EP1137793A2 - Novel nucleic acid transferring agents, compositions containing them and uses - Google Patents

Abstract

The invention concerns novel transfer agents, compositions containing them and their uses for transferring in vitro, in vivo or ex vivo nucleic acids into cells. More precisely, the invention concerns novel nucleic acid transfer agents comprising a hydrophobic spacer chemically bound to a polycation and to at least a hydrophilic substituent.

Description

 NOVEL NUCLEIC ACID TRANSFERS, COMPOSITIONS CONTAINING THEM, AND USES THEREOF

The present invention relates to new transfer agents, the compositions containing them and their uses for the in vitro, in vivo or ex vivo transfer of nucleic acids into cells.

With the development of biotechnology, the possibility of efficiently transferring nucleic acids into cells has become a basic technique with many biotechnological applications. It may be the transfer of nucleic acids into cells in vitro, for example for the production of recombinant proteins, or in the laboratory for the study of the regulation of gene expression, the cloning of genes or any other manipulation involving DNA. It can also involve the transfer of nucleic acids into cells in vivo, for example for the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of genes into cells taken from an organism, with a view to their subsequent re-administration, for example for the creation of transgenic animals.

Currently, the most common way to transfer genes into cells is through the use of viral vectors. But these are not completely without risks, several other methods based on the use of synthetic vectors have been proposed. These synthetic vectors have two main functions: to complex and compact the nucleic acid to be transfected, and to promote its passage through the plasma membrane and possibly through the two nuclear membranes.

Several families of synthetic vectors have been developed, such as, for example, polymers or biochemical vectors (made up of a cationic protein associated with a cellular receptor), but significant progress has mainly been made in non-viral transfection with the development of lipofectants, and more particularly cationic lipids. It has thus been demonstrated that lipids Because of their positive overall charge, cationic cells spontaneously interfered with globally negative DNA, forming nucleolipid complexes capable of fusing with cell membranes, and thus allowed intracellular release of DNA.

Different kinds of cationic lipids have thus been synthesized: lipids comprising a quaternary ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE, etc.), lipopolyamines such as, for example, DOGS, DC-Chol or the disclosed lipopolyamines in patent application WO 97/18185, lipids combining both a quaternary ammonium group and a polyamine such as DOSPA, or alternatively lipids comprising various other cationic entities, in particular amidinium groups (for example ADPDE, ADODE or the lipids of patent application WO 97/31935). In fact, the structural diversity of cationic lipids partly reflects the observation of the structure-activity relationship.

However, the use of these synthetic vectors still poses numerous difficulties, and their effectiveness remains to be improved. In particular, it would be desirable to be able to have non-cationic or less cationic vectors, for various reasons:

- the complexes formed between the nucleic acid and the transfer agents, because of their overall positive charge, tend to be taken up by the reticuloendothelial system which induces their elimination,

- due to the overall positive charge of the complexes formed, plasma proteins tend to adsorb on their surface, and this results in a loss of transfection power, - in a context of local injection, the presence of a large positive overall charge prevents the diffusion of the nucleic acid complexes outside the administration site, since the complexes adsorb on the extracellular matrices. The complexes can therefore no longer reach the target cells, which, by way of consequence, leads to a decrease in the transfer efficiency compared to the quantity of complexes injected,

- and finally, many players in the field of non-viral gene transfection have reported that cationic lipids or polymers have an inflammatory effect.

Furthermore, the stable formulation of the synthetic vectors developed until today at low charge ratios is generally difficult, if not impossible, and it has also been found that at low charge ratio, the transfer efficiency is often weak (Pitard et al., PNAS USA, 94, pp. 14412-14417, 1997). In the following, "charge ratio" means the ratio of the positive charges of the transfer agent to the negative charges of DNA. This ratio is often expressed in nmoles of transfer agent per μg of DNA.

These are the problems that the new transfecting agents developed by the applicant, and which are the subject of the present invention, propose to solve. Indeed, their particular structure forms a hydrophobic anchor linked on the one hand to a polycation which allows the formation of complexes with nucleic acids and on the other hand to at least one hydrophilic head which makes it possible to decrease the apparent overall charge density of these transfecting agents with respect to lipids or cationic polymers conventionally used in non-viral transfection. The presence of at least one hydrophilic head creates a sort of “charge screen” by reducing the zeta potential of the complexes formed with the nucleic acid. Thus, said complexes appear less cationic to the organism, with the beneficial consequences which result therefrom. In addition, it has been shown that the transfecting agents according to the present invention are particularly advantageous from a physicochemical point of view since they are particularly stable when they are brought into contact with nucleic acids at low charge ratios.

Thus, a first object of the invention relates to new nucleic acid transfer agents which comprise a hydrophobic spacer linked chemically on the one hand to a polycation and on the other hand to at least one hydrophilic substituent.

Polycation makes it possible to form complexes with nucleic acids by interactions with the anionic charges of nucleic acids. The hydrophobic spacer has a dual function. On the one hand, it allows passage through cell membranes, and on the other hand, it makes the complexes formed with nucleic acids viable in biological medium. Indeed, the hydrophobic spacer creates a physical constraint on the complexes which makes it possible to protect the nucleic acids from the external environment. The hydrophobicity necessary for the complexes to be viable can be easily determined by a person skilled in the art, by application of ordinary research methods or by the usual method of trial and error. Finally, the presence of the hydrophilic substituent (s) makes it possible to reduce the zeta potential of the complexes formed, which causes said less cationic complexes to appear in the outside environment.

Within the meaning of the invention, polycation is a linear or branched polycationic molecule capable of associating with nucleic acids. In the sense of the invention, the term “association with nucleic acid” means any type of bond, such as, for example, covalent bonds, electrostatic, ionic interactions, hydrogen bridges, etc. Preferably, the polycation is a linear or branched polyamine , each amino group being separated by one or more methylene groups. Optionally, the polyamine may also be substituted by other cationic functions, for example amidinium or guanidinium groups, cyclic guanidines, etc. It may especially be a polycation as defined in patent applications WO 96 / 17823, WO 97/18185, WO 97/31935, WO 98/54130 or also WO 99/51581, and more generally in all the literature concerning cationic lipid structures known to those skilled in the art. According to a preferred aspect of the invention, the polycation represents a polyamine of general formula (II) in which :

- R ,, R ₂ and R ₃ represent independently of each other a hydrogen atom or a group (CH ₂ ) _q NR'R "with q an integer which can vary from 1 to 6, this independently between the different groups R „R, and R ₃ , it being understood that at least one of R ,, R ₂ and R ₃ is different from a hydrogen atom,

- R 'and R "independently of one another represent a hydrogen atom or a group (CH ₂ ) _q NH ₂ with q defined as above,

- m represents an integer between 1 and 6, and - n and p independently represent whole numbers between 0 and 6, with when n is greater than or equal to 2, m can take values and R ₃ have different meanings within the general formula (II), and when n is equal to 0, at least one of the substituents R, and R ₂ is different from a hydrogen atom. Other possible polycations can also be chosen from spermine, spermidine, cadaverine, putrescine, hexamethylenetetramine (hexamine), methacrylamidopropyl-trimethylammonium chloride (AMBTAC), 3-acrylamido-3-methylbutyltrimethylammonium chloride ( AMBTAC), polyvinylamines, polyethyleneimines, or even ionenes. (references: Barton et al., Comprehensive Organic Chemistrv, Vol. 2, Ed. Pergamon Press, p. 90; Encyclopedia ofPolymer Science and Engineering, 2 ^nd Edition, Ed. Wiley Interscience, Vol. 1 1, p. 489; Mahler and Cordes, Biological Chemistrv, Harper International Edition, p. 124.)

The hydrophobic spacer can take very varied structures as long as it provides sufficient hydrophobicity to allow protection of the nucleic acids and passage through the membranes. This sufficient hydrophobicity can be determined by a person skilled in the art by applying research methods. ordinary. According to a preferred variant of the invention, the hydrophobic spacer consists of 2 or 3 linear fatty hydrocarbon chains (that is to say between 10 and 20 carbon atoms per chain, and preferably 12, 14, 15, 16, 17, or 18 carbon atoms per chain, each chain can be of different length). According to another variant, the hydrophobic spacer consists of a very long linear fatty hydrocarbon chain, that is to say comprising between 20 and 50 carbon atoms, and preferably between 40 and 50 carbon atoms and even more preferably between 44 and 50 carbon atoms).

Suitable hydrophilic substituents are for example chosen from hydroxy, amino substituents, polyols, sugars, or also hydrophilic peptides. Polyol is understood to mean any linear, branched or cyclic hydrocarbon molecules comprising at least two hydroxy functions. By way of example, mention may be made of glycerol, ethylene glycol, propylene glycol, tetritols, pentitols, cyclic pentitols (or quercitols), hexitols such as mannitol, sorbitol, dulcitols, cyclic hexitols or inositols etc. (Stanek et al., The Monosaccharidess Académie Press, pp. 621-655 and pp. 778-855).

According to an advantageous variant, the transfer agents according to the invention comprise at least one hydrophilic substituent which is a sugar. For the purposes of the invention, the term “sugar” means any molecule consisting of one or more saccharides. Mention may be made, by way of example, of sugars such as pyranoses and furanoses, for example glucose, mannose, rhamnose, galactose, fructose, or even maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorose, melibiose etc. Preferably, the sugar or sugars are chosen from glucose, mannose, rhamnose, galactose, fructose, lactose , sucrose and cellobiose. In addition, it can also be so-called "complex" sugars, that is to say several sugars covalently coupled to each other, each sugar preferably being chosen from the list cited above. As suitable polysaccharides, mention may be made of dextran, α-amylose, amylopectin, fructans. mannans, xylans and arabinans. Some sugars preferred can also interact with cellular receptors, such as certain types of lectins.

More particularly, the transfer agents according to the invention can be represented by the general formula (I):

for which :

- R represents a polycation,

- Z represents a hydrogen atom or a fluorine atom, the different Z being independent of each other, and - either x and y, independently of one another, represent integers between 10 and 22 inclusive, and X and Y, independently of one another, represent a hydrogen atom, an -OAlk group where Alk represents a straight or branched alkyl containing 1 to 4 carbon atoms, a hydroxy group, an amino group, a polyol, a sugar, a hydrophilic or non-hydrophilic peptide, or an oligonucleotide, it being understood that at least one of the substituents X and Y represents a hydrophilic group chosen from hydroxy, amino, polyols, sugars, or hydrophilic peptides,

- either x is equal to 0 or 1, y is an integer between 20 and 50, X is either a hydrogen atom or a group -OAlk where Alk represents a straight or branched alkyl containing 1 to 4 carbon atoms, and Y is a hydrophilic group chosen from hydroxy, amino, polyols, sugars, or hydrophilic peptides.

Within the meaning of the invention, the polycation, the polyols and the sugars of the general formula (I) are as defined above.

The terms x and y are defined in the general formula (I) so as to take any value between 10 and 22 inclusive or between 20 and 50 inclusive depending on the case. Of preferably, x and y, independently of one another, are between 12 and 18 inclusive. More preferably, x and y are, independently of one another, 14, 15, 16, 17 or 18. When x is equal to 0 or 1, then y is preferably between 30 and 50, or between 40 and 50. More preferably, it is between 44 and 50.

For the purposes of the invention, the term “oligonucleotide” means chains containing one or more nucleotides, deoxynucleotides, ribonucleotides and / or deoxyribonucleotides which are monomeric units which differ from one another by the presence of bases which can be chosen from adenine, guanine, cytosine, thymidine or uracil [see Lehninger Biochemistry, Flammarion Médecine Sciences, 2nd edition, p. 305-329]. Owing to their property in forming base pairs, oligonucleotides are widely used in molecular biology, for example as linkers or as probes. Furthermore, the oligonucleotides can also be used in the form of conjugates, that is to say coupled to one or more other molecules having distinct properties. By way of example, mention may be made of the coupling of an oligonucleotide with a reactive chemical group, with fluorescent or chemiluminescent groups, or even with groups capable of promoting intermolecular interactions so as to promote entry into cells. Such conjugates, described in Bioconjugate Chemistry [John Goodchild, Conjugates of Oligonucleotides and Modified Oligonucleotides: a Review oftheir Synthesis and properties, Vol. 1, No. 3, 1990, pp. 165-187], have many uses and advantages such as for example the capacity to improve the entry of complexes in the cells, to decrease the rate of degradation by nucleases, to increase the stability of the complex concerned, to follow the becoming oligonucleotides in an organism, etc. Thus, the oligonucleotide (s), when they are grafted onto the transfer agents according to the present invention, make it possible to provide an additional property to said transfer agents (for example targeting properties , marking etc.). The oligonucleotides can be obtained according to the conventional methods known to those skilled in the art, and it is also possible to synthesize oligonucleotides modified according to the methods described in Bioconjugate Chemistry, John Goodchild, Conjugates of Oligonucleotides and Modified Oligonucleotides: a Review of their Synthesis and properties, Vol. 1, No. 3, 1990, pp. 165-187 or in Tetrahedron, Beaucage et al., 772nd Synthesis of Modified} Oligonucleotides by the Phosphoramiditeb Approach and Their Application, Vol. 49, No. 28, pp. 6123-6194, 1993.

For the purposes of the invention, the term “peptide” is intended to mean chains containing one or more amino acids linked together by bonds of peptide nature [Lehninger Biochimie, Flammarion Médecine Sciences, 2nd edition]. These can be the 20 "classic" amino acids, that is to say those commonly found in the composition of proteins (Alamine, Valine, Leucine, Isoleucine, Proline, Phenylalamine, Tryptophan, Methionine, Aspartic Acid, Glutamine, Lysine, Arginine, Histidine, Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamic Acid), or it can also be the so-called "rare" amino acids such as 4-hydroxyproline, desmosine, 5 -hydroxylysine, N-methyllysine, 3-methylhistidine, episodesmosine etc. Finally, it may also be the amino acids appearing in different cells or various tissues in free or combined form and which generally derive from α acids -aminated (for example β-alamine, γ-aminobutyric acid, homocysteine, ornithine, canavanine, djenkalic acid, β-cyanoalamine etc.). Such peptides can for example allow targeting of certain cell types. In this context, mention may, for example, be made of the RGD or NLS peptides. It may also be peptide sequences having labeling properties, that is to say allowing identification, for example by analysis techniques such as fluorescence spectrometry, infrared spectrometry, nuclear magnetic resonance (NMR ) etc. For example, mention may be made of the linear or cyclic peptide or pseudopeptide sequences comprising the Arg-Gly-Asp epitope (Arginine-Glycine-Aspartic Acid) for recognition of the primary and / or secondary receptors for adhesion proteins of the integrin type. . The peptides according to the invention can also be substituted at the level of one or more of their functional groups, for example at the level of the α carboxyl, of the amino function in α and / or at the level of the functional groups of the side chain of each of the amino acids. By way of example, there may be mentioned substitutions with saturated or unsaturated, linear, branched or cyclic aliphatic groups containing 1 to 24 carbon atoms, such as for example cholesteryl, arachidonyl or retinoyl radicals, or alternatively mono- or polyaromatics such as, for example, substituted or unsubstituted benzyloxycarbonyl, benzylester or rhodaminyl derivatives. The interest of such substitutions is part of the modification of the chemical and possibly biological properties of said peptides, for example in order to mark them. When said peptides are used as hydrophilic substituent, these are chosen from hydrophilic peptides, that is to say peptides consisting only of hydrophilic amino acids or those partially composed of hydrophilic amino acids and the composition makes them generally hydrophilic.

According to a preferred variant of the invention, the Z groups all represent hydrogen atoms.

According to a more particularly advantageous aspect of the invention, the transfer agents have the general formula (III):

for which: - R represents a polycation, and

- either x and y, independently of one another, represent integers between 10 and 22 inclusive, and X and Y, independently of each other, represent a hydrogen atom or a sugar, being it being understood that at least one of the substituents X and Y represents a sugar, - either x is equal to 0 or 1, y is an integer between 20 and 50, X is a hydrogen atom and Y is a sugar. Within the meaning of the invention, the polycation, the sugars and x and y in the general formula (III) are as defined above for the general formula (I).

More particularly preferred transfer agents are of general formula (III) and x and y, independently of one another, represent integers between 10 and 22 inclusive, and one of X and Y represents an atom d hydrogen and the other a sugar. According to another advantageous variant, the transfer agents according to the invention have the general formula (III) and x is equal to 0, y is an integer between 40 and 50, X represents a hydrogen atom, and Y is a sugar.

It is understood that the present invention also relates to the isomers of the products of general formula (I) when they exist, as well as their mixtures, or their salts.

In particular, the compounds of the invention can be in the form of non-toxic and pharmaceutically acceptable salts. These non-toxic salts include the salts with mineral acids (for example hydrochloric, sulfuric, hydrobromic, phosphoric, nitric), with organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic or oxalic), with mineral bases (soda, potash, lithine, lime), or with organic bases (tertiary amines such as triethylamine, piperidine, benzylamine).

According to the invention, the preparation of the products of general formula (I) is carried out by implementing the following steps:

1) An alkyl chain containing x carbon atoms (x being defined as above), comprising a hydroxy function and an ester function, is firstly prepared by opening a corresponding lactone. The reaction is generally carried out in an alcohol, at basic pH and at a temperature between -10 ° C and room temperature. For example, the alcohol can be methanol or ethanol. 2) Then, group X is fixed to the bifunctional alkyl chain obtained in the previous step. When X represents a sugar, condensation is carried out in a chlorinated solvent, such as, for example, dichloromethane or chloroform, and in the presence of a Lewis acid, at a temperature between -5 ° C. and 10 ° C. Lewis acid can for example be chosen from tin chloride, iron chloride, p-toluene sulfonic acid (tsOH), trimethylsillyltrifluoromethane sulfonic (TMStf), boron trifluoride etherate etc. [Kazunobu Toshima et al., Recent Progress in O-glcosilation Methods and its Application to Natural Products Synthesis, Chem. Rev. 1993, Vol. 93, pp. 1503-1531]. When X represents a hydrophilic peptide group or not, peptide coupling is carried out according to conventional methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or by any analogous method known to those skilled in the art. In particular, the reaction is generally carried out in the presence of a non-nucleophilic base in suitable aprotic solvents, at a temperature between 0 and 100 ° C., the pH being adjusted between 9 and 1 1. By way of example, the chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as a solvent. The non-nucleophilic bases used are preferably tertiary amines, calcium carbonate or sodium dicarbonate. Even more preferably, the bases used are tertiary amines such as, for example, triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out between 0 and 50 ° C, and preferably between 10 and 30 ° C.

When it is desired that X represents a hydroxy group, this step is not carried out. When X represents an amino group, the reaction is carried out by nucleophilic substitution according to the conventional methods known to those skilled in the art which make it possible to obtain an amine from an alcohol.

When X represents an —OAlk group, the alcohol function is alkylated according to the conventional methods known to a person skilled in the art or by analogous methods. For example, a diazo compound of formula can be reacted general Alk-N ₂ optionally in the presence of a catalyst such as HBF ₄ or silica gel. We can also operate under the conditions of the Williamson Reaction which consists in reacting in basic medium a compound of general formula Alk-Hal where Hal represents a halogen atom such as chlorine, bromine or iodine, on the chain carrying an alcohol function.

The same Williamson type reaction can also be implemented when it is desired that X represents a polyol.

Finally, when X represents an oligonucleotide, it is coupled to the bifunctional chain according to the conventional methods known for covalently grafting an oligonucleotide. For example, said oligonucleotide can be grafted via a suitable linker.

3) Thirdly, the ester function present on the bifunctional chain is hydrolyzed to an acid function according to known methods. For example, one can operate in a basic medium in an alcohol with a high boiling point, at a temperature between 50 ° C. and the reflux temperature of the reaction mixture.

4) Then, a substituted or unsubstituted alkylamine chain of general formula (IV):

H ₂ N- (CH ₂ ) _y -Y (IV)

in which y and Y are defined as above, is coupled to the compound obtained in the previous step, according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or by any method analog known to those skilled in the art.

In particular, the reaction is generally carried out in the presence of a non-nucleophilic base in suitable aprotic solvents, at a temperature between 0 and 100 ° C., the pH being adjusted between 9 and 11. For example, chloroform , dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as solvent. The non-nucleophilic bases used are preferably tertiary amines, carbonate of calcium or sodium dicarbonate. Even more preferably, the bases used are tertiary amines such as, for example, triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out between 0 and 50 ° C, and preferably between 10 and 30 ° C. The group of general formula (IV) is either commercially available or it can be obtained by condensation of Y on the corresponding unsubstituted alkylamine according to a method analogous to that described previously in 2).

5) The amide obtained in the previous step is then reduced to an amine. This is done according to conventional methods known to those skilled in the art. For example, one operates in an anhydrous organic solvent such as anhydrous tetrahydrofuran, by the action of lithium aluminum hydride LiAlH ₄ . Other reducing agents which can be used are for example borane, borane in dimethylsulfide (BH ₃ - SMe ₂ ), sodium borohydride / titanium tetrachloride (NaBH ₄ , TiCl ₄ ), phosphorus oxide chloride on Zinc (POCl ₃ / Zn), phosphorus pentasulfide (P ₄ S ₁₀ ) on Raney nickel, etc. [Richard C. Larock, Comprehensive Organic Transformations, VCH Publishers Inc., 1989]. One can also operate by catalytic hydrogenation. Advantageously, the reduction is carried out by the action of lithium aluminum hydride LiAlH ₄ , in anhydrous tetrahydrofuran, at the reflux temperature of the mixture. A compound of general formula (V) is thus obtained:

for which X, Y, x and y are defined as above.

6) Finally, in a last step, the acid derivative corresponding to polycation R as defined above, is coupled to the compound of general formula (IV) obtained in the previous step, according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or by any analogous method known to those skilled in the art. In particular, the reaction is generally carried out in the presence of a non-nucleophilic base in suitable aprotic solvents, at a temperature between 0 and 100 ° C., the pH being adjusted between 9 and 11. For example, chloroform , dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as solvent. The non-nucleophilic bases used are preferably tertiary amines, calcium carbonate or sodium dicarbonate. Even more preferably, the bases used are tertiary amines such as, for example, triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out between 0 and 50 ° C, and preferably between 10 and 30 ° C.

The acid derivatives corresponding to the polycation are commercially available.

According to another variant, the transfecting agents according to the present invention can be prepared by operating as follows:

1) An alkyl chain containing x carbon atoms (x being defined as above), comprising a hydroxy function and an ester function, is firstly prepared by opening a corresponding lactone. The reaction is generally carried out in an alcohol, at basic pH and at a temperature between -10 ° C and room temperature. For example, the alcohol can be methanol or ethanol.

2) Then, this bifunctional alkyl chain is coupled with a substituted or unsubstituted alkylamine chain of general formula (IV):

H ₂ N- (CH ₂ ) _y -Y (IV)

in which y and Y are defined as above in the general formula (I).

The reaction is carried out at a temperature above the melting point of each product, with or without vacuum. The reaction can also be carried out at reflux temperature in the presence of an alcoholic solvent. For example, the solvent can be methanol or ethanol. One operates for example at a temperature between 45 ° C and 60 ° C.

The reaction can also be carried out at reflux temperature of the mixture in the presence of an alcohol such as methanol as solvent. Another alternative consists in coupling the compound of general formula (IV) directly with the lactone (in this case, the first step of opening the lactone is not carried out).

The group of general formula (IV) is either commercially available or it can be obtained by condensation of Y on the corresponding unsubstituted alkylamine according to a method analogous to that described above.

3) the bifunctional double-stranded amide obtained is then reduced to an amine. We operate for this according to conventional methods. For example, one operates in an anhydrous organic solvent such as anhydrous tetrahydrofuran, by the action of lithium aluminum hydride (LiAlH ₄ ). Other reducing agents which can be used are, for example, borane, boron hydride sulfide dimethyl (BH ₃ -SMe ₂ ), sodium borohydride / titanium tetrachloride (NaBH ₄ , TiCl ₄ ), chloride. phosphorus oxide on Zinc (POCl ₃ / Zn), phosphorus pentasulfide (P ₄ S ₁₀ ) on Raney nickel, etc. [Richard C. Larock, Comprehensive Organic Transformations, VCH Publishers Inc., 1989]. One can also operate by catalytic hydrogenation. Advantageously, the reduction is carried out by the action of lithium aluminum hydride LiAlH ₄ , in anhydrous tetrahydrofuran, at the reflux temperature of the mixture. A compound of general formula (VI) is thus obtained:

for which Y, x and y are defined as above. 4) Then, the group X is condensed on the amine of general formula (VI) obtained in the previous step. The condensation is carried out according to methods analogous to those described above for the first synthetic route.

5) Finally, in a last step, the acid derivative corresponding to polycation R as defined above, is coupled to the compound of general formula (VI) obtained in the previous step, according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or by any analogous method known to those skilled in the art. In particular, the reaction is generally carried out in the presence of a non-nucleophilic base in suitable aprotic solvents, at a temperature between 0 and 100 ° C., the pH being adjusted between 9 and 11. For example, chloroform , dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as solvent. The non-nucleophilic bases used are preferably tertiary amines, calcium carbonate or sodium dicarbonate. Even more preferably, the bases used are tertiary amines such as, for example, triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out between 0 and 50 ° C, and preferably between 10 and 30 ° C. The acid derivatives corresponding to the polycation are commercially available.

Naturally, when the substituents of X, Y and / or of the polycation can interfere with the reaction, it is preferable to protect them beforehand with compatible radicals which can be put in place and eliminated without touching the rest of the molecule. This is done according to the conventional methods known to those skilled in the art, and in particular according to the methods described in TW GREENE, Protective Groups in Organic Synthesis, 2nd Edition, Wiley-Interscience, in McOMIE, Protective Groups in Organic Chemistry, Plenum Press (1973), or in Philip J Kocienski, Protecting Groups, Thieme. Furthermore, each step of the preparation process can be followed, where appropriate, by steps of separation and purification of the compound obtained according to methods known to those skilled in the art.

As an illustrative example of advantageous nucleic acid transfer agents according to the invention, mention may be made of the following compounds:

OH

Another subject of the invention relates to the compositions comprising a nucleic acid transfer agent as defined above, and a nucleic acid. The respective amounts of each component can be easily adjusted by those skilled in the art depending on the transfer agent used, the nucleic acid, and the desired applications (in particular the type of cells to be transfected).

For the purposes of the invention, the term “nucleic acid” is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They may be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA

(CDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA

(RRNA), hybrid sequences or synthetic or semi-synthetic sequences, modified or unmodified oligonucleotides. These nucleic acids can be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries. They can be chemically modified.

As regards more particularly deoxyribonucleic acids, they can be single or double stranded as well as short oligonuleotides or longer sequences. In particular, the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes, etc. These deoxyribonucleic acids can carry an origin of functional or non-functional replication in the target cell, one or more marker genes, transcription or replication regulatory sequences, genes of therapeutic interest, modified or unmodified antisense sequences, regions to other cellular components, etc.

Preferably, the nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences, for example one or more promoters and a transcriptional terminator active in the target cells.

Within the meaning of the invention, the term “gene of therapeutic interest” is understood to mean any gene coding for a protein product having a therapeutic effect. The protein product thus coded can in particular be a protein or a peptide. This protein product can be exogenous homologous or endogenous with respect to the target cell, that is to say a product which is normally expressed in the target cell when the latter presents no pathology. In this case, the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein. The gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, activity modified, etc. The protein product can also be heterologous towards the target cell. In this case, an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.

Among the therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 92/03120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc. ) apolipoproteins (ApoAI, ApoAIV, ApoE, etc., FR 93/05125), dystrophin or a minidystrophin (FR 91/1 1947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc., FR 93/04745), genes coding for factors involved in coagulation (Factors VII, VIII, IX), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), genes of hemoglobin or other protein transporters, enzymes of metabolism, catabolism etc.

The nucleic acid of therapeutic interest can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308. The therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321,201).

As indicated above, the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response. In this particular mode of implementation, the invention makes it possible to carry out either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers. These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", d other viruses or tumor-specific antigenic peptides (EP 259,212).

Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the desired cell or organ. These may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the E1A, MLP, CMV, RSV, etc. genes. In addition, these expression sequences can be modified by adding activation, regulation sequences, etc. It can also be a promoter, inducible or repressible.

Furthermore, the nucleic acid can also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also include a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell. The compositions according to the invention may also comprise one or more adjuvants capable of associating with the transfer agent / nucleic acid complexes and of improving their transfecting power. In another embodiment, the present invention therefore relates to compositions comprising a nucleic acid, a nucleic acid transfer agent as defined above and at least one adjuvant capable of associating with the transfer agent complexes / nucleic acid and improve its transfecting power. The presence of this type of adjuvant (lipids, peptides or proteins for example) can advantageously make it possible to increase the transfecting power of the compounds. With this in mind, the compositions of the invention can comprise, as an adjuvant, one or more neutral lipids.

More preferably, the neutral lipids used in the context of the present invention are lipids with two fatty chains. In a particularly advantageous manner, natural or synthetic lipids are used, zwitterionic or devoid of ionic charge under physiological conditions. They can be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), distearoyl, - palmitoyl, -mirystoylphosphatidylethanolamines as well as their N-methylated derivatives, phosphatidyl glycols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelines) or also asialogangliosides (such as in particular asialoGMl and GM2).

These different lipids can be obtained either by synthesis or by extraction from organs (example: the brain) or eggs, by conventional techniques well known to those skilled in the art. In particular, the extraction of natural lipids can be carried out using organic solvents (see also

Lehninger, Biochemistry).

More recently, the applicant has demonstrated that it was also particularly advantageous to use, as an adjuvant, a compound intervening or not directly at the level of the condensation of said nucleic acid, such as those described in patent application WO 96/25508. The presence of such a compound, within a composition according to the invention, makes it possible to reduce the amount of transfecting agent, with the beneficial consequences which result therefrom from the toxicological point of view, without causing any damage to the activity. transfectant. By compound involved in the condensation of nucleic acid is meant to define a compacting compound, directly or indirectly, nucleic acid. More precisely, this compound can either act directly at the level of the nucleic acid to be transfected or intervene at the level of an annex compound which is directly involved in the condensation of this nucleic acid. Preferably, it acts directly at the nucleic acid level. In particular, the precompacting agent can be any polycation, for example polylysine. According to a preferred embodiment, the agent involved in the condensation of the nucleic acid derives in whole or in part from a protamine, a histone, or a nucleoline and / or one of their derivatives. Such an agent can also consist, in whole or in part, of peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of units can vary between 2 and 10. In the structure of the compound according to the invention, these units can be repeated continuously or not. Thus they can be separated by links of a biochemical nature, for example by one or more amino acids, or of a chemical nature.

Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant for one equivalent of nucleic acid in mol / mol and, more preferably, from 0.5 to 5.

In a particularly advantageous embodiment, the compositions according to the present invention further comprise a targeting element making it possible to direct the transfer of the nucleic acid. This targeting element can be an extracellular targeting element making it possible to direct the transfer of DNA to certain, cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells, etc.). It can also be a targeting element intracellular allowing to direct the transfer of the nucleic acid towards certain privileged cellular compartments (mitochondria, nucleus etc.). The targeting element can be linked to the nucleic acid transfer agent according to the invention, or also to the nucleic acid as has been specified above. When the targeting element is linked to the nucleic acid transfer agent of general formula (I), the latter preferably constitutes one of the substituents X or Y.

Among the targeting elements which can be used in the context of the invention, there may be mentioned sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives. Preferably, these are sugars, peptides or proteins such as antibodies or antibody fragments, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc. In particular, they may be ligands for growth factor receptors, cytokine receptors, cell lectin receptors, or ligands with RGD sequence with an affinity for adhesion protein receptors such as integrins. Mention may also be made of the transferrin, HDL and LDL receptors, or the folate transporter. The targeting element can also be a sugar making it possible to target lectins such as receptors for asialoglycoproteins or for syalydes such as sialyl Lewis X, or alternatively an Fab fragment of antibodies, or a single chain antibody (ScFv).

The association of the targeting elements with the nucleolipid complexes can be carried out by any technique known to those skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the transfer agent according to the invention, or to a group which interacts with the transfer agent according to the invention or with the nucleic acid. The interactions in question can be, according to a preferred mode, of ionic or covalent nature.

A subject of the invention is also the use of the compounds as defined above for the transfer of polynucleotides (and more generally of polyanions) in cells in vitro, in vivo or ex vivo. More specifically, the subject of the present invention is the use of the compounds as defined above for the preparation of a medicament intended for treating diseases, in particular diseases which result from a deficiency in a protein or nucleic product. The polynucleotide contained in said drug encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo.

For in vivo uses, for example in therapy or for the study of gene regulation or the creation of animal models of pathologies, the compositions according to the invention can be formulated for topical, cutaneous, oral administration. , rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc. Preferably, the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa). They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acids used for the injection as well as the number of administrations can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the pathology concerned, of the gene to be expressed, or else the duration of the treatment sought. As regards more particularly the mode of administration, it may be either a direct injection into the tissues, for example at the level of tumors, or into the circulatory tract, or a treatment of cultured cells followed of their reimplantation in vivo, by injection or graft. The tissues concerned in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissue, etc.

Another object of the present invention relates to a method of treatment of the human or animal body comprising the following steps: (1) bringing the nucleic acid into contact with a transfer agent as defined above, to form a complex , and

(2) bringing the cells of the human or animal body into contact with the complex formed in (l).

The invention further relates to a method for transferring nucleic acids into cells, comprising the following steps:

(1) bringing the nucleic acid into contact with a transfer agent as defined above, to form a complex, and

(2) bringing the cells into contact with the complex formed in (1).

The contacting of the cells with the complex can be carried out by incubation of the cells with the said complex (for uses in vitro or ex vivo), or by injection of the complex into an organism (for uses in vivo). The incubation is preferably carried out in the presence of, for example, from 0.01 to 1000 μg of nucleic acid per 10 ⁶ cells. For administration in vivo, doses of nucleic acid ranging from 0.01 to 10 mg can for example be used.

In the case where the compositions of the invention additionally contain one or more adjuvants as defined above, the adjuvant (s) are mixed beforehand with the transfer agent according to the invention and / or with the nucleic acid.

The present invention thus provides a particularly advantageous method for the transfer of nucleic acids in vivo, in particular for the treatment of diseases, comprising the administration in vivo or in vitro of a nucleic acid coding for a protein or capable of being transcribed into a nucleic acid able to correct said disease, said nucleic acid being associated with a compound of general formula (I) under the conditions defined above.

The nucleic acid transfer agents of the invention are particularly useful for the transfer of nucleic acids into primary cells or into established lines. They can be fibroblastic, muscular, nervous cells (neurons, astrocytes, glial cells), hepatic, hematopoietic (lymphocytes, CD34, dendritics, etc.), epithelial etc., in differentiated or pluripotent forms (precursors).

In addition to the foregoing provisions, the present invention also includes other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting its scope. In particular, the applicant proposes, without limitation, various operating protocols as well as reaction intermediates which may be used to prepare the transfer agents of general formula (I). Of course, it is within the reach of those skilled in the art to draw inspiration from these protocols or intermediate products to develop analogous processes with a view to producing these same compounds. It is also for those skilled in the art to draw inspiration from the synthesis methods described in the various patent applications cited above for the synthesis of polycation R included in the general formula (I) (WO 96/17823, WO 97/18185 , WO 97/31935 etc.).

FIGURES

Figure 1: Schematic representation of the plasmid pXL2774 used in DNA transfer experiments in cells.

Figure 2: In vitro gene transfer activity in HeLa cells of the complexes formed from compound 2 according to the invention without co-lipid, or else in the presence of cholesterol and in the presence of DOPE as co-lipids. The y-axis represents the expression of luciferase in pg / well. The absentee axis indicates the transfecting agent / DNA ratio in nmol / μg of DNA.

Figure 3: In vivo gene transfer activity after direct injection into the anterior muscle of the mouse tibia of complexes formed from compound 2 according to the present invention in the presence of DOPE (1: 1). The ordinate axis indicates the expression of luciferase in pg / muscle. The absentee axis indicates the compound 2 / DNA ratio in nmoles / μg of DNA.

EXAMPLES

A \ MATERIAL AND METHODS a) Material

• The starting polyamines, such as spermidine, spermine, tris- (2-aminoethyl) amine, phenylenediamine, diaminoalkane etc., are commercially available or they can be synthesized by conventional methods (for example by cyanoethylation of commercially available amines to obtain branched amines)

• many compounds such as, for example, triethylamine, benzotriazol-1-yloxytris (dimethyamino) phosphonium hexafluorophosphate (BOP), benzyl chloroformate, 1 1 -bromoundecanol, etc. are also commercial products.

• Amberlite IR 120 is a commercial ion exchange resin (BDH catalog). • Dimethyl sulfoxide (DMSO), treated beforehand with potassium hydroxide, was distilled over calcium hydride and then stored on a 4 A molecular sieve.

• Dichloromethane was distilled over phosphorus pentoxide and then stored on a 4 Â molecular sieve.

• Tetrahydrofuran (THF) was distilled over sodium in the presence of benzophenone. • For reactions requiring anhydrous conditions, all glassware is flame dried under a stream of nitrogen.

b) Methods - Spectroscopic analyzes

The nuclear magnetic resonance (NMR) spectra were recorded on a Brucker MSL 30 spectrometer at the frequency of 300 MHz for the proton and 75 MHz for the carbon. All chemical shifts are reported in ppm either relative to the frequency of tetramethylsilane (TMS), or relative to the solvent. The spectra were recorded using either the TMS or the residual solvent signal as an internal reference. The multiplicity of signals is designated by the following abbreviations: s (singlet), d (doublet), t (triplet), q (quadruplet) and m (multiplet).

- Chromatography techniques “The kinetics of the reactions were followed by thin layer chromatography (TLC) with silica gel containing a fluorescent indicator (Merck Slilicagel 60 F254) as support. The chromatograms were revealed by spraying with an alcoholic solution of anisaldehyde.

• All column chromatography was carried out under compressed air pressure with silica gel 60 as the stationary phase (0.05-0.02 mm). The mobile phase used differs according to the type of synthesis (medium pressure chromatography).

• HPLC analyzes (High Performance Liquid Chromatography) are carried out on a Waters LC 4000 device equipped with a C4 type analytical column marketed by Applied Biosystem ("Brownlee Columns" in stainless steel 3 cm long and 0.46 cm diameter) and a "Waters 486" detector at 220 nm. The stationary phase is 7 microns butyl aquapore, and the mobile phases are demineralized water (2500 cm ³ ) or acetonitrile (2500 cm ³ ) added with trifluoroacetic acid (2.5 cm ³ ). The flow rate is 1 ml per minute.

B \ SUMMARY OF TRANSFECTING AGENTS EXAMPLE 1 Synthesis of the α-D-mannopyrannoside of (3- [4- (3-amino-propyI-amino) -butyl-aminol-methylene-carbamoyl) -15-pentadecanyl-16-octadecyl (Compound 1)

a) Synthesis of 3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-amino] acetic acid (FRM375)

To a solution of spermine (5 g; 24.96 mmol) in methanol (125 ml), sodium cyanoborohydride NaBH ₃ CN (0.548 g; 8.74 mmol) is added. The solution is then subjected to vigorous stirring. By means of an isobaric bulb, a solution of glyoxylic acid (2.34 g; 25.46 mmol) in methanol (80 ml) is added over 100 minutes. After one night, triethylamine (3.86 ml; 27.71 mmol) and di-tert-butyl dicarbonate (27.67 g; 129.79 mmol) dissolved in tetrahydrofuran (55 ml) are added to the mixture. After one night, the mixture is concentrated on a rotary evaporator and then the residue is taken up in ethyl acetate (63 ml) and washing is carried out with potassium hydrogen sulphate and in brine. Then dried over magnesium sulfate and concentrated. The product obtained is purified by chromatography (CH ₂ Cl ₂ / MeOH 9: 1). The yield is 30%.

! H NMR (CDC1 ₃ ): δ (ppm) 1.42 (s, 36H, C (CH3) ₃ ), 1.45 (m, 4H, CH2), 1.60 (m, 4H, CH ₂ ), , 3.04-3.33 (m, 12H, CH2), 3.91 (s, 2H, NCH ₂ COO).

b) Synthesis of methyl 15-hydroxypentadecanoate To 10 g of pentadecalactone (41.60 mmol) in 41.60 cm ³ of methanol, 6.66 cm ³ of 2N sodium methylate (13.31 mmol) is added at 0 ° vs.

After 9 hours, 9.24 cm ³ of acetic acid are added and the mixture is left to act for 15 minutes. Then the solution is evaporated to dryness under vacuum, then taken up in dichloromethane, and a washing with sodium bicarbonate is carried out. The organic phase obtained is dried with magnesium sulfate and the solvent is evaporated on a rotary evaporator. The purification is carried out in a hexane / ethyl acetate mixture 6: 4. Methyl 1-ol-pentadecanoate is obtained with a yield of 80%. ! H NMR (CDCI ₃ ): δ (ppm) 1.26 (m, 12H, (CH ₂ ) \ o), 1.5-1.6 (m, 4H, H-2 and H- 13), 2 , 30 (t, 2H, J = 7.60 Hz, H-14), 3.64 (t, 1H, J≈ 5.84 Hz, Hl), 3.67 (s, 3H, H-16).

c) Synthesis of 2,3,4,6-tetra-O-acetyl-aD-mannopyranoside of methyl pentadecanoate At 0 ° C., 5.26 cm ³ of tin chloride (44.94 mmol) are added to 8 , 72 g of pentaacetylated mannose (22.47 mmol) in 56 cm ³ of dichloromethane for 30 minutes. Then 7.34 g of l-ol methyl pentadecanoate previously obtained in a) (26.96 mmol) are added. After 2 hours, the reaction mixture is diluted with ethyl ether and poured into a solution of sodium hydrogen phosphate (NaHPO ₄ ). The aqueous phases are extracted with diethyl ether and the organic phases are successively washed with a solution of sodium carbonate, brine, then dried over magnesium sulfate. The product obtained after evaporation under vacuum to dryness is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 7: 3. The yield is 53%. ^J H NMR (CDCI ₃ ): δ (ppm) 1.26 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH CH and H-

13), 2.01, 2.05, 2.12 and 2.17 (s, 3H, OCOCH3), 2.29 (t, 2H, J≈ 7.62 Hz, H-14), 3.40 ( m, IH, J = 7.89 Hz, OCH _a CH ₂ ), 3.66 (m, IH, J = 7.89 Hz, OCH _D CH ₂ ), 3.67 (s, 3H, COOCH3), 4.05 (ddd, IH, J = 9.56 Hz and 5.57 Hz, H-5), 4.1 (dd, IH, J≈ 5.57 Hz and 12.32 Hz, H-6a), 4.29 (dd, IH, J = 5.57 Hz and 12.32 Hz, H-6b), 4.8 (d, IH, J≈ 1.85 Hz, Hl), 5.23 (dd, IH, J = 1.85 Hz and 3.23 Hz, H-2), 5.27 (dd, IH, J = 9.97 Hz and 9.56 Hz, H-4), 5.35 (dd, IH, J≈ 9.97 Hz and 3.23 Hz, H-3).

d) Synthesis of methyl pentadecanoate -D-mannopyranoside

3.63 g of product obtained in the previous step (6.01 mmol) is treated in solution in 12 cm ³ of methanol with 3 cm ³ of 2N sodium methylate (6.01 mmol). When the reaction is complete, the latter is neutralized with Amberlite IR120 (1 equivalent weight / volume), filtered and evaporated to dryness under vacuum.

! H NMR (CDCI ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH ₂ ÇH ₂ and H- 13), 2.34 (t , 2H, J≈ 7.62 Hz, H- 14), 3.41 (m, IH, J≈ 6.71 Hz, OCH _a CH ₂ ), 3.74 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.67 (s, 3H, CH3OCO), 3.5-3.82 (m, 6H, H-2, H-3, H- 4, H-5 and H-6), 4.75 (d, 1H, J≈ 1.82 Hz, Hl).

e) Synthesis of 2,3,4,6-tetra-O-benzyl-aD-mannopyranoside of methyl pentadecanoate A 2 g (4.56 mmol) of product obtained in the previous step d) in solution in 20 cm ³ of anhydrous dimethylformamide (DMF), 4.54 g of potassium iodide (27.36 mmol), 1.09 g of 60% sodium hydride (27.36 mmol) and 3.25 cm ³ of benzyl bromide (27.36 mmol). After 12 hours, 18.24 cm ³ of a saturated ammonium chloride solution are added, and the mixture is left to act for 10 minutes. Then diluted with water and the organic phase is extracted with ethyl acetate. This is then washed with water and brine, and is finally dried with magnesium sulfate. Furthermore, an additional washing is carried out with a saturated sodium thiosulfate solution in order to remove the iodide ions. Evaporated in vacuo and the resulting oil is purified in a heptane / ethyl acetate mixture 9: 1. The product is obtained with a yield of 60%.

1 H NMR (CDCI ₃ ): d (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ ), 1.49 (m, 2H, OCH ₂ CH ₂ ), 1.59 (m, 2H, H- 13), 2.31 (t, 2H, J≈ 7.62 Hz, H-14), 3.34 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.63 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.67 (s, 3H, CH3OCO), 3.75 (m, IH, J = 8.97 Hz and 6.21 Hz, H-5) , 3.78 (s, 2H, CH ₂ Phe), 3.90 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6a), 3.97 (dd, IH, J ≈ 6.21 Hz and J≈ 11.82 Hz, H-6b), 4.07 (s, 2H, CH ^ Phe), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H -3), 4.57 (s, 2H, CH ^ Phe), 4.63 (s, 2H, ÇH ₂ Phe), 4.69 (dd, IH, J = 2.52 Hz and 2.91 Hz, H-2), 4.74 (IH, J = 2.52 Hz, Hl), 4.85 (dd, IH, J≈ 7.83 Hz and 8.97 Hz, H-4), 7.35 ( m, 20H, Phe).

f) Synthesis of 2,3,4,6-tetra-O-benzyl- -D-mannopyranoside of pentadecanoic acid

To 0.50 g (0.73 mmol) of product obtained in the preceding step e) dissolved in 7 cm ³ of methanol, 4.68 cm ³ of a 25% sodium hydroxide solution are added. The reaction mixture is heated at reflux for 30 minutes. Then, the mixture is neutralized at cold with 5% hydrochloric acid solution. The organic phase is extracted with ethyl acetate and evaporated to dryness under vacuum. The purification is carried out in a heptane / ethyl acetate mixture 4: 6. The product is obtained with a yield of 62%. ^Ï H NMR (CDClj): d (ppm) 1.28 (m, 20H, (CH ₂ ) _{Î 0} ), 1.49 (m, 2H, OCH ₂ CH ₂ ), 1.59 (m, 2H, H -13), 2.34 (t, 2H, J≈ 7.62 Hz, H-14), 3.34 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.63 (m , IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.75 (m, IH, J≈ 8.97 Hz and 6.21 Hz, H-5), 3.78 (s, 2H, CH ₂ Phe), 3.90 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H-6a), 3.97 (dd, IH, J = 6.21 Hz and J≈ 1 1 , 82 Hz, H-6b), 4.07 (s, 2H, CH ₂ Phe), 4.52 (dd, J≈ 2.91 Hz and 7.83 Hz, H-3), 4.57 (s , 2H, CH ₂ Phe), 4.63 (s, 2H, CH ^ Phe), 4.69 (dd, IH, J≈ 2.52 Hz and 2.91 Hz, H-2), 4.74 ( IH, J≈ 2.52 Hz, Hl), 4.85 (dd, IH, J≈ 7.83 Hz and 8.97 Hz, H-4), 7.35 (m, 20H, Phe).

g) Synthesis of 2,3,4,6-tetra-O-benzyl- -D-mannopyranoside of N-octadecyl-15-carbamoyl-pentadecanyl A 0.29 g (0.37 mmol) of a solution of product obtained in the preceding step f), in solution in 5 cm ³ of chloroform, 0.23 g of BOP (0.52 mmol), 0.21 cm ³ of diisopropylethylamine (1.48 mmol) and 0 are added successively 12 g octadecylamine (0.44 mmol). When the reaction is complete, dilute with dichloromethane and wash with water. Then, it is dried with magnesium sulfate and evaporated to dryness under vacuum. The product obtained is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 6: 4. The product is obtained with a yield of 98%.

* H NMR (CDCI ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.36 VLÏ, H-33), 1.27 (m, 50H, (CH) ₂ 5), 1.47 (m, 4H, OCH ₂ CH ₂ and H- 17), 1.58 (m, 2H, H- 13), 2.13 (t, 2H, J≈ 7.92 Hz, H- 14), 3, 23 (m, 2H, H-16), 3.34 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.63 (m, IH, J≈ 6, 71 Hz, OCH _h CH2 ), 3.75 (m, IH, J≈ 8.97 Hz and 6.21 Hz, H-5), 3.78 (s, 2H, CH ₂ Phe), 3.90 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H-6a), 3.97 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H-6b), 4.07 (s, 2H, CH ^ Phe), 4.52 (dd, J≈ 2.91 Hz and 7.83 Hz, H-3), 4.57 (s, 2H, CH ₂ Phe), 4.63 (s, 2H , CH ₂ Phe), 4.69 (dd, IH, J≈ 2.52 Hz and 2.91 Hz, H-2), 4.74 (IH, J≈ 2.52 Hz, Hl), 4.85 (dd, IH, J≈ 7.83 Hz and 8.97 Hz, H-4), 5.37 (band, IH, HNCO), 7.35 ( m, 20H, Phe).

h) Synthesis of 2,3,4,6-tetra-O-benzyl- -D-mannopyranoside of 15-octadecylamino-pentadecanyl A 0.77 g (0.75 mmol) of product obtained in the previous step g), 0.056 g of lithium aluminum hydride AlLiH ₄ (1.50 mmol) is added to 15 cm ³ of anhydrous tetrahydrofuran (THF). The mixture is heated at reflux for 10 hours. Then, the reaction mixture is cooled in an ice bath and 56 μl of water are added, then 1 12 μl of 2N sodium hydroxide after 10 minutes, and finally another 56 μl of water 10 minutes later. It is filtered and evaporated to dryness under vacuum. The product obtained is purified in a mixture of dichloromethane / methanol / ammonia 28% 9: 2: 0.5. The product is obtained with a yield of 86%. 1 H NMR (CDC1 ₃ ): δ (ppm) 0.88 (t, 3H, J ^≈ 6.36 Hz, H-33), 1.27 (m, 50H, (CH ₂ ) 25), 1.4- 1.6 (m, 9H, OCH ÇH2, H-17, H-14, H-17 and NH), 2.57 (t, 4H, J≈ 7.92 Hz, H-15 and H- 16), 3 , 34 (m, IH, J≈ 6.71 Hz, OCH _a CH ₂ ), 3.63 (m, IH, J≈ 6, 71 Hz, OCHhCH?), 3.75 (m, IH, J≈ 8 , 97 Hz and 6.21 Hz, H-5), 3.78 (s, 2H, CE ^ Phe), 3.90 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H -6a), 3.97 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H-6b), 4.07 (s, 2H, CH ₂ Phe), 4.52 (dd, J≈ 2.91 Hz and 7.83 Hz, H-3), 4.57 (s, 2H, CH ₂ Phe), 4.63 (s, 2H, CH ^ Phe), 4.69 (dd, IH , J≈ 2.52 Hz and 2.91 Hz, H-2), 4.74 (IH, J≈ 2.52 Hz, Hl), 4.85 (dd, IH, J≈ 7.83 Hz and 8 , 97 Hz, H-4), 7.35 (m, 20H, Phe).

i) Synthesis of 2,3,4,6-tetra-O-benzyl- -D-mannopyranoside of (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert - butoxycarbonyl-amino] -methylene-carbamoyl) -15-pentadecanyl-16-octadecyl

To 0.63 g (0.61 mmol) of a solution of product obtained previously in step h), in 10 cm ³ of chloroform, 0.38 g of BOP (0.85 mmol), 0.425 is successively added cm ³ of diisopropylethylamine (2.44 mmol) and 0.48 g of 3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-amino acid] - acetic acid (FRM 375) (0.73 mmol) obtained in step a). After 4 hours, we diluted with dichloromethane and washed with water. It is dried with magnesium sulphate and evaporated to dryness under vacuum. The product obtained is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 6: 4. The product is obtained with a yield of 80%. * H NMR (CDCI ₃ ): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 50H, (CH) 25), 1.4- 1.6 (m, 17H, OCH ÇH ₂ , H-17, H-14, H-17, H-37, H-40, H-41 and H-44), l, 46 (m, 36H, Boc ), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09-3, 33 (m, 12H, H-36, H-38, H- 39 , H-42, H-43 and H-45), 3.34 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.63 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.75 (m, IH, J≈ 8.97 Hz and 6.21 Hz, H-5), 3.78 (s, 2H, ÇH ₂ Phe), 3.90 (dd, IH, J≈ 6.21 Hz and J≈ 1 1.82 Hz, H-6a), 3.97 (dd, IH, J≈ 6.21 Hz and J≈ 11.82 Hz, H-6b), 4 , 07 (s, 2H, CH Phe), 4.52 (dd, J≈ 2.91 Hz and 7.83 Hz, H-3), 4.57 (s, 2H, CH ₂ Phe), 4.63 (s, 2H, ÇH ^ Phe), 4.69 (dd, IH, J≈ 2.52 Hz and 2.91 Hz, H-2), 4.74 (IH, J≈ 2.52 Hz, Hl) , 4.85 (dd, 1H, J≈ 7.83 Hz and 8.97 Hz, H-4), 7.35 (m, 18H, Phe).

j) Synthesis of the -D-mannopyranoside of (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-amino] -methylene-carbamoyl)! 5-pentadecanyl-l 6-octadecyl

To 0.63 g (0.38 mmol) of product obtained in the preceding step i), in 5 cm ³ of methanol, 10% of palladium on carbon (0.027 g) is added. The solution is stirred under hydrogen pressure at room temperature. After 6 hours, it is filtered and then evaporated to dryness under vacuum. The reaction is quantitative.

* H NMR (CD ₃ OD): δ (ppm) 0.88 (t, 3H, J≈ 6.36 Hz, H-33), 1.27 (m, 50H, (CH ₂ ) 25), 1, 4-1.6 (m, 17H, OCH9CH3 ,, H- 17, H- 14, H- 17, H-37, H-40, H-41 and H- 44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09-3, 33 (m, 12H, H- 36, H-38, H- 39, H-42, H-43 and H-45), 3.34 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.5-3.82 (m, 6H, H- 2, H-3, H-4, H-5 and H-6), 3.63 (m, IH, J≈ 6, 71 Hz, OCH CH ₂ ), 4.72 (IH, J≈ 2.52 Hz, Hl).

k) Synthesis of the α-D-mannopyranoside of (3- [4- (3-amino-propyl-amino) -butyl-amino] -methylene-carbamoyl) -15-pentadecanyl-16-octadecyl (compound 1) To 0.37 g (0.28 mmol) of product obtained in preceding step j), 21.50 cm ¹ of distilled tetrahydrofuran (TFA) is added. After 1 hour, the reaction mixture is concentrated to cold and lyophilized. The degree of purity of the product in solution in methanol is checked by HPLC as described in the section "Materials and Methods".

* H NMR (CD ₃ OD): 0.88 (t, 3H, J≈ 6.36 Hz, H-33), 1.27 (m, 14H, (CH ₂ ) 25), 1.4-1, 6 (m, 17H, OCH2ÇH2, H- 17, H- 14, H- 17, H-37, H-40, H-41 and H-44), 2.8-2.9 (m, 6H, H -15, H-16 and H-35), 2.92 (m, 2H, H-45), 2.92-3.17 (m, 12H, H-36, H-38, H-39, H -42, H-43), 3.34 (m, IH, J ^≈ 6, 71 Hz, OCH _a CH ₂ ), 3.5-3.82 (m, 6H, H-2, H-3, H -4, H-5 and H-6), 3.63 (m, 1H, J≈ 6, 71 Hz, OCH CH ₂ ), 4.72 (1H, J≈ 2.02 Hz, Hl).

EXAMPLE 2 Synthesis of 6-deoxy-α-L-mannopyranoside of (3- [4- (3-amino-propyl-amino) -butyl-amino] -methylene-carbamoyl) -15-pentadecanyI-16-octadecyl (compound 2)

a) Synthesis of 3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert-butoxγcarbonyl-amino] acetic acid (FRM 375) To a spermine solution (5 g; 24.96 mmol) in methanol (125 ml), sodium cyanoborohydride NaBH ₃ CN (0.548 g; 8.74 mmol) is added. The solution is then subjected to vigorous stirring. By means of an isobaric bulb, a solution of glyoxylic acid (2.34 g; 25.46 mmol) in methanol (80 ml) is added over 100 minutes. After one night, triethylamine (3.86 ml; 27.71 mmol) and di-tert-butyl dicarbonate (27.67 g; 129.79 mmol) dissolved in tetrahydrofuran (55 ml) are added to the mixture. After one night, the mixture is concentrated on a rotary evaporator and then the residue is taken up in ethyl acetate (63 ml) and washing is carried out with potassium hydrogen sulphate and in brine. Then dried over magnesium sulfate and concentrated. The product obtained is purified by chromatography (CH ₂ Cl ₂ / MeOH 9: 1). The yield is 30%.

* H NMR (CDCI ₃ ): δ (ppm) 1.42 (s, 36H, C (CH ₃ ) ₃ ), 1.45 (m, 4H, CH ₂ ), 1.60 (m, 4H, CH ₂ ),, 3.04-3.33 (m, 12H, CH ₂ ), 3.91 (s, 2H, NCH ₂ COO). b) Synthesis of 15-hydroxy ~ methyl pentadecanoate

To 10 g of pentadecalactone (41.60 mmol) in 41.60 cm ³ of methanol, 6.66 cm ³ of 2N sodium methylate (13.31 mmol) is added at 0 ° C. After 9 hours, 9.24 cm ³ of acetic acid are added and the mixture is left to act for 15 minutes. Then the solution is evaporated to dryness under vacuum, which is then taken up in dichloromethane, and washing is carried out with sodium bicarbonate. The organic phase obtained is dried with magnesium sulfate and the solvent is evaporated on a rotary evaporator. The purification is carried out in a hexane / ethyl acetate mixture 6: 4. Methyl 1-ol-pentadecanoate is obtained with a yield of 80%. * H NMR (CDCI ₃ ): δ (ppm) 1.26 (m, 12H, (CH ₂ ) ₁₀ ), 1.5-1.6 (m, 4H, H-2 and H-13), 2, 30 (t, 2H, J≈ 7.60 Hz, H-14), 3.64 (t, 1H, J≈ 5.84 Hz, Hl), 3.67 (s, 3H, H- 16).

c) Synthesis of 2,3,4-tri-0-acetyl-6-deoxy-a-L-mannopyranoside of methyl pentadecanoate

At 0 ° C., 2.49 cm ³ of tin chloride (21.30 mmol) is added to 3.55 g of tetraacetylated rhamnose (10.65 mmol) in 27 cm ³ of dichloromethane for 30 minutes. Then added 3.48 g of l-ol methyl pentadecanoate previously obtained (12.78 mmol). After 2 hours, the reaction mixture is diluted with ethyl ether and poured into a sodium phosphate solution (Na ₂ PO ₄ ). The aqueous phases are extracted with diethyl ether and the organic phases are successively washed with a sodium carbonate solution, brine, then dried with magnesium sulfate. After evaporation to dryness under vacuum, the mixture is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 7: 3. The product is obtained with a yield of 60%.

* H NMR (CDCI ₃ ): δ (ppm) 1.20 (d, 3H, J ^≈ 6.45 Hz, H-6), 1.26 (m, 20H, (CH ₂ ) io), 1.59 (m, 4H, OCH ₂ ÇH ₂ and H- 13), 1.98, 2.04 and 2.16 (s, 3H, OCOCH3), 2.29 (t, 2H, J≈ 7.62 Hz, H -14), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.66 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.67 ( s, 3H, COOÇH3), 3.88 (m, IH, J≈ 6.45 Hz and 9.97 Hz, H-5), 4.70 (d, IH, J≈ 1.72 Hz, Hl), 5.06 (dd, IH, J≈ 9.97 Hz and 9.97 Hz, H-4), 5.22 (dd, IH, J≈ 1, 72 Hz and 3.52 Hz, H-2), 5.30 (dd, IH, J≈ 3.52 Hz and 9.97 Hz, H-3). d) Synthesis of methyl pentadecanoate a-deoxy-L-6-mannopyranoside

5.08 g of product obtained in step c) (9.34 mmol) in solution in 20 cm ³ of methanol are treated with 9.34 ml of 2N sodium methylate (18.68 mmol). When the reaction is complete, the reaction mixture is neutralized with Amberlite IR120, filtered and evaporated to dryness under vacuum.

! H NMR (CDC1 ₃ ): δ (ppm) 1.20 (d, 3H, J≈ 6.45 Hz, H-6), 1.26 (m, 20H, (CH ₂ ) ιo), 1.59 (m, 4H, OCH9CH2 and H-13), 2.29 (t, 2H, J≈ 7.62 Hz, H- 14), 3.40 (m, IH, J≈ 6, 71 Hz, OCHbCH ₂ ) , 3.66 (m, IH, J ^≈ 6.71 Hz, OCH _b CH ₂ ), 3.67 (s, 3H, CH3OCO), 3.6-3.9 (m, 4H, H-2, H -3, H-4 and H-5), 4.70 (d, 1H, J≈ 1.72 Hz, Hl).

e) Synthesis of 2,3,4-tri-0-benzyl-6-deoxy-a-L-mannopyranoside of methyl pentadecanoate

To 2.09 g (5.00 mmol) of product obtained in preceding step d), in 30 cm ³ of anhydrous dimethylformamide (DMF), 3.32 g of potassium iodide (20.00 mmol) are successively added ), 0.80 g of 60% sodium hydride (20.00 mmol) and 2.38 cm ³ of benzyl bromide (20.00 mmol). After 12 hours, 20 cm ³ of a saturated ammonium chloride solution are added and the mixture is left to act for 10 minutes. Then diluted with water and the organic phase is extracted with ethyl acetate. This is then washed with water and brine, before being finally dried with magnesium sulfate. Furthermore, an additional washing with a saturated sodium thiosulfate solution is carried out in order to remove the iodide ions. It is evaporated to dryness under vacuum and the resulting oil is purified in a heptane / ethyl acetate mixture 9: 1. The product is obtained with a yield of 60%.

'H NMR (CDCI ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ιo),, 33 (d, 3H, J≈ 6.21 Hz, H-6), 1.59 ( m, 4H, OCH7CH7 and H- 13), 2.31 (t, 2H, J≈ 7.62 Hz, H- 14), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.61 (dd, IH, J≈ 8.96 Hz and 9.5 Hz, H-4), 3.66 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3, 67 (s, 3H, CH3OCO), 3.68 (m, IH, J≈ 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, IH, J≈ 2.01 Hz and 3.02 Hz, H-2), 3.88 (dd, J≈ 3.02 Hz and 8.96 Hz, H-3), 4.64 (s, 6H, CH ₂ Phe), 4.73 (IH, J≈ 2 , 01 Hz, Hl), 7.35 (m, 15H, Phe). f) Synthesis of 2,3,4-tri-0-benzyl-6-deoxy-aL-mannopyranoside of pentadecanoic acid

To 0.50 g (0.73 mmol) of a solution of product obtained in preceding step e), in 7 cm ³ of methanol, 4.68 ml of 25% sodium hydroxide is added. The reaction mixture is heated at reflux for 30 minutes. Then, the mixture is neutralized when cold with a 5% hydrochloric acid solution. The organic phase is extracted with ethyl acetate and evaporated to dryness under vacuum. The purification is carried out in a heptane / ethyl acetate mixture 4: 6. The product is obtained with a yield of 72%.

'H NMR (CDC13): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ι o), 1.33 (d, 3H, J≈ 6.21 Hz, H-6), 1.59 (m, 4H, OCH ₂ ÇH ₂ and H-13), 2.34 (t, 2H, J≈ 7.62 Hz, H-14), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.61 (dd, IH, J≈ 8.96 Hz and 9.5 Hz, H-4), 3.66 (m, IH, J≈ 6, 71 Hz, OÇH _b CH ₂ ), 3.68 (m, IH, J≈ 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, IH, J≈ 2.01 Hz and 3.02 Hz, H-2), 3.88 (dd, J≈ 3.02 Hz and 8.96 Hz, H-3), 4.64 (s, 6H, ÇH ^ Phe), 4.73 (IH, J≈ 2.01 Hz, Hl), 7.35 (m, 20H, Phe).

g) Synthesis of 2,3,4-tri-O-benzyl-6-deoxy-a-L-mannopyranoside of N-octadecyl-15-carbamoyl-pentadecanyl

To 0.70 g (1.04 mmol) of a solution of product previously obtained in step f), in 13 cm ³ of chloroform, there is successively added 0.69 g of BOP (1.56 mmol), 0 , 72 cm ³ of diisopropylethylamine (4.16 mmol) and 0.34 g of octadecylamine (1.25 mmol). When the reaction is complete, diluted with dichloromethane, washed with water, dried over magnesium sulfate, and evaporated to dryness under vacuum. The product obtained is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 6: 4. The product is obtained with a yield of 84%. 'H NMR (CDC1 ₃ ): δ (ppm 0.88 (t, 3H, J ^≈ 6.36 Hz, H-33), 1.27 (m, 50H, (CH ₂ ) 25), 1.33 ( d, 3H, J≈ 6.21 Hz, H-6), 1.47 (m, 4H, OCH7CH7 and H- 17), 1.58 (m, 2H, H- 13), 2.13 (t, 2H, J≈ 7.92 Hz, H- 14), 3.23 (m, 2H, H- 16), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3, 61 (dd, IH, J≈ 8.96 Hz and 9.5 Hz, H-4), 3.66 (m, IH, J≈ 6.71 Hz, OCH _b CH ₂ ), 3.68 (m, IH, J≈ 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, IH, J≈ 2.01 Hz and 3.02 Hz, H-2), 3.88 (dd, J≈ 3.02 Hz and 8.96 Hz, H-3), 4.64 (s, 6H, CH? Phe), 4.73 (IH, J≈ 2.01 Hz, Hl), 5.37 (band, 1H, HNCO), 7.35 (m, 15H, Phe).

h) Synthesis of 2,3,4-tri-O-benzyl-6-deoxy-aL-mannopyranoside of 15-octadecylam in o-pentadecanyl A 0.81 g (0.86 mmol) of product obtained with previous step g), in 15 cm ³ of anhydrous tetrahydrofuran (THF), 0.065 g of lithium hydride is added; aluminum AlLiH ₄ (1.72 mmol), and heated to reflux for 10 hours. Then, the reaction mixture is cooled in an ice bath and 65 μl of water are added, then 130 μl of 2N sodium hydroxide after 10 minutes, and finally again 65 μl of water after 10 minutes. It is filtered and evaporated to dryness under vacuum. The purification is carried out in a dichloromethane / methanol / 28% ammonia 9: 2: 0.5 mixture. The product is obtained with a yield of 93%.

'H NMR (CDC1 ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.36 Hz, H-33), 1.27 (m, 50H, (CH2) 25), 1.33 ( d, 3H, J≈ 6.21 Hz, H-6), 1.4-1.6 (m, 9H, OCH2ÇH2, H- 17, H- 14, H-17 and NH), 2.57 (t, 4H, J≈ 7.92 Hz, H-15 and H-16), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.61 (dd, IH, J≈ 8 , 96 Hz and 9.5 Hz, H-4), 3.66 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.68 (m, IH, J≈ 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, IH, J≈ 2.01 Hz and 3.02 Hz, H-2), 3.88 (dd, J≈ 3.02 Hz and 8.96 Hz , H-3), 4.64 (s, 6H, CH ^ Phe), 4.73 (1H, J≈ 2.01 Hz, Hl), 7.35 (m, 15H, Phe).

i) Synthesis of 2,3,4-tri-0-benzyl-6-deoxy-aL-mannopyranoside of (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino) -butyl- tert-butoxycarbonyl-amino] -methylene-carbamoyl) -15-pentadecanyl-16-octadecyl

To 0.78 g (0.86 mmol) of a solution of product obtained in the preceding step h), dissolved in 7 cm ³ of chloroform, 0.53 g of BOP (1.20 mmol) is successively added , 0.30 cm ³ of diisopropylethylamine (1.72 mmol) and 0.62 g of FRM 375 obtained in step a) (0.95 mmol). After 4 hours, diluted with dichloromethane, washed with water, dried over magnesium sulfate, and evaporated to dryness under vacuum. The product obtained is purified by "flash" chromatography in a heptane / ethyl acetate mixture 6: 4. The product is obtained with a yield of 72%. 'H NMR (CDC1 ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.36 Hz, H-33), 1.27 (m, 50H, (CH ₂ ) 25), 1.33 (d, 3H, J≈ 6.21 Hz, H-6), 1.4-1.6 (m, 17H, OCH7CH ?, H- 17, H- 14, H- 17, H-37, H- 40, H-41 and H-44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09 - 3.33 (m, 12H, H-36, H-38, H-39, H-42, H-43 and H-45), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.65 (s, 2H, CH ₂ Phe), 3.66 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.68 (m, IH, J≈ 9 , 5 Hz and 6.21 Hz, H-5), 3.99 (s, 2H, CH ₂ Phe), ⁴ , ⁰² ( ^dd , ^{i H} , ^{J =} 8.96 Hz and 9.5 Hz, H- 4), 4.32 (s, 2H, CH ^ Phe), 4.57 (dd, IH, J≈ 2.01 Hz and 3.02 Hz, H-2), 4.73 (IH, J≈ 2.01 Hz, Hl), 4.82 (dd, J≈ 3.02 Hz and 8.96 Hz, H-3), 7.35 (m, 18H, Phe).

j) Synthesis of 6-deoxy-aL-mannopyranoside of (3- [4- (3-tert-butoxycarbonyl- amino-propyl-tert-butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-amino] - methylene-carbamoyl) - ! 5-pentadecanyl-l 6-octadecyl

To 0.74 g (0.48 mmol) of product obtained in preceding step i), dissolved in 10 cm ³ of methanol, 10% (0.034 g) of palladium on carbon is added. The solution is stirred under hydrogen pressure at room temperature. After 4 hours, it is filtered and then evaporated to dryness under vacuum. The reaction is quantitative.

'H NMR (CD ₃ OD): δ (ppm) 0.88 (t, 3H, J≈ 6.36 Hz, H-33), 1.20 (d, 3H, J≈ 6.45 Hz,

H-6), 1.27 (m, 14H, (CH ₂ ) 25), 1.4-1.6 (m, 17H, OCH ₂ ÇH ₂ , H-17, H-14, H-17, H -37, H-40, H-41 and H-44), l, 46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35 ), 3.09-

3.33 (m, 12H, H-36, H-38, H-39, H-42, H-43 and H-45), 3.40 (m, IH, J≈ 6.71 Hz,

OCH _a CH ₂ ), 3.66 (m, IH, J≈ 6.71 Hz, OCH _b CH ₂ ), 3.6-3.9 (m, 4H, H-2, H-3, H-4 and

H-5), 4.73 (1H, J≈ 2.01 Hz, H-1).

k) Synthesis of the 6-deoxy-a-L-mannopyranoside of (3- [4- (3-amino-propyl-amino) -butyl-amino] -methylene-carbamoyl) -15-pentadecanyl-16-octadecyl (compound 2)

To 0.40 g (0.31 mmol) of product obtained in preceding step j), 24 cm ³ of distilled tetrahydrofuran (TFA) are added. After 1 hour, the reaction mixture is evaporated in vacuo to cold and to dryness, then is lyophilized. The degree of purity of the product in solution in methanol is checked by HPLC. 'H NMR (CD ₃ OD): δ (ppm) 0.88 (t, 3H, J ^≈ 6.36 Hz, H-33), 1.20 (d, 3H, J≈ 6.45 Hz, H- 6 '), 1.27 (m, 14H, (CH) ₂₅ ), 1, 4-1.6 (m, 17H, OCH ₂ CH ₂ , H- 17, H-14, Hl 7, H- 37, H-40, H-41 and H-44), 2.8-2.9 (m, 6H, Hl 5, H-16 and H-35), 2.92 (m, 2H, H-45), 2, 92-3.17 (m, 12H, H-36, H-38, H-39, H-42, H-43), 3.40 (m, IH, J≈ 6, 71 Hz, OCH _a CH ₂ ), 3.66 (m, IH, J≈ 6, 71 Hz, OCH _b CH ₂ ), 3.6-3.9 (m, 4H, H-2, H-3, H-4 and H-5 ), 4.73 (1H, J≈ 2.01 Hz, Hl).

Example 3 Synthesis of 6-deoxy-β-L-galactopyrannoside of l - [- (3- [4- (3- amino-propyl-amino) -butyl-amino-propyl-amino] -methylene-carbamoyl) -15 - pentadecanyl-16-octadecanyl (compound 3)

a) Synthesis of {3- [4- (3-benzyloxycarbonyl-amino-propyl-benzyloxycarbonyl-amino) -butyl-benzyloxycarbonyl-amino] -propylamino} acetic acid

To a solution of spermine (10 g; 49.91 mmol) in methanol (200 ml), sodium cyanoborohydride NaBH ₃ CN (1.10 g; 17.47 mmol) is added. The solution is then subjected to vigorous stirring. Using a isobaric ampoule, a solution of glyoxylic acid (4.59 g; 49.91 mmol) in methanol (120 ml) is added over 100 minutes. After overnight, the reaction mixture is placed in an ice bath and 2N sodium hydroxide (34 ml) and benzyl chloroformate (14.25 ml; 99.82 mmol) are successively added in 10 portions. Mix vigorously while maintaining the bath between 5 ° C and 10 ° C. After 2 hours at room temperature, the mixture is extracted with ether and neutralized with a 5N hydrochloric acid solution. The organic phase is then dried over magnesium sulphate and concentrated on a rotary evaporator. The product obtained is purified by chromatography (100% CH ₂ C1 ₂ then CH ₂ Cl ₂ / MeOH 9: 1). The yield is 52%.

! H NMR (CDCI ₃ ): δ (ppm) 1.28 (t, 4H, CH ₂ ), 1.60 (m, 4H, CH ₂ ),, 3.04-3.33 (m, 12H, CH ₂ ), 3.49 (s, 2H, NCH COO), 5.07 (s, 8H, CH ₂ ), 7.27 (m, 20H, Phe).

b) Synthesis of methyl 15-hydroxypentadecanoate Pentadecalactone (10 g; 41.6 mmol) dissolved in methanol (41.6 ml) is treated with 2N sodium methylate (6.656 ml; 13.31 mmol) at 0 ° C. After 9 hours, 9.24 ml of acetic acid are added and the mixture is left to act for 15 minutes. Then the solution is concentrated and the resulting oil is dissolved in dichloromethane and washed with sodium bicarbonate. After decantation, the organic phase is dried over magnesium sulfate and evaporated. The purification is carried out in a 6: 4 hexane / ethyl acetate (AcOEt) mixture to give methyl 15-hydroxypentadecanoate with a yield of 80%.

! H NMR (CDC1 ₃ ): δ (ppm) 1.29 (m, 20H, (CH ₂ ) ιo), 1.5-1.6 (m, 4H, H-2 and H- 13), 2, 30 (t, 2H, J≈ 7.60 Hz, H-14), 3.64 (t, 1H, J≈ 5.84 Hz, Hl), 3.67 (s, 3H, H-16).

c) Synthesis of N-octadecyl-15-hydroxypentadecanamide

10 g of methyl 15-hydroxypentadecanoate obtained in preceding step b) (36.85 mmol) and 19.86 g of octadecylamine (73.70 mmol) are melted at 150 ° C. under vacuum. After 24 hours, the mixture is cooled and diluted with dichloromethane. A precipitate is obtained which is filtered through Bϋchner. The solid obtained is then recrystallized from methanol to give N-octadecyl-15-hydroxypentadecanamide with a yield of 100%.

* H NMR (CDCI ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.96 Hz, H-33), 1.26 (m, 54H, (CH) 27), 1.4- 1.6 (m, 6H, H-2, H- 13 and H- 17), 2.30 (t, 2H, J≈ 7.60 Hz, H- 14), 3.25 (m, 2H, Hl 6) , 3.64 (t, 2H, J≈ 5.84 Hz, Hl), 5.39 (NHCO band).

¹³ C NMR (CDCI ₃ ): δ (ppm) 14.48 (C-33), 25.3 and 26.3 (C-2 and C-13), 29.72 ((CH ₂ ) 27)), 36.7 and 34.8 (C-14 and C-16), 63.6 (Cl), 174.31 (CO).

d) Synthesis of 15-octadecylamino-pentadecanol

2.98 g of lithium aluminum hydride LiAlH are added to a solution of 20 g of N-octadecyl-15-hydroxypentadecanamide obtained in the previous step c) (39.22 mmol) in anhydrous tetrahydrofuran (250 ml) ₄ (78.44 mmol). The reaction is carried out at reflux for 10 hours. After the reaction mixture has cooled, water (2.98 ml) and 2N sodium hydroxide (2.98 ml) are successively added. After 10 minutes, we add water (2.98 ml). The precipitate formed is filtered through Bϋchner and the filtrate is concentrated on a rotary evaporator to give 15-octadecylamino-pentadecanol.

* H NMR (CDC1 ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.96 Hz, H-33), 1.26 (m, 54H, (CH ₂ ) 27), 1, 43 -1.59 (m, 7H, H-2, H- 14, H- 17 and NH band), 1.5-1.6 (m, 4H, H-2 and H-13), 2.60 ( t, 4H, J≈ 6.50 Hz, H-15 and H- 16), 3.64 (t, 2H, J≈ 5.84 Hz, Hl).

¹³ C NMR (CDCI ₃ ): δ (ppm) 14.48 (C-33), 25.3 and 26.3 (C-2 and C-14), 29.72 ((CH ₂ ) 27)), 51.7 (C-15 and C-16), 63.6 (Cl).

e) Synthesis of N-fbenzyloxycarbonylJ-15-octadecylamino-pentadecanol

7.89 ml of benzyl chloroformate (55.26 mmol) are added dropwise to a solution cooled to 0 ° C of 15-octadecylamino-pentadecanol obtained in previous step d) (13.71 g; 27.63 mmol) and triethylamine (7.7 ml; 55.26 mmol) in dry dichloromethane (150 ml). After 10 minutes, the pH of the mixture is checked. The reaction mixture is then left at room temperature overnight. Then, the solution is washed with water, dried over magnesium sulfate (MgSO ₄ ) and concentrated. The reaction mixture is purified by chromatography (heptane / AcOEt 6: 4). N- [benzyloxycarbonyl] -15-octadecylamino-pentadecanol is obtained with a yield of 70%>.

* H NMR (CDCI ₃ ): δ (ppm) 0.88 (t, 3H, J≈ 6.96 Hz, H-33), 1.26 (m, 54H, (CH ₂ ) ₂₇ ), 1.43 -1.59 (m, 6H, H-2, Hl 4, Hl 7), 3.20-3.22 (m, 4H, H-15 and Hl 6), 3.64 (t, 2H, J≈ 5.84 Hz, Hl), 5.12 (s, 2H, OCH ₂ Phe), 7.34 (m, 5H, Phe).

¹³ C NMR (CDCI ₃ ): δ (ppm) 14.48 (C-33), 25.8, 26.9 and 31.94 (C-2, C-14 and Cl 7), 29.72 (( CH ₂ ) 27)), 47.26-48.04 (C-15 and C-16), 63.08 (Cl), 66.79 (OCH ₂ ), 128.40 (Phe).

f) Synthesis of 2,3,4-tri-0-acetyl-6-deoxy - $ - L-galactopyrannoside of 15-fN- (benzyloxycarbonyl) -octadécylamino] -pentadécanyl

1.5 g of tetraacetylated fucose (4.52 mmol) are reacted with 0.634 ml of tin tetrachloride (5.42 mmol) in dried acetonitrile (50 ml) for 30 minutes. Then 3,132 g of N- [benzyloxycarbonyl] -15-octadecylamino-pentadecanol obtained in previous step e) (4.97 mmol) are added. After 5 hours, the reaction is extracted and the product obtained is then purified by chromatography (heptane / ethyl acetate 6: 4). The yield is 69%. ! H NMR: δ (ppm) 0.87 (t, 3H, J≈ 6.96 Hz, H-33), 1.2 (d, 3H, J≈ 6.51 Hz, H-6), 1, 25 (m, 54H, (CH ₂ ) ₇ ), 1.52 (m, 6H, OCH ₂ CH ₂ , H-14 and H- 17), 1.95, 2.05 and 2.15 (s, 3H , OCOCH3), 3.14-3.25 (m, 4H, H-15 and H-16), 3.44 (m, IH, OCH _a CH ₂ ), 3.63 (m, IH, OCH _b CH ₂ ) , 3.79 (m, IH, H-5), 4.41 (d, IH, J≈ 7.98 Hz, Hl), 4.99 (dd, IH, J≈ 3.52 Hz and 10.46 Hz, H-3), 5.09 (s, 2H, OCH? Phe), 5.16 (dd, IH, J≈ 7.98 Hz and 10.46 Hz, H-2), 5.23 (dd , J≈ 3.52 Hz and 3.37Hz, H-4), 7.32 (m, 5H, Phe).

¹³ C NMR (CDCI ₃ ): δ (ppm) 14.68 (C-33), 17.31 (C-2), 20.75 (CH3COO), 27.29 (C-6), 29.72 ( (CH ₂ ) 27)), 25.89-31.98 (OCH7CH7, C-14, Cl 7), 47.25-48.04 (C-15 and C-16), 66.91 (CH ₂ Phe), 69.63 ( OCH7CH7), 69.45 (C-2), 70.57 (C-5), 70.85 (C-4), 71.44 (C-3), 96.25 (Cl), 128.43 (Phe) , 156.21 and 171.30 (CO).

g) Synthesis of 2,3,4-tri-O-acetyl-ό-deoxy-β-L-galactopyrannoside of 15-octadecylamino-pentadecanyl

To a solution of 2,3,4-tri-O-acetyl-6-deoxy-β-L-galactopyrannoside of 15- [N- (benzyloxycarbonyl) -octadécylamino] -pentadécanyl obtained in the previous step f) (2, 72 g; 4.23 mmol) in methanol (100 ml), 10% palladium on activated carbon (0.5 g) is added under hydrogen pressure. The reaction is quantitative.

! H NMR: δ (ppm) 0.87 (t, 3H, J≈ 6.96 Hz, H-33), 1.2 (d, 3H, J≈ 6.51 Hz, H-6), 1, 25 (m, 54H, (CH ₂ ) 27), 1.52 (m, 6H, OCH ₂ ÇH ₂ , H-14 and Hl 7), 1.88-1.93 (NH band), 1.95, 2.05 and 2.15 (s, 3H, OCOCH3), 2.64 (m, 4H, H-15 and Hl 6), 3.46 (m, 1H, OCH _a CH ₂ ), 3.63 (m, 1H, OCH _b CH), 3.79 (m, IH, H-5), 4.41 (d, IH, J≈ 7.98 Hz, Hl), 4.99 (dd, IH, J≈ 3.52 Hz and 10.46 Hz, H-3), 5.16 (dd, IH, J≈ 7.98 Hz and 10.46 Hz, H-2), 5.23 (dd, J≈ 3.52 Hz and 3, 31Hz, H-4).

¹³ C NMR (CDCI ₃ ): δ (ppm) 14.68 (C-33), 17.31 (C-2), 20.75 (CH3COO), 27.29 (C-6), 29.72 ( (CH ₂ ) 27)), 25.89-31.98 (OCH ₂ ÇH2, C-14, Cl 7), 47.75-48.04 (C-15 and C-16), 69.63 (OCH ₂ CH ₂ ), 69.45 (C-2), 70.57 (C-5), 70.85 (C-4), 71.44 (C-3) , 96.25 (Cl), 171.30 (CO).

h) Synthesis of 2,3,4-tri-0-acetyl-6-deoxy- -L-galactopyrannoside of (3- [4- (3- amino-propyl-amino) -butyl-amino-propyl-benzyloxycarbonyl-amino ] -methylene carbamoyl) -15-pentadecanyl-16-octadecanyl

To a solution of 0.60 g of the compound obtained in the preceding step g) (0.94 mmol) in chloroform (15 ml), diisopropylethylamine (0.491 ml; 2.82 mmol) and BOP are successively added (0.457 g; 1.03 mmol) and dev {{[4- (3- benzyIoxycarbonyI-amino-propyl-benzyIoxycarbonyl-amino) -butyI- benzyloxycarbonyl-amino] -propylamino} -acetic acid obtained in step a ) (0.748 g; 0.94 mmol). The resulting oil is purified by chromatography (heptane / ethyl acetate 4: 6). We obtain 2,3,4-tri- <9-acetyl-6-deoxy-β-L-galactopyrannoside from (3- [4- (3- amino-propyl-amino) -butyl-amino-propyl-benzyloxycarbonyl- amino] -methylene-carbamoyl) -15-pentadecanyl-16-octadecanyl with a yield of 45%. * H NMR: δ (ppm) 0.87 (t, 3H, J≈ 6.96 Hz, H-33), 1.2 (d, 3H, J≈ 6.51 Hz, H-6), 1, 24 (m, 54H, (CH ₂ ) 27), 1.39-1.67 (m, 15H, OCH7CH7, H- 14, Hl 7, NH, CH ₂ ), 1.95, 2.05 and 2, 15 (s, 3H, OCOCH3), 3.05-3.35 (m, 18H, H-15, H-16 and CH7N), 3.43 (m, 1H, OCH _a CH ₂ ), 3.67 ( m, IH, J≈ 6, 74 Hz, OCH _b CH ₂ ), 3.79 (m, IH, H-5), 4.41 (d, IH. J≈ 7.98 Hz, Hl), 4, 99 (dd, IH, J≈ 3.52 Hz and 10.46 Hz, H-3), 5.05 (s, 8H, CH2Phe), 5.16 (dd, IH, J≈ 7.98 Hz and 10 , 46 Hz, H-2), 5.23 (dd, J≈ 3.52 Hz and 3.37Hz, H-4), 5.47 (CONH band, IH), 7.32 (m, 20H, Phe).

1 ³ C NMR (CDCI ₃ ): δ (ppm) 14.84 (C-33), 20.75 (CH3COO), 27.29 (C-6 '), 29.72 ((CH) 27)), 25.89-31.98 (OCH ₂ CH ₂ , C-14, C-17 and CH ₂ ), 37.87-46.87 (C-15, C-16 and CN), 66.84 (CH ₂ Phe), 68 , 63 (OCH7CH7), 69.45 (C-2), 70.57 (C-5), 70.85 (C-4), 71, 44 (C-3), 96.25 (Cl), 128 , 31 (Phe), 157.01 and 171.30 (CO).

i) Synthesis of the 6-deoxy- -L-galactopyrannoside of l - [- (3- [4- (3-amino-propyl-amino) -butyl-amino-propyl-benzyloxycarbonyl-amino] -methylene -carbamoyl) -15 - pentadecanyl-16-octadecanyl To a methanolic solution (3 ml) containing the product obtained in the preceding step h) (0.60 g; 0.94 mmol) is added a methanolic solution (1 ml) saturated with ammonia. After one hour, it is concentrated.

* H NMR: δ (ppm) 0.87 (t, 3H, J≈ 6.96 Hz, H-33), 1.2 (d, 2H, J≈ 6.51 Hz, H-6), 1, 24 (m, 54H, (CH ₂ ) 27), 1.39-1.67 (m, 15H, OCH ₂ CH ₂ , H- 14, H-17, NH, CH), 3.05- 3.35 (m, 18H, H-15, H-16 and CH? N), 3.4-3.7 (m, 6H, OCH ₂ CH ₂ , H-3, H-4, H-5, H- 2 ), 4.73 (d, IH, J≈ 7.98 Hz, Hl), 5.05 (s, 8H, ÇH ₂ Phe), 5.47 (CONH band, IH), 7.32 (m, 20H, Phe ). j) Synthesis of the 6-deoxy- -L-galactopyrannoside of l - [- (3- [4- (3-amino-propyl-amino) -butyl-amino-propyl-amino] -methylene-carbamoyl) -15-pentadecanyl -16- octadecanyl (compound 3)

To a solution of the product obtained in previous step i) (0.072 g; 0.05 mmol), 10% palladium on carbon (0.032 g) is added in methanol. After one night, filter on glass paper and concentrate on a rotary evaporator. The product is then purified by HPLC on a preparative column of type C-4.

* H NMR: δ (ppm) 0.87 (t, 3H, J≈ 6.96 Hz, H-33), 1.2 (d, 2H, J≈ 6.51 Hz, H-6), 1, 24 (m, 54H, (CH ₂ ) ₂₇ ), 1.39-1.67 (m, 15H, OCH ₂ CH, H- 14, H- 17, CH ₂ ), 2.92-3.19 (m , 18H, Hl 5, H-16 and CH ₂ N), 3.4-3.7 (m, 6H, OCH ₂ CH ₂ , H-3, H-4, H-5, H-2), 4 , 73 (d, 1H, J = 7.98 Hz, Hl).

C \ USE OF TRANSFER AGENTS ACCORDING TO THE INVENTION

EXAMPLE 4 Preparation of Transfer Agent / Nucleic Acid Complexes with Compound 2 and Measurement of Their Size

This example illustrates the preparation of complexes between a transfer agent according to the invention and a nucleic acid, their size having then been measured.

The glycolipid used in this example and in the examples which follow is compound 2, in solution in chloroform, at a concentration of 10 mg / ml. In in some cases, a neutral co-lipid, cholesterol or DOPE, was previously mixed with compound 2.

The lipid solution is prepared as follows: a sample of the desired quantity is taken, the solvent is evaporated under a stream of argon and allowed to dry for 1 hour. Then, the lipid is rehydrated with a solution containing 5% dextrose and 10 mM sodium chloride overnight at 4 ° C. The next day, the lipid solutions are heated at 60 ° C for 5 minutes and then passed through ultrasound for 1 minute. The operation is repeated until the size of the lipid particles is stable.

The DNA used is the plasmid pXL3031 (FIG. 1) in solution in a mixture of 5% dextrose and 10 mM sodium chloride at a concentration of 0.5 mg / ml or 1.0 mg / ml. This plasmid contains the read gene coding for luciferase under the control of the P / E CMV promoter of the cytomegalovirus. Its size is 3671 bp. The diagram of this plasmid is shown in FIG. 1. The plasmid pXL3031 was purified according to the methods described in patent application WO 97/35002.

The compound 2 / DNA complexes are prepared by rapidly mixing appropriate volumes of plasmid DNA solution and of compound 2 (according to the desired charge ratio), at room temperature. The amount of transfecting agent varies between 0.25 nmol / μg of DNA and 12 nmol / μg of DNA. The size of the complexes was analyzed by measuring the hydrodynamic diameter by dynamic light scattering (Dynamic Laser Light Scattering) using a Coulter N4Plus device. The samples are diluted 20 times in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple diffusions. At a ratio of 3 nmoles of lipid / μg of DNA, the following results were obtained:

The term "micelles" indicates that compound 2 was used alone, that is to say without adding neutral co-lipid, and therefore it forms a micellar solution.

This table shows that the complexes obtained have a size of between 130 nm and 150 nm approximately, which is compatible with pharmaceutical use, in particular in injection.

Example 5: Behavior of the complexes formed from compound 2 at different charge ratios

This example illustrates the behavior of the transfer agent complexes according to the invention / nucleic acid when the charge ratio is varied. The impact of adding a co-lipid (cholesterol or DOPE) is also illustrated.

Conventionally, there are 3 physicochemical phases when the transfer agent / DNA charge ratio is increased (B. Pitard et al, Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote g ne transfer, PNAS, Vol. 94, pp. 14412-14417, 1997). These three phases determine the therapeutic potential of the transfer agent.

At a low charge ratio, the DNA is not saturated with the transfer agent. Uncomplexed DNA remains, and the complexes are generally negatively charged and small. This stable phase is called "Phase A". The fact that the DNA is not completely saturated with the transfer agent means that the DNA is not completely protected. DNA can therefore be subjected to degradation by nucleases. Furthermore, the complexes being generally negative, the passage of the cell membrane is difficult. For these reasons, the nucleolipid complexes of phase A are relatively inactive.

At an intermediate charge ratio, the DNA is completely saturated with the transfer agent, and the complexes are generally neutral or slightly positive. This phase is unstable because the ionic repulsions are minimal and an aggregation phenomenon can occur. Particle size is well above the limit detection by dynamic light scattering (much greater than 3 μ). This unstable phase is called "phase B". Such a complex size is not suitable for use in injection, although this does not mean that the complexes are inactive in phase B: they are only in a formulation which is not suitable for their injection for a purpose. pharmaceutical.

At a higher charge ratio, the DNA is supersaturated by the transfer agent, and the complexes are generally positive. Due to the strong repulsions between the positive charges, this phase is stable. It is referred to as "phase C". Unlike phase A, the complexes obtained are in a form such that DNA is very well protected against nucleases, and the generally positive charge of these complexes facilitates attachment to the cell membrane of an anionic nature and the passage of this membrane. The complexes of phase C are therefore particularly suitable for use for the transfer of nucleic acids into cells.

These 3 zones A, B and C have also been updated with compound 2 according to the invention as a transfer agent:

As shown in the table above, zone B, which is the zone of instability, is particularly small and is located at very low load ratios. Zone C starts from 2 nmoles of lipid / μg of DNA when compound 2 is used in conjunction with a co-lipid (Cholesterol or DOPE), and from 3 nmoles of lipid / μg of DNA when the compound is used alone. As has been specified previously, it is in this zone that it is particularly advantageous to be placed for pharmaceutical use. By way of comparison, it has been shown with one of the cationic lipids disclosed in application WO 97/18185 that zone C began to form at charge ratios at least equal to 2 depending on the concentration of sodium chloride in the solution (see Figure 3 A in B. Pitard et al., PNAS USA, 94, pp. 14412-14417, 1997).

Thus, compound 2 is a particularly advantageous transfer agent because it is stable at low charge ratios, which makes it possible to form stable complexes with low amounts of glycolipids, with the beneficial consequences which ensue in terms of toxicity.

Example 6: Use of Compound 2 for the In Vitro Transfer of DNA This example illustrates the capacity of the transfer agents according to the invention to transfect DNA in cells in vitro, at different charge ratios, in the absence and in the presence of a neutral co-lipid (cholesterol or DOPE).

24-well microplates are seeded with 60,000 HeLa cells per well, and are grown overnight. The number of cells after one night, and therefore at the time of transfection, is 100,000 cells per well.

Each well is brought into contact with the complexes formed with compound 2 and containing 1 μg of plasmid DNA in 0.5 ml of DMEM culture medium (Gibco / BRL without serum. The cells are incubated at 37 ° C. for 5 The medium containing the complexes is then removed and replaced with a DMEM culture medium and 10% fetal calf serum. Then the cells are again cultured for 24 hours. Finally, the cells are lysed and tested in using a luciferase test kit (Promega) and a Dynex MLX luminometer.

The results obtained are indicated on the histogram of FIG. 2. The efficiency of the transfer is represented by the expression of the luciferase in pg / well. It is noted that the maximum transfection is approximately 500 pg / well. In conclusion, this example clearly shows that it is possible to use the compound 2 according to the invention to form complexes capable of promoting the transfer of DNA into cells in vitro.

Example 7: Use of Compound 2 for the In Vivo Transfer of DNA This example illustrates the capacity of the transfer agents according to the invention to transfect DNA in cells in vivo.

In vivo gene transfer was performed on Balb / C mice by intratracheal, intravenous and intramuscular administration.

In the case of intramuscular injections, each mouse received 30 μl of formulation containing 15 μg of plasmid DNA in the anterior tibia muscle. The tissues are recovered 7 days after the injection, they are frozen and stored at -80 ° C while waiting to carry out the luciferase activity tests.

In the case of intravenous injections, each mouse received 200 μl of formulation containing 50 μg of plasmid DNA. The tissues are recovered 24 hours after the injection, then are frozen and stored in the same way as above.

FIG. 3 illustrates the activity of the complexes formed with compound 2 for gene transfer in vivo intramuscularly. These results clearly show that the formation of complexes with compound 2 according to the invention and DNA makes it possible to promote the transfer of said DNA into cells in vivo.

Likewise, any transfer agent as defined in the present invention can be used to promote the transfer of DNA into cells of any type of tissue.

Claims

1. Nucleic acid transfer agents characterized in that they comprise a hydrophobic spacer chemically linked on the one hand to a polycation and on the other hand to at least one hydrophilic substituent.

2. nucleic acid transfer agents according to claim 1, characterized in that said hydrophobic spacer consists of 2 or 3 linear hydrocarbon fatty chains comprising between 10 and 20 carbon atoms per chain, each chain being possibly of different length, or well, said hydrophobic spacer consists of a very long linear hydrocarbon fatty chain, comprising between 20 and 50 carbon atoms.

3. Nucleic acid transfer agents according to claim 1 characterized in that the hydrophilic substituent or substituents are chosen from hydroxy, amino substituents, polyols, sugars, or even hydrophilic peptides.

4. Nucleic acid transfer agents according to claim 1 or 3 characterized in that at least one of the hydrophilic substituents is a sugar.

5. Nucleic acid transfer agents according to claim 1 of general formula (I):

for which: - R represents a polycation,

Z represents a hydrogen atom or a fluorine atom, the different Z being independent of each other, and

- either x and y, independently of one another, represent integers between 10 and 22 inclusive, and X and Y, independently of each other, represent an atom hydrogen, an —OAlk group where Alk represents a straight or branched alkyl containing 1 to 4 carbon atoms, a hydroxy group, an amino group, a polyol, a sugar, a hydrophilic or non-hydrophilic peptide, or an oligonucleotide, it being understood that at least one of the substituents X and Y represents a hydrophilic group chosen from hydroxy, amino, polyols, sugars, or hydrophilic peptides,

- either x is equal to 0 or 1, y is an integer between 20 and 50, X is either a hydrogen atom or a group -OAlk where Alk represents a straight or branched alkyl containing 1 to 4 carbon atoms, and Y is a hydrophilic group chosen from hydroxy, amino, polyols, sugars, or hydrophilic peptides, where appropriate in their isomeric forms, as well as their mixtures, or their salts when they exist.

6. Nucleic acid transfer agents according to claim 1 or 5 of general formula (III):

for which :

- R represents a polycation, and

- either x and y, independently of one another, represent integers between 10 and 22 inclusive, and X and Y, independently of each other, represent a hydrogen atom or a sugar, being it being understood that at least one of the substituents X and Y represents a sugar,

- either x is equal to 0 or 1, y is an integer between 20 and 50, X is a hydrogen atom and Y is a sugar. where appropriate in their isomeric forms, as well as their mixtures, or their salts when they exist.

7. Nucleic acid transfer agents according to claim 6 characterized in that x and y, independently of one another, represent integers between 10 and 22 inclusive, and one of X and Y represents a hydrogen atom and the other a sugar.

8. Nucleic acid transfer agents according to one of claims 1 and 5 to 7 characterized in that said polycation is a linear or branched polyamine, each amino group being separated by one or more methylene groups.

9. Nucleic acid transfer agents according to claim 8, characterized in that said polycation has the general formula (II):

in which :

- R „R ₂ and R ₃ represent independently of each other a hydrogen atom or a group (CH ₂ ) _q NR'R" with q an integer which can vary from 1 to 6, this independently between the different R ,, R ₂ and R _{3 groups} , it being understood that at least one of R „R ₂ and R ₃ is different from a hydrogen atom, - R 'and R" independently represent one of the other a hydrogen atom or a group (CH ₂ ) _q NH ₂ with q defined as above,

- m represents an integer between 1 and 6, and

- n and p independently represent whole numbers between 0 and 6, with when n is greater than or equal to 2, m can take different values and R ₃ different meanings within the general formula (II), and when n is equal to 0, at least one of the substituents R, and R ₂ is different from a hydrogen atom.

10. Nucleic acid transfer agents according to one of claims 1 and 5 to 7 characterized in that said polycation is chosen from spermine, spermidine, cadaverine, putrescine, hexamethylenetetramine (hexamine), methacrylamidopropyl-trimethylammonium chloride (AMBTAC), 3-acrylamido-3-methylbutyltrimethylammonium chloride (AMBTAC), polyvinylamines, polyethyleneimines, or ionenes.

1 1. Nucleic acid transfer agents according to one of claims 3 to 7 characterized in that the sugar or sugars are mono-, oligo- or polysaccharide molecules.

12. Nucleic acid transfer agents according to claim 1 1, characterized in that the said sugar or sugars are chosen from glucose, mannose, rhamnose, galactose, fructose, maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarabiosis, gentiobiose, sophorose, melibiose, dextran, α-amylose, amylopectin, fructans, mannans, xylans and arabinans.

13. Nucleic acid transfer agents according to claim 5 characterized in that said oligonucleotide is any chain containing one or more nucleotides, deoxynucleotides, ribonucleotides and / or deoxyribonucleotides, optionally coupled to one or more molecules having distinct properties.

14. Nucleic acid transfer agents according to claim 5 characterized in that said peptide is any chain containing one or more amino acids linked together by bonds of peptide nature, optionally substituted by one or more aliphatic groups which may be saturated or unsaturated, and linear, branched or cyclic.

15. Transfer agent according to claim 1 of formula:

16. Transfer agent according to claim 1 of formula:

[7 Transfer agent according to claim 1 of formula:

18. Composition characterized in that it contains a nucleic acid transfer agent as defined in claims 1 to 17 and a nucleic acid.

19. Composition according to Claim 18, characterized in that the nucleic acid is a deoxyribonucleic acid or else a ribonucleic acid.

20. Composition according to claim 18 or 19 characterized in that said nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences.

21. Composition according to claims 18 to 20 characterized in that said nucleic acid is a gene or an antisense sequence.

22. Composition according to claim 18 characterized in that it also contains one or more adjuvants.

23. Composition according to claim 22 characterized in that the adjuvant is one or more neutral lipids.

24. Composition according to claim 23 characterized in that the neutral lipids are lipids with two fatty chains.

25. Composition according to Claims 23 and 24 characterized in that the neutral lipids are natural or synthetic lipids, zwitterionic or devoid of ionic charge under physiological conditions, chosen for example from dioleoylphosphatidylethanolamine (DOPE), oleylpalmitoylphosphatidylethanolamine (POPE) , di-stearoyl, -palmitoyl, - mirystoylphosphatidylethanolamines as well as their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), such as sphingides ) or asialogangliosides (such as in particular asialoGMl and GM2).

26. Composition according to claim 22 characterized in that the said adjuvant is a compound intervening directly or not at the level of the condensation of the nucleic acid.

27. Composition according to Claim 26, characterized in that the said adjuvant is derived wholly or in part from a protamine, a histone, or a nucleoline and / or one of their derivatives, or else consists, in whole or in part, of peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of units can vary between 2 and 10, and can be repeated continuously or not.

28. Composition according to claims 18 to 27 characterized in that it comprises a pharmaceutically acceptable vehicle for an injectable formulation.

29. Composition according to claims 18 to 27 characterized in that it comprises a pharmaceutically acceptable vehicle for application to the skin and / or the mucous membranes.

30. Use of a transfer agent as defined in claims 1 to 17 for the manufacture of a medicament intended to treat diseases.

31. Method for treating the human or animal body, comprising the following steps:

(1) bringing the nucleic acid into contact with a transfer agent as defined in claims 1 to 17, to form a complex, and (2) bringing the cells of the human or animal body into contact with the complex formed in (l).

32. Method for transferring nucleic acids into cells, characterized in that it comprises the following steps:

(1) bringing the nucleic acid into contact with a transfer agent as defined, to form a complex, and

(2) bringing the cells into contact with the complex formed in (1).

33. Method for transferring nucleic acids into cells according to claims 31 or 32 characterized in that said transfer agent and / or said nucleic acid are previously mixed with one or more adjuvant (s) as defined in claims 22 to 27.