CA2248186C - Compounds related to the amidinium family, pharmaceutical compositions containing same, and uses thereof - Google Patents

Abstract

Novel amidinium derivatives of formula (I), wherein R1 is a cholesterol derivative or an alkylamino-NR'R" grouping, and each of R2 and R3 is independently a hydrogen atom or a grouping of formula (II), wherein each of R4 and R5 is independently a hydrogen atom or a grouping of formula (III), are disclosed. The corresponding pharmaceutical compositions, which are particularly useful in gene therapy for transferring therapeutic genes into cells, are also disclosed.

Description

COMPOUNDS RELATED TO THE FAMILY OF AMIDINIUMS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND THEIR
APPLICATIONS
The present invention relates to new compounds related to the family amidiniums, in particular guanidiniums, compositions pharmaceutical products containing them and their applications.
More specifically, the present invention relates to compounds of formula I and their salts O

~ O-R1 in which:
R1 represents a derivative of cholesterol or an amino-alkyl group NR'R "with R 'and R" independently representing fun of (other a radical aliphatic, saturated or unsaturated, linear or branched in C12 to C22, R2 and R3 are independently of each other a hydrogen atom or a group of general formula II with at least fun of them different a hydrogen atom, L (CH 2) n-5 in which:
- n and m represent independently fun of each other and distinctly between the groups R2 and R3 an integer between 0 and 4,

2 - R4 and R5 represent independently of one another an atom hydrogen or a group of formula III, where at least R4 or R5 is different from hydrogen Nha - (CHa) p (X) r CHa) - C ~
q NHa x in which p and q represent, independently of one another, an integer inclusive between 0 and 4 and r is equal to 0 or 1, when r is equal to 1 X representing a group NH and x being then equal to 1 or X representing a nitrogen atom and x being then equal to 2 and with p, q and r being able to vary independently between the groups R4 and R5.
As a preferred subfamily, we will mention more particularly in the framework of the present invention the compounds of general formula I in which R2 or R3 represent, independently of one another, an atom of hydrogen or a group of formula IV
NI-i2 _ 'C (~~) n ~ m (CHI p ~ N
in which IV
n, m and p are defined as above and R4 represents an atom hydrogen or a group of formula

3 Nha - (CHa) - (NHr-Cy NHa P
V
with p and r defined as before, and with m, n, p, q and r that can vary independently between different groups R2 and R3.
These new products of general formula (I) can be presented under form of non-toxic and pharmaceutically acceptable salts. These non-salts toxic include salts with mineral acids (hydrochloric acid, sulfuric, hydrobromic, phosphoric, nitric) or with acids organic (acetic, propionic, succinic, maleic, hydroxymaleic acids, benzoic, fumaric, methanesulphonic or oxalic) or with bases mineral (soda, potash, lithium, lime) or organic (tertiary amines such as triethylamine, piperidine, benzylamine).
As a representative of the compounds according to the invention, it is possible mention particularly the compounds of the following general sub-formulas:
(H ~ N) ~ '' C- NH - (CH2) ~ -NH -COO- Rl VI
[(H ~ N) 2+ C- NH - (CH 2) 3] [(H ~ N) ~ C + - NH - (CH 2) a] -N-COO-RI VII
[(H ~ N) ~ + C- NH - (CH ~) ~] ~ - N- (CH ~ 2 -NH -COO-R1 VIII
[(HZN) 2 C- (CHa) a] ~ N-COO-R 1 IX
[[(HzN) ~ + C- (CH 2) z] a- N- (CH 2) 2 ZN-COO-R 1 X
in which R1 has the above definition.

4 As representative of the guanidiniums and amidiniums claimed, it is possible mention particularly the compounds of the preceding formulas VII, VIII and IX
with R1 representing a cholesteryl group. The three compounds will be respectively identified below under the designation BGSC (Bis Guanidino Spermidine Cholesterol) and BGTC (Bis Guanidino Tren Cholesterol) and BADC
(bis Bis Amidinium Hydrochloride Diethylenetriamine Cholesterol).
The claimed compounds are particularly interesting from the therapeutic for their non-toxic characters and their properties amphiphilic.
Having regard to these qualities, they may in particular be used for complexation of nucleic acids in the perspective of a cellular transfection of those this. These Compounds can therefore be advantageously employed in gene therapy.
The compounds VII (BGSC) and VIII (BGTC) are particularly advantageous for gene transfer in vivo. These two compounds complex with DNA and protect against degradation due to pH changes during transport to the cell to be treated.
The invention therefore relates to a pharmaceutical composition which comprises at least less a compound according to the invention. In a preferred embodiment of (invention the compound is the Bis Guanidino Spermidine Cholesterol (BGSC) and in another Preferred mode the compound is Bis Guanidino Tren Cholesterol (BGTC).
The main goal of gene therapy is to correct diseases associated with a lack of expression and / or abnormal expression, it's up to say deficient or excessive, of one or more nucleic acids. We try to compensate to this type of genetic abnormalities through cell expression in vivo or in in vitro of cloned genes.
Today, several methods are proposed for deliverance intracellular of this type of genetic information. One of the technologies currently implemented, relies precisely on (use of chemical vectors or biochemicals. These synthetic vectors have two main functions, compact (DNA to transfect and promote its cellular fixation and its passage through The plasma membrane and, where appropriate, across both membranes nuclear.
Positively charged cationic lipids such as N- [1- (2,3-) dioleyloxy) propyl) -N, N, N-trimethylammonium (DOTMA) have thus been proposed.

Advantageously, they interact in the form of liposomes or small vesicles, spontaneously with DNA, which is negatively charged to it, for form Lipid-DNA complexes, able to fuse with membranes cellular, and thus allow the intracellular delivery of DNA. However, in case DOTMA, its good efficiency in transfection remains unfortunately associated with a defect of biodegradability and a character toxic to (Regarding cells.
Since DOTMA, other cationic lipids have been developed on this structure model: lipophilic group associated with an amino group via a arm says "Spacer". Among these, we can particularly mention those comprising title of lipophilic group two fatty acids or a derivative of cholesterol, and comprising, in addition, where appropriate, as an amino group, an ammonium group quaternary. DOTAP, DOBT or ChOTB can be cited in particular representative of this class of cationic lipids.
Because of their chemical structure and their biodegradability, the compounds claimed precisely meet the requirements for a vector of transfection of nucleic acids.
The present invention therefore also aims at any application of these new compounds in in vitro, ex vivo and / or in vivo transfection of cells and especially for the vectorization of nucleic acids. It relates in particular to all pharmaceutical composition comprising, in addition to at least one compound according to (invention, a nucleic acid.
In the compositions of the present invention, nucleic acid associated with least one claimed compound can be as well a deoxyribonucleic acid one

6 ribonucleic acid. It can be oligonucleotide sequences or polynucleotides of natural or artificial origin, including DNA
genomics, cDNA, mRNA, tRNA, rRNA, hybrid sequences or sequences synthetic or semi-synthetic. These nucleic acids can be original human, animal, plant, bacterial, viral, etc. They include preferably a therapeutic gene.
Another object of (invention thus relates to a pharmaceutical composition further containing a nucleic acid. Preferably, this nucleic acid is either a deoxyribonucleic acid is a ribonucleic acid. In a favorite mode of embodiment of the invention the nucleic acid comprises a therapeutic gene.
In the meaning of (invention, the term "therapeutic gene" especially means gene encoding a protein product having a therapeutic effect. The product protein thus coded may be a protein, a peptide, etc. This product Protein can to be homologous to the target cell (that is, a product that is normally expressed in the target cell when it does not show any pathology). In this case, the expression of a protein makes it possible, for example, to to overcome insufficient expression in the cell or (expression of an inactive protein or weakly active due to modification, or overexpress said protein. The therapeutic gene can also encode a mutant of a protein cell, having increased stability, modified activity, etc. The product protein can also be heterologous vis-à-vis the target cell. In that case, a protein expressed can, for example, supplement or provide a deficient activity in the cell, allowing him to fight against a pathology, or to stimulate a reply immune.

7 Among the therapeutic products within the meaning of the present invention, it is possible mention more particularly enzymes, blood derivatives, hormones, ies lymphokines: interleukins, interferons, TNF, etc. (FR26885141, growth, neurotransmitters or their precursors or enzymes synthesis, Ies trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, HARP / pleiotrophin, etc. Dystrophin or a rninidystrophin (~ R268I7.86), the CFTR protein associated with cystic fibrosis, tumor suppressor genes:
p53 Rb, RaplA, DCC, k-rev, e. ~ C (FR2704234.), Genes encoding factors involved in coagulation: Factors VII, VIII, IX, genes involved in the repair of (DNA, suicide genes (thymidine kinase, cytosine deaminase), ies genes from (hemoglobin or other protein transporters, the genes corresponding proteins involved in lipid metabolism, such as apolipoprotein selected from apolipoproteins AI, A-II, A-IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a), metabolic enzymes such as lipoprotein lipase, Ia hepatic lipase, lecithin cholesterol acyltransferase, 7 alpha cholesterol hydroxylase, phosphatidic acid phosphatase, or lipid transfer as the transfer protein of cholesterol esters and the phospholipid transfer protein, an HDL binding protein or again a receiver chosen, for example, from LDL receptors, receptors chylornicrons-remnants and receivers.scavenger.etc.
The therapeutic nucleic acid can also be a gene or a sequence antisense. whose expression in the target cell allows you to control (expression of genes or transcription of cellular mRNAs. Such sequences can, for example,

8 for example, to be transcribed in the target cell into mRNA complementary RNA
cells and thus block their translation into protein, according to the technique described in EP 140,308. Therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively killing RNAs targets (EP 321 201).
As indicated above, the nucleic acid may also include one or several genes encoding an antigenic peptide, capable of generating the man or (animal an immune response.) In this particular mode of implementation artwork, (invention allows the realization of either vaccines or treatments immunotherapeutic agents applied to humans or animals, particularly against microorganisms, viruses or cancers. This may include peptides antigens specific for Epstein Barr virus, HIV virus, hepatitis B
(EP 185 573), viras of pseudo-rage, syncitia-forming virus, others virus or still specific for tumors (EP 259,212).
Preferably, the nucleic acid also comprises sequences allowing the expression of the therapeutic gene and / or the gene coding for the peptide antigen in the cell or (desired organ).
which are naturally responsible for the expression of the gene considered when these sequences likely to function in the infected cell. he can also this is sequences of different origin (responsible for (expression of others proteins, or even synthetic). In particular, they may be promoter sequences of Genoa eukaryotic or viral. For example, they may be promoter sequences from the

9 genome of the cell that we want to infect. Similarly, it may be sequences promoters from the genome of a virus. In this respect, we can mention example promoters of the genes ElA, MLP, CMV, RSV, etc. In addition, these sequences expression can be modified by the addition of activation sequences, regulation, etc. It can also be promoter, inducible or repressible.
Furthermore, the nucleic acid may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the product therapeutic synthesized in the secretory pathways of the target cell. This sequence signal can be the natural signal sequence of the therapeutic product, but it can also any other functional signal sequence, or a signal sequence artificial.
The nucleic acid may also include a signal sequence directing the product therapeutically synthesized to a particular compartment of the cell.
The cationic lipids described in the present invention, and, in particular, the BGTC and BGSC, are able to complex with fADN, and represent a an advantageous alternative to viral vectors for the transfer of acids nucleic of therapeutic interest, in vitro, ex vivo or in vivo. Due to the properties chemical guanidinium groups, and in particular their high pKa, these lipids cationic are able to protect DNA molecules against damage due to pH variations. The results presented in the examples show that these Compounds allow transfection of many cell types, with a big efficiency. The efficiency of transfection depends in particular on the ratio lipid cationic / DNA in the complexes formed. A relationship between the two compounds particularly favorable for transfection is to have 6 to 8 groups guanidinium for a phosphate group on DNA.

The transfection efficiency of the cationic lipid / DNA complexes can be outraged be improved by addition of neutral lipid, and formation of liposomes Cationic.
These liposomes are formed by a complex between the cationic lipid and the lipid neutral. This one can be chosen from 5 - dioleoylphosphatidylethanolamine (DOPE), oleoyl palmitoylphosphatidylethanolamine (POPE), - di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamine their N-methylated derivatives 1 to 3 times;
phosphatidylglycerols,

Diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) - and asialogangliosides (such as in particular asialoGMl and GM2).
DOPE is preferably used.
Preferably, the compound of the invention and the neutral lipid are present in a ratio between 1 and 5, more preferably 2 and 4. In addition the total lipid ratio (compound of the invention plus neutral lipid) on DNA is advantageously chosen so that the net ratio of positive charges is understood between 2 and 5. Particularly advantageously, the ratio is about 3.
The invention therefore also relates to any pharmaceutical composition comprising a compound according to (invention, a neutral lipid and an acid nucleic.
Preferably, the compound according to the invention is chosen from BGTC and BGSC.

11 Also preferentially the neutral lipid is DOPE. Nucleic acid is either a deoxyribonucleic acid is a ribonucleic acid. Preferably (acid nucleic acid comprises a therapeutic gene.
In addition to this application of the claimed amidinium derivatives, it is also possible to consider their valuation in the following applications:
interference level of interactions between nucleic acids and proteins resulting in a inhibition or stimulation of certain processes, for example expression regulation genetic or enzymatic activity (polymerase, transcriptase, etc.), complexation selective anionic species for their extraction, elimination or detection using probes / membrane electrodes for example, use as a reactive laboratory to perform in vitro transfection operations, etc.
Accordingly, the present invention also relates to any application therapeutically of amidinium derivatives as described above, either directly, either within pharmaceutical compositions.
Preferably, the pharmaceutical compositions of the invention contain also a pharmaceutically acceptable vehicle for a formulation injectable, in particular for direct injection into the desired organ, or for topical administration (on skin and / or mucosa). It could be in particular of sterile, isotonic or dry solutions, especially freeze-dried, which, according to the case of sterilized water or senim physiological, allow the constitution of injectable solutions.
The present invention will be more fully described with the help of examples and following figures, which should be considered as illustrative and not limiting.
FIGURES
Figure 1 . Schematic representation of operating protocols of preparation of BGSC and BGTC.

12 Figure 2 Schematic representation of operating protocols of preparation of the BADC.
Figure 3: Measurement of transfection efficiency by ratio compound lipid / DNA.
Figure 4: Measurement of transfection efficiency by ratio lipids totals / DNA.
Figure 5: Measurement of the serum effect on the transfection efficiency of cell HepG2 (5A), HeLa (5B), NIH 3T3 (5C) and 293 (SD). Gray bars: Presence of serum; Empty bars: transfections in the absence of serum for 2h.
Figure b: Marine trachea cryosections photographs showing reporter gene expression in the epithelium of after in vivo transfection with BGTC / DOPE liposomes. (bA) and (bB): Detection of the expression of beta-galactosidase in transfected cells, located at surface of epithelium (bA) and in the sub mucosal glands (bB); (bC):
Detection cells expressing beta-galactosidase in the submucosal glands by immunoperoxidase labeling using an anti-beta monoclonal antibody galactosidase; (bD): detection of cells expressing luciferase in glands sub-mucosal by immunoperoxidase labeling using an antibody polyclonal anti-luciferase; (bE): immunoperoxidase staining: negative control carried out absence of antibodies.
Figure 7: Measurement of In Vivo Gene Transfer Efficiency by Injection intravenous DNA / BGTC-liposome complex.
Figure 8: Measurement of In Vivo Gene Transfer Efficiency by Injection intraperitoneal complex of a DNA / BGTC-liposome complex.
A- PREPARATION OF DERIVATIVES ACCORDING TO THE INVENTION
MATERIAL AND METHODS

13 1. Physical measures Proton nuclear magnetic resonance spectra (1H NMR) were recorded on a Bruker AC200 spectrometer. Chemical shifts are expressed in ppm relative to the TMS.
2. Chromatosraphies on silica Thin layer chromatography (lCCM) was carried out on Merck silica gel plates 0.2 mm thick Column chromatographies were carried out on silica gel 60 Merck with particle size 0.040-0.063mm.

PREPARATION OF GUANIDINO SPERMIDINE CHOLESTEROL
BGSC
-1; Preparation of Di-Boc Carbamate (1) A solution of cholesteryl chloroformate (0.9 g, 2 mmol) and Et 3 N (0.2148, 2 mmol) in CH 2 Cl 2 (40 ml) to a solution of N, BN-Boc 2 spermidine (0.6908, 2mmol) in CH2Cl2 (20m1). After reflux for 5 hours, the mixture is washed with water (2x50m1), dried over NazSO4 and the solvent evaporated. The product gross is purified by chromatography on silica gel using CH 2 Cl 2 / MeOH 95/5 as eluent to give pure carbamate (1.38, 86%).
H NMR. 8lCDCl; t 200 Mhz); 0.5-2.5 (m, cholesteryl structure, Boc signal and central CH2 groups from spermidine), 3.1-3.3 (m, 8H, N-CHZ), 4.50 (m, 1H, H 2), 5.37 (d, 1H, H 6).
- 2. Preparation of the aminocarbamate (2)

14 The compound (1) (1.3 g) is dissolved in 20 ml of CH 2 Cl 2 and added to this cooled solution (ice bed) 2m1 of CF3COZH freshly distilled to release BOC protective groups. After stirring at room temperature for 3 hours 50 ml of N NaOH are added and the mixture is extracted with CH 2 Cl 2.
are washed with water, dried over NaS04 and evaporated to obtain crude aminocarbamate (2) (0.945 g, 98%).
H NMR 8 (CDCl 3 200 Mhz); 0.5-2.5 (m, cholesteryl structure and groups from spermidine), 2.7 (m, 4H, CH 2 -N-CO-), 3.25 (broad m, 4H, N-CHZ) 4.49 (m, 1H, H 2), 5.35 (d, 1H, H 6).
3.- Summary of the BGSC.
The crude carbamate (2) (0.94 g) is dissolved in 30 ml of 85/15 THF / MeOH. Then 1-pyrazolecarboxamidine (0.495 g, 3.4 mmol) is added and of isopropylethylamine (0.436, 3.4 mmol) in 30 ml of THF / MeOH 85/15. After stirring at room temperature for 18 hours 250 ml is added slowly of diethyl ether and the precipitate obtained by decantation is separated off. The crude compound is put in suspension three times in diethyl ether and separated by decantation for give some Pure GSC (0.570g, 47%).
1H NMR 8 (DMSO d6) 200 MHz; 0.5-2.5 (m, structure of cholesteryl and CHz central), 3.1 (m, 8H, N-CH 2), 4.3 (m, 1H, H 2), 5.3 (d, 1H, H 6); 7.2 (large s, 8H, NHz +), 7.8 {s, 2H, NH), Anal. Calc. for C₁ ,H6N₂O₂HCl, C, 62.15; H, 9.67; N, 13.71.
Elemental analysis result: C, 62.28; H, 9.81; N, 13.15.

PREPARATION OF GUANIDINO TREN CHOLESTEROL BGTC
Preparation of Aminocarbamate (S) A solution of cholesteryl chloroformate (1.8 g, 4 mmol) in 100 ml of CH 2 Cl 2 at a saturation of tris (2-aminoethyl) amine (TREN) (11.68 g, 80 mmol) in 400 ml of CH 2 Cl 2. After stirring at temperature ambient for two hours, the inert amine is liberated by washing with water (3 × 100 ml) and after Dry over Na 2 SO 4 and the solvent was evaporated. Isolation (5) in the form of trihydrochloride as follows: the crude product is dissolved in 10 ml of MeOH and added dropwise a saturated solution of methanolic HCl (Sm1); the precipitate got is separated by settling and, to obtain a pure compound, is put into suspension three in MeOH and separated by decantation. After vacuum drying trihydrochloride (6) pure is obtained (1.71g, 64%).
1H NMR 8 (CDCl 3 + ~ CD ~ OD 200MHz). 0.5-2.4 (m, cholesteryl structure), 2.5 (m, 2H, CH2NHCO), 2.8 (broad s, 4H, + HN-CHZ), 3.06 (broad s, 4H, CH2NH3 +), 3.20 (m, 2H, HN-CH 2), 4.4 (m, 1H, H 2), 5.3 (d, 1H, H 6), Anal. Calc. for C₁ ,H₁ NN40Oz, 3HCl, C, 61.10; H, 9.80; N, 8.38. Result: C, 61.25; H, 9.96;
N, 8.22.

2- Preparation of B.GTC
A solution of cholesteryl chloroformate (2.24 g, 5mmol) in 100m1 of CHZCIZ at a wide saturation of tris (2-aminoethyl) amine (TREN) (29.2g, 200mmol) in 250m1 of CH2Cl2. After stirring at at room temperature for 2 hours, the inert amine is liberated by washing at the water (3x100m1) and after drying over NaaSO4, the solvent is evaporated. The gross product (3.2 g) is dissolved in 50/50 THF / MeOH (20m1); then we add to the mixture of 1H-pyrazole-1-carboximidine (1.465 g, 10 mmol) and diisopropylamine (1.3 g, 10 mmol).
After stirring at room temperature for 18 hours, diethyl ether and the precipitate obtained is separated by decantation. In order to obtain a pure sample, the crude compound is naked in suspension three times in diethyl ether and separated by decantation (2.15 g), 60% after drying under vacuum.
1H NMR (DMSOd6 200MHz); 0.5-2 (m, cholesteryl structure), 2.2 (m, 2H, N-CH 2), 2.6 (broad s, 4H, N-CHI), 3.0 (d, 2H, N-CH 2), 3.2 (broad s, 4H),

16 4.3 (m, 1H, H 2), 5.3 (d, 1H, H 6), 7.3 (broad s, BH, NH 2 +), (7.8 (broad s, 2H, NH).
Calc.
for C ~ H ~ N802,2HC1; C, 60.39; H, 9.57; N, 15.65. Result: C, 60.38; H, 9.67;
N, 15.56.

PREPARATION OF BIS-CHLORHYDRATE OF BIS AMIDINIUM
BA D.
l.Preparation of dinitrile A solution of cholesteryl chloroformate (8.97 g, 20 mmol) is added.
in CH2Cl2 (100m1) dropwise to a solution of iminopropionitrile (2.468, 20 mmol) and Et 3 N (2.02 g, 20 mmol in CH 2 Cl 2 (100 ml).
the room temperature, for 6 hours, washed with water (2x 100m1) and dried over Na2S04. The solvent is evaporated and the crude product (10.5 g) is recrystallized in the methanol. (8.59 g, 80%).
1H NMR 8 (CDCl 3 200 MHz). 0.5-2.5 (m, cholesteryl structure), 2.7 (m, 4H;
-CH 2 CN), 3.62 (m, 4H, -CH 2 -N-), 4.50 (m, 1H, H 2), 5.39 (d, 1H, H 6).
2. Preparation of dithioamide In a solution of dinitrile, prepared as previously described, (0.3248, 6mmol) and diethylamine (0.8848, 12mmol) in DMF (30m1), maintained at 50 ° C, a slight current of HzS is bubbled for 2 hours. We pour the solution blue on ice and the precipitate thus obtained is separated by filtration.
We dissolve raw product in CHZCh and washed with water (2x50m1). After drying over Na 2 SO 4, the The solvent is evaporated and the product obtained recrystallized twice from methanol (1.868, 51.5%).
1H NMR (DMSOd6 200MHz); 0.5-2.5 (m, structure of cholesteryl>, 2.67 (t, 4H, N-CH 2), 3.50 (t, 4H, S = C-CH 2), 4.3 (m, 1H, H 2), 5.33 (d, 1H, H 6). .

3. Preparation of dithioamidate bis-iodide To a solution of dithioamide (0.6038, lmmol) in acetone (20mI), add methyl iodide (2ml) and let stand at room temperature ambient for 48 hours. The precipitate obtained (0.7418, 83.5%) is filtered. This product is used directly without additional purification.
4. Preparation of bis amidinium bishydrochloride BADC.
In a refluxing suspension of bis-iodide (0.7418, 0.83 mmol), as previously obtained, in isopropanol (10 ml), a current of NH3 for 2 hours and then continue heating at reflux 4 hours additional.
The isopropanol is evaporated and the residue is taken up in methanol (10 ml).
After passage of this solution on a basic ion exchange resin, one makes pass a hydrochloric acid stream in the boiling and evaporated to dryness. The bis hydrochloride thus obtained, is dissolved in the minimum of ethanol (about 5 ml). Filter a light insoluble and the filtrate is added a large excess of diethyl ether. We gets a powder that is filtered and dried (320 mg, 60%). A recrystallization in isopropanol makes it possible to isolate the expected product, in an analytically pure.
1H NMR (DMSOd6, 200MHz); 0.5-2.6 (m, cholesteryl structure and-CHZ-amidinium), 3.57 (s, broad 4H, N-CHr) 4.3 (m, 1H, H ~), 5.3 (d, 1H, H6), 8.64 (s, large;
2H; NH?), 9.07 (s; broad; 1H; NH), 9.18 (s; broad; 2H).
Anal. Cal. For C34H ~ N04a, 2HCl: C, 65.25. H, 9.32; N, 8.94. Result C, 65.40. H, 9.87; N, 9.55.
B: USE OF DERIVATIVES ACCORDING TO THE INVENTION FOR
GENE TRANSFER IN VITRO AND IN VIVO
MATERIALS AND METHODS
1- The following transfection assays are performed with BGSC and BGTC prepared according to the protocols described in Examples 1 and 2.

2 - Preparation of the smes A mixture of cationic lipid and DOPE (in a molar ratio of 3/2) in chloroform (CHCl3) is evaporated in vacuo and resuspended in a 20 mM HEPES buffer solution at pH 4, under N 2 atmosphere.
The final concentration of lipid is 1.2 mg / ml.
The mixture is vortexed for 5 minutes, then "sonicated" for 5 minutes with .un sonifier (Branson Ultrasonic 2210), finally stored at + 4 ° C for 24 hours h for hydration.
The resulting dispersion is "sonicated" again (Sonifier Branson 450) for 5 to 10 mm to form liposomes.
After centrifugation, the solution is filtered through a diameter filter.
pore 0.22p, (Millex GS, Millepore) and stored at + 4 ° C.
The size of liposomes containing BGSC or BGTC was studied using a laser diffraction apparatus (Autosizer 4? 00, Malvern Instruments). This study shows a single peak corresponding to a mean diameter of 50nm in multimodal analysis by numbers.
3 - Cell cultures The origin (species and tissues) of most of the cell lines used for the transfection experiments is given in Table I.
The cells of the HeLa cell line (P Briand, ICGM Paris) are derived of human carcinoma of cervical epithelial origin.
NIH 3T3 cells (C Lagrou, Institut Pasteur, Lille) are fibroblasts mouse.
* (marks of co ~~experce) The NB2A cell line (C. Gouget, Paris) is derived from a neuroblastoma mouse.
All cells except AtT-20 and PC12 cells were cultivated in modified Dulbecco's medium (DMEM, GIBCO) supplemented with 10% serum fetal calf (FCS, GIBCO), penicillin 100 units / ml (GIBCO) and streptomycin (GIBCO) at 100 ~ g / ml.
AtT-20 cells were cultured in DMEM / F12 (GIBCO) supplemented with 10% fetal calf serum.
PC12 cells were cultured in DMEM supplemented with 10%
FCS and 5% horse serum.
All the cells were kept in culture in plastic boxes (Falcon) under a humid atmosphere at 5% CO 2.
4 - Plasmids PRSV-Luc plasmids (O. BENSAUDE, ENS, Paris), and pRSV-nlsLacZ
were amplified in E. coli and prepared by CsCl gradient purification by standard techniques. In the plasmid pRSV-nlsLacZ, and the plasmid pRSV-Luc, the LacZ gene of E. coli and its nuclear localization signal sequence and the gene luciferase are respectively under the transcriptional control of the LTR / RSV (Rous Sarcoma Virus). The plasmid pXL2774 contains the gene coding for luciferase under control of the cytomegalovirus (CMV) promoter.
5 - Protocol of in vitro transfection The day before transfection 2 X 105 cells are deposited per well (Falcon 6-well plates).
Each well contains a different cell line.
Thus, on the day of transfection, the cells are half confluent.

The plasmid DNA (5 μg) and the desired amount of bis-guanidinium lipid were were each diluted in 2501 DMEM without FCS and vortexed. After about 5 minutes the two solutions were allowed to incubate at room temperature for l5min. The Transfection mixture is then added to the cells (0.5 ml per well) which have been Washed with serum-free medium. After 4 to 6 hours of incubation at 37 ° C, 1 ml of medium with serum is added to each well without removing the mixture of transfection. 24 hours after transfection, the medium is replaced by 1 ml middle fresh culture. Cells are collected 2 days after transfection to measure the expression of luciferase.
Control transfections were made using transfections marketed Transfectam lipopolyamine (JP Behr, Strasbourg, France) was used in alcoholic solution at an optimum ratio of ionic charges Transfectam ~ / DNA 6-8.
15 - Lipofectin ~ (Life Technologies Inc., Cergy Pontoise, France) which is a formulation of liposomes having DOPE and lipid as neutral lipid cationic the DOTMA. (N [1 (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride).
DNA / Liposome complexes were obtained under standard conditions recommended by the manufacturer.
Cells were transfected by phosphate precipitation from calcium.
(Keown, WA, Campbell, CR; Kucherlapati, RS (1990) Methods in Enzymology ;
Academic Press: New York, 185, 527-537).
6 - Measurement of luciferase for in vitro transfections The luciferase activity is measured 48 hours after transfection using variant the procedure of De Wet et al. (De Wet, JR, Wood, KV, DeLuca, M., Helinski, DR
& Subramani, S. (1987) Mol. Cell. Biol. 7, 725-37.) the culture medium is removed the cells are washed with a cold phosphate buffer saline solution - Then they are lysed by incubation in 250 ~ .1 of lysis buffer (25 mM
pH7 triphosphate, 8, 8 mM MgCl2, 1mM dithiotreitol, 15% glycerol and 1%
triton X-100).
a clear lysate is obtained after removal of the insoluble materials by centrifugation (15mm at + 4 ° C) in a microcentrifuge.
an aliquot (20 μl) of cell extract is diluted in 100 μl of buffer of lysis to which 9p, 1 of 25mM ATP (sigma) and 20μl of 25mM luciferin (sigma) are added.
the samples are placed in a luminometer (Lurmei LB950I Berthold, Nashua, NH) and the light emission is measured for 10 seconds.
The calibration of the RLU / Luciferase curve was done using different luciferase dilutions of purified firefly (sigma) and shows that the part linear of the curve goes from 10 ° to 10 'RLU (relative Iight units).
The protein concentration was measured by the BCA test (acid bicinchoninic) using bovine secumalbumin as a control.
Data for luciferase activity are expressed in RLU / mg protein.
7 - Prostocol ~ injection in vivo in the airways of the mouse The mice used are male OF1 mice (weight 30 g) derived from Iffa Creed (Lyon, France). BGTC / DOPE cationic liposomes (3: 2 molar ratio) are used. The transfection mixture is obtained from 10 μg of DNA
plasmid (in10u1 water), 20p.1 of cationic liposomes in 20mM Hepes medium (pH
7.4) at a total lipid concentration of 5 mg / ml. The aggregates DNA / lipids as well trained have a charge ratio of 6.
* (trade mark) After anesthesia of the mice with pentobarbital, the transfection mixture (30 ~, I) is injected into the respiratory tract by instillation intratrachealeale via a cannula inserted into the tracheal lumen. 48 hours after instillation the animals are sacrificed by overdose of pentobarbital and the lungs and tracheas taken for analysis.
$ - Coloring at fX-ga_1 To assay (expression of E. coli beta-galactosidase in cultures primary cells, the cells are impregnated for 15 minutes at 4 ° C. with solution of 4% paraformaldehyde and then incubated with fX-gal chromogenic galactosidase (Sigma). The blue coloration of the nuclei cell transfected is assayed by luminescence microscopy.
9-Assay of X-GaI Cells at Respiratory Traces Transfected in viv For the detection of X-gal cells in the respiratory tract, the lungs and tracheae are treated in 4% parafor- malaldehyde, immersed then overnight in PBS containing 30% sucrose and then incorporated into a middle freezing and frozen with liquid nitrogen. Cryostats sections of 5 ~.
are then stained for ~ 3-galactosidase activity by incubating an entire night with X-gal reagent (Sigma).
10- Determination of luciferase in the respiratory tract transfected in vivo In order to determine the luciferase activity after in vivo transfections, fragments of tissues are placed in the presence of 30.1 of lysis buffer (25 mM Tris-phosphate pH7.8, ImM dithiothreitol (DTT), 15% glycerol and 1% Triton X-100), lysed in about lmin with a homogenizer (Polylabo, Strasbourg, France) and kept sure ice cream. After centrifugation, 20.1 of lysate is used for the assay.
The activity luciferase is expressed in RLU per mg of protein.
*(trademark) 11- Immunohistochemistry protocol Cryosections of lung and tracheal specimens are incubated lh with either monoclonal anti-E.coli 3-galactosidase antibodies at 5 ~ g / ml (Genzyme, Cambridge, MA) or polyclonal rabbit antibodies 1: 400 dilution (Promega).
Escherichia coli 3-galactosidase protein is assayed by staining with immunoperoxidase using a secondary anti mouse Ig antibody marked at the biotin (Boehringer) and streptavidin-POD conjugate (Boehringer). Protein luciferase is assayed by immunoperoxidase staining using a antibody goat anti-rabbit (ICN Biomedicals, Inc.) and the peroxidase-system antiperoxidase rabbit (PAP) (Dako Glostrup, Denmark). The sections are examined by microscopy luminescent. Negative controls are performed without antibodies.

IN VITRO TRANSFECTIONS TO AGENT REPORTS
DIFFERENT TRANSFECTION / DNA
In order to optimize (transfection efficiency obtained with aggregates BGTC / DNA, the Applicant has studied (influence of the ratio BGTC / DNA on the rate of transfection into 3 different types of mammalian cells known to their relative sensitivity to conventional transfection methods. The results are presented in figure 3.
Transfection experiments were therefore performed with fibroblasts 3T3 cells of HeLa human epithelial cells and neuroblastomas mouse NB2A. During these experiments a certain fixed amount of plasmid pRSV-Luke has been contacted with varying amounts of BGTCen solution. For determine the reporting range to study the plaintiff is part of the principle that one g of DNA equals 3 nm of negatively charged phosphate and that only two guanidinium groups are positively charged to neutral formation pH
of the aggregates and transfection. The luciferase activity 48 hours after the transfection.
The expression of luciferase is maximal for aggregates containing 6 (cells HeLa) to 8 (3T3 cells, NB2A) guanidinium groups for a phosphate group DNA ie aggregates with a significant positive charge.

IN VITRO TRANSFECTIONS WITH TYPICAL DIFFERENTS
CELL.
Gene transfer through cationic lipids is a attractive transfection technique not only because it does not does not require intermediaries but also because it makes it possible to transfect a very wide variety tissue cells and different organisms. To study the extent of the effectiveness of BGTC as a transfection agent several cell lines of origins various have been tested. The results are shown in Table 1 below. The report loads was chosen between 6 and 8 for maximum efficiency. In order to establish a comparison, cells from the same lines were also transfected using a lipopolyamine on the one hand and on the other hand calcium phosphate. The results show that the BGTC is normally as effective as the Transfectam and two times more effective than calcium phosphate.
Table I. Expression of luciferase in different cell lines of mammals transfected with either BGTC, calcium phosphate or Transfectam ~.
IZLU / mg of proteins cell Tissue Lines BGTC Phosphate Transfectam ~

Calcium cell Human A549 Carcinoma 4 X 105 7 X 10 3.1 X 105 lung Monkey COS-7 Kidney Transform2.1 X 3.7 X 10 '8.4 X 10 by 10 Dog MDCK-1 Kidney 3 X 4.5 X 10 2.2 X 10 Rat RIN-m5 Tumor cell 2 X 2 X 10 '3.3 X 10' of pancreatic island10 ' ROS Osteosarcoma 4.0 4.5 X 105 2.3 X lOb PC12 Pheochromocytoma3 X 1.7 X 10 6 X 106 IO ' Mouse AtT-20 Pituitary Tumor2 X 8 X 10 '3 X 10' 10 ' All transfecdons were done at least twice (n? 2).

IN VITRO TRANSFECTIONS IN THE PRESENCE OF A NEUTRAL LIPID
The BGSC compound being less soluble in aqueous medium than the BGTC the Applicant used the first form of liposomes in the presence of a lipid neutral DOPE (dioleoylphosphatidylethanolamine). The formulations are prepared in a 3/2 molar ratio. A second preparation containing this time of BGTC has also been prepared with DOPE under the same conditions.
6.1 - Determination of the total lipid / DNA ratio.
The Applicant first determined the lipid / DNA ratio during of a transfection experiment on HeLa cells. The curve obtained is represented in FIG. 4. The transfection optima are obtained for reports BGSC / DNA liposomes between 1.5 and 5 with a 2.5-3 center. The groups guanidinium being protonated at neutral pH, the best aggregates BGSC-DOPE / ADN
15 have a positive charge of about 3 (Figure 4).
The best BGSC-DOPE / DNA ratios are therefore lower than those obtained with aggregates of BGTC / DNA type (between 6 and 8). Transfection in the presence of cationic liposomes is facilitated by the presence of DOPE and the properties fusogenic of it.

WO 97/31935 ~ PCT / FR97100364 6.2 - Transfection of different cell lines by liposomes Cationic.
The Applicant has carried out transfection experiments in different cell lines using cationic liposome formulations BGSC-DOPE
and BGTC-DOPE in 3/2 molar ratios. The charge ratio guanidinium the lipids / phosphates of fDNA is about 2.5 (~ 3) for these two systems of gene transfer. Transfection with Lipofectin is used as a control. The results are shown in Table 2 below.
These results show that cholesterol derivatives bearing groups guanidiniums as cationic liposomes are at least as effective for transfection as Lipofectin. These results can of course be optimized for each cell line.
Table 2 Expression of luciferase in various cell lines arvotiaues transfected with liposome liposomes BGSC / DOPE liposomes BGTC / DOPE and L ~ fectin RLU / mg of cellular proteins Liposome liposome lines BGTCLipofectin BGSC Cellular HeLa 4.6 X 106 7.7 X 106 3.3 X 106 COS-7 ND 1.4 X 10 '9.5 X 106 MDCK-1 1 X 106 7 X 106 1.9 X 106 NB2 Aa 1.5 X 10 '1.4 X 10' N

NIH 3T3a 7 X 106 1.5 X 106 ND

All transfections were done at least three times (n? 3) e: average RLU measurement of luciferase activity per 100 g / 500 g of lysate total (n? 4).

STUDY OF THE EFFECT OF PRESENCE OF SERIATED PROTEINS
ON THE EFFECTIVENESS OF GENE TRANSFER IN VITRO
Most cationic lipid transfection compositions have decreased efficacy in the presence of senim. This example object of test the sensitivity of the compounds of the invention to (serum effect.
7.1 - Formulations of cationic lipids The lipids described in the invention are solubilized in ethanol.
i) solutions of "liposomes" are obtained in the presence of DOPE as follows: DOPE (AVANTI) in solution in chloroform is added to solutions of lipid in fethanol, in a cationic lipid / DOPE molar ratio of 3/2.
After dry evaporation the mixtures are taken up in the water and heated to 50 ° C for 30 minutes ;
ü) "micelle" solutions are obtained by following the described protocol to obtain "liposomes" but the DOPE solution is replaced by of chloroform.
The solutions are all adjusted to 10 mM cationic lipid.
7.2 - Nucleic Acid The nucleic acid used is the plasmid pXL2774.
7.3 - Cytofectant mixtures (extemporaneous preparation) WO 97/31935 PCT / FR9? / 00364 The DNA is diluted to 20 ~ g / ml in 150 mM NaCl and the various cationic lipid formulations are diluted in water at 40, 80, 120 and 160 ~ .M. The solutions of DNA and lipid are mixed volume to volume which gives ratios in nmoles / ~ g DNA respectively of 2, 4, 6 and 8; saline concentration is mM
7.4. - Transfections The cells are cultured under the appropriate conditions in microplates of 24 wells (2 cm2 / well) and are transfected while they are in phase exponential growth and at 50-70% of the confluence. The cells are washed twice 500 pl of medium deprived of serum proteins and restored to growth either in medium without serum [transfection in the absence of serum], either in complete medium [transfection in presence of serum] and 50 ~ .1 of cytofectant mellate [0.5 μg DNA / well] are added to cells [3 wells / lipid-DNA condition].
When the cells are transfected in "absence of serum" the medium of growth is supplemented with the appropriate amount of serum 2 hours after the transfection.
Transfection efficiency is assessed by measuring the expression of the luciferase according to the recommendations given for the use of the Promega kit [Luciferase Assay System]. The toxicity of cytofectant mixtures is estimated by one measuring protein concentrations in cell lysates.
7.5 - Results (Figure 5) The results obtained with four cell types [NIH3T3 - 293 - HepG2 -HeLa] show that, under the conditions tested, formulations in the form of of "Liposomes" are more effective than those in the form of "micelles". By elsewhere, contrary to what is observed with most preparations cytofectantes containing cationic lipids, no significant inhibitory effects related to the presence of serum for BGTC in the form of "liposomes" or "micelles" could not be placed highlighted in our transfection conditions.

EXPRESSION IN VIVO OF TRANSGENE IN THE TRACKS
MOUSE RESPIRATORY WITH BGTC / DOPE LIPOSOMES.
To do this, we used the cationic liposomes BGTC / DOPE.
Obtaining the DNA / liposome aggregates and the administration protocol are performed as described in material and method.
X-gal cells are detected in the epithelium of the respiratory tract of the mice treated 48 hours after transfection at the level of the cells of the plasmid LacZ
has helped BGTC / DOPE liposomes. The results are presented in Figure 6.
Most transfected cells are located in the trachea and only some X-gal cells are observed in the respiratory tract secondary. This type of distribution has already been reported in other transfections cell.
We also performed an immunohistochemistry to identify the product of the reporter gene. Majority, it is the mature cells that are transfected to level of the surface of the epithelium (Figure 6A). These data show that cationic liposomes are able to induce gene transfection in cells non-proliferative and fully differentiated pathway epithelium respiratory.
Expression of the transgene is also detected in the sub-glands.
mucosal (Figure 6B). Detection by immunological staining of cells transfected, using a monoclonal antibody directed against E coli (3-galactosidase, confirmed this expression in the submucosal glands and the surface epithelium (Figure 6C). The transgene expression is also highlighted in the glands under-mucosal staining by immunoperoxidase staining with an antibody directed against the luciferase protein after transfection of p RSV-Luc with BGTC / DOP (FIG. 6D).
Negative controls are performed without antibodies (Figure 6E).

These results are of particular interest for gene therapy directed against muscavidosis. Indeed, to treat this pathology, it is necessary to target the submucosal glands which are the main site of the expression of the CFTR in the human bronchi.

QUANTITATIVE DETERMINATION OF TRANSFECTION EFFICIENCY
BGTC / DOPE IN VIVO
To do this, the lungs and trachea of mice, treated with liposome BGTC / DOPE and plasmid pRSV-Luc under the conditions described in Example 8, 10 are taken 48 hours after transfection and the luciferase activity assayed according to the protocol described in material and method.
The results are shown in Table 3 below.
Table 3 VECTORS ACTIVITY LUCIFERASE

(RLU / rn rotin) BGTC / DOPE Liposomes 2.8x105 pRSV-Luc (average: 3 x 104 - 8 x 105) RSV-Lake Z 0 Naked DNA

RSV-Luc 0 Luciferase activity is systematically detected in homogenates 15 trachea but not in those of the lungs. This observation is in agreement with the previous results obtained when examining the distribution of cells positive to Xgal. It is noted that the expression luciferase is directly linked to the plasmid of expression since no activity could be detected in the control containing a LacZ plasmid. As a witness, it was also realized a transfection of plasmid without vector. In this control trial, no expression of luciferase.
These data demonstrate the efficacy of BGTC / DOPE liposomes for In vivo gene transfection of the respiratory tract.

GENE TRANSFER IN VIVO BY INJECTION
INTRAVENOUS OR INTRAPERITONEAL
The formulations of the cationic lipids and the DNA are described in the example 7.
10.1 - Dose Effect of Cationic Lipids (Figures 7-8) BalbC mice, 5 weeks old, are injected intravenously (tail vein) or intraperitoneally by 200 ~ 1 of transfectant mixture solution in 37.5 mM NaCl, 5% glucose and the expression of luciferase is sought at hours post-transfection in the lung after intravenous injection, and in liver and spleen after intraperitoneal injection. The organs are harvested cold in lysis buffer [Promega E153A] supplemented with protease inhibitors [Boehringer 1697498] and homogenized with a Heidolph mill DIAX600. The activity luciferase is sought in the supernatant 140008 tissue extracts.
Intravenous injection Each mouse receives 50 μg of DNA complexed with different concentrations of BGTC in the form of "liposomes". The optimum ratio of lipid nanomoles cationic / ~ g DNA observed is 9 (Figure 7); no toxicity could be implementation evidence for doses up to 900 nanomoles of BGTC injected.
Intra-ritoneal injection BGTC in the form of "liposomes" was injected with 100 μg of DNA per mouse. In both the liver (Figure 8B) and the spleen (Figure 8A), the maximum expression is obtained for i, 5 nanomoles BGTC / ~, g of DNA.
10.2 - Kinetics of expression after intravenous injection We sought biodistribution of luciferase expression in organs in function of the time following the injection of 200 ~ 1 of mixture BGTC / DNA "liposomes" in 37.5 mM NaCl, 5% glucose [50 ~ g DNA and 9 nanomoles BGTC / ~, g DNA] in the vein of the tail of BalbC mice aged 5 weeks. The results are expressed in picogram of luciferase extracted by organ following a calibration curve with luciferase marketed under form crystallized. The detection limit is between 0.5 and 1 picogram of luciferase.
We highlight that, under the conditions tested, the maximum of expression is obtained at 23 hours post-transfection for 6 of the 7 organs studied, know the diaphragm, the gastrocnemius, the heart, the lung, the kidney and the missed.
Expression in the liver seems earlier. The maximum expression is obtained at the pulmonary level with a rate of 0.5 nanogram at 23 o'clock transfection.

Claims (22)

1. Compound of general formula I or a salt thereof in which:
- R1 represents a cholesterol derivative or an amino alkyl group -NR'R" with R' and R" independently representing a radical aliphatic, saturated or not, linear or branched at C12 to C22, R2 and R3 independently represent a hydrogen atom or a group of general formula II with at least one of them different of one hydrogen atom, in which:
- n and m represent independently of each other and distinctly between the groups R2 and R3 an integer between 0 and 4 - R4 and R5 independently represent an atom of hydrogen or a group of formula III, where at least R4 or R5 is different from hydrogen in which p and q represent independently of each other an integer comprised between 0 and 4 and r is equal to 0 or 1, when r is equal to 1 X representing an NH group and x then being equal to 1 or X representing a nitrogen atom and x then being equal to 2 with p, q and r being able to vary independently between the R4 groups and R5. 2. Compound according to claim 1, characterized in that R2 and R3 y represent independently of each other a hydrogen atom or a group of general formula IV

in which n, m and p being as defined in claim 1 and R4 represents a hydrogen atom or a group of formula V

with p and r being as defined in claim 1, and with m, n, p, q and r being able to vary independently between the different R2 and R3 groups. 3. Compound according to claim 1 or 2 characterized in that it is a compound represented by one of the general sub-formulae following:

(H2N)2+C-NH-(CH2)2-NH-COO-R1 VI
[(H2N)2+C-NH-(CH2)3[(H2N)2+C-NH-(CH2)4]-N-COO-R1 VII
[(H2N)2+C-NH-(CH2)2]2- N-(CH2)2 -NH -COO- R1 VIII
[(H2N)2+C-(CH2)2]2 N-COO-R1 or IX
[[(H2N)2+C-(CH2)2]2-N-(CH2)2]N-COO- R1 X
wherein R1 is defined according to claim 1. 4. Compound according to any one of claims 1 to 3 characterized in that it is chosen from [[(H2N)2+C-(CH2)2]2-N-(CH2)2]2N-COO-R1 [(H2N)2+C-NH-(CH2)3][(H2N)2+C-NH-CH2)4]-N-COO-R1 and [(H2N)2+C-(CH2)2]2N-COO-R1 in which R1 represents a cholesteryl group. 5. Compound according to any one of claims 1 to 4, characterized in that it is Bis Guanidino Spermidine Cholesterol (BGSC). 6. Compound according to any one of claims 1 to 4, characterized in that it is Bis Guanidino Tren Cholesterol (BGTC). 7. Pharmaceutical composition characterized in that it comprises at least one compound according to any one of claims 1 to 6, combined to a nucleic acid. 8. Pharmaceutical composition according to claim 7, characterized in that the compound is Bis Guanidino Spermidine Cholesterol (BGSC). 9. Pharmaceutical composition according to claim 7, characterized in that the compound is Bis Guanidino Tren Cholesterol (BGTC). 10. Composition according to any one of claims 7 to 9, characterized in that the nucleic acid is a deoxyribonucleic acid. 11. Composition according to any one of claims 7 to 9, characterized in that the nucleic acid is a ribonucleic acid. 12. Composition according to any one of claims 7 to 11, characterized in that the nucleic acid comprises a therapeutic gene. 13. Pharmaceutical composition comprising a compound according to claim 1, a neutral lipid and a nucleic acid. 14. Pharmaceutical composition according to claim 13, characterized in that the compound is chosen from BGTC and BGSC. 15. Pharmaceutical composition according to claim 13, characterized in that the neutral lipid is the dioleoylphosphatidylethanolamine (DOPE). 16. Pharmaceutical composition according to claim 13, characterized in that the nucleic acid is a deoxyribonucleic acid. 17. Pharmaceutical composition according to claim 13, characterized in that the nucleic acid is a ribonucleic acid. 18. Pharmaceutical composition according to claim 16 or 17, characterized in that the nucleic acid comprises a therapeutic gene. 19. Pharmaceutical composition according to any one of claims 7 to 18, characterized in that it comprises a vehicle pharmaceutically acceptable for an injectable formulation. 20. Pharmaceutical composition according to any one of claims 7 to 18, characterized in that it comprises a vehicle pharmaceutically acceptable for application to the skin and/or mucous membranes. 21. Use of a compound according to any of claims 1 to 6 for the in vitro, ex vivo and/or in vivo transfection of cells. 22. Method for transferring nucleic acid into cells comprising contacting said nucleic acid with a compound according to claim 1, and incubating the resulting mixture with said cells.