) was then added carefully to quench any unreacted BuLi, and the solvent evaporated. Ether (100 ml) was then added and the solution washed with water (2 x 50 mi) and brine (50 ml), dried (MgS04) and evaporated.

The crude product was purified by flash column chromatography (10-40% CH2CI2/hexane) to give oleyl tosylat 2 as a white solid (19.93 g, 63.0%). R, 0.4 (40% CH2CI2/hexane) ; 8H7. 79 (d, 2H, H4', H6', J=8.5 Hz), 7.34 (d, 2H, H3, H7.), 5.42-5.22 (m, 2H, H9, Hio), 4.02 (t, 2H, Hl, J=6. 5 Hz), 2.45 (s, 3H, H1'), 2.10-1.86 (m, 4H, H8, Hn), 1.70- 1.57 (m, 2H, H2), 1.40-1.16 (m, 22H, H3-H7, H, 2-H, 7), 0.88 (t, 3H, H, 4, J=6. 5 Hz).

HRMS (FAB) found molecular ion (M+H) @ 423.6742 (C25H4303S requires (M+H) # 423.6731); CHN found C 71.25, H 9.88 (C25H4203S requires C 71.04, H 10.02).

Example 3

N- (2, 3-propenyl) phthalimide (3) Potassium phthalimide (2.59 g, 15 mmoi) was added to a round-bottomed flask containing anhydrous DMF (100 ml). After dissolution the contents were degassed under vacuum. The solution was then flushed with nitrogen, and allyl bromide (1.21 g, 0.86 ml, 10 mmol) added. The reaction was stirred at 50°C overnight. The majority of the DMF was then in vacuo, and CH2CI2 (50 ml) was added to the remaining yellow liquid. The solution was then washed with water (2 x 50 ml) and brine (50 ml), dried over MgS04, and evaporated under reduced pressure. This yielded pale yellow crystals which were further purified by flash column chromatography (0-50% CH2CI2-hexane) to give N- (2, 3- propenyl) phthalimide as a white solid (1.50 g, 80.2%); Rf 0.5 (CH2CI2) ; mp 66-67°C ;'H- NMR (250 MHz), 8H ; 7.88 (dd, 2H, H3', H6', Jortho=5. 6 Hz, Jmeta=2. 9 Hz), 7.74 (dd, 2H, H4', H5,), 5.96-5.85 (m, 1 H, H2), 5.31-5.18 (m, 2H, H3), 4.33-4.29 (m, 2H, H,) ; 13C-NMR (250 MHz), bc ; 167.72 (C1', C8'), 133.83 (C4', C5'), 131.98 (C2, C7'), 131. 44 (C2) 123. 14 (C3', C6'), 117.58 (C3), 39.89 (C,) ; HRMS (FAB) found molecular ion (M+H) # 188.0712 (C11H10NO2 requires (M+H)# 188. 0715).

Example 4

N- (2, 3-dibromopropyl) phthalimide (4) A flask containing a solution of N- (2, 3-propenyl) phthalimide 3 (187mg, 1 mmol) in anhydrous CH2CI2 (10 ml) under argon was cooled to-5°C. A solution of bromine (1 mmol, 1.6g) in anhydrous CH2CI2 (5 ml) was then added dropwise over a period of 10 minutes, and the reaction stirred for a further 3 hours at room temperature. The reaction was washed with saturated Na2S203 (1 x 20 ml), water (1 x 20 ml) and brine (1 x 20 ml), dried (MgS04), and solvent removed under reduced pressure. The resulting solid was purified by column chromatography (20% CH2Cl2/hexane - CH2Cl2) to give N- (2, 3- dibromopropyl) phthalimide 4 as a pale brown solid (321 mg, 92.8%); R, 0.55 (CH2CI2) ; mp 106-107°C ;'H-NMR (250 MHz), 8H ; 7.86 (dd, 2H, H2',5', Jortho=5.5 Hz, Jmeta=3. 1 Hz), 7.73 (dd, 2H, H3 4'), 4.70-4.57 (m, 1 H, H2), 4.15 (ABX, 2H, H3A/B, JAB=14. 4, JAX=5. 4 JBX=8. 5), 3.78 (ABX, 2H, H1A/8, JAB=10.9, JAX=4. 8 JBX=8. 5) ;'3C-NMR (250 MHz), 8c ; 167.66 (Ci., C8'), 134. 22 (C4', C5), 131.65 (C2', C7'), 123.52 (C3', Ce'), 47.56 (C1), 43.03 (C2), 33.66 (C3) ; HRMS (FAB) found molecular ion (M+H) @ 345.9078 (C11H10Br2NO2 requires (M+H)# 345.9077).

Example 5

N-benzyloxycarbonylprop-2, 3-enylamine (5) To a flask containing CH2CI2 (20 mi) was added allyl amine (1.14 g, 1.5 mi, 20 mmol) and diisopropylamine (2.02g, 2.80 ml, 20 mmol), and the solution cooled to -18°C. Benzyl chloroformate (3.39g, 2.84 ml, 20 mmol) was then added dropwise over a period of 30 minutes, with the temperature of the solution maintained below 0°C. After a further 30 minutes, the cooling bath was removed and the reaction stirred for another 4 hours. The organic phase was then washed with water (2 x 20 ml) and brine (1 x 20 ml), dried over MgSO4, and evaporated under reduced pressure. The product was purified by chromatography (30-100% CH2CI2/hexane) to give N-benzyloxycabonylprop-2, 3- enylamine 5 as a pale yellow oil (3.23g, 84.6%); Rf 0.5 (CH2CI2) ; 1 H-NMR (250 MHz), 8H 7.40-7.25 (m, 5H, H2'-6'), 5.93-5.73 (m, 1 H, H2), 5.46-5.29 (m, 1H, NH), 5.25-5.06 (m, 4H, H3, 7'), 3.86-3.74 (m, 2H, H,) ;'3C-NMR (250 MHz), 8c ; 156.89 (C8), 137.09 (C,), 135.07 (C2), 128.91 (C3', 5.), 128.48 (Cz, C4', C6'), 116.25 (C3), 67.07 (C7'), 43.89 (C1) ; HRMS (FAB) found molecular ion (M+H) # 192.1025 (C"H, 4NO2requires (M+H) # 192.1031).

Example 6

N-benzyloxycarbonyl-2, 3-dibromopropylamine (6) Prepared analogously to N- (2, 3-dibromopropyl) phthalimide 4, starting with N-benzyloxycarbonylprop-2, 3-enylamine 5 (1.91g, 10 mmol) to give after chromatography (20% CH2Cl2/hexane - CH2Cl2), N-benzyloxycarbonyl-2, 3-dibromopropylamine 6 as a white solid (3.02g, 86.0%); R, 0.55 (CH2CI2) ; mp 80-83°C ; 1 H-NMR (250 MHz), #H7. 40- 7.26 (m, 5H, H2'-6'), 5.30-5.05 (m, 3H, NH, H,7 '), 4.37-4.22 (m, 1H, H2), 3.95-3.73 (m, 2H, H3), 3.73-3.42 (m, 2H, H1) ;'3C-NMR (250 MHz), 8c ; 156.62 (C8), 136.06 (Ci.), 128. 52 (C3', C5), 128.25 (C2', C4, Ce). 67.14 (C7), 51.12 (C2), 45.69 (C1), 33.09 (C3) ; HRMS (FAB) found molecular ion (M+H) # 349. 9391 (C11H14Br2NO2 requires (M+H) @ 349.9400).

Example 7

N- (2, 3-epoxypropyl) phthalimide (7) A solution of N- (2, 3-propenyl) phthalimide 3 (187 mg, 1 mmol) in CH2CI2 (2 ml) was prepared under argon. In a separate flask, a solution of m-chloroperbenzoic acid (57- 86%, 602 mg, 2 mmol min.) in CH2CI2 (3 ml) was prepared, also under argon, and then added to the solution of 1. The solution was stirred for three days, and was then evaporated under reduced pressure. The product, a white solid, was redissolved in THF (10 ml) and a solution of 4% sodium dithionite (6 ml) added to remove excess peracid.

The solution was stirred for 20 minutes, and then EtOAc (50 ml) added. It was then washed with water (40 ml), saturated aqueous NaHC03 (2 x 40 ml) water again (40 ml) and brine (40 ml) dried over MgS04, and the solvent evaporated under reduced pressure.

This yielded N- (2, 3-epoxypropyl) phthalimide 7 as a white solid (186 mg, 92%); Rf 0.4 (CH2CI2) ; mp 98-100°C ;'H-NMR (250 MHz), 8H ; 7.85 (dd, 2H, H2, H5 Jortho=5. 6 Hz, Jmeta=2. 9 Hz), 7. 72 (dd, 2H, H3, H4'), 3.86 (ABX, 2H, H1A/B, JAB=14.2, JAX=5. 1 JBX=5. 6), 3.30-3.20 (m, 1 H, H2), 3.84-2.65 (m, 2H, H3), HRMS (FAB) found molecular ion (M+H)#204. 0663 (C11H10NO3 requires (M+H)# 204. 0661); CHN found C 64.83, H 4.39, N 6.87 (C11H9NO3 requires C 65.02, H 4.46, N 6.89).

Example 8

N- (2-hydroxy-3-lauryloxypropyl) phthalimide (8) To a flask under argon containing N- (2, 3-epoxypropyl) phthalimide 3 (20.1 g, 100 mmol) and lauryl alcohol (27.9 g, 150 mmol) was added CH2CI2 (50 ml). After complete dissolution, boron trifluoride diethyl etherate (BF3. Et2O) (1.42 g, 1.23 ml, 10 mmol) was added dropwise at room temperature with stirring. Almost immediately translucence was observed and the reaction was then stirred at 40°C for 18 hours. The mixture was then diluted with CH2CI2 to a final volume of 300 ml, washed with saturated aqueous NaHC03 (150 ml), water (150 ml) and brine (150 ml) and the solvent evaporated under reduced pressure to give a colourless oil. Flash column chromatography needed to be carried out twice, once with hexane/EtOAc (1: 9) to remove excess lauryl alcohol, and then with CH2CI2 to remove unreacted starting product. This yielded N- (2-hydroxy-3- lauryloxypropyl) phthalimide 8 as a white solid (27.60 g, 72.1%); R, 0.35 (30% EtOAc-70% hexane); mp 72-74°C ;'H-NMR (250 MHz), 8H ; 7.87 (dd, 2H, H3, He., Jortho=5. 4 Hz, Jmeta=3. 0 Hz), 7.73 (dd, 2H, H4', H5'), 4.14-3.98 (m, 1 H, H2), 3.85 (ABX, 2H, H1A/B, JAB=14. 0 Hz, JAX=7. 3 Hz, JBX=4. 4 z), 3.58-3.37 (m, 4H, H1, H1'',), 2.69 (d, 1 H, OH, J=6. 2 Hz), 1.63- 1.45 (m, 4H, H2''), 1.37-1.17 (m, 18H, H3''-H11''), 0.88 (t, 3H, Hi2.., J=6.6 Hz); HRMS (FAB) found molecular ion (M+H) @ 390.2620 (C23H36NO4 requires (M+H) #390. 2644); CHN found C 70.79, H 9.10, N 3.60 (C23H35NO4 requires C 70.92, H 9.06, N 3.60).

Example 9 N- (2-hydroxy-3-myristyloxypropyl) phthalimide (9)

Prepared analogously to N- (2-hydroxy-3-lauryloxypropyl) phthalimide 8 (Example 8) on a 100 mmol scale to give after 2 step chromatography (hexane/EtOAc (1: 9), CH2Cl2), N-(2- hydroxy-3-myristyloxypropyl) phthalimide 9 as a white solid (29.05 g, 70.7%); R, 0.35 (30% EtOAc-70% hexane); mp 78-79°C ;'H-NMR (250MHz), #H ; 7. 87 (dd, 2H, H3', He., Jortho=5.4 Hz, Jeta=3 0 Hz), 7. 73 (dd, 2H, H4', H5'), 4.15-3.98 (m, 1 H, H2), 3.86 (ABX, 2H, H1A/B, JAB=14. 0 Hz, JAX=7. 3 Hz, JBX=4. 4 Hz), 3.58-3.36 (m, 4H, H1, H1''), 2.69 (d, 1 H, OH, J=6.1 Hz), 1.62-1.44 (m, 4H, H2''), 1.37-1.13 (m, 22H, H3''-H13''), 0.88 (t, 3H, H14'', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H) 0 418. 2940 (C25H4oN04 requires (M+H)#418. 2957); CHN found C 71. 52, H 9. 39, N 3. 32 (C25H39NO4 requires C 71.91, H 9.41, N 3. 35).

Example 10 N- (2-hydroxy-3-palmityloxypropyl) phthalimide (10) Prepared analogously to N-(2-hydroxy-3-lauryloxypropyl)phthalimide 8 (Example 8) on a 50 mmol scale, to give on purification the product N- (2-hydroxy-3- palmityloxypropyl)phthalimide 5 as a white solid (16.26 g, 74.1%); R, 0.35 (30% EtOAc- 70% hexane); mp 87-88°C ;'H-NMR (250 MHz), 8H ; 7.87 (dd, 2H, Ha., He., Jortho=5. 5 Hz, Jmeta=3 0 Hz), 7.73 (dd, 2H, H4', H5'), 4.14-3.99 (m, 1 H, H2), 3.85 (ABX, 2H, H1A/B, JAB=14. 0 Hz, JAX=7. 3 Hz, JBX=4. 4 Hz), 3.59-3.39 (m, 4H, H1, H1''), 2.70 (d, 1 H, OH, J=6.2 Hz), 1.62- 1.45 (m, 4H, H2''), 1.37-1.12 (m, 26H, H3''-H15''), 0.88 (t, 3H, H16'', J=6. 6 Hz); HRMS (FAB) found molecular ion (M+H) o 446.3255 (C27H44NO4 requires (M+H) @ 446.3270); CHN found C 72.68, H 9.81, N 3.10 (C27H43NO4 requires C 72.77, H 9.73, N 3.14).

Example 11 N-(2-hydroxy-3-stearyloxypropyl)phthalimide (11)

Prepared analogously to N- (2-hydroxy-3-lauryloxypropyl) phthalimide 8 (Example 8) on a 50 mmol scale, to give after chromatography (hexane/EtOAc (1: 9), CH2Cl2), N-(2- hydroxy-3-stearyloxypropyl) phthalimide 11 as a white solid (17.59 g, 75.2%); R, 0.35 (30% EtOAc-70% hexane); mp 88-89°C ;'H-NMR (250 MHz), 8H ; 7.86 (dd, 2H, H3, He, Jorhto=5.4 Hz, Jmeta=3. 0 Hz), 7.73 (dd, 2H, H4', H5'), 4.13-3.99 (m, 1H, H2), 3.85 (ABX, 2H, H1A/B, JAB=14. 1 Hz, JAX=7. 3 Hz, JBX=4. 4 Hz), 3.59-3.40 (m, 4H, H1, H1''), 2.74 (d, 1H, OH, J=6.0 Hz), 1.62-1.46 (m, 4H, H2''), 1.37-1.16 (m, 30H, H3''-H17''), 0.88 (t, 3H, H18'', J=6.5 Hz); HRMS (FAB) found molecular ion (M+Na)#496. 3420 (C29H47NO4Na requires (M+Na) @ 496.3403); CHN found C 73.45, H 10.16, N 2.84 (C29H47NO4 requires C 73.53, H 10.00, N 2. 96).

Example 12 N- (2-hydroxy-3-oleyloxypropyl) phthalimide (12)

Prepared analogously to N- (2-hydroxy-3-lauryloxypropyl) phthalimide 8 (Example 8) on a 100 mmol scale. Following purification by chromatography (hexane/EtOAc (1: 9), the compound N- (2-hydroxy-3-oleyloxypropyl) phthalimide 12 was yielded as a colourless low melting point solid (31.20 g, 67%); R, 0.35 (30% EtOAc-70% hexane); mp-25°C ;'H- NMR (250 MHz), SOH ; 7.83 (dd, 2H, H3', H6', Ortho=5. 6 Hz, Jmeta=3. 1 Hz), 7.73 (dd, 2H, H4', H 5'), 5.41-5.24 (m, 2H, Hg, H10''), 4.13-3.98 (m, 1 H, Hz), 3.82 (ABX, 2H, Hi, JAB=14. 0 Hz, JAX=7. 3 Hz, JBX=4.5 Hz), 3.55-3.35 (m, 4H, Hl, Hl,,), 2.89 (d, 1 H, OH, J=6.0 Hz), 2.07-1.91 (m, 4H, H8'', Hn..), 1.60-1.45 (m, 2H, H2''), 1.41-1.18 (m, 30H, H3-H7, H12''-H17''), 0.86 (t, 3H, H18'', J=6.5 Hz); HRMS (FAB) found molecular ion (M+H)# 472.3400 (C29H45NO4 requires (M+H)# 472. 3427); CHN found C 73.54, H 9.64, N 2.93 (C29H47NO4 requires C 73.85, H 9.62, N 2.97).

Example 13 Lauryl triflate (13)

Prior to carrying out the example, the CH2CI2 to be used as the solvent was distilled over P205 to purify and dry it (see, e. g., reference 150). To an ice cooled flask containing distilled CH2CI2 (100 ml) under argon was added trifluoromethanesulfonic anhydride (18.33 g, 10.94 ml, 65 mmol), followed by anhydrous pyridine (5.14g, 5.26 ml, 65 mmol).

Fuming was observed and a white precipitate formed. The cooling bath was removed, and a solution of lauryl alcohol (9.317g, 50 mmol) in distilled CH2CI2 (40 ml) was added dropwise over a period of 10 mins with stirring. The solution was stirred for 2 hours at room temperature, and then water (20 ml) was added to quench the reaction. CH2CI2 (200 mi) was added and the solution washed twice with water (2 x 100 ml). The aqueous layers were backwashed with CH2CI2 (20 ml) and the organic layers combined, washed with brine (100 ml), dried over MgS04, and evaporated under reduced pressure to yield a pale brown oil. The oil was dissolved in hexane (20 ml) and loaded onto a 1-inch bed of silica.

The product was eluted with hexane and diethyl ether, taking care not to co-elute the more polar, coloured by-products. The hexane was removed under reduced pressure to yield lauryl triflate 13 as a colourless oil (15.26 g, 96.0%); R, 0.7, (hexane/ether 1: 1) ;'H- NMR (250 MHz), 5H 4. 56 (t, 2H, H"J=6. 5 Hz), 1.91-1.79 (m, 2H, H2), 1.53-1.20 (m, 18H, Hs-Hn), 0.91 (t, 3H, His, J=6.6 Hz). Instability of the product prevented further characterisation.

Example 14

Myristyl triflate (14) This was prepared analogously to lauryl triflate 13 (Example 13) on a 50 mmol scale.

Myristyl triflate 14 was yielded as a colourless oil (15.53 g, 90%); Rf 0.7, (hexane/ether 1: 1);'H-NMR (250 MHz), #H4. 54 (t, 2H, Hl, J=6. 5 Hz), 1.89-1.77 (m, 2H, H2), 1.50-1.20 (m, 22H, H3-H, 3), 0.89 (t, 3H, H, 4, J=6. 5 Hz).

Example 15

Palmityl triflate (15) This was prepared analogously to lauryl triflate 13 (Example 13) on a 75 mmol scale.

Palmityl triflate 15 was yielded as a white, waxy solid (20.95 g, 81.4%); R, 0.7, (hexane/ether 1: 1) ;'H-NMR (250 MHz), #H4. 55 (t, 2H, H1, J=6. 5 Hz), 1.90-1.69 (m, 2H, H2), 1.54-1.12 (m, 26H, H3-H, 5), 0.89 (t, 3H, H16, J=6. 5 Hz).

Example 16 Stearyl triflate (16)

This was prepared in an almost identical way to lauryl triflate 13 (Example 13) on a 100 mmol scale. Due to stearyl alcohol being particularly unreactive, slightly larger equivalents of trifluoromethanesulphonic anhydride and pyridine were used (125 mmol).

Also, due to its lower solubility, the stearyl alcohol was added in solid form under an argon blanket. Stearyl triflate 16 was yielded as a waxy white solid (29.45 g, 73.6%); R, 0.7, (hexane/ether 1: 1) ;'H-NMR (250 MHz), 8H4. 55 (t, 2H, Hi, J=6. 5 Hz), 1.91-1.68 (m, 2H, H2), 1.5-1.1 (m, 30H, H3), 0.87 (t, 3H, H4, J=6.5 Hz).

Example 17 0 8 1 3 5 7 9 10 12 14 16 18 2 8 11 F3C 0 Oleyl triflate (17)

Prepared analogously to lauryl trifiate 13 (Example 13) on a 77 mmol scale. Oleyl alcohol was first distilled under vacuum (bp 182-184°C/1. 5mm). Oleyl triflate 17 was yielded as a colourless liquid (22.20 g, 72.1%); Rf 0.7, (hexane/ether 1: 1) ;'H-NMR (250 MHz), 8H 5.42-5.33 (m, 2H, Hg, H, o), 4.54 (t, 2H, Hl, J=6. 5 Hz), 2.13-1.93 (m, 4H, H8, H") 1.90-1.69 (m, 2H, H2), 1.54-1.12 (m, 26H, H3-H7, H12 - H17), 0.89 (t, 3H, Hie, J=6.5 Hz).

Example 18

N- (2, 3-dilauryloxypropyl) phthalimide (18) To a mixture of N- (2-hydroxy-3-lauryloxypropyl) phthalimide 8 (3.40 g, 8.7 mmol), lauryl triflate 13 (5.00 g, 15.7 mmol) and 1,8-bis (dimethylamino)-naphthalene (proton sponge- 3.36 g, 15.7 mmol) under argon was added anhydrous distilled CH2CI2 (30 ml). The yellow solution was refluxed under argon for 78 hours. TLC (50% ether, 50% hexane) suggested that some starting materials were still present, but no further reaction was taking place.

During this time, the reaction had turned dark orange/brown, and a precipitate had formed. CH2CI2was then removed under reduced pressure to give a dark orange oil, and hexane added (100 ml). The mixture was sonicated to ensure dissolution of product, filtered through a 1-inch bed of Celite 521, and the filtrate evaporated under reduced pressure to yield an orange oil. Flash column chromatography (1-5% ether/hexane) gave N-(2,3-dilauryloxypropyl)phthalimide 18 as a colourless solid (3.79g, 77.9%); R, 0.5, (hexane/ether 1: 1); mp 38-39°C ;'H-NMR (250 MHz), #H7. 84 (dd, 2H, H3', He., Jortho=5 5 Hz, Jeta=3 3 Hz), 7.70 (dd, 2H, H4', H5'), 3.90-3.70 (m, 3H, Hi, H2), 3.65-3.30 (m, 6H, H3, H1'', H1'''), 1.60-1.36 (m, 4H, H2'', H2'''), 1.35-1.05 (m, 36H, H3'' - H11'', H3-H"), 0. 89 (t, 6H, H12'', H12''', J=6.3 Hz). HRMS (FAB) found molecular ion (M+H) 9558. 4541 (C35H60NO4 requires (M+H) # 558.4522); CHN found C 75.27, H 10.74, N 2.44 (C3sH59No4 requires C 75.36, H 10.66, N 2. 51).

Example 19

N- (2-myristyloxy-3-lauryloxypropyl) phthalimide (19) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 5 mmol scale to give after chromatography (1-5% ether/hexane), N- (2-myristyloxy-3- lauryloxypropyl) phthalimide 19 as colourless solid (2.10g, 71.7%); R, (hexane/ether 1: 1); mp 39-40°C ;'H-NMR (250 MHz), #H7. 85 (dd, 2H, H3', He., Jortho=5. 4 Hz, Jmeta=3. 1 Hz), 7.73 (dd, 2H, H4', H5'), 3.90-3.70 (m, 3H, H1, H2), 3.65-3.35 (m, 6H, H3, H1'', H1'''), 1.55-1.36 (m, 4H, H2, H2'''), 1.35-1.10 (m, 40H, H3'' - H11'', H3''' - H13'''), 0.89 (t, 6H, H12'', H14''', J=6.2 Hz). HRMS (FAB) found molecular ion (M+H) # 586. 4811 (C37H64NO4 requires (M+H)#586. 4835); CHN found C 75.78, H 10.92, N 2.33 (C37H63NO4 requires C 75.85, H 10.84, N 2. 39).

Example 20 N-(2-palmityloxy-3-lauryloxypropyl)phthalimide(20) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 6 mmol scale to give after chromatography (1-5% ether/hexane), N-(2-palmityloxy-3- lauryloxypropyl) phthalimide 20 as a colourless solid (2.65 g, 72.0%); R, 0.5, (hexane/ether 1: 1); mp 46-47°C ; 1 H-NMR (250 MHz) #H7. 86 (dd, 2H, H3', He., Jortho=5 5 Hz, Jeta=3 2 Hz), 7.73 (dd, 2H, H4', H5'), 3.93-3.73 (m, 3H, Hi, H2), 3.67-3.35 (m, 6H, H3, H1'', H1'''), 1.60-1.37 (m, 4H, H2'', H2'''), 1.36-1.10 (m, 44H, H3'' - H11'', H3''' - H15'''), O. 89 (t, 6H, H, H16''', J=6. 5 Hz). HRMS (FAB) found molecular ion (M+H)# 614. 5192 (C39H68NO4 requires (M+H)#614. 5148); CHN found C 76.18, H 11.09, N 2.21 (C39H67NO4 requires C 76.30, H 11. 00, N 2.28).

Example 21

N- (2, 3-dimyristyloxypropyl) phthalimide (21) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 21 mol scale to give after chromatography (1-5% ether/hexane), N- (2, 3- dimyristyloxypropyl) phthalimide 21 as a colourless solid (9.87 g, 76.6%); R, 0.5, (hexane/ether 1: 1); mp 47-48°C ; 1H-NMR (250 MHz), #H7. 85 (dd, 2H, H3', H6', Jortho=5 5 Hz, Jmeta=3. 3 Hz), 7.73 (dd, 2H, H4'. H5'), 3.95-3.65 (m, 3H, Hl, H2), 3.65-3.35 (m, 6H, H3, H,, H1'''), 1.60-1.37 (m, 4H H2'', H2'''), 1.36-1.05 (m, 44H, H3'' - H13'', H3''' - H13'''), 0.88 (t, 6H, H14'', H14''', J=6.5 Hz). HRMS (FAB) found molecular ion (M+H) #614. 5136 (C39H68NO4 requires (M+H)#614. 5148); CHN found C 76.12, H 10.99, N 2.22 (C39H67NO4requires C 76.30, H 11.00, N 2. 28).

Example 22 N- (2-palmityloxy-3-myristyloxypropyl) phthalimide (22) Prepared analogously to N-(2,3-dialuryloxypropyl)phthalimide 18 (Example 18) on a 6 mmol scale to give after chromatography (1-5% ether/hexane) N-(2-palmityloxy-3- myristyloxypropyl) phthalimide 22 as a colourless solid (2.97 g, 77.2%); R, 0.5, (hexane/ether 1: 1); mp 50°C ; 1 H-NMR (250 MHz), 8H 7. 85 (dd, 2H, H3', He., Jortho=5. 6 Hz, Jmeta=3. 1 Hz), 7. 71 (dd, 2H, H4', H5'), 3.95-3.65 (m, 3H, H1, H2), 3.65-3.35 (m, 6H, H3, Hi.., H1'''), 1.60-1.37 (m, 4H, H2, H2'''), 1.36-1.05 (m, 48H, H3'' - H13'', H3''' - H15'''), 0.88 (t, 6H, H14'', H16''', J=6.5 Hz). HRMS (FAB) found molecular ion (M+H) #642. 5434 (C4, H72NO4 requires (M+H) #642. 5461); CHN found C 76.66, H 11.30, N 2.10 (C41H71NO4 requires C 76.70, H 11.15, N 2. 18).

Example 23

N- (2-stearyloxy-3-myristyloxypropyl) phthalimide (23) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 13 (Example 13) on a 4 mmol scale to give after chromatography (1-5% ether/hexane) N- (2-stearyloxy-3- myristyloxypropyl)phthalimide 23 as a colourless solid (1.80 g, 67.0%); R, 0.5, (hexane/ether 1: 1); mp 54-55°C ; 1H-NMR (250 MHz), #H7. 85 (dd, 2H, H3', H6', Jortho=5.6 Hz, Jeta=3. 1 Hz), 7.71 (dd, 2H, H4', H5,), 3.95-3.70 (m, 3H, Hl, H2), 3.70-3.35 (m, 6H, H3, H1'', H1'''), 1.60-1.36 (m, 4H, H2'', H2'''), 1.35-1.08 (m, 52H, H3'' - H13'', H3''' - H17'''), O. 88 (t, 6H, H14'', H18''', J=6.6 Hz). HRMS (FAB) found molecular ion (M+H) #670. 5787 (C43H76NO4 requires (M+H)# 670.5774); CHN found C 77.02, H 11.44, N 2.01 (C43H75NO4 requires C 77.08, H 11. 28, N 2. 09).

Example 24 N- (2-oleyloxy-3-myristyloxypropyl) phthalimide (24) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 4 mmol scale to give after chromatography (1-5% ether/hexane) N- (2-stearyloxy-3- myristyloxypropyl) phthalimide 24 as a colourless waxy solid (2.40 g, 64.8%); R, 0.5, (hexane/ether 1: 1); mp 34-36°C ; 1H-NMR (250 MHz), 8H 7. 86 (dd, 2H, H3', H6', Jortho=5. 4 Hz, Jeta=3. 0 Hz), 7.72 (dd, 2H, H4', H5'), 5.43-5.25 (m, 2H, Hg, Hio 3.90-3.70 (m, 3H, Hl, H2), 3.70-3.35 (m, 6H, H3, H1'', H1'''), 2.10-1.90 (m, 4H, H8''', H11''') 1.60-1.41 (m, 4H, H2", H2'''), 1. 40-1.12 (m, 44H, H3'' - H13'', H3''' - H7''', H12''' - H17'''), O. 88 (t, 6H, H, 8'', H18''', J=6.6 Hz). HRMS (FAB) found molecular ion (M+H) #668.5592 (C43H74NO4 requires (M+H) #668. 5618); CHN found C 77.31, H 11.01, N 2.10 (C43H73NO4 requires C 77.19, H 11.16, N 2. 01).

Example 25

N- (2, 3-dipalmityloxypropyl) phthalimide (25) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 5 mmol scale to give after chromatography (1-5% ether/hexane) N- (2, 3- dipalmityoxypropyl) phthalimide 25 as a colourless solid (2.63 g, 78.6%); R, 0.5, (hexane/ether 1: 1), ether 50%; mp 61°C ; 1H-NMR (250 MHz), #H 7. 86 (dd, 2H, H3', H6', Jortho=5 6 Hz, Jmeta=3. 1 Hz), 7. 71 (dd, 2H, H4', H5'), 3.95-3.73 (m, 3H, H1, H2), 3.66-3.38 (m, 6H, H3, H1'', H1'''), 1.60-1.40 (m, 4H, Hz, H2'''), 1.38-1.10 (m, 52H, H3'' - H15'', H3''' - H15'''), 0.88 (t, 6H, H16'', H16''', J=6.6 Hz). HRMS (FAB) found molecular ion (M+H)#670. 5787 (C43H76NO4 requires (M+H) #670. 5774); CHN found C 77.05, H 11.42, N 2.01 (C43H75NO4 requires C 77.08, H 11.28, N 2.09).

Example 26 N- (2-stearyloxy-3-palmityloxypropyl) phthalimide (26) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 4 mol scale to give after chromatography (1-5% ether/hexane) N- (2-stearyloxy-3- palmityloxypropyl) phthalimide 26 as a colourless solid (1.77 g, 63.4%); R, 0.5, (hexane/ether 1: 1); mp 62°C ; 1H-NMR (250 MHz), #H7. 86 (dd, 2H, H3', H6', Jortho=5. 6 Hz, Jmeta=3. 1 Hz), 7.71 (dd, 2H, H4', H5'), 3.95-3.73 (m, 3H, H1, H2), 3.65-3.38 (m, 6H, H3, H1'', H,), 1.60-1.39 (m, 4H, H2'', H2), 1.38-1.10 (m, 56H, H3'' - H17'', H3''' - H15'''), 0.89 (t, 6H, H18'', H16''', J=6.5 Hz); HRMS (FAB) found molecular ion (M+H) #698. 6113 (C45H8oN04 requires (M+H) # 698.6087; CHN found C 77.25, H 11.56, N 1.92 (C45H79NO4 requires C 77.42, H 11. 41, N 2. 01).

Example 27

N-(2-oleyloxy-3-palmityloxypropyl)phthalimide (27) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 5 mmol scale to give after chromatography (1-5% ether/hexane) N-(2-stearyloxy-3- myristyloxypropyl) phthalimide 27 as a colourless waxy solid (2.44 g, 70.1%); R, 0.5, (hexane/ether 1: 1); mp 37-40°C ; 1H-NMR (250 MHz), #H 7. 86 (dd, 2H, H3', H6', Jortho-5. 4 Hz, Jmeta=3. 1 Hz), 7.72 (dd, 2H, H4', H5'), 5.43-5.27 (m, 2H, H9, Ho-), 3. 95-3.70 (m, 3H, Hl, H2), 3.68-3.35 (m, 6H, H3, H1'', H1'''), 2.10-1.85 (m, 4H, He-, H, 1) 1.56-1.39 (m, 4H, H2, H2'''), 1.39-1.07 (m, 48H, H3'' - H15'', H3''' - H7''', H12''' - H17'''), 0. 88 (t, 6H, H18'', H18''', J=6. 5 Hz). HRMS (FAB) found molecular ion (M+H)# 696. 5890 (C45H78NO4 requires (M+H) #696. 5931); CHN found C 77.58, H 11.24, N 1.96 (C45H77NO4 requires C 77.64, H 11.15, N 2. 01).

Example 28 N- (2-lauryloxy-3-stearyloxypropyl) phthalimide (28) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 8.7 mol scale to give after chromatography (1-5% ether/hexane) N- (2-stearyloxy-3- lauryloxypropyl) phthalimide 28 as a colourless solid (3.87g, 79.8%); R, 0.5, (hexane/ether 1: 1); mp 51-53°C ; 1H-NMR (250 MHz), #H 7. 86 (dd, 2H, H3', He., Jortho=5. 5 Hz, Jmeta=3. 0 Hz), 7.71 (dd, 2H, H4', H5'), 3.94-3.72 (m, 3H, Hi, H2), 3.66-3.36 (m, 6H, H3, H1'', H1'''), 1.57-1.37 (m, 4H, H2'', H2'''), 1.35-1.10 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.89 (t, 6H, H, B, H12''', J=6.5 Hz); HRMS (FAB) found molecular ion (M+H)#642. 5434 (C41H72NO4 requires (M+H)# 642. 5461; CHN found C 76.59, H 11. 20, N 2. 13 (C41H71NOR requires C 76.70, H 11.15, N 2. 18).

Example 29

Preparation of N- (2, 3-distearyloxypropyl) phthalimide (29) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 4 mmol scale to give after chromatography (1-5% ether/hexane) N- (2, 3- distearyloxypropyl) phthalimide 29 as a colourless solid (2.29 g, 789.8%); R, 0.5, (hexane/ether 1: 1); mp 68°C ; 1H-NMR (250 MHz), #H 7. 86 (dd, 2H, H3', H6', Jortho=5. 3 Hz, Jmeta=3. 1 Hz), 7.71 (dd, 2H, H4', H5'), 3.94-3.70 (m, 3H, Hi, H2), 3.67-3.35 (m, 6H, H3, H,, H1'''), 1.57-1.37 (m, 4H, H2'', H2'''), 1.36-1.10 (m, 60H, H3'' - H17'', H3''' - H17'''), 0.83 (t, 6H, H18'', H18''', J=6. 5 Hz); HRMS (FAB) found molecular ion (M+H)#726. 6383 (C47H84NO4 requires (M+H)#726. 6400; CHN found C 77.76, H 11.69, N 1.86 (C47H83NO4 requires C 77.74, H 11.52, N 1.93).

Example 30 N-(2-oleloxy-3-stearyloxypropyl)phthalimide (30) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 3 mol scale to give after chromatography (1-5% ether/hexane) N- (2-oleyloxy-3- stearyloxypropyl) phthalimide 30 as a colourless waxy solid (1.47 g, 68.9%); R, 0.5, (hexane/ether 1: 1); mp 39-40°C ; 1 H-NMR (250 MHz), #H7. 86 (dd, 2H, H3', H6', Jortho=5. 4 Hz, Jme, a=3. 2 Hz), 7.72 (dd, 2H, H4', H5'), 5.46-5.26 (m, 2H, Hg, Hie), 3.93-3.68 (m, 3H, Hl, H2), 3.66-3.33 (m, 6H, H3, H1'', H1'''), 2.10-1.85 (m, 4H, H8''', H") 1. 57-1.38 (m, 4H, H2'', H2), 1. 38-1.05 (m, 52H, H3'' - H17'', H3''' - H7''', H12''' - H17'''), 0. 88 (t, 6H, H18'', H18''', J=6. 5 Hz). HRMS (FAB) found molecular ion (M+H)#724. 6270 (C47H82NO4 requires (M+H) #724. 6244); CHN found C 77.79, H 11.37, N 1.87 (C4sH77NO4 requires C 77.95, H 11.27, N 1.93).

Example 31

N- (2-lauryloxy-3-oleyloxypropyl) phthalimide (31) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on an 8.7 mmol scale to give after chromatography (1-5% ether/hexane) N- (2-lauryloxy-3- oleyloxypropyl) phthalimide 31 as a colourless oil (4.25 g, 76.2%); Ri 0.5, (hexane/ether 1: 1); 1H-NMR (250 MHz), #H7. 86 (dd, 2H, H3', H6', Jortho=5. 6 Hz, Jmeta=3. 1 Hz), 7.71 (dd, 2H, H4', H5'), 5.43.5.25 (m, 2H, H9'', Hio), 3.95-3.66 (m, 3H, H1, H2), 3.66-3.33 (m, 6H, H3, H,, H1'''), 2.10-1.85 (m, 4H, H8'', Hn..), 1.60-1.38 (m, 4H, H2'', H2'''), 1.38-1.00 (m, 40H, H3'' - H7'', H12'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H, 8'', H18''', J=6.1 Hz). HRMS (FAB) found molecular ion (M+H) #640. 5320 (C41H70NO4 requires (M+H)#640. 5305); CHN found C 76.60, H 10.87, N 2.14 (C41H69NO4 requires C 76.94, H 10.87, N 2. 19).

Example 32 N-(2,3-dioleyloxypropyl)phthalimide (32) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on an 10 mmol scale to give after chromatography (1-5% ether/hexane N-(2, 3- dioleyloxypropyl) phthalimide 32 as a colourless oil (5.95 g, 82.5%); R, 0.5, (hexane/ether 1: 1); 1H-NMR (250 MHz), 8H 7. 82 (dd, 2H, H3, He., Jortno=5. 5 Hz, Jeta=3. 0 Hz), 7.68 (dd, 2H, H4', H5'), 5.39.5.22 (m, 4H, Hg, H10'', H9''', H10'''), 3.91-3.65 (m, 3H, Hl, H2), 3.65-3.31 (m, 6H, H3, H,, H1'''), 2.09-1.86 (m, 8H, He", H11'', H8''', H11'''), 1.55-1.36 (m, 4H, H2'', H2), 1.36-1.03 (m, 44H, H3'' - H7'', H12'' - H17'', H3''' - H7''', H12''' - H17'''), 0. 85 (t, 6H, H, 8'', H18''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)#722.6102 (C47H80NO4 requires (M+H) 722.6087); CHN found C 78.10, H 11.19, N 1.85 (C47H79NO4 requires C 78.17, H 11. 03, N 1.94).

Example 33

2,3-dilauryloxypropylamine (33) To a stirred, warmed (-40°C) solution of N- (2,3-dilauryloxypropyl) phthalimide 18 (4.0g, 7.17 mmol) in ethanol (75 ml) was added hydrazine monohydrate (8.97 g, 8.70 ml, 179 mmol,). The solution was refluxed for 18 hours, during which time a colourless precipitate developed. The suspension was filtered, and the filtrate evaporated under reduced pressure. This gave a yellow oil, which was purified by flash column chromatography (0.5-5% MeOH/CH2CI2) to give 2,3-dilauryloxypropylamine 33 as a colourless oil (2.73 g, 89.0%); R, 0.3, (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 8H 3.65-3.58 (m, 1 H, H2), 3.55-3.35 (m, 6H, H3, H1', H,), 2.89 (ABX, 2H, H, A/B, JAB=13. 3 Hz, JAX=3. 5 Hz, JBX=6. 4 Hz), 1.85-1.72 (m, 2H, NH2), 1.72-1.55 (m, 4H, H2', H2''), 1.55-1.20 (m, 36H, H3'-H11', H3'' -H"), 0.90 (t, 6H, H12', H12'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 80.20 (C2), 72.07 (C3), 71.61 (cl), 70.73 (C,), 43.90 (C1), 32.30 (Clo,, Clo,,), 30.56-29.72 (C2', C2'', C4' - H9', C4'' - Cg), 26.54 (C3', C3''), 23.05 (C11', C11''), 14.46 (C12', C12'') ; HRMS (FAB) found molecular ion (M+H) # 428. 4468 (C27H58NO2 requires (M+H) @ 428.4485).

Example 34 2-myristyloxy-3-lauryloxypropylamine (34)

Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.2 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-palmityloxy-3- lauryloxypropylamine 34 as a pale yellow oil (1.19 g, 82.6%); R, 0.3, (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), #H 3. 70-3.55 (m, 1 H, H2), 3.55-3.30 (m, 6H, H3, H1', H1'',), 2.80 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAX=3. 6 Hz, JBX=6. 4 Hz), 1.65-1.45 (m, 6H, H2', 2", NH2), 1.45-1.15 (m, 40H, H3'-H11', H3''-H13''), 0.88 (t, 6H, H12', H14'', J=6.6 Hz) ;'3C-NMR (250 MHz), 8c ; 80.15 (C2), 72.10 (C3), 71.61 (C1'), 70.76 (C1''), 43.88 (C1), 32.31 (C10', C, 2), 30.57-29.75 (Cy, C2", C4'-H9', C4''-C11''), 26.55 (C3', C3), 23.07 (C11', C13''), 14.48 (C2', C14'') ; HRMS (FAB) found molecular ion (M+H) #456. 4766 (C29H62NO2 requires (M+H)# 456. 4781).

Example 35 2-palmityloxy-3-lauryloxypropylamine (35) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.2 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-palmityloxy-3- lauryloxypropylamine 35 as a pale yellow oil/wax (1.30 g, 84.1%); Rf 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), lah3. 67-3.55 (m, 1H, H2), 3.55-3.30 (m, 6H, H3, H1', H,), 2.79 (ABX, 2H, H1A/B, JAB=13.3 Hz, JAX=3. 6 Hz, JBX=6. 3 Hz), 1.65-1.47 (m, 4H, H2, 2"), 1. 47-1. 38 (m, 2H, NH2), 1.38-1.15 (m, 44H, H3'-H11', H3''-H15''), 0. 88 (t, 6H, H12', H16'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 80.05 (C2), 72.10 (C3), 71.62 (Ci.), 70.75 (C,), 43.83 (C1), 32.31 (C10', C14''), 30.57-29.75 (C2', C2", C4'-H9', C4''-C13''), 26.55 (C3, C3''), 23.07 (C11', Ci5"), 14.46 (C12', C16'') ; HRMS (FAB) found molecular ion (M+H) @ 484.5094 (C31H66NO2 requires (M+H) # 484.5085).

Example 36

Preparation of 2,3-dimyristyloxypropylamine (36) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 7.3 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2,3-dimyristyloxypropylamine 36 as a pale yellow wax (3.12 g, 88.2%); R, 0.3 (10% MeOH, 90% CH2Cl2) ; 1H-NMR (250 MHz), 8H 3.61-3.55 (m, 1 H, H2), 3.55-3.32 (m, 6H, H3, H1', H1'',), 2. 77 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAX=3. 6 Hz, JBX=6. 4 Hz), 1.62-1.40 (m, 6H, Hz, 2'', NH2), 1.40-1.10 (m, 44H, H3'-H13', H3''-H13''), 0.86 (t, 6H, H14', H14'', J=6.6 Hz) ;'3C-NMR (250 MHz), 8c ; 80.33 (C2), 72.10 (C3), 71.62 (Ci.), 70.77 (C,), 43.95 (C1), 32.33 (C12', C12''), 30.58-29.77 (C2', C2, C4' - H11', C4'' - C11''), 26.57 (C3', Ca"), 23.10 (C, C13''), 14.52 (C14', C14'') ; HRMS (FAB) found molecular ion (M+H)# 484. 5094 (C3, H66NO2 requires (M+H)# 484. 5086).

Example 37 2-palmityloxy-3-myristyloxypropylamine (37)

Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.84 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-palmityloxy-3- myristyloxypropylamine 37 as a pale yellow wax (1.81 g, 92.1 %) ; R, 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 8H 3.67-3.51 (m, 1 H, H2), 3.51-3.33 (m, 6H, H3, H1', H,,), 2.80 (ABX, 2H, H1A/B, JAB=13.2 Hz, JAX=3. 5 Hz, JBX=6. 3 Hz), 1.65-1.44 (m, 6H, H2', 2'', NH2), 1.44-1.18 (m, 48H, H3' - H13', H3'' - H15''), 0.89 (t, 6H, H14', H16'', J=6. 5 Hz) ; 13C- NMR (250 MHz), 8c ; 80.17 (C2), 72.09 (C3), 71.62 (Ci.), 70.75 (C,), 43.88 (C1), 32.32 (C, Ci4..), 30.57-29.75 (C2', C2'', C4' - H11', C4'' - C13''), 26.56 (C3', C3'') 23.07 (C, Cis"), 14.48 (C14', C16'') ; HRMS (FAB) found molecular ion (M+H) zu 512.5407 (C33H70NO2 requires (M+H) # 512.5425).

Example 38 2-stearyloxy-3-myristyloxypropylamine (38) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 2.43 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-stearyloxy-3- myristyloxypropylamine 38 as a pale yellow wax (0.98 g, 74.7%); R, 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 8H 3.60-3.50 (m, 1 H, H2), 3.50-3.30 (m, 6H, H3, H, H,), 2.79 (ABX, 2H, H1A/B, JAB=12.9 Hz, JAX=2. 1 Hz, JBx=5. 6 Hz), 2.40-2.25 (m, 2H, NH2), 1.60-1.35 (m, 4H, H2', 2''), 1.35-1.10 (m, 52H, H3' - H13', H3'' - H17''), 0. 89 (t, 6H, H14', H18'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 79.75 (C2), 72.10 (C3), 71.52 (Ci.), 70.74 (C,), 43.70 (C1), 32.33 (C12', C16''), 30.55-29.77 (C2', C2', ', C4' - H11', C4'' - C15''), 26.55 (C3, C3''), 23.08 (C, C17''), 14.49 (C14', C18'') ; HRMS (FAB) found molecular ion (M+H) 540.5735 (C35H74NO2 requires (M+H) # 540.5720).

Example 39 2-oleyloxy-3-myristyloxypropylamine (39)

Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 1.5 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-oleyloxy-3- myristylpropylamine 39 as a pale yellow oil/wax (0.657 g, 81.4%); R, 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 6H 5.43-5.28 (m, 2H, Hg, H10''), 3.70-3.55 (m, 1 H, H2), 3.50-3.33 (m, 6H, H3, H, H,,), 2.80 (ABX, 2H, H, A/B, JAB=L 3. 2 Hz, JAX=3. 6 Hz, JBX=6. 3 Hz), 2.11-1.89 (m, 4H, H8', H11'), 1.66-1.46 (m, 6H, H2', 2'', NH2), 1.46-1.18 (m, 44H, H3' - H13', H3'' - H7'', H12'' - H17''), 0.88 (t, 6H, H14', H18'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 130.31,130.20 (C9'', C10''), 80.23 (C2), 72.11 (C3), 71.62 (Ci.), 70.75 (C,), 43.93 (C1), 32.32 (C, Cive"), 30.58-29.67,27.60 (C2', C4' - H11', C2'', C4'' - C8'', C11'' - C15''), 26.56 (C3', C3''), 23.08 (C, Ci7..), 14.49 (C14', C18'') ; HRMS (FAB) found molecular ion (M+H) @ 538.5540 (C35H72NO2 requires (M+H) # 538.5563).

Example 40 2, 3-dipalmityloxypropylamine (40) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.15 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2,3-palmityloxypropylamine 40 as a pale yellow wax (1.36 g, 79.9%); Rf 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), #H3. 67-3.56 (m, 1 H, H2), 3.56-3.34 (m, 6H, H3, H1', H,), 2.81 (ABX, 2H, H1A/B, JAB=13. 3 Hz, JAX=3. 5 Hz, JBX=6. 4 Hz), 1.67-1.43 (m, 6H, H2', 2'', NH2), 1.41-1.15 (m, 52H, H3' - H15', H3'' - H15''), 0.89 (t, 6H, H16', H16'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 80.14 (C2), 72.10 (C3), 71.62 (Ci.), 70.76 (C1''), 43.90 (C1), 32.32 (C14', C14''), 30.58-29.76 (C2', C2, C4' - Whizz C4'' - C13''), 26.56 (C3', C3), 23.08 (C15', C15''), 14.48 (C16', C16'') ; HRMS (FAB) found molecular ion (M+H) @ 540.5730 (C35H74NO2 requires (M+H) # 540.5720).

Example 41 2-stearyloxy-3-palmityloxypropylamine (41)

Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 2.30 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-stearyloxy-3- palmityloxypropylamine 41 as a pale yellow wax (0.98 g, 76.4%); R, 0.3 (10% MeOH, 90% CH2CI2) ; 1H-NMR (250 MHz), #H 3. 66-3.52 (m, 1H, H2), 3.52-3.32 (m, 6H, H3, H1', H,,), 2.80 (ABX, 2H, H1A/B, JAB=13.1 Hz, JAX=2. 9 Hz, JBX=5. 8, Hz), 2.22-2.08 (m, 2H, NH2), 1.62-1.41 (m, 4H, H2', 2"), 1. 41-1. 10 (m, 56H, H3'-H15', H3'' - H17''), 0. 86 (t, 6H, H16', H18'', J=6.5 Hz) ;'3C-NMR (250 MHz), 5c ; 79.84 (C2), 72.13 (C3), 71.56 (Ci.), 70.77 (C1''), 43.78 (C1), 32.34 (C14', C16''), 30.56-29.78 (C2', C2'', C4' - H13', C4'' - C15''), 26. 56 (C3', C3''), 23.10 (C15', C17''), 14.52 (C16', C18'') ; HRMS (FAB) found molecular ion (M+H)# 568. 6055 (C37H78NO2 requires (M+H) # 568.6033).

Example 42 2-lauryloxy-3-stearyloxypropylamine (42) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 4.58 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-lauryloxy-3- stearyloxypropylamine 42 as a pale yellow wax (2.15 g, 91.8%); Rf 0.3 (10% MeOH, 90% CH2CI2) ; 1H-NMR (250 MHz), #H 3. 68-3.56 (m, 1 H, H2), 3.56-3.32 (m, 6H, H3, H1', H1'',), 2.80 (ABX, 2H, HIA/B, JAB=L 3. 2 Hz, JAX=3. 7 Hz, JBX=6. 4 Hz), 1.67-1.44 (m, 6H, H2', 2'', NH2), 1.38-1.15 (m, 48H, H3' - H17', H3'' - H11''), 0.88 (t, 6H, H18', H12'', J=6.6 Hz) ;'3C-NMR (250 MHz), 8c ; 80.11 (C2), 72.11 (C3), 71.61 (C1'), 70. 76 (Ci..), 43. 89 (C1), 32. 32 (C16', C10''), 30.57-29.75 (C2', C2", C4' - H15', C4'' - C9''), 26.56 (C3, C3''), 23.07 (C, 7', C11''), 14.49 (C18', C12'') ; HRMS (FAB) found molecular ion (M+H) # 512. 5407 (C33H70NO2 requires (M+H)# 512. 5422).

Example 43

2,3-distearyloxypropylamine (43) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 1.37 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2,3-distearyloxypropylamine 43 as a pale yellow wax (0.65 g, 79.6%); Rf 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 8H 3.65-3.52 (m, 1 H, H2), 3.52-3.32 (m, 6H, H3, H1', H1'',), 2.78 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAx=3. 5 Hz, JBX=6. 4 Hz), 1.70-1.60 (m, 2H, NH2) 1. 60-1.40 (m, 4H, H2', 2''), 1.40-1.10 (m, 60H, H3' - H17', H3'' - H17''), 0.86 (t, 6H, H18', H18'', J=6.4 Hz) ;'3C-NMR (250 MHz), 8c ; 80.24 (C2), 72.10 (C3), 71.61 (Ci.), 70.77 (C,), 43.92 (C1), 32.34 (C16', C16''), 30.58-29.78 (C2', C2", C4' - H15', C4'' - C15''), 26.57 (C3, C3"), 23.10 (C17', C17''), 14.52 (C18', C18'') ; HRMS (FAB) found molecular ion (M+H) @ 596.6330 (C39H82NO2 requires (M+H) 596.6346).

Example 44 2-oleyloxy-3-stearyloxypropylamine (44) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 0.83 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-oleyloxy-3- stearyloxypropylamine 44 as a colourless wax (0.454 g, 92.1%); R, 0.3 (10% MeOH, 90% CHzCb) ; 1 H-NMR (250 MHz), #H 5. 41-5.28 (m, 2H, H9'', H10''), 3.68-3. 55 (m, 1 H, H2), 3.55- 3.33 (m, 6H, H3, H1', H1'',), 2.79 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAX=3. 5 Hz, JBX=6. 3 Hz), 2.07-1.80 (m, 6H, He., H11', NH2), 1.63-1.45 (m, 4H, H2', 2''), 1.40-1.13 (m, 52H, H3' - H17', H3'' - H7'',H12'' - H17''), 0.86 (t, 6H, H18', H18'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 130.30, 130.20 (Cg, C10''), 80.11 (C2), 72.12 (C3), 71.59 (Ci.), 70.78 (Ci..), 43.87 (C1), 32.34 (Cis., de"), 30.58-29.78,27.61 (C2', C4' - H12', C2'', C4'' - C8'', C11'' - C15''), 26.53 (C3', C3''), 23.10 (C13', C17''), 14.52 (C18', C18'') ; HRMS (FAB) found molecular ion (M+H) 594.6173 (C39H8oNO2 requires (M+H) # 594.6189).

Example 45

2-lauryloxy-3-oleyloxypropylamine (45) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.89 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-lauryloxy-3- oleyloxypropylamine 45 as a pale yellow oil (1.99 g, 90.5%); R, 0.3 (10% MeOH, 90% CH2CI2) ; 1H-NMR (250 MHz), #H 5. 37-5.23 (m, 2H, Hg, H10'), 3.65-3.50 (m, 1H, H2), 3.50- 3.30 (m, 6H, H3, H1', H1'',), 2.76 (ABX, 2H, HiA/B, JAB=13. 2 Hz, JAX=3. 4 Hz, JBX=6. 4 Hz), 2.02-1.87 (m, 6H, He", H11'', NH2,) 1.85-1.75 (m, 2H, NH2), 1.60-1.45 (m, 4H, H2', 2''), 1.35- 1.11 (m, 40H, H3' - H7', H12' - H17', H3' - H11''), 0.89 (t, 6H, H18', H12'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 130.28,130.18 (Cg, Ciao'), 80.14 (C2), 72.07 (C3), 71.58 (Ci), 70.74 (C,), 43.86 (Ci), 32.32 (C, C10''), 30.56-29.51,27.59 (C2', C4' - C8', C11' - C15', C2'', C4'' - H9'',), 26.56 (C3', C3''), 23.08 (C17', C11''), 14.49 (Cl8', C12") ; HRMS (FAB) found molecular ion (M+H)# 510.5250 (C33H68NO2 requires (M+H) @ 510.5235).

Example 46 2-palmityloxy-3-oleyloxypropylamine (46)

Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 3.0 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-palmityloxy-3- oleyloxypropylamine 46 as a pale yellow wax (1.26 g, 74.1%); R, 0. 3 (10% MeOH, 90% CH2CI2) ; 1H-NMR (250 MHz), 8H5. 41-5.30 (m, 2H, Hg, Halo,), 3.69-3.56 (m, 1 H, H2), 3.56- 3.35 (m, 6H, H3, H, H,), 2.82 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAX=3. 7 Hz, JBX=6. 2 Hz), 2.12-1.93 (m, 4H, H8', H11'), 1.78-1.64 (m, 2H, NH2), 1.64-1.49 (m, 4H, H2', 2''), 1. 38-1.17 (m, 48H, H3-H7, H12' - H17', H3' - H15''), 0.89 (t, 6H, H18', H16'', J=6.5 Hz) ;'3C-NMR (250 MHz), 8c ; 130.31,130.21 (C9', C10'), 79.77 (C2), 72.13 (C3), 71.58 (Ci.), 70.77 (C,), 43.76 (C1), 32.32 (Cl6', C14''), 30.56-29.72,27.61 (C2', C4' - C8', C11' - C15', C2'', C4'' - H13'',), 26.56 (C3', C3''), 23.08 (C17', Ci5"), 14.49 (C18', C16'') ; HRMS (FAB) found molecular ion (M+H) @ 566.5861 (C37H76NO2 requires (M+H)# 566. 5876).

Example 47 2,3-dioleyloxypropylamine (47) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 1.5 mmol scale to give after chromatography (0.5-5% MeOH/CH2CI2) 2-oleyloxy-3- lauryloxypropylamine 47 as a pale yellow oil (1.92 g, 87.5%); Ri 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), #H 5. 44-5.24 (m, 4H, Hg, H10', H9'', H10''), 3.68-3.54 (m, 1 H, H2), 3.54-3.33 (m, 6H, H3, H1', H1'',), 2.80 (ABX, 2H, H1A/B, JAB=13.2 Hz, JAX=3. 5 Hz, JBX=6. 4 Hz), 2.12-1.88 (m, 4H, H8', H11', H8'', H11'), 1.61-1.45 (m, 4H, H2', 2"), 1.45-1.38 (m, 2H, NH2), 1.38-1.20 (m, 48H, H3' - H7', H12' - H17', H3'' - H7'', H12'' - H17''), 0. 88 (t, 6H, H18', His", J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)# 592.6010 (C39H78NO2 requires (M+H)# 592. 6033); CHN found C 78.74, H 13.31, N 2.57 (C47H79NO4 requires C 79.12, H 13.11, N 2. 37).

Example 48 N-benzyloxycarbonyl-2, 3-dilauroyloxy-1-propyl-amine (48)

A flask containing N-benzyloxycarbonyl-1-aminopropan-2, 3-diol 1 (2.7 g, 12 mmol) and DMAP (146 mg, 1.2 mmol) was flushed with nitrogen before the addition of anhydrous CH2CI2 (120 ml). Triethylamine (2.42mg, 3.34 ml, 24 mmol) was added, the solution cooled to-10°C, and a solution of lauroyl chloride (5.26g, 5.56 ml, 24 mmol) in CH2CI2 (30 ml) added dropwise over a period of 10 minutes. The cooling was removed, and the reaction stirred overnight, by which time the solution had turned orange and a precipitate formed. A further 100 ml of CH2CI2 were added and the mixture washed with citric acid (50 ml), water (2 x 50 ml) and brine (50 ml). The aqueous layers were extracted with CH2CI2 (50 ml), and the organic layers then combined, dried (MgSO4), and evaporated under reduced pressure. The crude product was purified by chromatography (5-40% ether/hexane) to give N-benzyloxycarbonyl-2, 3-dilauroyloxypropylamine 48 as a white crystalline solid (5.13 g, 72.6%); Rf 0.5 (40% ether, 60% hexane); mp 34°C ; 1H-NMR (250 MHz), #H 7. 39-7.27 (m, 5H, H2'-6'), 5.16-5.0 (m, 4H, H2, H7,, NH), 4.20 (ABX, 2H, H3A/B, JAB=12.0 Hz, JAX=4. 2 Hz, JBX=5. 6 Hz), 3.52-3.34 (m, 2H, H,), 2.36-2.23 (m, 4H, H2, H2'''), 1.68-1.52 (m, 4H, H3'', H3'''), 1.34-1.16 (m, 32, H4'' - H11'', H4''' - H11''') 0.87 (t, 6H, H12'', H12'', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)# 612. 4222 (C35H59NO6Na requires (M+H) # 612.5876); CHN found C 71.19, H 10.21, N 2.37 (C35H59NO6 requires C 71.27, H 10.08, N 2. 37).

Example 49

N-benzyloxycarbonyl-2,3-dimyristoyloxy-1-propyl- amine (49) Prepared analogously to N-benzyloxycarbonyl-2, 3-dilauroyloxypropylamine 48 (Example 44) on a 12 mmol scale to give after chromatography (5-40% ether/hexane) N- benzyloxycarbonyl-2, 3-dimyristoyloxypropylamine 49 as a white crystalline solid (6.15 g, 79.4%); Rf 0.5 (40% ether, 60% hexane); mp 48°C ; 1 H-NMR (250 MHz), #H 7. 38-7.30 (m, 5H, H2 6), 5.15-4.95 (m, 4H, H2, H7', NH), 4.20 (ABX, 2H, H3A/B, JAB=11. 9 Hz, JAX=4. 3 Hz, JBX=5. 5 Hz), 3.54-3.31 (m, 2H, Hi), 2.34-2.23 (m, 4H, H2'', H2'''), 1.67-1.52 (m, 4H, H3'', H3'''), 1.36-1.17 (m, 40H, H4'' - H13'', H4''' - H13''') 0.88 (t, 6H, H14'', H14''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)# 648. 4866 (C39H67NO6Na requires (M+H) 648.4873); CHN found C 72.22, H 10.55, N 2.22 (C39H67NO6 requires C 72.52, H 10.45, N 2.17).

Example 50 N-benzyloxycarbonyl-2,3-dipalmitoyloxy-1-propyl- amine (50) Prepared analogously to N-benzyloxycarbonyl-2, 3-dilauroyloxypropylamine 48 (Example 44) on a 12 mmol scale to give after chromatography (5-40% ether/hexane) N- benzyloxycarbonyl-2, 3-dipalmitoyloxypropylamine 50 as a white crystalline solid (6.41 g, 76.1%); R, 0.5 (40% ether, 60% hexane); mp 53°C ; 1 H-NMR (250 MHz), bH 7. 35-7.28 (m, 5H, H'), 5.15-4.99 (m, 4H, H2, H7', NH), 4.20 (ABX, 2H, H3A/B, JAB=12. 0 Hz, JAX=4. 2 Hz, JBX=5. 6 Hz), 3.50-3.32 (m, 2H, H,), 2.35-2.25 (m, 4H, H2'', H2), 1.67-1.52 (m, 4H, H3'', H3), 1. 35-1.17 (m, 48, H4'' - H15'', H4''' - H15''') 0. 88 (t, 6H, H16'', H16''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)# 724. 5492 (C43H75NO6Na requires (M+H) # 724.5468); CHN found C 73.46, H 10.92, N 1.99 (C43H75NO6 requires C 73.56, H 10.77, N 2.00).

Example 51 N-benzyloxycarbonyl-2, 3-distearoyloxy-1-propyl-amine (51)

Prepared analogously to N-benzyloxycarbonyl-2, 3-dilauroyloxypropylamine 48 (Example 44) on a 3.3 mol scale to give after chromatography (5-40% ether/hexane) N- benzyloxycarbonyl-2, 3-distearoyloxypropylamine 51 as a white crystalline solid (1.79 g, 71.6%); Rf 0.5 (40% ether, 60% hexane); mp 62-63°C ; 1 H-NMR (250 MHz), #H 7. 40-7.27 (m, 5H, H2'-6'), 5.18-4.98 (m, 4H, H2, H7', NH), 4.20 (ABX, 2H, H3A/B, JAB=12. 0 Hz, JAX=4. 2 Hz, JBX=5. 6 Hz), 3.52-3.34 (m, 2H, H1), 2.34-2.21 (m, 4H, H2, H2'''), 1. 70-1. 47 (m, 4H, H3'', H3'''), 1.36-1.13 (m, 56H, H4'' - H17'', H4''' - H17''') 0.88 (t, 6H, H18'', H18''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H) # 780.6118 (C47H83NO6Na requires (M+H)# 780.6119) ; CHN found C 74.06, H 11. 15, N 1.84 (C47H83NO6 requires C 74.46, H 11. 03, N 1.85).

Example 52

2,3-dilauroyloxypropylamine hydrobromide (52) To a solution of N-benzyloxycarbonyl-2, 3-dilauroyloxypropylamine 48 (590 mg, 8 mmol) in chloroform (5 ml) and glacial acetic acid (1 ml) was added a solution of hydrogen bromide in glacial acetic acid (45% w/v, 0.5 ml). The reaction was then stirred at room temperature for 1 hour, when TLC indicated that all starting material had reacted. During this time the product formed as a dense white precipitate. The mixture was then plunged into cold diethyl ether (30 ml), shaken, and centrifuged. The pellet was washed in this fashion 4 more times, and then dried under vacuum over potassium hydroxide. This yielded 2,3-dilauroyloxypropylamine hydrobromide 52 as a colourless solid (480 mg, 89.7%). mp; initially appeared to soften at 75°C, melted at 158°C ; 1 H-NMR (250 MHz, 45°C), #H 8. 26-8.08 (m, 3H, NH3), 5.52-5.38 (m, 1 H, Hz), 4.28 (ABX, 2H, H1A/B, JAB=12. 3 Hz, JAX=3. 9 Hz, JBX=4. 8 Hz), 3.53-3.20 (m, 2H, H3), 2.59-2.27 (m, 4H, H2', H2''), 1.70-1.50 (m, 4H, H3', H3''), 1.33-1.15 (m, 32H, H4' - H11', H4'' - H11''), 0.88 (t, 6H, H12'', H12', J=6.6 Hz); '3C-NMR (250 MHz, 45°C), 8c 174.24 (Ci.), 173.52 (Ci.-), 68.45 (C2), 63.11 (C3), 41.32 (C1), 35.09 (C2''), 34.39 (C2X), 32.25 (C10', C10''), 30.04-29.56 (C4'-C9', C4'' - C9''), 25.19 (C3, C3''), 22.89 (C11', C11''), 14.33 (Ci2', Ci2") ; HRMS (FAB) found molecular ion (M+H) 512.4679 (C31H62NO4 requires (M+H) # 512.4660).

Example 53 2,3-dimyristoyloxypropylamine hydrobromide (53)

Prepared analogously to 2,3-dilauroyloxypropylamine hydrobromide 52 (Example 52) on a 1 mmol scale to give 2,3-dimyristoyloxypropylamine hydrobromide 53 as a colourless solid (532 mg, 90.1%); mp; initially appeared to soften at 78°C, melted at 156°C ; 1H- NMR (250 MHz, 45°C), 6H 8.24-8.05 (m, 3H, NH3), 5.53-5.42 (m, 1 H, H2), 4.41-4.08 (m, 2H, Hi), 3.50-3.20 (m, 2H, H3), 2.60-2.22 (m, 4H, H2', H2''), 1.70-1.50 (m, 4H, H3', H3''), 1.38-1.12 (m, 40H, H4' - H13', H4'' - H13''), 0.94-0.80 (m, 6H, H14', H14''); 13C-NMR (250 MHz, 45°C), 8c 174.44 (Ci.), 173.68 (Ci..), 68.33 (C2), 63.10 (C3), 41-32 (Ci), 35-13 (C2"), 34.40 (C2), 32.36 (C12', C12''), 30.18-29. 62 (C4' - C11', C4'' - C11''), 25.22 (C3', C3''), 23.12 (C13', C13''), 14.54 (C14', C14) ; HRMS (FAB) found molecular ion (M+H) @ 512.4679 (C3, H62NO4 requires (M+H) # 512.4660).

Example 54 2,3-dipalmitoyloxypropylamine hydrobromide (54) Prepared analogously to 2,3-dilauroyloxypropylamine hydrobromide 52 (Example 52) on a 1 mmol scale to give 2,3-dipalmitoyloxypropylamine hydrobromide 54 as a colourless solid (596 mg, 92.0%); mp; initially appeared to soften at 80-82°C, melted at 150°C ; 1 H- NMR (250 MHz, 45°C), 8H 8.23-8.05 (m, 3H, NH3), 5.56-5.45 (m, 1 H, H2), 4.34 (ABX, 2H, HiA/e, JAB=12. 2 Hz, JAX=4. 0 Hz, JBX=5. 0 Hz), 3.55-3.34 (m, 2H, H3), 2.60-2.30 (m, 4H, H2', H2''), 1.73-1.54 (m, 4H, H3', H3''), 1.40-1.18 (m, 48H, H4'-H15,, H4"-H15"), 0.92 (t, 6H, Hie., H16'', J=6.6 Hz) ; 13C-NMR (250 MHz, 45°C), 8c 174.19 (Ci.), 173. 50 (Ci..), 68. 40 (C2), 63.04 (C3), 41.29 (C1), 35.06 (C2''), 34.39 (C2), 32.27 (Ci4', C14"), 30. 08-29. 57 (C4' - C13', C4'' - C13''), 25.20 (C3', C3''), 22.99 (C15, C15), 14.34 (Ci6., C16) ; HRMS (FAB) found molecular ion (M+H)# 568. 5305 (C35H70NO4 requires (M+H)# 568. 5300).

Example 55

2,3-distearoyloxypropylamine hydrobromide (55) Prepared analogously to 2,3-dilauroyloxypropylamine hydrobromide 52 (Example 52) on a 1 mmol scale to give 2,3-distearoyloxypropylamine hydrobromide 55 as a colourless solid (596 mg, 92.0%); mp; initially appeared to soften at 84-86°C, melted at 148°C ; 1 H- NMR (250 MHz, 45°C), 5H 8.27-8.09 (m, 3H, NH3), 5.51-5.40 (m, 1 H, H2), 4.30 (ABX, 2H, HiA/B, JAB=12. 2 Hz, JAX=4. 1Hz, JBX=4. 9 Hz), 3.51-3.20 (m, 2H, H3), 2.59-2.29 (m, 4H, H2, H2''), 1.70-1.50 (m, 4H, H3', H3''), 1.39-1.17 (m, 56H, H4' - H17', H4'' - H17''), 0.89 (t, 6H, H18', His", J=6.6 Hz) ; 13C-NMR (250 MHz, 45°C), #c 174. 19 (C1'), 173.50 (Ci.), 68.39 (C2), 63.02 (C3), 41.28 (C1), 35.05 (Cz"), 34.40 (C2'), 32. 27 (C16', C16''), 30.07-29.56 (C4' - C15', C4'' - C15''), 25.20 (C3, C3''), 22.99 (Civ, C17''), 14.34 (C14', C14'') ; RMS (FAB) found molecular ion (M+H) # 624.5931 (C39H78NO4 requires (M+H) # 624.5946).

Example 56

N- (2-hydroxy-3-cholesteryloxypropyl) phthalimide (56) This was carried out analogously to the preparation of N- (2-hydroxy-3- lauryloxypropyl) phthalimide 8 (Example 8) on a 14.8 mmol scale, although reaction was refluxed at 80°C. On purification (2 step chromatography (hexane/EtOAc (1: 9), CH2CI2), the product N-(2-hydroxy-3-cholesteryloxypropyl)phthalimide 56 was obtained as a white solid (6.17 g, 70.7%); R, 0.35, (30% EtOAc-70% hexane); mp 65-68°C ;'H-NMR (250 MHz), 8H ; 7.86 (dd, 2H, H3, H6'', Jortho=5. 5 Hz, Jmeta=3. 0 Hz), 7.72 (dd, 2H, H4'', H5''), 5.34 (d, 1 H, H6 J=5. 2 Hz), 4.15-4.00 (m, 1H, H2'), 4.00-3.73 (m, 2H, H3'), 3.64-3.45 (m, 2H, Hr), 3.26-3.10 (m, 1 H, H3), 2.81 (d, 1H, OH, J=6.2 Hz), 2.39-2.08 (m, 2H, H4), 2.08-1.71 (m, 5H, H2, H7, H8), 1.65-1.00 (m, 21 H, H1, H9, H11, H12, H14 - H17, H20, H22 - H25), 0.97 (s, 3H, Hie), 0.91 (d, 3H, H21, J=6. 5 Hz), 0.87 (d, 3H, H26, J=6. 6 Hz), 0.86 (d, 3H, H27, J=6.6 Hz), 0.67 (s, 3H, H18) ; HRMS (FAB) found molecular ion (M+Na)# 612. 4050 (C38H55NO4Na requires (M+Na) # 612.4029); CHN found C 77.33, H 9.55, N 2.39 (C38H55NO4 requires C 77.38, H 6.40, N 2.37).

Example 57

N- (2-lauryloxy-3-cholesteryloxypropyl) phthalimide (57) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 3.4 mmol scale to give after chromatography (0-10% ether/hexane) N- (2-lauryloxy-3- cholesteryloxypropyl) phthalimide 57 as an off-white wax (2.09 g, 81.0%); Rf 0.5, (50% hexane-50% ether);'H-NMR (250 MHz), 5H ; 7.85 (dd, 2H, H3''-6', Jortho=5. 4 Hz, Jmeta=3. 1 Hz), 7.70 (dd, 2H, H4'', H5), 5.33-5.24 (m, 1 H, H6), 3.92-3.69 (m, 3H, H2', H3'), 3.63-3.39 (m, 4H, H1', H1'''), 3.20-3.03 (m, 1 H, H3), 2.37-1.81 (m, 7H, H2, H4, H7, H8), 1. 65-021, J=6. 4 Hz), 0.87 (d, 3H, H26, J=6.6 Hz), 0.86 (d, 3H, H27, J=6. 6 Hz), 0.67 (. 96 (m, 45H, Hi, Hg, H11, H12, H14 - H17, H20, H22 - H25, H2''' - H12'''), 0.95 (s, 3H, H19), 0.91 (d, 3H, Hs, 3H, H, 8) ; 13C-NMR (250 MHz), #c ; 168.74 (Ci.., Ca"), 141.31 (C5), 134.21 (C4'', C5''), 132.62 (C2, C7''), 123.56 (C3'', Ce"), 121.91 (C6), 80.10 (C3), 76.30 (C2), 71.01 (C3), 69.43 (C,), 57.17 (C14), 56.57 (C17), 50.57 (C9), 42.72 (Ci.), 40.19 (C4), 40.06 (C16), 39.93 (C24), 39.39 (C13), 37.57 (C1), 37.24 (C10), 36.60 (C22), 36.19 (C20), 32.34,32.30 (C8, C10'''), 30.37-29.78 (C2''', C4''' - C9''', C2, C7), 28.53 (C12), 28.41 (C25), 26.41 (C3'''), 24.69 (C15), 24. 23 (C23), 23.22,23.10,22.97 (C11''', C27, C26), 21.46 (C11), 19.76 (C, g), 19.12 (C21), 14.53 (C12'''), 12.25 (C, 8) ; HRMS (FAB) found molecular ion (M+Na)# 780. 5933 (C50H79NO4Na requires (M+Na) 780.5907).

Example 58

N- (2-stearyloxy-3-cholesteryloxypropyl) phthalimide (58) Prepared analogously to N- (2, 3-dilauryloxypropyl) phthalimide 18 (Example 18) on a 3.4 mmol scale to give after chromatography (0-10% ether/hexane) N- (2-lauryloxy-3- cholesteryloxypropyl) phthalimide 58 as an off-white wax (2.41 g, 84.3%); R, 0. 5, (50% hexane-50% ether) ;'H-NMR (250 MHz), 5H ; 7.85 (dd, 2H, H3'', H6'', Jortho=5.4 Hz, Jmeta=3. 1 Hz), 7.70 (dd, 2H, H4'', H5), 5.33-5.24 (m, 1 H, H6), 3.91-3.69 (m, 3H, H2', 3'), 3.65-3.38 (m, 4H, H1', H1'''), 3.20-3.05 (m, 1 H, H3), 2.38-1.85 (m, 7H, H2, H4, H7, H8), 1.63-0.98 (m, 57H, H1, H9, H11, H12, H14 - H17, H20, H22 - H25, H2''' - H18'''), 0. 95 (s, 3H, H19), 0.91 (d, 3H, H21, J=6.5 Hz), 0.87 (d, 3H, H26, J=6. 7 Hz), 0.86 (d, 3H, H27, J=6. 6 Hz), 0.67 (s, 3H, H18); 13C- NMR (250 MHz), 8c ; 168.72 (C1'', C8''), 141.29 (C5), 134.20 (C4'', C5''), 132.62 (Cz", C7), 123.56 (C3, Ce"), 121.90 1 (C6), 80.10 (C3), 76.30 (C2), 71.00 (C3), 69.43 (C1'''), 57.18 (C14), 56.58 (C17), 50.57 (C9), 42.72 (Ci.), 40.20 (C4), 40.05 (C16), 39.93 (C24), 39.39 (C13), 37.57 (C1), 37.23 (C10), 36.60 (C22), 36.19 (C20), 32.34,32.30 (C8, Cie".), 30.37-29.78 (C2''', C4''' - C15''', C2, C7), 28.53 (C, 2), 28.41 (C25), 26.42 (C3'''), 24. 69 (C15), 24. 24 (C23), 23.22,23.10,22.97 (C17''', C27, C26), 21.47 (C11), 19.76 (Cig), 19.12 (C21), 14.52 (C18'''), 12.25 (C, 8) ; HRMS (FAB) found molecular ion (M+H) #864. 6816 (C4, H72NO4 requires (M+H) @ 864.6816).

Example 59

2-lauryloxy-3-cholesteryloxypropylamine (59) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 1.7 mmol scale to give after chromatography (0.5-3% MeOH/CH2CI2) 2-lauryloxy-3- cholesteryloxypropylamine 59 as a pale yellow oil (0.93 g, 87.0%); Rf 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), #H 5. 33 (d, 1 H, H6, J=5.3 Hz), 3.68-3.43 (m, 5H, H2', H3', H,), 3.24-3.05 (m, 1 H, H3), 2.80 (ABX, 2H, H1'A/B, JAB=13. 2 Hz, JAX=3. 9 Hz, JBX=6. 5 Hz), 2.42-2.08 (m, 2H, H4), 2.08-1.64 (m, 5H, H2, H7, H8), 1.64-1.54 (m, 4H, NH2, H2''), 1.54-1.00 (m, 45H, H1, H9, H11, H12, H14 - H17, H20, H22 - H25, H2'' - H12''), 0.99 (s, 3H, H19), 0.91 (d, 3H, H21, J=6. 5 Hz), 0.87 (d, 3H, H26, J=6. 7 Hz), 0.86 (d, 3H, H27, J=6.6 Hz), 0.67 (s, 3H, H18); 13C-NMR (250 MHz), 8c ; 141.27 (C5), 122.001 (C6), 80.31,80.11 (C3, C2'), 70.76 (C3), 68.68 (C1''), 57.18 (Ci4), 56.57 (C17), 50.60 (C9), 43.90 (Ci.), 42.72 (C4), 40.20 (C16), 39.92 (C24), 39.54 (Cis), 37.62 (C1), 3727 (C, o), 36.60 (C22), 36.19 (C20), 32.34, 32.30 (C8, Ciao"), 30.57-29.77 (C2, C4"-C9", C2, C7), 28.63 (Cis), 28.41 (C25), 26.58 (C3''), 24.69 (Cis), 24.23 (C23), 23.21,23.09,22.96 (C", C27, C26), 21.48 (Cil), 19.78 (C, g), 19.12 (C21), 14.52 (C12''), 12.25 (C18) ; HRMS (FAB) found molecular ion (M+H) # 628.6003 (C42H78NO2 requires (M+H)# 628.6033).

Example 60

2-stearyloxy-3-cnoiesteryloxypropylamine (60) Prepared analogously to 2,3-dilauryloxypropylamine 33 (Example 33) on a 2.7 mmol scale to give after chromatography (0.5-3% MeOH/CH2CI2) 2-stearyloxy-3- cholesteryloxypropylamine 60 as a white solid (1.75 g, 91.0%); R, 0.3 (10% MeOH, 90% CH2CI2) ; 1 H-NMR (250 MHz), 8H 5. 33 (d, 1 H, H6, J=5.4 Hz), 3.67-3.30 (m, 5H, H2', H3', H1''), 3.23-3.05 (m, 1H, H3), 2.80 (ABX, 2H, H1A/B, JAB=13. 2 Hz, JAX=3. 9 Hz, JBX=6. 5 Hz), 2.43-2.09 (m, 2H, H4), 2.05-1.70 (m, 5H, H2, H7, H8), 1.62-1.52 (m, 4H, NH2, H2''), 1.52- 1.00 (m, 57H, Hi, Hg, H11, H12, H14 - H17, H20, H22 - H25, H2''' - H18''), 0.99 (s, 3H, H19), 0.91 (d, 3H, H21, J=6. 5 Hz), 0.87 (d, 3H, H26, J=6. 7 Hz), 0.86 (d, 3H, H27, J=6. 6 Hz), 0.67 (s, 3H, H18); 13C-NMR (250 MHz), 8c ; 141.25 (C5), 121.98 1 (C6), 80.41,80.09 (C3, C2), 70.74 (C3), 68.68 (C,), 57.18 (C14), 56.58 (C17), 50.60 (Cg), 43.91 (Ci.), 42.72 (C4), 40.20 (C, 6), 39.92 (C24), 39.54 (C13), 37.62 (C1), 37.26 (C10), 36.60 (C22), 36.19 (C20), 32.34,32.30 (C8, Ci6..), 30.58-29.77 (C2'', C4'' - C15'', C2, C7), 28.84 (Ci2), 28.71 (C25), 26.58 (C3''), 24.69 (C15), 24.24 (C23), 23. 21,23.09,22.96 (Cl7", C27, C26), 21.48 (C11), 19.77 (C19), 19.12 (C21), 14.51 (C18''), 12.25 (C, 8) ; HRMS (FAB) found molecular ion (M+H)# 712.6950 (C48H9oN02 requires (M+H) @ 712.6972).

Example 61

N-a, e-di-tert-butyloxycarbonyl-L-lysine-a- (2, 3-dilauryloxy) propylamide (61) Prior to carrying out this example, the THF to be used as solvent was distilled over potassium. A flask containing N-a, #-di-tert-butyloxycarbonyl-L-lysine N- hydroxysuccinimide ester (399 mg, 0.9 mmol) and 2,3-dilauryloxypropylamine 33 (257 mg, 0.6 mmol) was flushed with argon. THF (10 ml) was added and the reaction refluxed at 85°C overnight. The solvent was then removed and CH2CI2 (30 ml) added. The solution was washed with NaHC03 (1 x 20 ml), water (1 x 20 ml) and brine (1 x 20 ml), and each of the aqueous layers backwashed with CH2CI2 (10 ml). The organic layers were combined, dried (MgSO4), and evaporated under reduced pressure to give a yellow solid.

The solid was purified by column chromatography (10-30% ethyl acetate/hexane) to give of N-a, E-di-tert-butyloxycarbonyl-L-lysine-a- (2, 3-dilauryloxy) propylamide 61 as a white solid (420 mg, 92.6%). Rf 0.35 (30% ethyl acetate/hexane); mp 58.60°C ;'H-NMR (250 MHz), #H 6. 47-6.35 (m, 1H, NHamjde), 5.16-5.02 (m, 1 H, oc,-NHcarbamate), 4. 67-4.51 (m, 1H, E-NHCarbama, e), 4.10-3.93 (m, 1 H, H2), 3.62-3.19 (m, 9H, H1'-H3', H1'', H1'''), 3.15-3.01 (m, 2H, H6), 1.90-1.48 (m, 10H, H3 - H5, H2'', H2'''), 1.43 (s, 18H, Ht-butyl), 1.36-1.16 (m, 36H, Ha- H11'', H3''' - H11'''), 0.86 (t, 6H, H12'', H12''' J=6.6 Hz) ;'3H-NMR (250 MHz), 8c 172.30 (C1), 156.52 (C7), 156.00 (C12), 80.33 (C2'), 79.59 (C8, C, 3), 72.26 (C3'), 71.83 (C1''), 70.71 (C1'''), 54.98 (C2), 41.11 (Ci.), 40.53 (C6), 32.76 (C3, C5), 32.32 (C, Calo), 30.45-29.75 (Cz", C4'' - C9'', C2''', C4''' - C9'''), 28.84,28.74 (C9 - C11, C14 - C16), 26.51 (C3'', C3'''), 23.08 (C11'', C11'''), 22.99 (C4), 14.50 (C12'', C12''') ; HRMS (FAB) found molecular ion (M+H)# 756. 6466 (C43H86NO7 requires (M+H)# 756. 6486).

Example 62 N-a, e-di-tert-butyloxycarbonyl-L-lysine-a- (2-lau ryloxy-

3-stearyloxy)propylamide (62) Prepared analogously to N-α,#-di-tert-butyloxycarbonyl-L-lysine-&alpha ;-(2-lauryloxy-3- stearyloxy) propylamide 61 (Example 61) on a 0.4 mol scale to give, after chromatography (10-30% ethyl acetate/hexane), N-a, #-di-tert-butyloxycarbonyl-L-lysine- a- (2-lauryloxy-3-stearyloxy) propylamide 62 as a white solid (297 mg, 88.5%). Rt 0.35 (30% ethyl acetate/hexane); mp 62-64°C ;'H-NMR (250 MHz), #H 6.47-6.36 (m, 1 H, Amide), 5.15-5.03 (m, 1H, α-NHcarbamate), 4. 65-4.51 (m, 1H, #-NHcarbamate), 4. 09-3.94 (m, 1H, H2), 3.61-3.20 (m, 9H, H1'-H3', H1'', H1'''), 3.16-3.01 (m, 2H, H6), 1.90-1.49 (m, 10H, H3 - H5, H2'', H2'''), 1.43 (s, 18H, Ht-butyl), 1. 36-1.16 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.87 (t, 6H, H18'', H12''', J=6.6 Hz); 13H-NMR (250 MHz), 8c172. 30 (Ci), 156.51 (C7), 156.01 (Cis), 80.35 (C2'), 79.58 (C8, C, 3), 72.26 (C3), 71.84 (Ci..), 70.71 (C1'''), 54.97 (C2), 41.13 (Ci.), 40.51 (C6), 32.78 (C3, C5), 32.32 (C, Cl ()), 30.46-29.76 (C2' ', C4'' - C15'', C2''', C4''' - C9'''), 28.84, 28.74 (C9 - C11, C14 - C16), 26.51 (C3, C3'''), 23.06 (C17'', Ciel), 23.01 (C4), 14. 51 (C18'', Ci2"') ; HRMS (FAB) found molecular ion (M+H)# 840.7405 (C49H98NO7 requires (M+H)# 840. 7396).

Example 63 L-lysine-a- (2, 3-dilauryloxy) propylamide (63)

To a flask containing 95% TFA (20 ml) was added N-a, E-di-tert-butyloxycarbonyl-L-lysine- a- (2, 3-dilauryloxy) propylamide 61 (420 mg, 0.56 mmol) and the solution stirred for 1.5 hours. The solvent was then removed under reduced pressure, and the resulting white solid purified by column chromatography (CH2Cl2 - CH2Cl2:MeOH:NH3 96.5: 3: 0.5), to give L-lysine-α-(2,3-dilauryloxy)propylamide 63 as a white solid (273 mg, 88. 1%). Rf 0.30 (CH2CI2 : MeOH: NH3 92: 7: 1); mp 53°C ;'H-NMR (250 MHz), Sn 7. 60-7.50 (m, 1H, NHamide), 3.69-3.20 (m, 10H, H2, H1'-H3', H1'' H1'''), 2.77-2.65 (m, 2H, H6), 1.71-1.39 (m, 14H, H3-H5, H2'' H2''', 2NH2), 1.39-1.16 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.88 (t, 6H, H12'', H12''', J=6. 5 Hz) ; 13H-NMR (250 MHz), #c 175.54 (C1), 72.23 (C3'), 71.91 (C,), 70.68 (C1'''), 55.38 (C2), 41.33 (Ci.), 40.79 (C6), 35.11 (C3), 32.32 (C10'', C10'''), 31.53 (C5), 30.48-29.76 (C2'', C4"- C9", C2''', C4'' - C9'''), 26.52 (C3'', C3'''), 23.28 (C4), 23.08 (C11'', C1'''), 14.50 (C12'', C12''); HRMS (FAB) found molecular ion (M+H) @ 556.5417 (C33H70N303 requires (M+H)# 556. 5422).

Example 64 L-lysine-a- (2-lauryloxy-3-stearyloxy) propylamide (64)

Prepared analogously to L-lysine-α-(2,3-dilauryloxy)propylamide 63 (Example 63) on a 0.35 mmol scale to give, after chromatography (CH2CI2-CH2CI2 : MeOH : NH3 96.5: 3: 0.5), L-lysine-α-(2-lauryloxy-3-stearyloxy)propyl-amide 64 as a white solid (170 mg, 75.9%). Rf 0.30 (CH2CI2 : MeOH: NH3 92: 7: 1); mp 56°C ;'H-NMR (250 MHz), #H 7. 60 (t, 1H, NHamide J=5.1 Hz), 3.63-3.17 (m, 10H, H2, H1' - H3', H1'', H1'''), 3.17-3.04 (m, 4H, 2NH2), 2.84-2.72 (m, 2H, H6), 1.63-1.36 (m, 14H, H3-H5, H2'' H2''', 2NH2), 1.36-1.17 (m, 48H, H3'' - H17'', H3''' - Hie), 0.86 (t, 6H, H18'', H12''', J=6. 6 Hz) ;'3H-NMR (250 MHz), 8c 175.55 (C1), 72.24 (C3), 71.93 (C,), 70.67 (C1'''), 55.48 (C2), 41.56 (Ci.), 40.79 (C6), 35.21 (C3), 32. 32 (Ci6", Cio 31.99 (C5), 30.48-29.76 (C2", C4'' - C15'', C2''', C4''' - C9'''), 26.52 (C3'', C3'''), 23.31 (C4), 23.08 (C17'', C11'''), 14.50 (C18'', C12'''); HRMS (FAB) found molecular ion (M+H) @ 640.6356 (C39H82N303 requires (M+H) # 640.6363).

Example 65 <BR> <BR> N-&alpha;-fluorenylmethoxycarbonyl-L-tryptophan-&alp ha;-

(2,3-dilauryloxy) propylamide (65) Prior to carrying out this example, the THF to be used as solvent was distilled over potassium. A flask containing N-a-fluorenylmethoxycarbonyl-L-tryptophan pentafluorophenyl ester (889 mg, 1.5 mmol) and 2,3-dilauryloxypropylamine 33 (428 mg, 1.0 mmol) was flushed with argon. THF (15 ml) was added and the reaction refluxed at 85°C for one hour. The solvent was then removed and CH2CI2 (30 ml) added. The solution was washed with NaHC03 (1 x 20 ml), water (1 x 20 ml) and brine (1 x 20 ml), and each of the aqueous layers backwashed with CH2CI2 (10 ml). The organic layers were combined, dried (MgSO4), and evaporated under reduced pressure to give a yellow solid. The solid was purified by column chromatography (CH2CI2-0. 5% MeOH/CH2CI2) to give N-a-fluorenylmethoxycarbonyl-L-tryptophan-a- (2, 3-dilauryloxy) propylamide 65 as a pale yellow oil (598 mg, 71.7%). Rf 0.50 (2% MeOH/CH2CI2) ;'H-NMR (250 MHz), 8H 8.11-8.03 (m, 1 H, NH), 7.76 (d, 2H, H, 9, H22, J=7.4 Hz), 7.74-7.63 (m, 1 H, H6), 7.59-7.49 (m, 3H, NHamide, H14, H25), 7.42-7.00 (m, 8H, H7-Hg, Hlly H17, H18, H23, H24), 5.58-5.45 (m, 1H, NHcarbamate), 4.56-4.25 (m, 3H, H2, H13), 4.19 (t, 1H, H14, J=7.0 Hz), 3.50-3.07 (m, 11 H, H3, H1' - H3', H1'', H1'''), 1.55-1.30 (m, 4H, H2'', H2), 1.30-1.09 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.88 (t, 6H, H12'', H12''', J=6.5 Hz) ;'3C-NMR (250 MHz), 5c171. 59 (C1), 156.33 (C12), 144.23 (C20, C21), 141.70 (C15, C26), 136.69 (C10), 128.13 (C16, C25), 127.85 (C5), 127. 50 (Cl7, C24), 125.51 (Cl8, C23), 123.50 (C11), 122.80 (C7), 120.39,120.350 (C6, C19, C22), 119.24 (C8), 111.68 (C9), 111.06 (C4), 72.17 (C3), 71.57 (Ci.), 70.59 (C1'''), 67.56 (Cis), 56.37 (C2), 47.57 (C14), 41.25 (C,), 32.34 (C10'', C10'''), 30.38-29.78 (C2'', C4'' - C9'', C2''', C4''' - C9'''), 29.27 (C3), 26.44 (C3'', C3'''), 23.10 (C11'', C11'''), 14.53 (C, Ci2-) ; HRMS (FAB) found molecular ion (M+H) o 858.6125 (C54H8, N304Na requires (M+H) # 858.6122).

Example 66 <BR> <BR> N-&alpha;-fluorenylmethoxycarbonyl-L-tryptophan-&alp ha;-

(2-lauryloxy-3-stearyloxy) propylamide (66) Prepared analogously to N-&alpha;-fluroenylmethoxycarbonyl-L-tryptophan-&alp ha;-(2, 3- dilauryloxy) propylamide 65 (Example 65) on a 1 mol scale to give, after chromatography (CH2CI2-0. 5% MeOH/CH2Cl2), N-&alpha;-fluorenyl-methoxycarbonyl-L- tryptophan-a- (2-lauryloxy-3-stearyloxy) propylamide 66 as a yellow wax (750 mg, 81.7%).

Rf 0.50 (2% MeOH/CH2Ct2) ; 1H-NMR (250 MHz), lah8. 11-8.03 (m, 1H, NH), 7.76 (d, 2H, H19'', H22'', J=7.3 Hz), 7.73-7.63 (m, 1H, H6), 7.59-7.49 (m, 2H, Hi4", H25"), 7.43-7.00 (m, 9H, NHamide, H7 - H9, H11, H17, H18, H23, H24), 5.59-5.45 (m, 1H, NHcarbamate), 4. 53-4.25 (m, 3H, H2, H13), 4.20 (t, 1 H, H14, J=7.1 Hz), 3.52-3.04 (m, 11 H, H3, H1' - H3', H1'', H1'''), 1.53- 1.30 (m, 4H, H2, H2'''), 1.30-1.11 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6. 6 Hz) ;'3C-NMR (250 MHz), 6c 171.46 (C1), 156.30 (C12), 144.23 (C20, C21), 141.70 (C15, C26), 136.66 (C10), 128.12 (C16, C25), 127.85 (C5), 127.45 (C17, C24), 125.51 (C18, C23), 123.50 (C11), 122.83 (C7), 120.38, (C6, C19, C22), 119. 28 (C8), 111. 62 (C9), 111. 10 (C4), 72.17 (C3'), 71.58 (C,), 70.58 (C1'''), 67.53 (C, 3), 56.33 (C2), 47.58 (C, 4), 41.22 (C1'), 32.33 (C16'', C10'''), 30.37-29.77 (C2'', C4'' - C15', C2''', C4''' - C9'''), 29.30 (C3), 26.48 (C3'', C3'''), 23.10 (C, C11'''), 14.52 (C18'', C12''') ; HRMS (FAB) found molecular ion (M+H)# 920.7244 (C60H94N3O4 requires (M+H) # 920.7196).

Example 67

l-tryptophan-&alpha;-(2,3-dilauryloxy)propylamide (67) To a solution of N-a-fluorenylmethoxycarbonyl-L-tryptophan-a- (2, 3- dilauryloxy) propylamide 65 (598 mg, 0.97 mmol) in DMF (8 ml) was added piperidine (2 ml), and the reaction stirred for 45 mins. The solvent was then removed under high vacuum, and the resulting yellow oil purified by column chromatography (50% EtOAc/hexane-100% EtOAc). Freeze-thawing under vacuum was used to obtain the product L-tryptophan-&alpha;-(2,3-dilauryloxy)propylamide 67 as a pale yellow wax (462 mg, 80.8%). Rf 0.20 (EtOAc) ; 1H-NMR (250 MHz), 8H 8. 22-8.12 (m, 1H, NHindole), 7.67 (d, 1H, H6, J=7. 5 Hz), 7.56-7.44 (m, 1 H, NHamide), 7.36 (d, 1 H, Hg, J=7.7), 7.23-7.06 (m, 3H, H7, H8, H"), 3.79-3.70 (m, 1 H, H2), 3.58-3.18 (m, 9H, H,-H3, H,, H1'''), 3. 01-2.88, (m, 2H, H3), 1.97-1.70 (m, 2H, NH2), 1.60-1.41 (m, 4H, H2'', H2'''), 1.36-1.12 (m, 36H, H3"-H 1,,, H3 -H"), 0.88 (t, 6H, H, 2'', H12''', J=6. 5 Hz); HRMS (FAB) found molecular ion (M+H)# 614.5261 (C38H68N303 requires (M+H)# 614.5253); CHN found C 74.10, H 11.07, N 6.70 (C38H67 N3O3 requires C 74.34, H 11.00 N 6.84).

Example 68

L-tryptophan-&alpha;-(2-lauryloxy-3-stearyloxy)propyl-am ide (68) Prepared analogously to L-tryptophan-&alpha;-(2,3-dilauryloxy)propylamide 67 (Example 67) on a 0.74 mol scale to give, after chromatography (50% EtOAc/hexane-100% EtOAc), L- tryptophan-&alpha;-(2-lauryloxy-3-stearyloxy)propylamide 68 as a yellow wax (310 mg, 77.3%).

R, 0.20 (EtOAc) ; 1H-NMR (250 MHz), #H 8. 28-8.18 (m, 1H, NHindole), 7.68 (d, 1H, H6, J=7.7 Hz), 7.61-7.49 (m, 1 H, NHamjde), 7.36 (d, 1H, H9, J=7.5 Hz), 7.23-7.03 (m, 3H, H7, H8, Hn), 3.77-3.67 (m, 1H, H2), 3.61-3.20 (m, 9H, H1' - H3', H1'', H1'''), 3.00-2.84, (m, 2H, H3), 1.87-1.62 (m, 2H, NH2), 1.62-1.40 (m, 4H, H2'', H2'''), 1.40-1.12 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H) 698.6200 (C44H8oN303 requires (M+H) &commat; 698.5422); CHN found C 75.13, H 11.46, N 5.90 (C44H79 N303 requires C 75.30, H 11.41 N 6.02).

Example 69

N-a-butyloxycarbonyl-N-im-trityl-L-histidine a-pentafluorophenyl ester (69) This compound was prepared analogously to N-t-butyloxycarbonyl-L-aspartic acid ß- benzyl ester pentafluorophenol ester 52 (Example 52) on a 4 mmol scale to give, after chromatography (10% EtOAc/hexane) N-a-butyloxycarbonyl-N-im-trityl-L-histidine a- pentafluorophenyl ester 69 (1.42 g, 53.5%) as a white foam. Rf 0.85 (40% EtOAc/hexane). The product was found to be unstable by 2D TLC, and was used immediately in the next step.

Example 70 N-a-butyloxycarbonyl-N-im-trityl-L-histidine-a- (2,3-dilauryloxy) propylamide (70)

Prior to carrying out this example, the THF to be used as solvent was distilled over potassium. A flask containing N-a-butyloxycarbonyl-N-im-trityl-L-histidine a- pentafluorophenyl ester 69 (400 mg, 0.63 mmol) and 2,3-dilauryloxypropylamine 33 (193 mg, 0.45 mmol) was flushed with argon. THF (5 ml) was added and the reaction refluxed at 85°C overnight. The solvent was then removed and CH2CI2 (20 ml) added. The solution was washed with NaHC03 (1 x 10 ml), water (1 x 10 ml) and brine (1 x 10 ml), and each of the aqueous layers backwashed with CH2CI2 (5 ml). The organic layers were combined, dried (MgSO4), and evaporated under reduced pressure to give a colourless oil. The solid was purified by column chromatography (10-50% ether/hexane) to give N-a- butyloxycarbonyl-N-im-trityl-L-histidine-a- (2, 3-dilauryloxy) propylamide 70 as a pale yellow oil (309 mg, 75.7%). R, 0.25 (70% ether/hexane) ;'H-NMR (250 MHz), #H 7. 41-7.36 (m, 1 H, H6), 7.46-7.28 (m, 9H, Htrityl - meta, para), 7. 14-7.06 (m, 6H, Htrity-ortho), 7.06-6.95 (m, 1H, <BR> <BR> NH. mid.), 6.63 (s, 1 H, H5), 6.44-6.30 (m, 1 H, NHcarbamate), 4.48-4.34 (m, 1 H, H2), 3.60-2.90 (m, 11H, H3, H1' - H3', H1'', H1'''), 1.61-1.47 (m, 4H, H2'', H2), 1.42 (s, 9H, Ht-butyl), 1.35-1.12 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.88 (t, 6H, H12'', H12''', J=6.6 Hz) ;'3C-NMR (250 MHz), 8c 171.98 (C1), 156.02 (C26), 142.70 (C8, C14, C20), 138.74 (C6), 137.32,137.25 (C4), 130.15 (Ctrityl - metal), 128. 48 (Ctrityl - ortho, para), 120.04 (Cs), 80.04 (C27), 72.22 (C3), 71.69,71.63 (C,), 70.93,70.78 (C1'''), 55.37 (C2), 41.17,40.90 (Ci.), 32.32 (C, Calo), 30.72 (C3), 30.52-29.76 (C2'', C4'' - C9'', C2''', C4''' - C9'''), 28. 79 (C28 - C30), 26.51,26.48 (C3'', C3'''), 23.08 (C1'', C11'''), 14.51 (Cis", Ci2.") ; HRMS (FAB) found molecular ion (M+Na)# 929.6496 (C57H86N405Na requires (M+Na)# 929. 6513).

Example 71 N-a-butyloxycarbonyl-N-im-trityl-L-histidine-a-

(2-lauryloxy-3-stearyloxy) propylamide (71) Prepared analogously to N-a-butyloxycarbonyl-N-im-trityl-L-histidine-a- (2-lauryloxy-3- stearyloxy) propylamide 70 (Example 70) on a 0.52 mol scale to give, after chromatography (10-50% ether/hexane) N-a-butyloxycarbonyl-N-im-trityl-L-histidine-a- (2,3-dilauryloxy) propylamide 71 as a pale yellow oil (396 mg, 76.9%). Rf 0.25 (70% ether/hexane); 1 H-NMR (250 MHz), #H 7. 49-7.39 (m, 1 H, H6), 7.39-7.29 (m, 9H, Htrityl - meta, para), 7.17-7.07 (m, 6H, Htrityl - ortho), 7. 07-6.98 (m, 1H, NHamide), 6. 65 (s, 1 H, H5), 6.37-6.26 (m, 1H, NHcarbamate), 4. 48-4.35 (m, 1 H, H2), 3.58-2.92 (m, 11H, H3, H1' - H3', H11', H1'''), 1.61- 1.47 (m, 4H, H2, H2'''), 1.42 (s, 9H, Ht-b, tyl), 1. 37-1. 13 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6.6 Hz) ;'3C-NMR (250 MHz), #c 171. 72 (C1), 156.13 (C26), 142.14 (C8, C14, C20), 138. 45 (C6), 137.01,136.88 (C4), 130.10 (Ctrityl - meta), 128. 75,128.64 (C ortho, para), 120. 20 (C5), 80.05 (C27), 72.19 (C3), 71.70 (Ci.), 70.92,70.80 (C1'''), 55.62, 55.41 (C2), 41.15,40.98 (Ci.), 32.33 (C, Calo), 30.72,30.51 (C3), 30.51-29.76 (C2'', C4" - C15'', C2''', C4''' - C9'''), 28.76 (C28 - C30), 26.51,26.48 (C3'', C3'''), 23.09 (C17'', C11'''), 14.52 (C18'', C12''') ; HRMS (FAB) found molecular ion (M+Na)# 1013. 7435 (C63Hg8N405Na requires (M+Na) &commat; 1013.7412).

Example 72

L-histidine-&alpha;-(2,3-dilauryloxy)propylamide (72) To a flask containing TFA: triisopropylsilane : water (95: 2.5: 2.5,20 ml) was added N- a-butyloxycarbonyl-N-im-trityl-L-histidine-a- (2, 3-dilauryloxy) propyl-amide 70 (300 mg, 0.33 mmol) and the solution stirred for 1.5 hours. The solvent was then removed under reduced pressure, and the resulting white solid purified by column chromatography (CH2Cl2 - CH2Cl2 : MeOH: NH3 92: 7: 1) to give L-histidine-&alpha;-(2,3-dilauryloxy)propylamide 72 as a colourless glassy solid (141 mg, 75.8%). Rf 0.50 (CH2CI2 : MeOH: NH3 92: 7: 1); mp 55- 56°C ; 1H-NMR (250 MHz), #H 7. 82-7.67 (m, 1H, NHamide), 7.56-7.51 (m, 1 H, H6), 6.85 (s, 1 H, H5), 3.71-2.90 (m, 14H, H2, H3, H1' - H3', H1'', H1''', NH2), 1.61-1.45 (m, 4H, H2, H2'''), 1.37-1.14 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.87 (t, 6H, H12'', H12''', J=6. 6 Hz) ;'3C-NMR (250 MHz), #c 175.20, 175.12 (C1), 135.68,135.56 (C6), 132.64,131.84 (C4), 121.26,120.84 (C5), 77.22 (C2'), 72.25 (C3), 71.65 (C,), 70.80,70.66 (C1'''), 55.57,55.21 (C2), 40.82, 40.56 (Ci.), 32.30 (C10'', C10'''), 32.15 (C3), 30.43-29.74 (C2", C4'' - C9'', C2''', C4''' - C9'''), 26.49 (C3'', C3'''), 23.07 (C11'', C11'''), 14.49 (C12'', C12''') ; HRMS (FAB) found molecular ion (M+H) # 565.5057 (C33H65N403 requires (M+H) # 565.5042).

Example 73 L-histidine-&alpha;-(2-lauryloxy-3-stearyloxy)propylamid e (73)

Prepared analogously to give L-histidine-&alpha;-(2,3-dilauryloxy)propylamide 72 (Example 72) on a 0.36 mol scale to give, after chromatography (CH2CI2-CH2CI2 : MeOH: NH3 92: 7: 1), L-histidine-&alpha;-(2,3-dilauryloxy)prlopylamide 73 as a white solid (207 mg, 88.9%). Rf 0.50 (CH2CI2 : MeOH: NH3 92: 7: 1); mp 56-58°C ; 1H-NMR (250 MHz), #H 7. 82-7.68 (m, 1H, NHamide), 7.55-7.50 (m, 1 H, H6), 6.85 (s, 1 H, H5), 3.72-2.90 (m, 14H, H2, H3, H1' - H3', H,, H,, NH2), 1.60-1.45 (m, 4H, H2'', H2'''), 1.36-1.13 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.87 (t, 6H, H18'', H12''', J=6. 6 Hz) ;'3C-NMR (250 MHz), #c 175. 11,175.01 (C1), 135.67,135. 51 (C6), 131.29,130.39 (C4), 121.85,120.81 (C5), 77.20 (C2'), 72.25 (C3), 71.65 (Ci..), 70.81, 70.67 (C1'''), 55.47,55.07 (C2), 40.85,40.51 (Ci.), 32.32 (C16'', C10'''), 32.18,32.00 (C3), 30.44-29.75 (C2'', C4'' - C15'', C2''', C4''' - C9'''), 26.51 (C3'', C3'''), 23.08 (C17'', C11'''), 14.50 (C18'', C12''') ; HRMS (FAB) found molecular ion (M+H) &commat; 649.5996 (C39H77N403 requires (M+H)# 649. 5983).

Example 74 <BR> <BR> N-a-fluorenylmethoxycarbonyl-NG-4-methoxy- 2,3,6-trimethylbenzene sulphonyl-L-arginine-

&alpha;-(2,3-dilauryloxy)propyl-amide (74) Prior to carrying out this example, the THF to be used as solvent was distilled over potassium. A flask containing N-&alpha;-fluorentylmethoxycarbonyl-NG-4-methoxy-2, 3,6- trimethylbenzene sulphonyl-L-arginine pentafluorophenyl ester (1.162 g, 1.5 mmol) and 2,3-dilauryloxypropylamine 33 (428 mg, 1.0 mmol) was flushed with argon. THF (15 ml) was added and the reaction refluxed at 85°C for one hour. The solvent was then removed and CH2CI2 (30 ml) added. The solution was washed with NaHC03 (1 x 20 ml), water (1 x 20 ml) and brine (1 x 20 ml), and each of the aqueous layers backwashed with CH2CI2 (10 ml). The organic layers were combined, dried (MgS04), and evaporated under reduced pressure to give a colourless oil, which was purified by column chromatography (CH2CI2-1% MeOH/CH2CI2). This gave N-a-fluorenylmethoxycarbonyl-NG-4-methoxy- 2,3, 6-trimethylbenzene sulphonyl-L-arginine-&alpha;-(2,3-dilauryloxy)propylamid e 74 as a pale yellow oil (750 mg, 74.0%). Rf 0.30 (3% MeOH/CH2CI2) ;'H-NMR (250 MHz), #H 7. 74 (d, 2H, H24, H27, J=7.6 Hz), 7.56 (d, 2H, H21, H30, J=7.2), 7.37 (t, 2H, H23, H28, J=7.5 Hz), 7.27 (d, 2H, H22, H29, J=7.4 Hz), 6.94-6.82 (m, 1 H, NHamide), 6.49 (s, 1 H, Hg), 6.23-5.90 (m, 3H, NHguanidine), 5.86 (d, 1H, NHcarbamate), 4.35 (t, 2H, Hie, J=7.0 Hz), 4.25-4.10 (m, 2H, H2, H19), 3.78 (s, 3H, H, 4), 3.54-3.30 (m, 9H, H1', H3', H1'', H1'''), 3.30-3.10 (m, 2H, H5), 2.69, 2.61 (2s, 6H, H, 3, H, 6), 2.10 (s, 3H, H15), 1.78-1.40 (m, 8H, H3, H4, H2, H2'''), 1.30-1.10 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.87 (t, 6H, H12'', H12''' J=6.6 Hz) ;'3C-NMR (250 MHz), 8c 172.05 (C1), 158.99 (C10), 156.54 (C6), 144.16 (C25, C26), 141.69 (C20, C31), 139.04 (C12), 137.16 (C7), 133.73 (C8), 128.15 (C21, C30), 127.50 (C22, C29), 125.46 (C23, C28), 125.19 (C11), 120.34 (C24, C27), 112.12 (C9), 72.23 (C3), 71.69 (Ci..), 70.68 (C1'''), 67.54 (C18), 55-81 (Cl4), 54. 64 (Cz), 47.53 (do), 41.00 (C5), 32.33 (C10'', C10'''), 30.81-29.77 (C2", C4-

C9", C2''', C4''' - C9'''), 26.50 (C3", C3'''), 25.51 (C3), 25.24 (C4), 24.52 (C13), 23.10 (C11'', C11'''), 18.74 (C16), 14.52 (C12'', C12'''), 12.35 (C15) ; HRMS (FAB) found molecular ion (M+H) &commat; 1016.6874 (C59Hg4N507S requires (M+H) # 1016.6835).

Example 75 N-a-fluorenylmethoxycarbonyl-NG-4-methoxy- 2,3,6-trimethylbenzene sulphonyl-L-arginine- &alpha;-(2-lauryloxy-3-stearyl-oxy)propylamide (75) Prepared analogously to N-a-fluorenylmethoxycarbonyl-NG-4-methoxy-2, 3,6- trimethylbenzene sulphonyl-L-arginine-&alpha;-(2,3-dilauryloxy)propylamid e 74 (Example 74) on a 1 mmol scale to give, after chromatography (CH2CI2-1% MeOH/CH2CI2) N-a- butyloxycarbonyl-N-im-trityl-L-histidine-a- (2, 3-dilauryloxy)-propylamide 75 as a pale yellow oil (809 mg, 73.5%). Rf 0.3 (3% MeOH/CH2CI2) ; 1 H-NMR (250 MHz), 8H 7. 73 (d, 2H, H24, H27, J=7.5 Hz), 7.56 (d, 2H, H21, H30, J=7.2 Hz), 7.37 (t, 2H, H23, H28, J=7.7 Hz), 7.26 (d, 2H, H22, H29, J=6. 8 Hz), 6.96-6.82 (m, 1 H, NHamide), 6.49 (s, 1H, H9), 6.25-5.96 (m, 3H, NHguanidine), 5. 90 (d, 1H, NHcarbamate, J=6. 8 Hz), 4.35 (t, 2H, His, J=7.0 Hz), 4.25- 4.10 (m, 2H, H2, H19), 3.79 (s, 3H, H14), 3.54-3.30 (m, 9H, H1'-3', H1'', H1'''), 3.30-3.11 (m, 2H, H5), 2.69,2.62 (2s, 6H, H13, 16), 2.10 (s, 3H, H, 5), 1.79-1.40 (m, 8H, H3, H4, H2'', H2), 1.30-1.10 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.87 (t, 6H, H18'', H12''', J=6.6 Hz) ;'3C-NMR (250 MHz), #C 172. 33 (C1), 158.96 (C10), 156.63 (C6), 144.17 (C25, C26), 141.65 (C20, C31), 139.01 (C12), 137.10 (C7), 133.63 (C8), 128.13 (C21, C30), 127.50 (C22, C29), 125.50 (C23, C28), 125.24 (C11), 120.36 (C24, C27), 112. 13 (C9), 72.18 (C3), 71.66 (C1''), 70.75 (C1'''), 67.56 (C, 8), 55.80 (C, 4), 54.79 (C2), 47.51 (C19), 40.98 (C5), 32.33 (C, C10'''), 30. 56- 29.77 (C2", C4-C, 5, C2, C4-Cg), 26.50 (C3'', C3'''), 25.70 (C3), 25.44 (C4), 24.54 (C13) 23.10 (C17'', C11'''), 18.75 (C16), 14.52 (C, Ciz"'), 12.35 (C15) ; HRMS (FAB) found molecular ion (M+Na) &commat; 1122.7632 (C65H105N5O7SNa requires (M+Na)# 1122. 7653).

Example 76 NG-4-methoxy-2, 3,6-trimethylbenzenesulphonyl-L-arginine-

&alpha;-(2,3-dilauryloxy)propylamide (76) Prepared analogously to L-tryptophan-&alpha;-(2,3-dilauryloxyl)propylamide 67 (Example 67) on a 0.7 mmol scale to give, after chromatography (CH2CI2-CH2CI2 : MeOH: NH3 92: 7: 1) NG- 4-methoxy-2,3,6-trimethylbenzenesulphonyl-L-arginine-a- (2, 3-dilauryloxy) propylamide 76 as a colourless oil (501 mg, 89.9%). R, 0.6 (CH2CI2 : MeOH : NH3 92: 7: 1); 1H-NMR (250 MHz), #H 7. 71-7.61 (m, 1H, NHamide), 6.52 (s, 1 H, Hg), 6.42-6.22 (m, 3H, NHguanidine), 3.82 (s, 3H, H, 4), 3.63-3.33 (m, 9H, H1' - H3', H1'', H1'''), 3.33-3.10 (m, 3H, H2, 5), 2.69,2.61 (2s, 6H, His, 16), 2.17 (s, 3H, His), 1.94-1.81 (m, 2H, NH2), 1.69-1.40 (m, 8H, H3, H4, H2'', H2'''), 1.40-1.15 (m, 36H, H3'' - H11'', H3"'-Hll), 0.87 (t, 6H, H12'', H12''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H)# 796.6028 (C43H82N506S requires (M+H)# 796. 5986); CHN found C 64.51, H 10.42, N 8.70 (C43H81 N506S requires C 64.87, H 10.25, N 8.80).

Example 77 NG-4-methoxy-2,3,6-trimethylbenzenesulphonyl-L-arginine-

&alpha;-(2-lauryloxy-3-stearyloxy)propylamide (77) Prepared analogously to L-tryptophan-a- (2, 3-dilauryloxy) propylamide 67 (Example 69) on a 0.7 mmol scale to give, after chromatography (CH2CI2-CH2CI2 : MeOH: NH3 92: 7: 1) NG- 4-methoxy-2, 3, 6-trimethylbenzenesulphonyl-L-arginine-&alpha;-(2-lauryl oxy-3- stearyloxy) propylamide 77 as a white solid (559 mg, 90.7%). Rf 0.6 (CH2CI2 : MeOH: NH3 92: 7: 1); mp 75-77°C ;'H-NMR (250 MHz), #H 7. 72-7.60 (m, 1H, NHamide) 6. 52 (s, 1H, Hg), 6.42-6.19 (m, 3H, NHguanidine), 3.82 (s, 3H, H14), 3.62-3.35 (m, 9H, H1' - H3', H,, H1'''), 3.35- 3.11 (m, 3H, H2, 5), 2.69,2.62 (2s, 6H, H13, 16), 2.17 (s, 3H, H15), 2.09-1.89 (m, 2H, NH2), 1.73-1.41 (m, 8H, H3, H4, H2, H2'''), 1.38-1.11 (m, 48H, H3'' - H17'', H3''' - H11'''), 0. 88 (t, 6H, H18'', H12''', J=6.4 Hz) ;'3C-NMR (250 MHz), 8c 173.60 (Ci), 157.83 (C6), 72.23 (C3), 71.38 (C,), 70.71 (C1'''), 54.04 (C2), 41.09 (C5), 32.33 (C10'', C10'''), 31.07 (C3), 30.39-29.78 (C2, C4'' - C9'', C2''', C4''' - C9''') 26.47 (C3'', C3'''), 24.98 (C4), 23.08 (C11'', C11'''), 14.49 (C12'', C12'''); HRMS (FAB) found molecular ion (M+H) &commat; 880.6925 (C49H94N506S requires (M+H)# 880.6937). CHN found C 66.91, H 10.81, N 7.91 (C49H93N5O6S requires C 66.85, H 10.65, N 7.96).

Example 78

L-arginine-&alpha;-(2,3-dilauryloxy)propylamide (78) To a flask containing a mixture of TFA, phenol, water 1,2-ethane-dithiol, thioanisole, and triisopropylsilane (81: 5: 5: 5: 2.5: 1) (20 ml) was NG-4-methoxy-2, 3,6- trimethylbenzenesulphonyl-L-arginine-&alpha;-(2,3-dilaur yloxy)propylamide 76 (199 mg, 0.25 mmol) and the solution stirred for 8 hours. The solvent was then removed under reduced pressure, and the product purified by column chromatography (CH2CI2- CH2CI2 : MeOH: NH3 92: 7: 1), to give L-arginine-&alpha;-(2,3-dilauryloxy)propylamide 78 as a colourless oil (120 mg, 82.2%). Rf 0.1 (CH2CI2 : MeOH : NH3 92: 7: 1);'H-NMR (250 MHz), #H 8. 01-7.88 (m, 2H, Amide, C5-NH), 7.18-6.92 (m, 3H, NHguanidine), 3.86-3.63 (m, 3H, NH2, H2), 3.63-3.10 (m, 11H, H5, H1' - H3', H1'', H1'''), 1.86-1.40 (m, 8H, H3, H4, H2'', H2), 1.40-1.17 (m, 36H, H3'' - H11'', H3''' - H11'''), 0.86 (t, 6H, H12'', H12''', J=6. 6 Hz) ;'3C-NMR (250 MHz), 8c 173.63 (C1), 157.90 (C6), 72.22 (C3), 71.42 (C,), 70.71 (C1'''), 54.03 (C2), 41.07 (Cs), 32.33 (C16'', C10'''), 31.07 (C3), 30.41-29.77 (C2'', C4'' - C15'', C2''', C4''' - C9'''), 26. 48 (C3, C3'''), 25.03 (C4), 23.09 (C17'', C11'''), 14.50 (C18'', C12''') ; HRMS (FAB) found molecular ion (M+H) # 584.5479 (C33H7oN503 requires (M+H)# 584.5490).

Example 79

L-arginine-a- (2-lauryloxy-3-stearyloxy) propylamide (79) Prepared analogously to L-arginine-&alpha;-(2,3-dilauryloxy)propylamie 78 (Example 78) on a 0.25 mmol scale to give, after chromatography (CH2CI2-CH2CI2 : MeOH : NH3 92: 7: 1) L- arginine-&alpha;-(2-lauryloxy-3-stearyloxy)propylamide 79 as a colourless oil (140 mg, 83.8%).

Rf 0.1 (CH2CI2 : MeOH: NH3 92: 7: 1); 1H-NMR (250 MHz), #H 8. 08-7.90 (m, 2H, NHamide, C5- NH), 7.16-6.99 (m, 3H, NHguanidine), 3.73-3.62 (m, 1H, H2), 3.62-3.12 (m, 11H, H5, H1' - H3', H,, H1'''), 2.70-2.58 (m, 2H, NH2), 1.83-1.41 (m, 8H, H3, H4, H2'', H2'''), 1.34-1.15 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6.6 Hz); HRMS (FAB) found molecular ion (M+H) o 668.6418 (C39H82N503 requires (M+H) # 668.6426).

Example 80

3ß[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) (80) To an ice cooled, stirred solution of cholesteryl chloroformate (20.2 g, 45 mmol) in anhydrous CHCl3 (150 ml) under argon, was added dropwise a solution of N, N- dimethyidiamine (9.9 ml, 90 mmol) in CHC13 (150 ml) over a period of 20 minutes. When addition was complete, the cooling bath was removed and reaction stirred at room temperature for a further 5 hours. The solution was washed with water (2 x 150 ml) and brine (1 x 150 ml), aqueous washes extracted with CHC13 (100 ml), and the organic layers combined and dried (MgS04). The solvent was removed and the product recrystallised from acetone to give 3ß [N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol 84 as a white solid (18. 6g, 82.7%); Rf 0.3 (Titanic); mp 110°C (Lit. 108 °C, see. e. g, reference 38); 'H-NMR (250 MHz), 8H ; 5.35 (d, 1H, H6J=5. 0 Hz), 5.23-5.09 (m, 1H, NH), 4.56-4.40 (m, 1 H, H3), 3.23 (q, 2H, H1', J=5.6 Hz), 2.51-2.23 (m, 4H, H4, H2'), 2.22 (s, 6H, 2 x NCH3), 2.05-1.71 (m, 5H, H2, H7, H8), 1.60-1.00 (m, 21 H, H"Hg, H9, H11, H12, H14 - H17, H20, H22 - H25), 0.99 (s, 3H, H19), 0.90 (d, 3H, H21, J=6. 5 Hz), 0.88-0.80 (m, 6H, H26, H27), 0.66 (s, 3H, H18) ; HRMS (FAB) found molecular ion (M+H) # 501.4420 (C32H57N202 requires (M+H)# 501.4425); CHN found C 76.74, H 11.34, N 5.58 (C32H56N202 requires C 76.75, H 11.27, N 5.59).

Example 81-A N-&alpha;-t-butyloxycarbonyl-L-aspartic acid ß-benzyl ester

pentafluorophenol ester (81) Prior to carrying out the example, the 1,4-dioxane to be used as solvent was distilled over calcium hydride to remove residual water and other impurities. To a stirred, cooled (0°C) solution of N-a-t-butyloxycarbonyl-L-aspartic acid ß-enzyl ester (6 mmol, 1.94 g) and pentafluorophenol (6 mmol, 1.10 g) in dry, distilled 1,4-dioxane was added a 1 M solution of dicyclohexylcarbodiimide in CH2CI2 (6 mmol, 6 ml). Stirring was continued for 45 minutes at 0°C, and then for a further hour at room temperature. The dicyclohexylurea by- product was then filtered off and the solvent removed under reduced pressure. The resulting pale yellow oil was triturated with n-hexane and the solid filtered off and recrystallized from 1: 1 ethyl acetate/hexane. This gave N-a-t-butyloxycarbonyl-L-aspartic acid ß-benzyl ester pentafluorophenol ester 81 as a white solid (2.02 g, 68.8%); Rf 0.5 (CH2CI2) ; mp 82°C ;'H-NMR (250 MHz), #H 7. 40-7.33 (m, 5H, Benzyl) 5.62 (d, H, NH, J=8.9 Hz) 5.18 (s, 2H, H5), 5.03-4.91 (m, 1H, H2), 3.13 (ABX, 2H, H3A/B, JAB=17. 5 Hz, JAX=4. 6 Hz, JBX=4. 4 Hz), 1.47 (s, 9H, Ht-butyl) ; HRMS (FAB) found molecular ion (M+H) &commat; 490.1289 (C22H2, F5NO6 requires (M+H)# 490. 1283); CHN found C 53.95, H 4.09, N 2.90 (C22H2oF5NO6 requires C 53.99, H 4.12, N 2.86).

Example 81-B N-t-butyloxycarbonyl-L-aspartic acid (3-benzyl ester pentafluorophenol ester (81) To a flask containing pentafluorophenol (5 mmol, 0.92 g) and N-t-butyloxycarbonyl-L- aspartic acid ß-benzyl ester (4 mmol, 1.29 g) under nitrogen was added CH2CI2 (20 ml). A solution of EDC (5 mmol, 0.96 g) in CH2CI2 (20 ml) was added dropwise, and the solution stirred for two hours at room temperature. It was then washed with NaHC03 (1 x 20 ml), water (1 x 20 ml) and brine (1 x 20 ml), dried (MgSO4), and evaporated under reduced pressure. The product was purified by column chromatography (5-20% ethyl acetate/hexane) to give N-t-butyloxycarbonyl-L-aspartic acid ß-benzyl ester pentafluorophenol ester 81 as a white solid (1.91 g, 97.7%), spectroscopically identical to previously prepared material (Example 81-A).

Example 82 N-t-butyloxycarbonyl-L-aspartic acid p-benzyt ester N- (2-lauryloxy-3-stearyloxy) propylamide (82)

Prior to carrying out this example the THF was distilled over potassium. @ o a tiask under nitrogen containing N-t-butyloxycarbonyl-L-aspartic acid ß-benzyl ester pentafluorophenol ester 82 (3 mmol, 1.47 g), 2-lauryloxy-3-stearyloxypropylamine 42 (2 mmol, 1.02 g) and 4-dimethylaminopyridine (0.3 mmol, 37 mg) was added THF (20 ml), and the resulting solution refluxed at 80°C overnight. The solvent was then removed and CH2CI2 (50 ml) added. The solution was washed with NaHC03 (1 x 30 ml), NH4Cl (1 x 30 ml), water (2 x 30 ml) and brine (1 x 30 ml). The aqueous layers were backwashed with CH2CI2, and the organic phases combined, dried (MgSO4) and evaporated under reduced pressure. The product, a pale yellow solid, was purified by column chromatography (0-40% ether/hexane) to give N-t-butyloxycarbonyl-L-aspartic acid (3-benzyl ester-a- (2-lauryloxy- 3-stearyloxy) propylamide 82 as a white solid (1.58g, 96.5%); R, 0.6 (50% ether/hexane); mp 68-70°C ; 1 H-NMR (250 MHz), 8H 7. 42-7.32 (m, 5H, Hbenzy , 6.88-6.76 (m, H, NHamide), 5.65 (d, H, NHcarbamate, J=8. 6) 5.16 (d, 1H, H5A, J=12. 7 Hz), 5.10 (d, 1H, H5B, J=12. 7 Hz), 4.69-4.44 (m, 1H, H2), 3.61-3.19 (m, 9H, H1'-H3', H1'', H1'''), 3.17-3.03 (m, 1 H, H3A), 2.79- 2.63 (m, 1H, H3B), 1.67-1.50 (m, 4H, H2'', H2'''), 1.45 (s, 9H, Ht -butyl), 1.38-1.15 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6.5 Hz); HRMS (FAB) found molecular ion (M+H)# 839. 6489 (C49H88N207Na requires (M+H) &commat; 839.6499); CHN found C 72.02, H 10.93, N 3.41 (C49H88N207 requires C 72.01, H 10.85, N 3.43).

Example 83 N-t-butyloxycarbonyl-L-aspartic acid- &alpha;-(2-lauryloxy-3-stearyloxy)propylamide (83)

A flask containing N-t-butyloxycarbonyl-L-aspartic acid ß-benzyl ester-&alpha;-(2-luaryloxy-3- stearyloxy) propylamide 83 (410 mg, 0.5 mmol) and 5% palladium on charcoal (106 mg, 0.05 mmol, 0. 1eq.) and ethanol (10 mi) was flushed with hydrogen and stirred for 1 hour.

The reaction mixture was filtered through a 1-inch bed of Celite 521 and evaporated under reduced pressure to yield a colourless oil. This was redissolved in ether and cold evaporated several times to give N-t-butyloxycarbonyl-L-aspartic acid-a- (2-lauryloxy-3- stearyloxy) propylamide 83 as a white solid (345 mg, 94.7%) R, 0.55 (Ethyl acetate); mp 59-61 °C ; 1 H-NMR (250 MHz), #H 7.03-6.91 (m, H, NHamide), 5.68 (d, 1 H, NHcarbamate, J=7. 6 Hz), 4.60-4.45 (m, 1H, H2), 3.64-3.24 (m, 9H, Hi,-H3', H1'', H1'''), 2.88 (ABX, 2H, H3A/B, JAB=17. 3 Hz, JAX=3. 9 Hz, JBX=5. 9 Hz), 1.64-1.48 (m, 4H, H2, H2), 1. 46 (s, 9H, Ht-butyl butyl) 1.38-1.19 (m, 48H, H3-H17, H3-H1,), 0.88 (t, 6H, H18'', Hiz..., J=6.6 Hz); HRMS (FAB) found molecular ion (M+Na) &commat; 749.6020 (C42H82N207Na requires (M+Na) &commat; 749.5999); CHN found C 69.15, H 11.44, N 3.83 (C42H82N207 requires C 69.38, H 11.37, N 3.85).

Example 84 N-t-butyloxycarbonyl-L-aspartic acid P-pentafluorophenyl ester- a- (2-lauryloxy-3-stearyloxy) propylamide (84)

This was prepared using the same method as N-t-butyloxycarbonyl-L-aspartic acid p- benzyl ester pentafluorophenol ester 84 (Example 84) on a 1.3 mmol scale to give, after purification (column chromatography, 0-15% ethyl acetate/hexane), N-t-butyloxycarbonyl- L-aspartic acid p-pentaftuorophenyt ester-a- (2-lauryloxy-3-stearyloxy) propylamide 84 (0.97 g, 83.5%). R, 0.6 (CH2CI2) ; mp 64°C ; 1 H-NMR (250 MHz), #H 6.81-6.70 (m, H, NHamide), 5.43 (d, 1H, NHcarbamate, J=8. 97 Hz), 4.71-4.56 (m, 1 H, H2), 3.66-3.01 (m, 11 H, H1' - H3', H3, H1'', H1'''), 1.65-1.51 (m, 4H, H2'', H2'''), 1.47 (s, 9H, Ht-butyl), 1.38-1.18 (m, 48H, H3'' - H17'', H3''' - H11'''), 0.88 (t, 6H, H18'', H12''', J=6. 5 Hz); HRMS (FAB) found molecular ion (M+Na) &commat; 915. 5862 (C48H81F5N2O7Na requires (M+Na)# 915. 5842); CHN found C 64.50, H 9.20, N 3.14 (C48H8, F5N207 requires C 64.55, H 9.14, N 3.14).

General Peptide/Peptidolipid Synthesis All peptides were synthesised on an Applied Biosystems 433A solid phase peptide synthesiser using the 9-fluorenylmethoxycarbonyl (Fmoc) coupling strategy. Solvents and reagents were purchased from Applied Biosystems unless otherwise stated. Amino acids for the synthesiser were purchased in pre-packed cartridges from Applied Biosystems, apart from the Fmoc-L-glutamic acid a-allyl ester, which was purchased from Calbiochem-Novabiochem (Nottingham, UK). Wang (p-benzyloxybenzyl alcohol) resin (100-200 mesh) was also purchased from Calbiochem-Novabiochem.

The cleavage solution used to cleave the peptides from their resin support depended upon the peptide sequence in question. A standard solution (mixture A) of TFA (95% v/v) and H20 (5% v/v) was used, unless the sequence contained arginine. Since arginine can undergo side chain alkylation, a solution containing triisopropyl silane (TIS) was then used. This was composed of TFA (95% v/v), H20 (2.5% v/v) and triisopropylsilane (2.5% v/v)-mixture B.

Once cleaved from the solid support, crude peptides were analysed and purified using an AKTA Purifier FPLC system (Pharmacia Biotech, Uppsala, Sweden). The system comprises of two solvent pumps (P-910), variable wavelength UV-absorption monitor (UV-900), single chamber mixer (M-925) and sample delivery pump (P-910). Attached were an autosampler (A-900) and fraction collector (Frac-900). The reversed-phase columns used for analytical work were a Source 15RPC (4.6 x 100 mm, 5 um, Pharmacia Biotech) and a Jupiter C18 (4.6 x 250 mm, 5 m, Phenomenex Ltd.). For semi- preparative work a Jupiter C4 (10 x 250 mm, 5 m, Phenomenex Ltd.) was used.

Products were eluted using a binary system of H20 containing 0.1% TFA and acetonitrile (MeCN), also containing 0.1 % TFA.

Intermediate precursors to final products were characterised by mass spectrometry and analytical FPLC. Peptides sequences were confirmed by amino acid sequence analysis (AASA) once fully deprotected (automated Edman degradation)). Final products were characterised by high resolution mass spectrometry, and analytical FPLC.

Example 85 Arg-Gly-Asp-Glu-Lys-Lys and subsequent coupling to N-t-butyloxycarbonyl-L-aspartic acid-pentafluorophenyl ester-

a- (2-lauryloxy-3-stearyloxy) propylamide 84 (85) Pre-loading of Wang resin with Fmoc-Glu a-allyl ester (OAII) to be used in peptide synthesis was carried out as follows. Prior to loading, the resin was dried under vacuum overnight. 3.94 g of resin were then suspended in DMF and nitrogen passed through to agitate and swell the resin. In a separate flask, anhydrous CH2CI2 (50 ml) was added to Fmoc-Glu-OAII under nitrogen (4.38 g, 10.7 mmol, 3.6 eq compared to resin loading) and the solution cooled to 0°C. Diisopropylcarbodiimide (0.63 g, 0.78 ml, 5 mmol, 1.66 eq) was then added and the solution stirred gently for 20 minutes. After this time the solvent was removed under reduced pressure, DMF (10 mi) added under nitrogen, and the resulting solution added to the resin in the bubble. DMAP (37 mg, 0.3 mmol, 0.1 eq) and a further 40 ml of DMF were added, and the entire suspension agitated with a steady stream of nitrogen for 2 hours. Resin loading was determined by taking a precise weight of resin (-5 mg), swirling in a solution of 20% piperidine in DMF (0.5 ml), diluting 100-fold with DMF, and measuring the absorbance of the N- (9-fluorenyl-methyl) piperidine adduct at 301 nm.

Synthesised on Lys-preloaded Wang resin (143mg, 0.70 mmol g-1, 0.1 mmol), the initial peptide had the sequence RGDEKK. AASA results were entirely consistent with this sequence. Once complete (including N-terminus deprotection with 20% piperidine in DMF), peptidoresin was washed with MeOH (30 ml) and dried under vacuum. 85 mg (0.02 mmol) of the peptidoresin were then put in a dry flask with N-t-butyloxycarbonyl-L- aspartic acid p-pentafiuoropheny) ester-a- (2-lauryloxy-3-stearyloxy) propylamide 84 (89mg, 0.1 mmol, 5 eq.) and DMAP (12 mg, 0.1 mmol, 5 eq.). The flask was flushed with argon, anhydrous DMF (1 ml) added, and the suspension agitated at 38°C overnight. The product was then cleaved from the resin by stirring in cleavage solution B (5 ml) for 1

hour. The resin was then filtered off and washed (1 ml TFA), and the filtrate evaporated to give the crude peptido-lipid 85. FPLC analysis using the Source 15RPC column showed one major peak eluting at 67% MeCN, Retention Time 12.68 minutes; m/z found [M+H] &commat; 1340.9 (C66H125N13O15 requires 1341.7), [M+2H] 2&commat; 671.3 and [M+3H] 3&commat; 447.9.

Example 86 Glu- (Lys) B and subsequent coupling to 2-lauryloxy-3-stearyloxypropylamine 42 (86) This was synthesised starting with Lys-preloaded Wang resin (357 mg, 0.70 mmol g', 0.25 mmol). The initial peptide had the sequence EKKKKKKKK, although the Glu residue was coupled via the carboxyl side chain. The Glu a-carboxyl group was orthogonally protected with the allyl ester. Once complete (including N-terminus deprotection with 20% piperidine in DMF), peptidoresin was washed with MeOH (50 ml), dried under vacuum, and a sample cleaved from the resin to check the integrity of the peptide. 10 mg of peptidoresin were added to 1 ml of cleavage solution A, and the reaction stirred for 45 minutes. The resin was then filtered off and washed (1 ml TFA), and the filtrate evaporated to give the crude peptide 86a. FPLC analysis using the Jupiter C18 column showed one major peak eluting at 47% MeCN, Retention Time 7.95 minutes; m/z found [M+H] &commat;1212. 7 (Cs6Hio9Ni70i2 requires 1212.5) and [M+2H] 2&commat;607. 1 N-Terminus Protection with t-butyloxycarbonyl The remainder of the peptidoresin (0.25 mmol) was then suspended in DMF and di-tert- butyl dicarbonate (1.09 g, 5 mmol, 20 eq.) and triethylamine (506 mg, 5 mmol, 20 eq.) added. The suspension was shaken overnight under argon, then the resin collected, washed thoroughly with DMF (50 ml) and MeOH (50 ml), and dried under vacuum to give the N-terminus BOC-protected peptide 86b.

Removal of OAII-protecting group Peptidoresin (0.25 mmol) was then put in a flask and flushed with argon. In a separate flask, containing tetrakis (triphenylphosphine) palladium (0) (866 mg, 0.75 mmol, 3 eq.), was added a mixture of chloroform, acetic acid and N-methylmorpholine (37: 2: 1,10 ml).

The resulting solution was then transferred to the peptidoresin, and the suspension gently agitated for 2 hours. After this the resin was transferred to a sintered glass funnel and washed alternately with 0.5% diisopropylamine in DMF (v/v, 50 ml) and 0.5% sodium diethyidithiocarbamate in DMF (w/w, 50 ml) to remove the catalyst. Finally, the resin was washed with MeOH (30 ml), dried under vacuum, and a sample (10 mg) cleaved (cleavage solution A, 1 ml) to check the integrity of the product, peptide 86c. FPLC analysis using the Source 15RPC column showed one major peak eluting at 14% MeCN, Retention Time 3.82 minutes; m/z found [M+H] &commat; 1172.7 (C53H105N, 7O, 2 requires 1172.5).

AASA results were entirely consistent with the sequence EKKKKKKKK.

Formation of activated DHBt ester The THF used as solvent was distilled over sodium prior to use. To a dry flask containing the peptidoresin (0.25 mmol) under argon was added dry, distilled THF (10 ml) and the suspension cooled to-15°C. Diisopropyl carbodiimide (DIC) (126 mg, 1 mmol, 4 eq.) was added and, after stirring for 20 minutes, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (367 mg, 2.25 mmol, 9 eq.) was added under a blanket of argon. A further 5 ml of THF were added and the suspension then stirred overnight at room temperature. The resin was then transferred to a sintered glass funnel, washed with MeOH (10 ml) and CH2CI2, and dried exhaustively under vacuum to give the activated peptide 86d.

Coupling of peptide to 2-lauryloxv-3-stearvloxvpropylamine To a flask containing peptidoresin (50 mg,-0.02 mmol) and 2-lauryloxy-3- stearyloxypropylamine 42 (51 mg, 0.1 mmol,- 5 eq.) under argon was added anhydrous dimethlyacetamide (DMA) (2 ml) and the solution shaken gently at 40°C overnight. The resin was then filtered off, washed with DMA (30 ml) and MeOH (20 ml), and dried under vacuum. The product was cleaved from the resin by stirring in cleavage solution A (10 ml) for 1 hour, after which time the resin was filtered off, washed with TFA (10 ml) and the combined filtrate evaporated to give the crude peptidolipid. The product was purified by FPLC using the semi-preparative Jupiter C4 column, with the product eluting from 74-

83% MeCN and the relevant fractions being combined and lyophilised. This yielded peptido-lipid 86 as a low-density, colourless solid (15 mg, 45%); Analytical FPLC (Source 15RPC) Elution at 60% MeCN, Retention Time 11.22 minutes; HRMS (FAB) found molecular ion (M+H) &commat; 1666.336 (C86H173N18O13 requires (M+H) &commat; 915.5842).

Example 87 Glu-(Lys) 8-Arg-Gly-Asp and subsequent coupling to 2-lauryloxy-3-stearyloxypropylamine 42 (87) This was synthesised starting with Lys-preloaded Wang resin (357 mg, 0.70 mmol g-1, 0.25 mmol). The initial peptide had the sequence EKKKKKKKKRGD. Again, the Glu residue was coupled via the carboxyl side chain, with the Glu a-carboxyl group orthogonally protected with the allyl ester. A sample was deprotected and cleaved from the resin to confirm the success of the synthesis. 10mg of peptidoresin were cleaved as for peptide 86a, except cleavage solution B was used instead. FPLC analysis using the Source 15RPC column showed one major peak (peptide 87a) eluting at 34% MeCN, Retention Time 7.03 minutes; m/z found [M+H] &commat; 1540. 9 (C65H125N23O17 requires 1540.9), [M+2H] 29 771.2, [M+3H] 39 514. 6 and [M+4H] 4&commat; 386.2.

N-Terminus Protection with t-butyloxycarbonvl The peptidoresin was treated with di-tert-butyl dicarbonate as for peptide 86b to give the N-terminus BOC-protected peptide 87b.

Removal of OAII-protecting group The allyl group was removed in analogous fashion to the formation of 86c, and a sample (10 mg) cleaved (cleavage solution B, 1 ml) to check peptide (87c) integrity. FPLC analysis using the Source 15RPC column showed one major peak eluting at 15% MeCN, Retention Time 3.94 minutes; mlz found [M+H]# 1500. 9 (C65H, 26N24016 requires 1500.9)

and [M+2H] 2"' 751.2. AASA results were entirely consistent with the sequence EKKKKKKKKRGD.

Formation of activated DHBt ester The peptide 87c was converted to the corresponding activated DHBt ester 87d in an analogous fashion to the formation of 86d.

Coupling of peptide to 2-lauryloxv-3-stearvloxvpropylamine Peptide 87d was coupled to 2-lauryloxy-3-stearyloxypropylamine 42 on a-0. 02 mmol scale to give peptido-lipid 87 in analogous fashion to the formation of 86e. The product was purified by FPLC using the Jupiter C4 semi-prep column, with the bulk of the product eluting from 76-83% MeCN and the relevant fractions being combined and lyophilised.

This yielded peptido-lipid 87 as a low density, colourless white solid (20 mg,-61%) ; Analytical FPLC (Source 15RPC) Elution at 61 % MeCN, Retention Time 11.48 minutes; HRMS (FAB) found molecular ion (M+H) &commat; 1994.466 (C98Hl93N24018 requires (M+H) &commat; 1994.484).

Example 88 Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Glu-Glu-Gly-Glu and subsequent coupling to 2-lauryloxy-3-stearyloxy-propylamine 42 (88) This was synthesised starting with Glu- (OAII)-preloaded Wang resin (403mg, 0.62 mmol g-1, 0.25mmol). The initial peptide had the sequence TGRGDSPASEEGE. Once the synthesis and Fmoc deprotection were complete a sample was cleaved (solution B) and analysed to confirm the success of the synthesis. FPLC analysis using the Source 15RPC column showed one major peak (peptide 88a) eluting at 23% MeCN, Retention

Time 5.44 minutes; m/zfound [M+H] &commat;1331. 6 (C52Hg3N,7024 requires 1331.3), and [M+2H] 2&commat; 666. 5.

N-Terminus Protection with t-butytoxycarbonyi The peptidoresin was treated with di-tert-butyl dicarbonate as for peptide 86b to give the N-terminus BOC-protected peptide 88b.

Removal of Aloc-protectin group The allyl group was removed in analogous fashion to the formation of 86c, and a sample (10mg) cleaved (solution B) to check peptide (88c) integrity. FPLC analysis using the Source 15RPC column showed one major peak eluting at 19% MeCN, Retention Time 4.64 minutes; m/z found [M+H] o 1291. 8 (C49H79N17024 requires 1291.3). AASA results were entirely consistent with the sequence TGRGDSPASEEKE.

Formation of activated DHBt ester The peptide 88c was converted to the corresponding activated DHBt ester 88d in an analogous fashion to the formation of 86d.

Coupling of peptide to 2-lauryloxv-3-stearyloxvpropvlamine Peptide 88d was coupled to 2-lauryloxy-3-stearyloxypropylamine 37 on a-0. 02 mmol scale to give peptido-lipid 88 in analogous fashion to the formation of 86. The product was purified by FPLC using the Jupiter C4 semi-prep column, with the bulk of the product eluting at 100% MeCN and the relevant fractions being combined and lyophilised. This yielded peptido-lipid 88 as a low-density, colourless solid (18 mg,-50%) ; Analytical FPLC (Source 15RPC) Elution at 76% MeCN, Retention Time 13.98 minutes. HRMS (FAB) found molecular ion (M-H+2Na)# 1829. 026 (C82H144N17O26Na2 requires (M- H+2Na)# 1829.035).

Biological Data Liposome Formulation DOPE (48 NI of a 12.5 mg ml-'CHC13 solution, i. e. 0.6 mg, 0.8 pmol) was added to a solution containing cationic lipid component (1.2 umol ; 3: 2 ratio cationic: DOPE) in CHC13 (0.5 ml) in a flask under argon. 20 mM HEPES buffer (pH 7.8) was sterilised by passing through a 200 nm filter, and 1 ml added to the lipid solution. The mixture was bath- sonicated for 5 minutes until it became opalescent, and then the organic solvent removed carefully on a rotary evaporator with no external heating. During this time, the material sometimes formed a viscous gel. Once complete, the liposome formulation was sonicated for a further 5 minutes, in which time any gel reverted to the liquid suspension state.

Liposomes were then extruded through a LiposoFast Basic manual extruder (Avestin Inc., Ottawa, Canada) at 37°C, by immersing the loaded apparatus in a warm water bath.

Then, liposomes were passed 5 times through a filter with a porosity of 400 nm, and a further 9 times through a filter with a porosity of 200 nm (unless otherwise stated), using the LiposoFast Basic Extruder (Avestin Inc.), prior to transfection experiments. The LiposoFast Extruder is based on the design of MacDonald et al (reference 122). It is a simple hand held device comprising a disposable polycarbonate filter, held between two teflon supports, which have Luer lock at each end. The supports are then pressed firmly together inside a stainless steel housing. Two 1 ml gas-tight syringes (Hamilton Inc.) (the first containing the liposome formulation, the other empty) are fixed to the Luer locks and the formulation passed back and forth through the filter 11 times. An odd number of passages were performed to avoid contamination of the sample by large vesicles that might not have passed through the filter (reference 122).

The extrusion of liposomes was first described by Olson et al in 1979 (reference 121).

The process involves passing the liposomal formulation back and forth through polycarbonate membrane filters with pores of a fixed diameter. Typically membranes of a relatively large porosity are used at the start, and then liposomes can be further downsized by reducing pore (tears may develop in the membrane if the smallest porosity is used at the start). This gives large, unilamellar vesicles (LUVs) of a more uniform size, slightly above that of the pore diameter (reference 122). Liposomes formulated without

performing this procedure tend to consist of multilamellar, large vesicles (MLVs) with a wide range of diameters, typically from 100-2000 nm (reference 121).

Cell Lines and Cultures Transfection studies were carried out using the folowing cell lines: V79-Chinese hamster lung cell line (European Collection of Cell Cultures, Salisbury, Wiltshire, UK).

HT29-Human colon adenocarcinoma (European Collection of Cell Cultures, Salisbury, Wiltshire, UK).

All tissue culture media and reagents were obtained from Life Technologies Inc. (Paisley, UK) unless otherwise stated. V79/HT29 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS) and 0.5% Ciproxin Infusion (Bayer, Newbury, UK). Cells were cultured in T-75 and T-175 flasks respectively and were passaged when complete confluence was achieved. SCC4/SCC25 cells were cultured in a 1: 1 mixture of Ham's F12 Medium and DMEM, supplemented with 10% FBS, 0.5% Ciproxin Infusion and 400 ng ml 1 hydrocortisone. T-175 flasks were used for culturing, and the medium was renewed every 2-3 days until confluence was achieved and cells were passaged. All cell lines were incubated at 37°C in an atmosphere with 60% relative humidity and 5% C02 in a TC2323 incubator (BoroLabs, Aldermaston, UK).

Cells were dissociated and disaggregated by washing with Phosphate Buffered Saline (PBS), followed by incubation with 0.25% trypsin and 1 mmol EDTA in Hank's Balanced Salt Solution (HBSS).

Transfection Procedure 24 hours before transfection, cells were dissociated and disaggregated and centrifuged to form a pellet. This was resuspended in DMEM and dispensed into 6-well plates (35mm well diameter). Each well received 15,000 cells and 2 ml of the corresponding growth medium. Cells were then incubated in conditions as described above for 24 hours.

To form the lipoplexes for transfection, plasmid DNA containing the lacZ gene under the Elongation Factor promoter (pEFlacZ) was diluted to a concentration of 0.1 mg mu 1 in PBS. DNA solution (4 ul) was then dispensed into a 500 NI Eppendorf tube for each well of cells to be transfected. The corresponding amount of liposome formulation to be used

in the experiment (typically 3-5 psi, at a total lipid concentration of 1.2 mg mol 1) was then added to the DNA, and the total volume made up to 32 ul with PBS. Each tube was then vortexed briefly and left to stand for 15-20 minutes.

The medium on the cells was then removed, and each well washed with serum free (SF) medium (2 x 0.8 ml) before receiving a final aliquot of SF medium. The lipoplex formulations were then taken up in 168 NI of SF medium, added to the cells, and incubated for a period of 6 hours. After this time, SF medium was replaced with 2 ml of fully supplemented medium, and cells were incubated for a further 18 hours (V79, HT29) or 42 hours (SCC4, SCC25).

Harvest and ß-Galactosidase Assay The purpose of the ß-galactosidase (P-gal) assay is to assess how successful different vectors have been at transfection. Cells that have been transfected successfully will produce the p-gai enzyme. When lysed and dosed with the compound ortho-nitrophenyl- P-galactopyranoside (ONPG), the enzyme will react with the compound to produce ortho- nitrophenol, which has a strong yellow colour. Intensity of colour produced is therefore a measure of success of transfection. This assay was based on the procedure outlined in Sambrook's Molecular Cloning laboratory manual (reference 148).

To harvest, cells were first washed with cold PBS (2 x 0.5 ml), and then 200 pi of Extraction Buffer (Table 1) was added. Cells were left until lysis was complete (typically 15 minutes), transferred to 1.5 ml Eppendorf tubes, and centrifuged for 5 minutes at 10,000 rpm using a Sigma 112 microcentrifuge to pellet any undissolved cellular matter.

To determine the quantity of p-gai present, 80 pi of the cellular extract was mixed with 520 ul of lacZ Buffer B (see Table 2), which contains ONPG, and the mixture left for a time of 20-60 minutes. The reaction was then stopped by addition of 1 M Na2CO3 (250 ul). The intensity of the yellow colour that developed was measured at a wavelength of 420 nm using a Uvikon 922 spectrophotometer (Kontron Instruments, Milan, Italy).

Protein content must be determined since some wells may contain more cells than others. Activity needs to be calculated as a fraction of successfully transfected cells over the total number of cells, rather than merely the number of successfully transfected cells.

The overall protein content of the cellular extract was determined by the Bio-Rad Protein

Assay Reagent (Bio-Rad Laboratories Ltd., Munich, Germany) (reference 149). The protein assay was calibrated every time it was performed with Bovine Serum Albumin (BSA).

IacZ Buffer A (Table 3), a component of Buffer B, could be made in advance, while Buffer B was always made freshly on the day of use.

Table 1-Extraction Buffer Reagent Amount 250mM Tris/HCI pH 7. 8 89% (v/v) Glycerol 10% Triton X100 1 % Table 2-LacZ Buffer B Reagent Amount lacZ Buffer A 22 ml ONPG/Buffer A @ 5 mg/ml 4 mi -Mercaptoethanol 54 µl Table 3-lacZ Buffer A Reagent Amount 1 M Na2HP04 60 ml 1 M NaH2PO4 40 ml 1 M KCI 10 ml 1 M mg2S04 1 ml 1 M Tris/HCI pH 7. 8 20 ml Distilled water 869 ml Cvtotoxicity Assay 24 hours before dosing with liposomes, V79 cells were dissociated and disaggregated, resuspended in DMEM, and dispensed into 96-well plates (5 mm well diameter) using a Multidrop 8 channel peristaltic pump (Thermo Labsystems, Helsinki, Finland).

Each of the first 11 well columns received approximately 500 cells in 100 pi of medium, the last was left as a blank. Cells were then incubated for 24 hours, and then 150 pi of liposome formulation was dispensed into the first well of a row. The medium and liposomes were gently agitated using a pipette, before 150 pi of the resulting mixture was removed and added to the next well. This was repeated across the row to give a dilution series, except the 11'"well which was left as a control and received no liposomes. Each well was supplemented with 100 NI of medium, and the plates then incubated for a period of 72 hours.

After this time, the medium was removed and the wells were filled with a 10% solution of trichloroacetic acid (TCA) and left to stand for 10 minutes. The plates were then plunged into water, rinsed several times, and left to dry. 50 uL of dye solution (0.1% sulforhodamine B in 1% acetic acid) was dispensed per well, and the plates left to stand for a further 10 minutes. Excess dye agent was then washed off under a stream of 1% acetic acid, and the plates left to dry. 150 NI of 10 mM Tris/HCI (pH 8) was dispensed per well, plates were shaken for 10 minutes, and absorption was measured at 540 nm using a Multiskan plate reader (Thermo Labsystems, Helsinki, Finland).

Example 89 Comparison of Diether Lipid 43 to DC-Chol Nine liposome formulations were prepared as described above (without extrusion), with the lipid compositions shown in Table 4.1. The first contained DC-Chol as the cationic lipid and the neutral co-lipid DOPE in a 3: 2 molar ratio. The remaining formulations substituted a steadily increasing amount of DC-Chol for the cationic 18/18 diether lipid 43, so that the final formulation contained 43 and DOPE alone (still in a 3: 2 molar ratio) and no DC-Chol.

Table 4. 1 Liposome Molar Ratio of Cationic Lipid (s) formulation DC-Chol 43 1 1- 2 0. 85 0. 15 3 0. 7 0. 3 4 0. 6 0. 4 5 0. 5 0. 5 6 0. 4 0. 6 7 0. 3 0. 7 8 0. 15 0. 85 9-1 The formulations were then used, both unextruded and after extrusion (200 nm), to form lipoplexes with the pEFlacZ plasmid, by mixing 5 ul of liposomes with 0.4 ug of DNA (a (+/-) charge ratio of 3: 1).

V79 cells were transfected (as described herein), and (3-gal activity subsequently measured (as described herein). The results are illustrated in Figure 1.

The data showed that a mixture of the two lipids appears to give the best results. The transfection peak occurs at approximately a 40: 60 or 50: 50 mixture of the two, and transfection is 3-4 times more effective than the DC-Chol control liposomes.

Example 90 The Effect of Extrusion Nine liposomal formulations were made from DC-Chol, DOPE and compound 43, with lipid compositions as described in Table 4.1.

The formulations were extruded (200 nm) to prepare liposomes (as described herein) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 3: 1).

V79 cells were transfected (as described herein), and 0-gal activity subsequently measured (as described herein). The example was performed in triplicate. The results are illustrated in Figure 2.

The same pattern was observed as in the previous example, with neither DC-Chol nor the diether lipid 43 being better alone in the formulation with DOPE. The results indicate that maximum transfection is achieved when the cationic component of the formulation is a 50: 50 mixture of diether lipid and DC-Chol. This formulation is 3-4 times more effective than the DC-Chol control. Statistical analysis of these results shows them to be more reproducible than those in the previous example. Table 4.2 shows the difference in the Standard Error of the Mean (SEM) for the more significant points of the two examples.

Table4.2 % Compound 43 in Non-extruded Extruded Cationic Component S.E.M. Mean S.E.M. Mean 30 0.079 0.526 0.051 0.590 40 0. 136 0. 513 0.043 0.764 50 0.134 0. 492 0.019 0.686 60 0. 114 0.381 0. 030 0. 492 70 0. 073 0.151 0.041 0.195

Example 91 Comparison of Ether Lipid 47 to DC-Chol The dioleyl diether lipid 47 was assayed in an identical manner to 43. Nine formulations were made with the same steadily increasing molar percentages of 47, and commensurately decreasing amounts of DC-Chol, as described in Table 4.3. All contained the neutral helper lipid DOPE in a 2: 3 molar ratio with respect to the cationic lipid (s).

Table 4.3 Molar Ratio of Cationic Liposome Lipid(s) Conc. (mM) formulation DC-Chol 47 Total Cat. Lipid DOPE 1 1 - 1.2 0.8 2 0.85 0.15 1.2 0.8 3 0.7 0.3 1.2 0.8 4 0. 6 0. 4 1.2 0. 8 5 0. 5 0.5 1.2 0.8 6 0. 4 0.6 1. 2 0.8 7 0. 3 0. 7 1.2 0. 8 8 0. 15 0.85 1. 2 0.8 9 - 1 1.2 0. 8 The formulations were then used, both unextruded and after extrusion (200 nm), to form lipoplexes with the pEFlacZ plasmid (as described herein).

V79 cells were transfected (as described herein), and ß-gal activity subsequently measured (as described herein). The example was performed in triplicate. The results for unextruded liposomes are illustrated in Figure 3, and the results for extruded liposomes are illustrated in Figure 4.

Again, neither cationic lipid proved more efficient than the other as the sole cationic agent. The maximum on the graphs occurs at the point corresponding to the ternary formulation with a 50: 50 mix of the two cationic lipids. Again, this formulation is approximately 3-4 times more efficient than DC-Chol.

Example 92 Comparison of Ether Lipid 33 to DC-Chol The shorter 12/12 diether lipid 33 was assayed in an identical manner to 43. Nine liposome formulations were prepared, with cationic lipid ratios analogous to those in the previous examples (see Tables 4.1 and 4.3).

Again, the formulations were used, both before and after extrusion (200 nm) to form lipoplexes with the pEFlacZ plasmid (as described herein).

V79 cells were transfected (as described herein), and a-gal activity subsequently measured (as described herein). The results for unextruded liposomes are illustrated in Figure 5, and the results for extruded liposomes are illustrated in Figure 6.

Again, a maximum occurs on the graph corresponding to an approximate 50% substitution of 33 for DC-Chol.

Note, however, that the transfection peak corresponds to an 8 or 9-fold improvement on the DC-Chol control liposomes. The difference in SEM values for extruded and unextruded liposomes in these examples are shown in Table 4.4 and Table 4.5.

Table 4.4 % Compound 47 in Non-extruded Extruded Cationic Component S. E. M. Mean S. E. M. Mean 30 0. 010 0.584 0.079 0.530 40 0.093 0.631 0.063 0.765 50 0.003 0.703 0.080 0.955 60 0. 058 0.434 0. 098 0.409 70 0. 012 0.188 0. 114 0.084 Table 4. 5 % Compound 33 in Non-extruded Extruded Cationic Component S. E. M. Mean S. E. M. Mean 30 0. 154 0. 378 0. 085 0.783 40 0. 199 0.732 0. 100 1.541 50 0. 072 1.403 0.171 1.691 60 0. 180 1.234 0. 143 1.593 70 0. 413 1. 238 0. 161 1.035

In the following examples, the SEM values are not quoted, but in the majority of cases, they are less than 10% of the value of the mean.

Example 93 Pore Size of Extruder Membrane In the literature, different groups have reported the use of liposomes of various sizes typically ranging from 100-400 nm (see, e. g., references 124-126); however, there is no real agreement on an optimum size.

The effect of liposome size was examined further by comparing several formulations that had been extruded to different sizes. 2,3-Dilauryloxypropylamine 33 was used, and was substituted in increasing amounts in liposome DC-Chol/DOPE formulations to give the six formulations shown in Table 4.6. These were then either left unextruded or extruded to one of three different sizes, using membranes with a 100,200, or 400 nm porosity, to give 6x4 or 24 formulations.

Table 4.6 Liposome formulation Molar Ratio of Cationic Lipid (s) DC-Chol 33 1 1- 2 0. 6 0. 4 3 0. 5 0. 5 4 0. 4 0. 6 50307 6-1 The resulting 24 formulations were then used to form lipoplexes with the pEFlacZ plasmid (as described herein).

V79 cells were transfected (as described herein), and ß-gal activity subsequently measured (as described herein). The results are illustrated in Figure 7. Each line refers to a set of liposomes extruded to a particular porosity.

Altering extrusion porosity does not significantly affect the transfection efficiency of the liposomes. In all subsequent examples, liposomes were extruded through membranes to a 200 nm size (unless otherwise stated).

Example 94 Ratio of Liposomes to DNA in Lipoplex For in vitro experiments reported in the literature, optimal liposome : DNA (+/-) charge ratios ranging from 0.3: 1 to 5: 1 have been reported, although typically a ratio of approximately 1.1: 1 is used (see, e. g., references 127-132).

The effect of the liposome : DNA ratio was examined further by comparing the three liposome formulations described in Table 4.7, which were used to form lipoplexes with pEFlacZ DNA, with the following (+/-) charge ratios: 1: 1,2: 1,3: 1,5: 1,8: 1 and 12: 1.

Table 4. 7 Molar Ratio of Cationic Lipid (s) DC-Chol 33 43 1 1-- 20. 50. 5 3cul505 (+/-) charge ratio was calculated according to the following equation: Ratio tig Lipid x 325 x N ug DNA M. W. Lipid In the above formula, N is the net number of positive charges per molecule of lipid. 325 is the average molecular weight of a single nucleotide. M. W. Lipid is the molecular weight of the cationic lipid used.

V79 cells were transfected (as described herein) using these lipoplexes, and (3-gal activity subsequently measured (as described herein). The results are illustrated in Figure 8.

The optimum (+/-) charge ratio for the two formulations containing DOPE and a 50: 50 mix of DC-Chol and diether lipid is at 3: 1. This ratio, which was used in the previous examples, is also used in the following examples (unless otherwise stated).

The DC-Chol/DOPE lipoplexes do not show such a large differential, although the transfection peak corresponds to a ratio of 5: 1. In the following examples, DC-Chol/DOPE control liposomes were mixed at a (+/-) charge ratio of 5: 1 (unless otherwise stated).

Example 95 Transfection Activity of the Diether Lipids Transfection data was collected for each of the diether lipids.

Each of the 15 diether lipids was used to make four formulations with the general compositions shown in Table 4.8. This gave a total of 61 formulations, including a DC- Chol/DOPE liposome to be used as a control, all of which were extruded to a 200 nm specification.

Table 4.8 The formulations were used, after extrusion (200 nm), to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 3: 1, apart from the control (DC-Chol/DOPE), which was mixed at a (+/-) charge ratio of 5: 1).

V79 cells were transfected (as described herein), and ß-gal activity subsequently measured (as described herein). The example was performed in quadruplicate. The results are illustrated in Figure 9 and Figure 10.

The data indicate that maximum transfection was obtained when the diether lipids were substituted for between 40 to 70% of the DC-Chol in the control DC-Chol/DOPE liposomes.

The results suggest that the shorter lipids, such as 12/12 (Compound 33), 12/14 (34), and 14/14 (35) appear to perform far better than the longer ones, such as (18/18 (43), 18/oleyl (44), and oleyl/oleyl (47)).

The results also suggest that the more asymmetric lipids (e. g. 12/18 (42) and 12/Oleyl (45)) appear to perform better than less asymmetric lipids. This may be due to asymmetric lipids having a more fusogenic character, leading to their increased

transfection efficacy. Another possibility is that the structure of the more asymmetric lipids allows a greater overlap of the alkyl chains within the lipid bilayer. This would give rise to greater van der Waals forces being created between them, meaning bilayer containing asymmetric lipids are subtly different in nature from those containing only symmetric lipids. In any case, both 42 and 45 reach their maximum transfection efficiency at a 40% substitution for DC-Chol. Optimal transfection represents a 7-and 10- fold improvement over DC-Chol respectively.

Also, all of the lipids appear to attain maximum transfection efficiency when mixed in approximately a 50: 50 ratio with DC-Chol. A possible reason for these observations is that the lipids were formulated as ternary mixtures with DC-Chol as well as DOPE. DC- Chol is based on cholesterol, and is a large, rigid tetracyclic. It thus forms relatively strong intermolecular bonds, due to van der Waals forces, resulting in low bilayer fluidity (see, e. g., reference 134). These may inhibit the bilayer disintegrating quickly enough when the lipoplex is inside the endosome, which in turn would result in some degradation of the plasmid being carried. The diethers 33-47 have a much more flexible structure than DC-Chol, and including a proportion of them in the formulation would result in the bilayer also being less rigid. This would allow them, and thus the lipoplex, to fragment more easily. The fusogenic lipid can then proceed to break down the lysosomal membrane, releasing the plasmid into the cytoplasm before it is degraded. Including too much of the flexible diether lipid could make the bilayer too unstable. This would render it susceptible to breakdown before it was required to do so, i. e. before it was inside the lysosome. The observation of an optimal transfection efficiency between 100% DC-Chol and 100% diether lipid might be explained by the required balance between rigidity and fluidity in the bilayer.

Example 96 Transfection Activity of the Diester Lipids The four diester lipids, 52,53,54, and 55, were each incorporated into five liposome formulations according to the general compositions shown in Table 4.9, to give 20 formulations.

Table 4. 9

The four formulations that included no DC-Chol would not form liposomes and transfection was recorded as zero. The remaining 16 formulations, plus a DC-Chol/DOPE control, were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 3: 1, apart from the control (DC-Chol/DOPE), which was mixed at a (+/-) charge ratio of 5: 1).

V79 cells were transfected (as described herein), and ß-gal activity subsequently measured (as described herein). The example was performed in quadruplicate. The results are illustrated in Figure 11.

The results show that the maximum (3-gal transfection of the esters is somewhat lower than the corresponding ether analogues. This may be due to the fact that esters are more easily metabolised than ethers. The transfection is nonetheless comparable, and if the esters offer lower cytotoxicity, and cytotoxicity is an issue, the small reduction in transfection efficiency may be acceptable. Note that direct comparisons may be difficult since the diesters are salts and the ethers free bases.

The lipid with the shortest alkyl chains (Cl2) performs the best, and transfection steadily decreases as alkyl chain length increases. Also, the maximum for all lipids occurs when approximately 40-60% of the DC-Chol is replaced by the ester.

The optimal DNA/lipid mixing ratio for peak transfection was subsequently determined to occur when (+/-) charge ratio was 3: 1.

Example 97 Cholesterol based lipids As shown above, mixtures of DC-Chol and the diether lipids prove more effective than either cationic lipid alone. With the hypothesis that the alkyl chains give the rigid,

cholesterol-based bilayer some beneficial degree of flexibility, these structural elements were incorporated into single structures, for example, compounds 59 and 60. Although there are many of examples in the literature of lipids with the cholesterol ring alone as the hydrophobic domain (see, e. g., references 39,40,135,136), none include an alkyl chain as well.

Compounds 59 and 60 were used to prepare six different formulations, with the compositions described in Table 4.10. A further five were also prepared using compound 33 (12/12 diether) for comparison, as well as a DC-Chol/DOPE control.

Table 4. 10 The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 3: 1, which was confirmed to be optimal).

V79 cells were transfected (as described herein), and ß-gal activity subsequently measured (as described herein). The example was performed in quadruplicate. The results are illustrated in Figure 12.

At the optimal formulation ratio, compound 59 (which contains the C12 chain) exhibits a 3-fold improvement over the DC-Chol control. Compound 60, which contains the C18 chain, is not significantly better than the control. Neither lipid performs as well as compound 33. This may be due to the large hydrophobic region, especially for a cationic lipid containing only one positive charge. Analogues with an increasing number of positive charges may address this problem.

It is possible that the cholesteryl and alkyl moieties are held in a more rigid position in space with respect to one another. The short-O-CH2-CH2-O-link may reduce the freedom of movement each has, in comparison to separate DC-Chol/diether molecules.

Corresponding cholesteryl-based lipids with linkers that have been extended by several- CH2-units may address this problem and improve transfection.

Example 98 Alternative Head Groups Compounds 33 and 42 (12/12 and 12/18 respectively), were coupled to each of the four naturally occurring amino acids that possess a basic side chain (lysine, arginine, histidine, and tryptophan). This generated lipid head groups with two positive charges rather than one, as well as introducing functionalities other than primary amines.

Initially, it was attempted to incorporate, individually, each of these compounds into four formulations with general compositions shown in Table 4.8. Neither of the compounds with the indole head group (67 and 68, incorporating tryptophan) formed liposomes at any formulation ratio (apart from that containing 40% of 67), and consequently were not assayed. Compound 33 was also used in formulations at these four ratios, along with a standard DC-Chol/DOPE formulation, as controls.

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1).

V79 cells were transfected (as described herein), and p-gai activity subsequently measured (as described herein). The example was performed in quadruplicate. The results are illustrated in Figure 13.

Unlike the diethers/diesters, none of these amino acid compounds appeared to reach maximum transfection between 40 and 70% substitution. Instead, many appear to continue improving up to 70%. Therefore, optimal transfection efficiency had possibly not been attained, and formulations with up to 100% substitution of DC-Chol may be better.

Also, the three compounds based on the 12/12 diether lipid 33 (compounds 63,72 and 78) are all better than their 12/18 analogues (compounds 64,73 and 79), at all comparable ratios of substitution.

Example 99 Alternative Head Group Lipids at other formulation ratios Formulations containing 63,72 or 78 were prepared to examine transfection efficiency at DC-Chol substitutions above 70%. The formulation compositions are described in Table 4.11.

Table 4. 11 Liposome Molar Ratio of Cationic Lipid (s) formulation DC-Chol Diether Lipid 1 0. 8 0. 2 2 0. 4 0. 6 3 0. 2 08 401 Controls were made up from several ratios of DC-Chol, DOPE and 33 as described in the previous example.

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1).

V79 cells were transfected (as described herein), and p-ga) activity subsequently measured (as described herein). The results are illustrated in Figure 14.

The formulations containing 100% of the cationic lipids produced the highest transfection.

Those containing 63 and 78 were particularly good and proved to be as efficient as the diether lipid 33 at optimal ratios, exhibiting approximately a 9 or 1 0-fold improvement over the standard DC-Chol/DOPE control. The increase using 72 was not so pronounced, but still resulted in a 3-fold improvement over the control.

Example 100 Optimal (+/-) charge ratio for Compounds 63,72 and 78 The optimal (+/-) charge ratios for compounds 63,72 and 78 were examined. The electrostatic properties of the head groups of these compounds may differ from those of the monovalent diether/diester lipids, and possibly also from each other.

Formulations for each of the three lipids, 63,72 and 78, in a 3: 2 molar ratio with DOPE were prepared.

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at variuos (+/-) charge ratios).

V79 cells were transfected (as described herein), and p-gai activity subsequently measured (as described herein). The results are illustrated in Figure 15.

All three formulations show peak transfection activity when a (+/-) charge ratio of 5: 1 was used.

Example 101 Cytotoxicity Assays Various lipid formulations were assessed for cytotoxicity (as described herein), in quadruplicate. The ICso values of the various formulations assayed are shown in Table 4. 12.

Table 4. 12 CationicLipid Component IC50 (µM DC-Cholcontrol 38 33 (12/12 diether) : DC-Chol (1 : 1) 75 42 (12/18 diether) : DC-Chol (1 : 1) 76 43 (18/18 diether) : DC-Chol (1 : 1) 67 52 (12/12 diester) : DC-Chol (1 : 1) 77 55 (18/18 diester) : DC-Chol (1 : 1) 91 63 (12/12 Lys) 34 78 12/12 Ar 20 Each formulation also contained DOPE in a 2: 3 molar ratio with the respective cationic lipid (s).

All formulations are at least comparable with DC-Chol/DOPE controls. The diesters and diethers have a slightly lower cytotoxicity than the other formulations. The fact that the ratio of lipid to DNA required for optimal transfection is lower than for DC-Chol makes them even better candidates. For this cell line, there was no difference in cytotoxicity between the esters and ethers.

Example 102 Lipids assayed on HT29 cells Three lipid formulations were prepared containing either DC-Chol, 63, or 78 in a 3: 2 molar ratio with DOPE. The fourth formulation contained a 50: 50 mixture of 33 and DC-Chol as the cationic component, at an equivalent ratio with DOPE.

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at various (+/-) charge ratios).

HT29 cells were cultured and transfected in an fashion identical to that for V79 cells (as described herein), and ß-gal activity subsequently measured (as described herein, 18 hours after transfection). The results are illustrated in Figure 16.

Formulations with 63,78, or 33 were significantly better than formulations with only DC- Chol. Notably, the optimal DNA mixing ratio is different from the corresponding example when carried out on V79 cells, as shown in Table 4.13.

Table 4. 13 Cationic Lipid Component Peak +/-Ratio V79 Peak +/-Ratio Cells HT29 Cells DC-Chol 5:1 3: 1 DC-Chol : 33 (1 : 1) 3:1 5:1 12/12Lys 5 : 1 8 : 1 12/12 Arg 5 : 1 8 : 1 The significantly lower level of transfection in comparison with the V79 cell line may be due to one or more of a number of reasons. The lipoplexes may be transported into the cells less efficiently, for example, or lysosomal activity within the cell may be higher.

Another possibility is the slower rate at which the cells are proliferating; HT29 cells have a much longer cell cycle (approximately 24 hours) compared to V79 cells (approximately 10 hours).

This example was repeated, but the HT29 cells were given an extra 24 hours to produce the ß-gal (but this doesn't solve any cytoplasmic retention problems). However while this did increase the amount of p-gat detected, the increase was only proportional to the

protein count (number of cells present), and thus the final enzyme activity was no different from the original example.

Example 103 Peptidolipids 85 in V79 Cells The peptidolipid, 85, was substituted for DC-Chol at various formulation ratios in standard DC-Chol/DOPE formulation (DC-Chol : DOPE is 3: 2) as described in Table 4.14.

Table 4. 14 Liposome Molar Ratio of Cationic Lipid (s) formulation DC-Chol Peptidolipid 1 control 1 0 2 0. 95 0. 05 3 0. 85 0. 15 4 0. 70 0. 30 5 0. 25 0. 75 6 0 1 The six formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1).

V79 cells were transfected (as described herein), and 0-gal activity subsequently measured (as described herein). The example was performed in quadruplicate. The results are illustrated in Figure 17.

At a 5% substitution of the peptidolipid, transfection is about 2-fold higher than the control. However, as the amount of substitution increases further, transfection decreases sharply. This may well be due to the peptide head group being rather bulky ; at high levels of substitution this might prevent correct formation of the bilayer, so reducing efficacy.

Example 104 Peptidolipids 86,87, and 88 in V79 Cells The peptidolipids 86,87, and 88 were assayed as in the previous example. Compound 86 had a poly- (8)-lysine peptide headgroup. Compound 87 contained 8 lysines plus the RGD motif. Compound 88 contained the sequence from fibronectin.

Each of the peptidolipids was used to make up three formulations, being substituted for 2, 5, or 10 molar % of the DC-Chol in a standard DC-Chol/DOPE formulation, as described in Table 4.14. This gave 10 formulations, including the DC-Chol/DOPE control.

Table 4. 15 Liposome Molar Ratio of Cationic Lipid (s) formulation DC-Chol Peptidolipid 1 control 1 0 2 0. 98 0. 02 3 0. 95 0. 05 4 0. 9 0. 1 The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1; the different charges of peptide groups were also taken into account).

V79 cells were transfected (as described herein), and 0-gal activity subsequently measured (as described herein). The example was performed in triplicate. The results are illustrated in Figure 18.

Example 105 Peptidolipids in V79 Cells The following three lipid formulations were considered: (i) 33: DC-Chol : DOPE (1.5: 1.5: 2 molar ratio), (ii) 63: DOPE (3: 2 molar ratio), and (iii) 78: DOPE (3: 2) as controls.

The cationic lipid content in each of these formulations was then substituted by one of the 3 peptidolipids, 86,87, and 88, in exactly the same manner as the DC-Chol in Table 4.15 to give 30 formulations (i. e., 2%, 5%, 10% substitution for each of 86,87, and 88, for each of (i), (ii), and (iii), plus three controls for each of (i), (ii), and (iii)).

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1; the different charges of peptide groups were also taken into account).

V79 cells were transfected (as described herein), and p-gat activity subsequently measured (as described herein). The results are illustrated in Figure 19.

Example 106 Peptidolipids in HT29 Cells The following four lipid formulations were considered: (i) DC-Chol : DOPE (3: 2 molar ratio), (ii) 33: DC-Chol : DOPE (1.5: 1.5: 2 molar ratio), (iii) 63: DOPE (3: 2 molar ratio), and (iv) 78: DOPE (3: 2) as controls.

The cationic lipid content in each of these formulations was then substituted by one of the 3 peptidolipids, 86,87, and 88, in exactly the same manner as the DC-Chol in Table 4.15 to give 40 formulations (i. e., 2%, 5%, 10% substitution for each of 86,87, and 88, for each of (i), (ii), (iii), and (iv) plus four controls for each of (i), (ii), (iii) and (iv)).

The formulations were extruded (200 nm) and used to form lipoplexes with the pEFlacZ plasmid (as described herein) (at a (+/-) charge ratio of 5: 1; the different charges of peptide groups were also taken into account).

HT29 cells were cultured and transfected in an fashion identical to that for V79 cells (as described herein), and p-ga) activity subsequently measured (as described herein, 18 hours after transfection). The example was performed in triplicate. The results are illustrated in Figure 20 and Figure 21.

The formulations containing RGD peptidolipids appear to perform slightly better than do the ones with poly- (L)-lysine. There is some improvement over the control formulations at a 2% substitution of these peptidolipids. Again, at 10% substitution, transfection begins to decrease in almost every case, possibly due to bilayer disruption.

REFERENCES A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

(1) I. Niculescu-Duvaz, Robert Spooner, Richard Marais and Carline Springer, Bioconjugate Chem. 1998,9,4-22.

(2) A. D. Miller, Angew. Chem. Int. Ed. 37,1998,1768-1785.

(3) C. J. Springer and 1. Niculescu-Duvaz, Adv. Drug Delivery Rev. 1996,22,351- 364.

(4) B. W. O'Malley, K. A. Cope, S. H. Chen, D. Li, M. R. Schwartz, S. L. C. Woo, Cancer Res. 1996,56,1737-1741.

(5) I. Niculescu-Duvaz, C. J. Springer, Exp. Opin. Invest. Drugs, 1997,6,685-703.

(6) K. W. Culver, Z. Ram, S. Wallbridge, H. Ishii, E. H. Oldfield, R. M. Blaese, Science, 1992,256,1550-1552.

(7) M. Caruso, Y. Panis, S. Gagandeep, D, Houssin, J-L. Salzmann, D. Klatzmann, Proc. Natl. Acad. Sci. USA, 1993,90,7024-7028.

(8) A. M. Sandmair, M. Turunen, K. Tyynela, S. Loimas, P. Vainio, R. Vanninen, M.

Vapalahti, R. Bjerkvig, J. Janne, S. Yla-Herttuala, Cancer Gene Ther. 2000,7, 413-421.

(9) B. E. Huber, E. A. Austin, C. A. Richards, S. T. Davis, S. S. Good, Proc. Natl. Acad.

Sci. USA 1994,91,8302-8306.

(10) M. Mesnil, H. Yamasaki, Cancer Res. 2000,60,3989-3999.

(11) M. A. Kay, J. C. Glorisio, L. Naldini, Nature Med., 2001,7,33-40.

(12) R. G. Crystal Science, 1995,270,404-410.

(13) I. M. Pope, G. J. Poston, A. R. Kinsella, Eur. J. Cancer, 1997,33,1005-1016.

(14) I. MacLachlan, P. Cullis, R. W. Graham, Current Opin. Mol. Ther., 1999,1,252- 259.

(15) S. L. Hart, Exp. Opin. Ther. Patents, 2000,10,199-208.

(16) G. Y. Wu, C. H. Wu, J. Biol. Chem. 1987,262,4429-4432.

(17) J. M. Benns, J. S. Choi, R. I. Mahato, J. S. Parks, S. W. Kim, Bioconjugate Chem.

2000,11,637-645.

(18) M. Monsigny, A. C. Roche, P. Midoux, R. Mayer, Adv. Drug Delivery Rev. 1994, 14,1-24.

(19) O. Boussif, F. Lezoualch, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. USA, 1995,92,7297-7301.

(20) I. Fajac, J. C. Allo, E. Souil, M. Merten, C. Pichon, C. Figarella, M. Monsigny, P.

Briand, P. Midoux, J. Gene Med. 2000,2,368-378.

(21) D. W. Pack, D. Putnam, R. Langer, Biotech. Bioeng. 2000,67,217-223.

(22) P. Erbacher, J-S Remy, J-P Behr, Gene Ther., 1999,6,289-295.

(23) C. Plank, M. X. Tang, A. R. Wolfe, F. C. Szoka, Human Gene Ther. 1999,10,319- 332.

(24) M. Colin, R. P. Harbottle, A. Knight, M. Kornprobst, R. G. Cooper, A. D. Miller, G.

Trugnan, J. Capeau, C. Coutelle, M. C. Brahimi-Horn, Gene Ther. 1998,5,1488- 1498.

(25) R. P. Harbottle, R. G. Cooper, S. L. Hart, A. Ladhoff, T. McKay, A. Knight, E.

Wagner, A. D. Miller, C. Coutelle, Human Gene Ther. 1998,9,1037-1047.

(26) R. Pasqualini, E. Koivunen, R. Kain, J. Lahdenranta, M. Sakamoto, A. Stryhn, R. A. Ashmun, L. H. Shapiro, W. Arap, E. Ruoslahti, Cancer Res. 2000,60,722- 727.

(27) W. Mier, R. Eritja, A. Mohammed, U. Haberkorn, M. Eisenhut, Bioconjugate Chem. 2000,11,855-860.

(28) N. K. Subbarao, R. A. Parente, F. C. Szoka, L. Nadasdi, K. Pongracz, Biochemistry, 1987,26,2964-2972.

(29) C. Ciolina, G. Byk, F. Blanche, V. Thuillier, D. Scherman, P. Wils, Bioconjugate Chem. 1999,10,49-55.

(30) M. A. Zanta, P. Belguise-Valladier, J-P. Behr, Proc. Natl. Acad. Sci. USA, 1999, 96,91-96.

(31) P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P.

Northrop, G. M. Ringold, M. Danielsen, Proc. Natl. Acad. Sci. 1987,84,7413- 7417.

(32) L. Stamatatos, R. Leventis, M. J. Zuckerman, J. R. Silvius, Biochemistry, 1988,27, 3917-3925.

(33) C. J. Wheeler, P. L. Felgner, Y. J. Tsai, J. Marshall, L. Sukhu, S. G. Doh, J. Hartikka, J. Nietupski, M. Manthorpe, M. Nichols, M. Plewe, X. W. Liang, J. Norman, A.

Smith, S. H. Cheng, Proc. Natl. Acad. Sci. USA, 1996,93,11454-11459.

(34) T. Ren, Y. K. Song, G. Zhang, D. Liu, Gene Ther. 2000,7,764-768 (35) P. Hawley-Nelson, V. Ciccarone, G. Gebeyehu, J. Jesse, P. L. Felgner, Focus, 1993,15,73-79.

(36) J-P. Behr, B. Demeneix, J-P. Loeffler, J. Perez-Mutul, Proc. Natl. Acad. Sci. USA, 1989,86,6982-6986.

(37) S. Obika, W. Yu, A. Shimoyama, T. Uneda, K. Miyashita, T. Doi, T. Imanishi, Bioorg. Med. Chem. Lett., 1997,147,1817-1820.

(38) X. Gao, L. Huang, Biochem. Biophys. Res. Comm. 1991,179,280-285.

(39) R. G. Cooper, C. J. Etheridge, L. Stewart, J. Marshal, S. Rudginsky, S. H. Cheng, A. D. Miller, Chem. Eur. J., 1998,4,137-151.

(40) J-P. Vigneron, N. Oudrhiri, M. Fauquet, L. Vergely, J-C. Bradley, M. Basseville, P.

Lehn, J-M. Lehn, Proc. Natl. Acad. Sci. USA, 1996, 93,9682-9686.

(41) D. Moradpour, J. l. Schauer, V. R. Zurawski, J. R. Wands, R. H. Boutin, Biochem.

Biophys. Res. Comm., 1996,221,82-88.

(42) J. H. Felgner, R. Kumar, C. N. Sridar, C. J. Wheeler, Y. J. Tsai, R. Border, P.

Ramsey, M. Martin, P. L. Felgner, J. Biol. Chem., 1994,269,2550-2561.

(43) F. Tang, J. A. Hughes, Biochem. Biophys. Res. Comm., 1998,242,141-145.

(44) F. Tang, J. A. Hughes, Bioconjugate Chem., 1999,10,791-796.

(45) V. Floch, S. Loisel, E. Guenin, A-C. Herve, J-C. Clement, J-J. Yaouanc, H. des Abbayes, C. Ferec, J. Med. Chem., 2000,43,4617-4628.

(46) F. Tang, J. A. Hughes, J. Control. Release, 1999,345-358.

(47) D. D. Lasic, Liposomes in Gene Delivery, CRC Press, 1997, pp67.

(48) P-A. Monnard, T. Oberholzer, P. Luisi, Biochim. Biophys. Acta, 1997,1329,39-50.

(49) G. Gregoriadis, B. McCormack, M. Obrenovic, R. Saffie, B. Zadi, Y. Perrie, Methods, 1999,19,156-162.

(50) R. Leventis, J. R. Silvius, Biochim Biophys Acta, 1990,1023,124-132.

(51) Y. K. Song F. Liu, S. Y. Chu, D. X. Liu, Human. Gene Ther. 1997,8,1585-1594.

(52) S. Obika, W. Yu, A. Shimoyama, T. Uneda, T. Minami, K. Miyashita, T. Doi, T.

Imanishi, Biol. Pharm. Bull., 1999,22,187-190.

(53) W. Yu, A. Shimoyama, T. Uneda, S. Obika, K. Miyashita, T. Doi, T. Imanishi, J.

Biochem., 1999, 125,1034-1038.

(54) G. Byk, B. Wetzer, M. Frederic, C. Dubertret, B. Pitard, G. Jaslin, D. Scherman, J.

Med. Chem., 2000,43,4377-4387.

(55) E. Guenin, A-C. Herve, V. Floch, S. Loisel, J-J. Yaouanc, J-C. Clement, C. Ferec, H. des Abbayes, Angew. Chem. Int. Ed., 2000,39,629-631.

(56) P. Pinnaduwage, L. Schmitt, L. Huang, Biochim. Biophys. Acta, 1989,985,33-37.

(57) P. L. Felgner, G. M. Ringold, Nature, 1989,337,387-388.

(58) D. K. Struck, D. Hoekstra, R. E Pagano, Biochemistry, 1981,20,4093-4099.

(59) H. Gershon, R. Ghirlando, S. B. Guttman, A. Minsky, Biochemistry, 1993,32, 7143-7151.

(60) B. Sternberg, F. L. Sorgi, L. Huang, FEBS Lett., 1994,356,361-366.

(61) J. Gustafsson, G. Arvidson, G. Karlsson, M. Almgren, Biochim. Biophys. Acta, 1995,1235,305-312.

(62) J. Zabner, A. J. Fasbender, T. Moninger, K. A. Poellinger, M. J. Welsh, J. Biol.

Chem., 1995,270,18997-19007.

(63) J. O. Radler, 1. Koltover, T. Salditt, C. R. Safinya, Science, 1997,275,810-814.

(64) D. D. Lasic, H. Strey, M. C. A. Stuart, R. Podgornik, P. M. Frederik, J. Am. Chem.

Soc., 1997,832-833.

(65) I. Koltover, T. Salditt, J. O. Radler, C. R. Safinya, Science, 1997,281,78-81.

(66) D. D. Lasic, Liposomes in Gene Delivery, CRC Press, 1997, pp125.

(67) J. J. Wheeler, L. Palmer, M. Ossanlou, l. MacLachlan, R. W. Graham, Y. P. Zhang, M. J. Hope, P. Scherrer, P. R. Cullis, Gene Ther., 1999,6,271-281.

(68) K. W. C. Mok, A. M. I. Lam, P. R. Cullis, Biochim. Biophys. Acta, 1999,1419,137- 150.

(69) Y. P. Zhang, L. Sekirov, E. G. Saravolac, J. J. Wheeler, P. Tardi, K. Clow, E. Leng, R. Sun, P. Scherrer, P. R. Cullis, Gene Ther., 1999,6,1438-1447.

(70) P. Tam, M. Monck, D. Lee, O. Ludkovski, E. C. Leng, K. Clow, H. Stark, P.

Scherrer, R. W. Graham, P. R. Cullis, Gene Ther., 2000,7,1867-1874.

(71) X. Zhou, L. Huang, Biochim. Biophys. Acta, 1994,1189,195-203.

(72) F. Labat-Moleur, A. M. Steffan, C. Brisson, H. Perron, O. Feugeas, Gene Ther., 1996,3,1010-1017.

(73) P. C. Ross, S. W. Hui, Gene Ther., 1999,6,651-659.

(74) A. Fasbender, J. Marshall, T. O. Moninger, T. Grunst, S. Cheng, M. J. Welsh, Gene Ther., 1997,4,716-725.

(75) H. Farhood, N. Serbina, L. Huang, Biochim. Biophys. Acta, 1995,1235,289-295.

(76) D. C. Litzinger, L. Huang, Biochim. Biophys. Acta, 1992,1113,201-227.

(77) Y. Xu, F. C. Szoka, Biochemistry, 1996,35,5616-5623.

(78) I. Mortimer, P. Tam, l. MacLachlan, R. W. Graham, E. G. Saravolac, P. B. Joshi, Gene Ther., 1999,6,403-411.

(79) D. Lechardeur, K. J. Sohn, M. Haardt, P. B. Joshi, M. Monck, R. W. Graham, B.

Beatty, J. Squire, H. O'Brodovich, G. L. Lukacs, Gene Ther., 1999,6,482-497.

(80) M. R. Cappechi, Cell, 1980,22,479-488.

(81) K. Weiss, Trends Biochem. Sci., 1998,23,185-189.

(82) X. Gao, L. Huang, Nucleic Acids Res., 1993,21,2867-2872.

(83) X. Gao, D. Jaffers, P. D. Robbins, L. Huang, Biochem. Biophys. Res. Comm., 1994,200,1201-1206.

(84) M. Brisson, Y. He, S. Li, J. P. Yang, L. Huang, Gene Ther., 1999,6,263-270.

(85) G. Byk, C. Dubertret, V. Escriou, M. Frederic, G. Jaslin, R. Rangara, B. Pitard, J.

Crouzet, P. Wils, B. Schwartz, D. Scherman, J. Med. Chem., 1998,41,224-235.

(86) R. P. Balasubramaniam, M. J. Bennett, A. M. Aberle, J. G. Malone, M. H. Nantz, R. W. Malone, Gene Ther., 1996,3,163-172.

(87) G. Byk, Letters in peptide science. 1997,4,263-267.

(88) M. D. Pierschbacher, E. Ruoslahti, J. Sundelin, P. Lind, P. A. Petersen, J. Biol.

Chem., 1982,257,9593-9597.

(89) M. D. Pierschbacher, E. Ruoslahti, Nature, 1984,309,30-33.

(90) E. Ruoslahti, M. D. Pierschbacher, Science, 1987,238,491-497.

(91) R. O. Hynes, Cell, 1992,69,11-25.

(92) T. J. Vanderwaak, M. Wang, J. Gomez-Navarro, C. Rancourt, l. Dimitriev, V.

Krasnykh, M. Barnes, G. P. Siegal, R. Alvarez, D. T. Curiel, Gynecol. Oncol., 1999, 74,227-234.

(93) R. Pasqualini, E. Koivunen, E. Ruoslahti, Nature Biotech., 1997,15,542-546.

(94) W. Arap, R. Pasqualini, E. Ruoslahti, Science, 1998,279,377-380 (95) M. Gurrath, G. Muller, H. Kessler, M. Aumailley, R. Timpl, Eur. J. Biochem., 1992, 210,911-921.

(96) J. M. Healy, M. Haruki, M. Kikuchi, Prot. Pepf. Lett., 1996,3,23-30.

(97) M. Bergman, L. Zervas, Chem. Ber., 1932,65,1192-1201.

(98) J. March, Advanced Organic Chemistry, 4' edition, John Wiley & Sons, 1992, pp 386-387.

(99) F. J. Soday, C. E. Boord, J. Am. Chem. Soc., 1933,55,3293-3302.

(100) G. Sennyev, G. Barcelo, J-P. Senet, Tetr. Lett., 1986, 27,5375-5376.

(101) M. Yamaguchi, l. Hirao, Tetrahedron Lett., 1983,24,391-394.

(102) J. March, Advanced Organic Chemistry, 4'"edition, John Wiley & Sons, 1992, pp826.

(103) P. N. Guivisdalsky, R. Bittman, J. Org. Chem., 1989,54,4637-4642.

(104) T. Aoki, C. D. Poulter, J. Org. Chem., 1985,50,5634-5636.

(105) D. H. Thompson, C. B. Svensden, C. Di Meglio, V. C. Anderson, J. Org. Chem., 1994,59,2945-2955.

(106) H. R. Ing, R. H. F. Manske, J. Chem. Soc., 1926,2348-2349.

(107) S. S. Wang, J. P. Tam, B. S. H. Wang, R. B. Merrifield, Int. J. Peptide Protein Res., 1981,18,459-467.

(108) G. M. Anantharamaiah, K. M. Sivanandaiah, J. Chem. Soc. Perkins Trans. I, 1977, 490-491.

(109) R. M. Silverstein, F. X. Webster, Spectrometric Identification of Organic Compounds, 6'"edition, John Wiley & Sons, 1998, pp211.

(110) E. Kaiser et al, Anal. Biochem., 1970,34,595-599.

(111) T. Ren, D. Liu, Tetrahedron Lett., 1999,40,209-212.

(112) S. M. Ansell, L. D. Kojic, J. S. Hankins, L. Sekirov, A. Boey, D. K. Lee, A. R. Bennett, S. K. Klimuk, T. O. Harasym, N. Dos Santos, S. C. Semple, Bioconjugate Chem., 1999,10,653-666.

(113) L. Kisfaludy, 1. Schon, Synthesis, 1983,4,325-327.

(114) J. C. Sheehan, P. A. Cruickshank, G. L. Boshart, J. Org. Chem., 1961,26,2525- 2528.

(115) D. A. Pearson, M. Blanchette, M. L. Baker, C. A. Guindon, Tetrahedron Lett., 1989, 30,2739-2742.

(116) W. C. Chan, P. D. White, Fmoc Solid Phase Peptide Synthesis, ls'edition, Oxford University Press, 2000, pp 65.

(117) A. Kates, S. B. Daniels, N. A. Sole, G. Barany, F. Albericio, Proc. 13th American Peptide Symposium, 1994,113-115.

(118) W. Konig, R. Geiger, Chem. Ber., 1970,103,2034.

(119) E. Atherton, J. L. Holder, M. Meldal, R. C. Sheppard, R. M. Valerio, J. Chem. Soc.

Perkin Trans./, 1988,2887-2894.

(120) NovaBiochem Catalogue and Peptide Synthesis Handbook, 1999, pp S41-S42.

(121) F. Olson, C. Hunt, F. Szoka, W. Vail, D. Papahadjopoulos, Biochim. Biophys.

Acta, 1979,557,9-23.

(122) R. C. MacDonald, R. I. MacDonald, B. M. Menco, K. Takeshita, N. K. Subbarao, L- R. Hu, Biochim. Biophys. Acta, 1991,1061,297-303.

(123) D. D. Lasic, Liposomes in Gene Delivery, CRC Press, 1997, pp91.

(124) N. S. Templeton, D. D. Lasic, P. M. Frederik, H. H. Strey, D. D. Roberts, G. N.

Pavlakis, Nature Biotech., 1997,15,647-652.

(125) O. Zelphati, C. Nguyen, M. Ferrari, J. Felgner, Y. Tsai, P. L. Felgner, Gene Ther., 1998,5,1272-1282.

(126) R. I. Mahato, K. Anwer, F. Tagliferri, G. Meaney, P. Leonard, M. S. Wadhwa, M.

Logan, M. French, A. Rolland, Human Gene Ther., 1998,9,2083-2099.

(127) R. Banerjee, P. K. Das, G. V. Srilakshmi, A. Chaudhuri, N. M. Rao, J. Med. Chem., 1999,42,4292-4299.

(128) A. Colosimo, A. Serafino, F. Sanguilo, S. Di Sario, E. Bruscia, P. Amicucci, G.

Novelli, B. Dallapiccola, G. Mossa, Biochim. Biophys. Acta., 1999,1419,186-194.

(129) A. J. Fasbender, J. Zabner, M. J. Welsh, Am. J. Physio, 1995,13, L45-L51.

(130) J. S. Matsumura, R. Kim, V. P. Shively, R. C. MacDonald, W. H. Pearce, J. Surg.

Res., 1999,85,339-345.

(131) A. R. Holmes, A. F. Dohrman, A. R. Ellison, K. K. Goncz, D. C. Gruenert, Pharm.

Res., 1999,16,1020-1025.

(132) A. Cao, D. Briane, R. Coudert, J. Vassy, N. Leivre, E. Olsman, E. Tamboise, J. L.

Salzman, J. P. Rigaut, E. Taillandier, Antisense and Nucleic Acid Drug Delivery, 2000,10,369-380.

(133) V. Floch, M. P. Audrezet, C. Guillaume, E. Gobin, G. Le Bloch, J. C. Clement, J. J.

Yaouanc, H. Des Abbayes, B. Mercier, J. P. Leroy, J. F. Abgrall, C. Ferec, Biochim.

Biophys. Acta, 1998,1371,53-70.

(134) D. Voet, J. G. Voet Biochemistry, 15t Edition, 1990, pp 284.

(135) Y. K. Ghosh, S. S. Visweswariah, S. Battacharya, Gene Ther., 2000,7,341-344.

(136) T. Fichert, A. Regeln, U. Massing, Bioorg. Med. Chem. Lett., 2000,10,787-791.

(137) M. Wilke, Gene Ther., 1996,3,1133-1142.

(138) B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J. D. Watson, Molecular Biology of the Cell, 3rd Edition, Garland Publishing, NY, 1994, pp 917-918.

(139) S. Brunner, T. Sauer, S. Carotta, M. Cotton, M. Saltik, E. Wagner, Gene Ther., 2000,7,401-407.

(140) B. Alerts, D. Bray, J. Lewis, M. Raff, K. Roberts, J. D. Watson, Molecular Biology of the Cell, 3rd Edition, Garland Publishing, NY, 1994, pp995.

(141) (142) K. Kasono, J. L. Blackwell, J. T. Douglas, 1. Dimitriev, T. V. Strong, P. Reynolds, D. A. Kropf, W. R. Carroll, G. E. Peters, R. P. Bucy, D. T. Curiel, V. Krasnykh, Clin.

Canc. Res., 1999,5,2571-2579.

(143) A. M. I. Lam, P. R. Cullis, Biochim. Biophys. Acta, 2000,1463,279-290.

(144) P. T. Jain, D. A. Gewirtz, Intl. J. Mol. Med., 1998, 1,609-611.

(145) W. C. Chan, B. W. Bycroft, D. J. Evans, P. D. White, J. Chem. Soc., Chem.

Commun., 1995,2209-2210.

(146) E. Koivunen, B. Wang, E. Ruoslahti, J. Cell Biol., 1994, 124,373-380.

(147) F. Szoka, D. Papahadjopoulos, Proc. Natl. Acad. Sci. USA, 1978,75,4194-4198.

(148) J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning-A Laboratory Manual, 2"d Edition, Cold Spring Harbor Laboratory Press, 1989, pp 16.66-16.67.

(149) Bio Rad Catalog.

(150) W. L. F. Amarego, D. D. Perrin, Purification of Laboratory Chemicals, 4'"Edition, The Bath Press, 1996, pp 176.

(151) Heyes, J. A., et al., 2002,"Synthesis of Novel Cationic Lipids: Effect of Structural Modification on the Efficiency of Gene Transfer,"J. Med. Chem., Vol. 45, pp. 99- 114 (Published on Web: 06 Dec 2001).