This application claims the priority benefit of Argentine Patent Application No. 00-01-04921 filed on September 20,2000, which is incorporated herein as reference in its entirety.

Field of the Invention This invention relates to the description and synthesis of lipids.

Background of the Invention The cationic lipids are characterized by having a hydrophilic domain and a hydrophobic domain. These domains are formed by long carbon chains. The most active molecules are positively charged and are associated to laminar structures containing multiple positive charges on their surfaces.

At the present, two main groups of cationic lipids are known in the art.

According to their structure, these groups are classified into cholesterol or glycerin derivatives. The glycerin group may be divided further in diacylglyceryl or dietherglyceryl sub-groups.

The amphiphilic cationic lipids (cytofectins) are used currently in gene therapy to transfer DNA and RNA to cultured animal cells. These compounds facilitate an efficient non-viral transference (transfection, lipofection) of nucleic acids into cells.

The amphiphilic cationic lipids are lipophilic molecules charged positively that form complexes easily with DNA and other anionic polynucleotides.

The cationic lipids known in the art require mixing with variable amounts of neutral, zwitterionic lipids such as DOPE (dioleoylphosphatidylethanolamine) to stabilize their aqueous emulsion and optimize transfection. The mixture of the cationic lipid with a neutral phospholipid increases its activity. This combination results in cationic liposomes which are usually more efficient in cell transfection than its individual components. These liposomes are characterized by preserving all the properties of both amphipathic subrogates of the precursor cationic lipid.

Cationic liposomes have a net polycationic surface capable of strong interaction with the polyanionic phosphate backbone of polynucleotides. Such interaction occurs spontaneously. The complex formed by the polynucleotide, the cationic lipid and the neutral phospholipid-referred to as lipoplex-interacts strongly with the negative charge prevailing in the cell surface. This phenomenon favors the lipoplex-plasmatic membrane fusion and the effective transference of the polynucleotide into the cellular cytoplasm.

Amphiphilic cationic lipids are characterized by containing an amino group, a spacer arm, a linking bond and a lipophilic group. The amino group is charged positively at neutral pH. The amino group may be primary, secondary, tertiary or quaternary.

The lipophilic group is a hydrophobic unit or portion that allows the insertion of the cationic amphiphile into the membrane of the cell or liposome. The lipophilic group serves as an anchor for the cationic ammonium group to attach to the surface of a cell or liposome.

The spacer group is usually hydrophilic and has a variable length and composition. The function of the spacer group is to connect the amino group to the lipophilic group by means of a chemical bond. It is preferable that the chemical bond be hydrolizable, i. e. esters, amides, carbonate or carbamate.

The molecule cationic region and the hydrophobic lipophilic region of the cationic lipid are biocompatible. That is, the hydrophobic and cationic units either occurs naturally or is well tolerated by cells.

The connecting union of cationic lipids is biodegradable. A biodegradable union is that which degrades or hydrolyzes under certain conditions or during normal processes in cells or organisms.

The biodegradation of a cationic lipid during its application into a cell or organism results in two biocompatible components. This characteristic confers the amphiphilic cationic lipids less toxicity as compared to other lipids characterized by the presence of an ether bond, which is more difficult to degrade. Cationic lipids must be non-toxic or minimally toxic and biodegradable in order to avoid adverse side effects in the treated cells.

The relationship between the efficiency of transfection and the presence of a polar or hydrophobic domain in the transfecting lipid has been described in the art.

However, the physical and chemical characteristics of cationic lipids affecting polynucleotide transfer into cells is not well known at the present. The connection between the chemical structure of the cationic lipid and its biological activity is little known.

At the present, several liposome formulations using either synthetic or naturally occurring lipid molecules have been disclosed. In this sense, clear advantages have been achieved. See WO 96/17063 (Hobart et als) ; WO 96/40067 (Wyse et als) and WO 98/10649 (Shefter et als). However, no cholesterol derived cationic lipids, containing a 2-hydroxyethyl group as quaternizing radical of the corresponding tertiary amine in the hydrophilic group, are known.

Summary of the Invention The present patent claims amphiphilic cationic lipids not presently known in the art. The present patent also describes the synthesis of said amphiphilic cationic lipids.

The claimed compounds have the following advantages, among others: low toxicity, biocompatibility of the hydrophilic and hydrophobic domains and biodegradability of its connecting bond. Because of these characteristics, the claimed lipids may be used for in vivo, in vitro and ex vivo gene transfer and for medical treatments based on gene therapy.

Several of the lipids claimed in the present patent have the additional advantage of preserving their chemical integrity in solution while hydrolyzing when in contact with tissue. This characteristic makes the claimed lipids more stable and extends their half-life without altering their therapeutical utility.

Another advantage of the lipids claimed herein is their rigid planar hydrophobic backbone that does not alter the hydrocarbonated packing in the bilayer membrane. The claimed lipids include also a positively charged tertiary or quaternary amine group that enhances further the bilayer stability.

The following abbreviations are used in the present invention to identify the compounds indicated next: Table A Abbreviation Claimed Products HDC-Chol (II) Cholesteryl-3 ß-N-[2-[N'-(2-hydroxyethyl)-N', N'- (dimethylammonio) ] ethyl] carbamate bromide HCC-Chol (IV) Cholesteryl-3p- [2- [N- (2-hydroxyethyl)-N, N- (dimethylammonio)] ethyl] carbonate bromide AB-Chol (V) Cholesteryl 3ß-4-(N, N-dimethylamino) butanoate HAB-Chol (VI) Cholesteryl 3p-4- [N (2-hydroxyethyl) -N, N- dimethylammonio)] butanoate bromide HDS-Chol (IX) Cholesteryl 3ß-4 [[2-[N-(2-hydroxyethyl)-N, N- (dimethylammonio) ] ethyl] amine] butanedioate bromide CS-Chol (X) Cholesteryl 3p-4- [2- (dimethylamine) ethyl] butanedioate HCS-Chol (XI) Cholesteryl 3ß-4-[2-[N-(2-hydroxyethyl)-N, N- (dimethylammonio) ethyl butanedioate bromide

Table B Abbreviation Products Known in the Art DC-Chol Cholesteryl 3ß-N,N-(dimethylaminoethyl) carbamate CC-Chol Cholesteryl 3-ß-N, N- (dimethylaminoethyl) carbonate DS-Chol Cholesteryl ß-4[[2-(dimethylamino) ethyl] amino] oxobutanoate

The present invention claims amphiphilic cationic lipids derived from cholesterol characterized by the following chemical structure: 1) an hydrophilic domain which includes a dimethylamine group, 2) a spacer group (Ri) which includes at least 2 carbon atoms linked to the hydrophilic domain, 3) one linking bond (L) that

includes a carboxyl ester, carbamate or carbonate group linked to the spacer group and 4) one lipophilic group derived from cholesterol (Chol) attached to the linking bond.

In one embodiment of the present invention, the hydrophilic domain of the claimed amphiphilic cationic lipids is a tertiary dimethylamine group. In such cases, the present invention has the following overall structure:

In another embodiment of the present invention, the hydrophilic domain of the claimed amphiphilic cationic lipids is a quaternary dimethylamine group. Preferably, the quaternary dimethylamine group is formed by binding a tertiary dimethylamine group to a 1,2-halohydrin or a 1,3-halohydrin. Preferably, the tertiary dimethylamine group is linked to a 1,2-halohydrin ; more preferably, to 2-hydroxyethyl. In such cases, the present invention has the following overall chemical structure:

wherein: Rl represents a spacer group composed by at least two carbon atoms, R2 represents either a 2-hidroxyalkyl group or 3-hydroxyalkyl group, L represents a linking bond between a lipophilic group (Chol) and the Ri chain, and L includes a carboxyl ester, carbamate or carbonate group,

Chol represents the cholesterol tetracyclic core without the ß hydroxyl at C-3 substituted in said position by a linking bond (L), and X represents an anion.

In an embodiment of the present invention, the linking bond includes a carbamate group and has the following overall chemical structure: In another embodiment of the present invention, the linking bond includes a carbonate group and has the following overall chemical structure: In another embodiment of the present invention, the linking bond includes a carboxyl ester group and has the following overall chemical structure: The Rl spacer group may be an aliphatic chain or contain an aromatic radical.

According to the present invention, the aliphatic chain may be linear or branched. In one embodiment of the present invention, the aliphatic chain is linear and saturated. In another embodiment of the present invention, the aliphatic chain is unsaturated. In some embodiments of the present invention, the spacer group contains a heteroatom, i. e. nitrogen or oxygen. In such cases, the heteroatom forms an additional ester, amide, amino or ether group. Preferably, the aromatic radical is a phenyl group. Preferably, the spacer group has more than 2 carbon atoms. More preferably, the spacer group has less than 10 carbon atoms.

According to one of the embodiments of the present invention, the quaternary dimethylamine group forms bonds with negatively charged ions. Examples of anions

that may form the quaternary salts claimed in the present invention are chloride, bromide, iodine, acetate, sulfate, bisulfate, trifluoroacetate, trifluoromethanesulfonate and monophosphate. Preferably, the anion linked to the quaternary dimethylamine group is bromide.

Brief Description of the Figures Figure 1 illustrates the synthesis of HDC-Chol (II) and represents its chemical structure.

Figure 2 illustrates the synthesis of HCC-Chol (IV) and represents its chemical structure.

Figure 3 illustrates the syntheses of AB-Chol (V) and HAB-Chol (VI) and represents their chemical structures.

Figure 4 illustrates the synthesis of HDS-Chol (IX) and represents its chemical structure.

Figure 5 illustrates the syntheses of CS-Chol (X) and HCS-Chol (XI) and represents their chemical structures.

Detailed Description of the Preferred Embodiments General Procedures DC-Chol (I) was prepared from cholesteryl chloroformate and N, N- dimethylethylenediamine applying techniques known in the art. See Gao et al., Biochem. and Biophys. Res. Comm., 179 (1) : 280-285 (1991). CC-Chol (in) was obtained by reaction of cholesteryl chloroformate with 2- (dimethylamino) ethanol according to the technique described in WO 98/10649 (Shefter et als).

The 2-bromoethanol quaternization was produced according to the Moss technique. See Moss et al., J. Amer. Chem. Soc., 109 (19) : 5740-5744 (1987).

Catalytic amounts of N, N-diisopropylethylamine were used to fix the hydrobromic acid formed during the thermal break down of 2-bromoethanol. The reaction took place at reflux in a nitrogen atmosphere using anhydrous acetone as solvent.

The melting points were determined with a Mel-Temp II equipment and were uncorrected.

The 13C nuclear magnetic resonance spectra (NMR) were determined with a Brucker MSL 300 spectrophotometer at 75.47 MHz. Chemical shifts were expressed as parts per million (ppm) relative to tetramethylsilane used as internal standard.

Deuterated chloroform or deuterated dimethylsulfoxide were used as solvent.

The mass spectra (MS) were determined with a Quatro II-Micromass instrument using the FAB mode. The infrared spectra were determined with a FT-IR Perkin Elmer Spectrum BX equipment utilizing a KBr solid dispersion.

Evaporations were performed in a Rotavapor Buchi R-114.

TLC was performed using silica gel plates 60G. The plates were developed by spraying with sulfuric acid in methanol 10% (v/v) and heating at 110 °C.

The column chromatographies were performed in silica gel 60 (0.040-0. 063 mm) utilizing a 230-400 mesh ASTM (Merck).

Example 1 <BR> <BR> <BR> <BR> <BR> <BR> Description and synthesis of Cholesteryl-3$N-[2-[N'-(2-hydroxyethyl)-<BR> <BR> <BR> <BR> N', N'-(dimethylammonio) lethyllcarbamate bromide<BR> <BR> <BR> <BR> HDC-Chol (II) 3.6 g (28.8 mmol) of 2-bromoethanol and 0.32 ml (1.87 mmol) of N, N- diisopropylethylamine were added to a solution containing 3.0 g (6.0 mmol) of DC- Chol (I) in 150 ml of anhydrous acetone. The reaction mixture was heated then at reflux and stirred magnetically for 24 hours under a nitrogen atmosphere.

The almost complete disappearance of the substrate and the appearance of a stain (Rf 0.47) were observed by TLC (CHCl3 : MeOH: H20 65: 25: 4). White crystals were obtained by cooling the reaction mixture. These crystals were then filtered, rinsed with anhydrous acetone and exposed to air.

The quaternary salt thus obtained was purified by column chromatography.

The salt was eluted with a CHCl3 : MeOH (9: 1) mixture. The pure fractions obtained were evaporated to dryness. 1.96 g of a yellowish white solid identified as HDC-Chol was thus obtained. The reaction yield was 52.3%. See Fig. 1.

The following information was obtained from the analyses applied to the resulting HDC-Chol:

1. The mass spectrum test showed a peak for [M-Br] + at m/e 545 corresponding to the molecular formula C34H61BrN203 and to a molecular weight of 625.77 g/mol.

2. The NMR spectrum of 13C in C13CD showed peaks at: 156.6 ; 139.6 ; 122.6 ; 75.0 ; 66.1 ; 64.0 ; 56.6 ; 56.2 ; 55.8 ; 52.8 ; 49.9 ; 42.2 ; 39.7 ; 39.4 ; 38.5 ; 36.9 ; 36.5 ; 36.2 ; 35.8 ; 35.4 ; 31.8 ; 29.6 ; 28.2 ; 27.9 ; 24.2 ; 23.9 ; 22.7 ; 22.5 ; 21.0 ; 19.3 ; 18. 7 and 11. 8 ppm.

3. The IR spectrum showed peaks at 3405 (OH), 2935,2908, 2868,2851, 1714 (C=O) cari'.

4. The melting point was 233-236 °C.

All the test results corresponded to the expected values for HDC-Chol.

Example 2 Description and synthesis of Cholesteryl-3, B [2-[N-(2-hydroxyethyl)- N,N-(dimethylammonio)]ethyl]carbonate bromide HCC-Chol (IV) 4.90 g (39.2 mmol) of 2-bromoethanol and 0.4 ml (5.25 mmol) of N, N- diisopropylethylamine were added to a solution containing 4.04 g (8.05 mmol) of CC- Chol (ici) in 190 ml of anhydrous acetone. The reaction mixture was heated then at reflux and stirred magnetically under a nitrogen atmosphere. The reaction progress was monitored by TLC (CHCl3 : acetone 2: 1) and by observing the partial disappearance of CC-Chol. 4.90 g (39.2 mmol) of 2-bromoethanol and 0.4 ml (5.25 mmol) of N, N-diisopropylethylamine were added again to the reaction mixture at 24 and 48 hours after the beginning of reflux. The reaction was completed 72 hours after the beginning of reflux.

The reaction mixture was cooled in an ice-water bath. The cooling of the reaction mixture resulted in a yellowish white wax-like precipitate. The resulting precipitate was centrifuged, rinsed with anhydrous acetone and dried with air. 3.82 g of a product identified as HCC-Chol was thus obtained. The reaction yield was 75.5%.

See Fig. 2.

The following information was obtained from the analyses applied to the resulting HCC-Chol:

1. The mass spectrum test showed a peak for [M-Br] + at m/e 546 corresponding to the molecular formula C34H6, BrN203 and a molecular weight of 626.77 g/mol.

2. The NMR spectrum of 13C in C13CD showed peaks at: 153.5 ; 139.1 ; 123.1 ; 78.8 ; 66.9 ; 63.7 ; 61.1 ; 56.6 ; 56.2 ; 55.8 ; 52.9 ; 49.9 ; 42.3 ; 39.7 ; 39.5 ; 37.9 ; 36.8 ; 36.5 ; 36.2 ; 35.8 ; 31.9 ; 31.8 ; 28.2 ; 28.0 ; 27.6 ; 24.3 ; 23.9 ; 22.8 ; 22.5 ; 21.0 ; 19.2 ; 18. 7 and 11. 8 ppm.

3. The IR spectrum showed peaks at 3400 (OH), 2938,2901, 2864,1749 (C=O) cm~'.

All the test results corresponded to the expected values for HCC-Chol.

Example 3 Description and synthesis of Cholesteryl 33-4- <BR> <BR> (dimethylamino) butanoate<BR> <BR> <BR> AB-Chol (V) A solution containing 1.76 g (25.85 mmol) of imidazole in 20 ml of anhydrous dichloromethane was added to a suspension of 4.0 g (23.86 mmol) of 4- (dimethylamine) butyric acid hydrochloride in 40 ml of anhydrous dichloromethane.

The reaction mixture was stirred magnetically for 30 minutes at room temperature.

5.8 g (35.77 mmol) of 1, 1'-carbonyldiimidazole dissolved in 100 ml of anhydrous dichloromethane were added slowly to the acid liberated in the preceding step at a temperature of 5 to 8 °C. A complete solution was obtained after 1 hour.

Thereafter, 9.28 g (24.00 mmol) of cholesterol dissolved in 40 ml of anhydrous chloroform was added to the reaction mixture. The reaction progress was monitored by TLC (CHC13). The reaction mixture was stirred magnetically at room temperature and completed after 20 hours.

The reaction mixture was evaporated to dryness and a semisolid residue was obtained. The resulting semisolid residue was treated further with acetonitrile (120 ml) and cooled using an ice-water bath. A wax-like (Rf 0. 25) product with an impurity (Rf 0. 79) was thus obtained.

The semisolid residue was purified by a silica gel column chromatography to separate the wax-like product according to the following protocol. The semisolid

residue was treated with CHC13 and first eluted with a mixture of CHCl3 : MeOH 3% (v/v) to remove the Rf 0.79 impurity. Then, the eluting mixture was increased with CHCl3 : MeOH 5% (v/v) and the Rf 0.25 product was eluted. The resulting pure fractions were evaporated to dryness and an oily product was obtained. The oily product solidified later into a yellowish white wax. 10.6 g of a product identified as AB-Chol was thus obtained. The reaction yield was 88.8%. See Fig. 3.

The following information was obtained from the analyses applied to the resulting AB-Chol: 1. The mass spectrum test showed a peak for [MH] + at m/e 500 corresponding to the molecular formula C33H57NO2 and a molecular weight of 499.81 g/mol.

2. The NMR spectrum of 3C in C13CD showed peaks at: 171.8 ; 139.3 ; 122.6 ; 74.3 ; 57.3 ; 56.5 ; 56.0 ; 49.8 ; 43.4 ; 42.1 ; 39.5 ; 39.4 ; 37.9 ; 36.8 ; 36.4 ; 36.0 ; 35.6 ; 31.7 ; 31.6 ; 31.3 ; 29.6 ; 28.1 ; 27.8 ; 27.6 ; 24.1 ; 23.7 ; 22.7 ; 22.4 ; 20.9 ; 20.4 ; 19.2 ; 18. 6 and 11. 7 ppm.

3. The IR spectrum showed peaks at 2950,2938, 2869,2849, 1731 (C=O) cm All the test results corresponded to the expected values for AB-Chol.

Example 4 <BR> <BR> <BR> <BR> <BR> <BR> Description and synthesis Cholesteryl 33-4-N (2-hydroxyethyl)-N, N<BR> <BR> <BR> <BR> <BR> dimethylammonio) lbutanoate bromide<BR> <BR> <BR> <BR> HAB-Chol (VI) 1.23 g (9.87 mmol) of 2-bromoethanol and 0.1 ml (0.58 mmol) of N, N- diisopropylethylamine were added to a solution containing 1.0 g (2.00 mmol) of AB- Chol (V) in 50 ml of anhydrous acetone. The reaction mixture was heated then at reflux and stirred magnetically under a nitrogen atmosphere. The reaction progress was monitored by TLC (CHCl3 : MeOH 7: 3) and the partial conversion of AB-Chol was observed. 1.23 g (9.87 mmol) of 2-bromoethanol and 0.1 ml (0.58 mmol) of N, N- diisopropylethylamine were added again to the reaction mixture at 24 and 48 hours

after the beginning of reflux. The reaction was completed 72 hours after the beginning of reflux. The appearance of a main stain (Rf 0. 47) was observed.

The reaction mixture was evaporated to dryness and the resulting residue was purified by silica gel column chromatography. A CHCl3 : MeOH (4: 1 v/v) mixture was utilized as eluent. The evaporation to dryness of the product pure fractions yielded a yellowish white wax-like residue. 1.04 g of a product identified as HAB-Chol was thus obtained. The reaction yield was 83.2%. See Fig. 3.

The following information was obtained from the analyses applied to the resulting HAB-Chloe : 1. The mass spectrum test showed a peak for [M-Br] + at m/e 544 corresponding to the molecular formula C35H62BrNO3 and a molecular weight of 624.78 g/moll.

2. The NMR spectrum of 3C in C13CD showed peaks at: 171.4 ; 139.4 ; 130.9 ; 128.8 ; 122.9 ; 74.9 ; 56.6 ; 56.1 ; 55.9 ; 54.1 ; 52.3 ; 50.0 ; 42.3 ; 39.7 ; 39.5 ; 36.9 ; 36.5 ; 36.1 ; 35.7 ; 31.9 ; 29.6 ; 29.3 ; 28.0 ; 27.7 ; 23.8 ; 22.8 ; 22.6 ; 22.5 ; 21.0 ; 19.2 ; 18.7 ; 18.1 ; 17.3 ; 12.0 and 11.8 ppm.

3. The IR spectrum showed peaks at 3427 (OH), 2975,2950, 2869,2850, 1735 (C=O) cm-1.

All the test results corresponded to the expected values for HAB-Chol.

Example 5 <BR> <BR> <BR> <BR> <BR> <BR> <BR> Description and synthesis of Cholesteryl 3, B 4 [[2-[N-(2-hydroxyethyl)-<BR> <BR> <BR> <BR> <BR> <BR> N, N-(dimethylammonio) JethyllamineJbutanedioate bromide<BR> <BR> <BR> <BR> <BR> HDS-Chol (IX) 1.93 g (15.44 mmol) of 1-bromoethanol and 0.35 ml (2.04 mmol) of N, N- diisopropylethylamine were added to a solution containing 1.75 g (3.14 mmol) of DS- Chol in 75 ml of anhydrous acetone. The reaction mixture was heated then at reflux and stirred magnetically under a nitrogen atmosphere. The reaction progress was monitored by TLC (CHCl3 : MeOH 9: 1) and the partial conversion of DS-Chol substrate was observed. 1.93 g (15.44 mmol) of 2-bromoethanol and 0.35 ml (2.04 mmol) of N, N-diisopropylethylamine were added again to the reaction mixture at 24 and 48 hours after the beginning of reflux. After 72 hours of reflux, the presence of

non-reacting DC-Chol traces (Rf 0.48) in the reaction mixture, and the appearance of a main stain (Rf 0. 21) and impurity traces (Rf 0.70) were observed.

Thereafter, the reaction mixture was evaporated to dryness. The resulting oily residue was purified using a silica gel column chromatography. A CHCl3 : MeOH (9: 1 v/v) mixture was utilized as eluent. Pure fractions of this product were treated next with methanol. Then, the evaporation to dryness of the product pure fractions yielded a wax-like yellowish white solid. 0.86 g of a product identified as HDS-Chol was thus obtained. The reaction yield was 40. 1 %. See Fig. 4.

The following information was obtained from the analyses applied to the resulting HDS-Chol: 1. The mass spectrum test showed a peak for [MH-Br] + at m/e 602 corresponding to the molecular formula C37H65BrN204 and a molecular weight of 681.84 g/mol.

2. The NMR spectrum of 13C in C13CD showed peaks at: 171.8 ; 171.6 ; 139.5 ; 125.8 ; 122.2 ; 73.4 ; 65.2 ; 62.4 ; 56.2 ; 55.7 ; 55.0 ; 51.3 ; 49.5 ; 48.7 ; 41.9 ; 38.0 ; 37.7 ; 37.1 ; 36.5 ; 36.1 ; 35.7 ; 35.3 ; 32.9 ; 31.4 ; 29.8 ; 29.0 ; 27.8 ; 27.5 ; 23.9 ; 23.3 ; 22.7 ; 22.5 ; 20.6 ; 19.0 ; 18.8 ; 18.6 and 11.7 ppm.

3. The IR spectrum showed peaks at 3396 (OH), 2959,2936, 2869,2850, 1734 (C=O) cm-1.

All the test results corresponded to the expected values for HDS-Chol.

Example 6 Description and synthesis of Cholesteryl 3f3-4-2- (dimethylamine) ethylJ butanedioate CS-Chol (X) A solution containing 2.0 g (12.33 mmol) of 1, 1'-carbonyldiimidazole in 30 ml of anhydrous dichlorometane was slowly added to a solution containing 4.0 g (8.22 mmol) of cholesteryl hemisuccinate (S-Chol) (VIE in 30 ml of anhydrous dichloromethane. The reaction was performed using an ice-water bath at 5°C-8°C.

The reaction mixture was stirred magnetically for 8 hours. A solution of 2.8 ml (27.86 mmol) of 2- (dimethylamino) ethanol in 30 ml of anhydrous dichloromethane

was added then drop by drop to the reaction mixture. The reaction mixture was left stirring overnight at room temperature.

The reaction progress was monitored by TLC (CHCl3 : acetone 2: 1) until full conversion of the substrate (S-Chol) (Rf 0.48). The appearance of a stain (Rf 0.29) and impurity traces (Rf 0. 69) were observed.

The reaction mixture was evaporated to dryness. The resulting oily residue is prone to solidify in storage. The residue was treated with acetonitrile (40 ml) and cooled in an ice-water bath. A yellowish white wax-like solid resulted. This solid was centrifuged and rinsed thrice with acetonitrile. The solid was dried then with air. 4.01 g of a product identified as CS-Chol was thus obtained. The reaction yield was 87.5%.

See Fig. 5.

The following information was obtained from the analyses applied to the resulting CS-Chol: 1. The mass spectrum test showed a peak for [MH] + at m/e 558 corresponding to the molecular formula C35H59NO4 and a molecular weight of 557.86 g/mol.

2. The NMR spectrum of 13C in C13CD showed peaks at: 172.4 ; 171.6 ; 139.4 ; 122.6 ; 74.2 ; 62.2 ; 57.6 ; 56.5 ; 56.0 ; 49.9 ; 45.5 ; 42.2 ; 39.6 ; 39.4 ; 37.9 ; 36.8 ; 36.4 ; 36.1 ; 35.7 ; 31.8 ; 31.7 ; 29.3 ; 29.0 ; 28.1 ; 27.9 ; 27.6 ; 24.2 ; 23.7 ; 22.7 ; 20.9 ; 19.2 ; 18. 6 and 11. 8 ppm.

3. The IR spectrum showed peaks at 2938,2909, 2890,2868, 1727 (C=O) cm All the test results corresponded to the expected values for CS-Chol.

Example 7 Description and synthesis of Cholesteryl 3i-4 2 N (2-hydroxyethyl)- N, N-(dimethylammonio) ethyl butanedioate bromide HCS-Chol (XI) 3.33 g (26.66 mmol) of 2-bromoethanol and 0.27 ml (1.57 mmol) of N, N- diisopropylethylamine were added to a solution containing 3.0 g (5.38 mmol) of CS- Chol (X) in 135 ml of anhydrous acetone. The reaction mixture was heated then at reflux and was stirred magnetically under a nitrogen atmosphere.

The reaction progress was monitored by TLC (CHCl3 : MeOH: H20 65: 25: 4).

3.33 g (26.66 mmol) of 2-bromoethanol and 0.27 ml (1.57 mmol) of N, N- diisopropylethylamine were added again to the reaction mixture at 24 and 48 hours after the beginning of reflux. After 72 hours of reflux, the partial conversion of substrate (Rf. 0.68), and the appearance of a main stain (Rf 0. 41) and impurity traces (Rf 0.85) were observed.

The reaction mixture was evaporated then to dryness. The resulting oily residue was purified by a silica gel column chromatography according to the following protocol. The oily residue was grown in CHC13. The substrate and the impurity traces were eluted with a CHCl3 : MeOH (9: 1 v/v) mixture. The product of interest was eluted then with a CHCl3 : MeOH (8: 2 v/v) mixture. The evaporation of product pure fractions resulted in a yellowish white wax-like solid. 0.41 g of a product identified as HCS-Chol was thus obtained. The reaction yield was 63.8%. See Fig. 5.

The following information was obtained from the analyses applied to the resulting HCS-Chol: 1. The mass spectrum test showed a peak for [MH-Br] + at m/e 603 corresponding to the molecular formula C37H64BrNO5 and a molecular weight of 682.82 g/mol.

2. The NMR spectrum of'3C in C13CD showed peaks at: 130.9 ; 128.8 ; 77.2 ; 66.2 ; 53.1 ; 36.5 ; 31.9 ; 31.4 ; 30.1 ; 29.7 ; 29.4 ; 29.3 ; 29.2 ; 29.0 ; 27.9 ; 22.9 ; 22.8 ; 22.5 ; 19.3 ; 19.1 ; 18.7 ; 14.1 and 11.8 ppm.

3. The IR spectrum showed peaks at 3419 (OH), 2959,2926, 2873,2853, 1734 (C=O) cm-'.

All the test results corresponded to the expected values for HCS-Chol.

* * * * * All publications mentioned hereinabove are incorporated hereby in their entirety as references.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.