KR20010101116A - Novel nucleic acid transferring agents, compositions containing them and uses - Google Patents

Abstract

The present invention relates to novel delivery agents, compositions containing them and their use for delivery of nucleic acids in vitro, in vivo or ex vivo. More precisely, the present invention relates to novel nucleic acid delivery agents comprising a polycation and a hydrophobic spacer chemically bonded to at least one hydrophilic substituent.

Description

Novel nucleic acid delivery agents, compositions containing them and uses {NOVEL NUCLEIC ACID TRANSFERRING AGENTS, COMPOSITIONS CONTAINING THEM AND USES}

With the development of biotechnology, the potential delivery of nucleic acids into cells has become a fundamental technology with many biotechnological applications. This may include the delivery of nucleic acids into cells in vitro, for example for the production of recombinant proteins or for the study of regulatory expression of genes in the laboratory, gene cloning or other manipulations involving DNA. It may also include the delivery of nucleic acids into cells in vivo, for example for the production of vaccines, the conduct of labeling studies or the implementation of therapeutic methods. It may also include, for example, for the production of transgenic animals, from the point of subsequent re-administration, the transfer of genes into cells harvested from the organism.

Currently, the most widespread means for gene transfer into cells is the use of viral vectors. However, since the latter is not entirely without risk, several other methods based on the use of synthetic vectors have been proposed. This synthetic vector has two main functions: complexing and compacting the nucleic acid to be transfected, and facilitating its passage across the plasma membrane, and possibly across the two nuclear membranes.

For example, several strains of synthetic vectors, such as polymers or biochemical vectors (consisting of cationic proteins associated with cell receptors) have been developed, but with the development of lipofectants, more specifically cationic lipids, non-viral transfection In particular significant progress has been made in. Thus, it has been demonstrated that cationic lipids, due to their overall positive charge, spontaneously interfere with the overall negatively charged DNA, thereby forming a nuclear lipid complex that can fuse with the cell membrane, thus allowing intracellular release of DNA.

Thus, various cationic lipids have been synthesized: lipids containing quaternary ammonium groups (e.g., DOTMA, DOTAP, DMRIE, DLRIE, etc.), for example lipois disclosed in DOGS, DC-Chol or patent application WO 97/18185. Lipopolyamines such as polyamines, for example lipids containing both quaternary ammonium groups and polyamines, such as DOSPA, or lipids containing various other cationic entities, in particular amidinium groups (e.g. ADPDE, ADODE or patent applications) Lipids of WO 97/31935). In fact, the structural diversity of the cationic lipids is partly a reflection of the structure-activity relationship observation.

However, the use of such synthetic vectors still presents many challenges, and there is room for improvement in efficiency. In particular, it would be desirable to be able to construct non-cationic or low cationic vectors, and the reasons vary as follows:

The complex formed between the nucleic acid and the delivery agent is generally positively charged and therefore tends to be absorbed by the reticuloendothelial system, leading to its removal.

Due to the overall positive charge of the complexes formed, plasma proteins tend to adsorb to the surface, from which a loss of transfection occurs.

In the case of topical injection, the presence of a significant amount of total positive charge prevents the diffusion of the nucleic acid complex outside the site of administration because the complex is adsorbed to the extracellular matrix. Thus, the complex no longer reaches the target cell, resulting in a reduction in delivery efficiency for the dose of the complex.

Finally, many of those involved in the domain of non-viral transfection of genes pointed to an inflammatory effect on cationic lipids or polymers.

Moreover, it has been noted that stable formulations of synthetic vectors developed to date are generally difficult, or even impossible, at low charge ratios and often have poor transfer efficiency at low charge ratios (Pitard et al., PNAS USA, 94). , pp. 14412-14417, 1997). In the remainder of the text, the term "charge ratio" refers to the ratio of the positive charge of the transfer agent to the negative charge of the DNA. This ratio is often expressed in nmol of delivery agent per μg DNA.

These are the problems that the applicant proposes to solve and the novel transfection agents that make up the subject of the present invention. Specifically, their specific structures bind primarily to polycations that allow complex formation with nucleic acids, and secondly those transfections to cationic lipids or polymers commonly used for non-viral transfections. It forms a hydrophobic anchor coupled to at least one hydrophilic head that can reduce the apparent overall charge density of the agent. The presence of at least one hydrophilic head creates a kind of "charge barrier" by reducing the zeta potential of the complex formed with the nucleic acid. Thus, these complexes appear to be less cationic to the organism, and thus have beneficial results. Furthermore, the transfection agents according to the invention are particularly advantageous from a physicochemical point of view because they are particularly stable when contacted with nucleic acids at low charge ratios.

The present invention relates to novel delivery agents, compositions containing them and their use for in vitro, in vivo or ex vivo delivery of nucleic acids in cells.

1: Schematic diagram of plasmid pXL2774 used for DNA delivery experiments into cells.

Figure 2: Gene transfer activity in HeLa cells in vitro of complexes formed from compound 2 according to the invention in the absence of co-lipids, or in the presence of cholesterol and in the presence of DOPE as co-lipids. The y axis shows luciferase expression (pg / well). The x-axis shows the transfection agent / DNA ratio (nmol) per μg DNA.

Figure 3: In vivo gene transfer activity after direct injection into the mouse anterior tibial muscle of the complex formed from compound 2 according to the invention in the presence of DOPE (1: 1). The y axis shows luciferase expression (pg / muscle). The x axis shows the Compound 2 / DNA ratio (nmol) per μg DNA.

Accordingly, a first subject of the invention relates to novel nucleic acid transfection agents, which primarily comprise a polycation and a hydrophobic spacer chemically bonded to at least one hydrophilic substituent.

Polycations allow the formation of complexes with nucleic acids by interaction with the anionic charge of the nucleic acid. Hydrophobic spacers have a dual function. First, it allows cell membrane passage, and secondly, the complex formed with the nucleic acid makes it viable in a biological medium. Specifically, hydrophobic spacers create a physical bond on the complex that enables protection of the nucleic acid against an external medium. The hydrophobicity required to allow the complex to survive can be readily determined by one skilled in the art using conventional investigative methods or using conventional trial and error methods. Finally, the presence of hydrophilic substituents makes it possible to reduce the zeta potential of the formed complex, making the complex appear less cationic to the external medium.

For the purposes of the present invention, polycations are linear or branched polycation molecules capable of associating with nucleic acids. For the purposes of the present invention, the expression "association with nucleic acid" means other forms of attachment, such as, for example, covalent attachment, electrostatic or ionic interaction, hydrogen bridge, and the like. Preferably the polycation is a straight or branched polyamine, with each amino group separated by one or more methylene groups. Optionally, the polyamines may also be substituted with other cationic functional groups such as amidinium or guanidinium groups, cyclic guanidines and the like. This is, in particular, defined in the patent applications WO 96/17823, WO 97/18185, WO 97/31935, WO 98/54130 or WO 99/51581, and more generally in the entire literature on cationic lipid structures known to those skilled in the art. It may be a polycation. According to a preferred aspect of the invention, the polycation represents a polyamine of formula (2).

In the above formula,

R ₁ , R ₂ and R ₃ independently of one another represent a hydrogen atom or a (CH ₂ ) _q NR′R ″ group, where q is an integer ranging from 1 to 6, which is various R ₁ , R ₂ and Independent of the R ₃ groups, at least one of R ₁ , R ₂ and R ₃ is not a hydrogen atom,

R ′ and R ″ are independently of each other a hydrogen atom or q is a (CH ₂ ) _q NH ₂ group as defined above,

m is an integer from 1 to 6,

n and p are each independently an integer from 0 to 6, when n is 2 or more, m may have a different value and R ₃ has a different meaning in formula (2), and when n is 0, R ₁ and R At least one of the _two substituents is not a hydrogen atom.

Other possible polycations also include spermine, spermidine, cadaverine, putrescine, hexamethylenetetramine (hexamine), methacrylamidopropyltrimethylammonium chloride (AMBTAC), 3-acrylamido-3-methylbutyl Trimethylammonium chloride (AMBTAC), polyvinylamine, polyethyleneimine, or ionone (see Barton et al., Comprehensive Organic Chemistry, Vol. 2, Ed.Pergamon Press, P. 90; Encyclopedia of Polymer) Science and Engineering, 2nd Edition, Ed. Wiley Interscience, Vol. 11, p. 489; Mahler and Cordes, Biological Chemistry, Harper International Edition, p. 124).

Hydrophobic spacers can take a wide variety of structures as long as they provide sufficient hydrophobicity to allow for the protection of nucleic acids and the passage of membranes. Such sufficient hydrophobicity can be measured by one skilled in the art using conventional irradiation methods. According to a preferred alternative of the invention, the hydrophobic spacer comprises two or three hydrocarbon-based straight chain fatty acids (ie 10 to 20 carbon atoms per chain, preferably 12, 14, 15, 16, 17 or 18 carbon atoms per chain, respectively). Chains may vary in length). According to another alternative, the hydrophobic spacer consists of a very long hydrocarbon-based straight fatty chain, ie comprises from 20 to 50 carbon atoms, preferably 40 to 50 carbon atoms, even more preferably 44 to 50 carbon atoms.

Suitable hydrophilic substituents are for example selected from hydroxyl or amino substituents, polyols, sugars or hydrophilic peptides. The term "polyol" refers to any straight, branched or cyclic hydrocarbon-based molecule comprising at least two hydroxyl functional groups. For example, glycerol, ethylene glycol, propylene glycol, tetritol, pentitol, cyclic pentitol (or queritol), hexitol, for example mannitol, sorbitol, dulitol, cyclic hexitol or inositol, etc. (Stanek et al., The Monosaccharides Academic Press, pp. 621-655 and pp. 778-855).

According to an advantageous alternative, the delivery agent according to the invention comprises at least one hydrophilic substituent which is a sugar. For the purposes of the present invention, the term "sugar" means any molecule consisting of one or more saccharides. For example, sugars such as pyranose and furanose, for example glucose, mannose, rhamnose, galactose, fructose or maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminara Biose, genthiobiose, sophorose, melibiose, etc. are mentioned. Preferably the sugar is selected from glucose, mannose, rhamnose, galactose, fructose, lactose, saccharose and cellobiose. In addition, they may be “complex” sugars, ie several sugars that are covalently coupled to each other, each sugar being preferably selected from the list above. Suitable polysaccharides include dextran, α-amylose, amylopectin, fructans, mannan, xylan and arabinane. Certain preferred sugars may also interact with cellular receptors such as certain lectins.

More specifically, the delivery agent according to the present invention may be represented by the formula (1).

In the above formula,

R represents a polycation,

Z represents a hydrogen atom or a fluorine atom, the various Z are mutually independent,

x and y independently of each other represent an integer of 10 to 22, X and Y independently of each other a hydrogen atom, an -OAlk group (wherein Alk represents a straight or branched chain alkyl of 1 to 4 carbon atoms), hydroxyl Group, amino group, polyol, sugar, hydrophilic or non-hydrophilic peptide, or oligonucleotide, at least one of the X and Y substituents represents a hydrophilic group selected from hydroxyl group, amino group, polyol, sugar or hydrophilic peptide ,

Or x is 0 or 1, y is an integer from 20 to 50, X is a hydrogen atom or an -OAlk group, where Alk represents straight or branched chain alkyl of 1 to 4 carbon atoms, and Y is hydroxyl Hydrophilic group selected from the group, amino group, polyol, sugar or hydrophilic peptide.

For the purposes of the present invention, polycations, polyols and sugars of formula (I) are as defined above.

In formula (1) x and y are defined to have any value of 10 to 22 or 20 to 50 in some cases. Preferably, x and y are 12 to 18 independently of each other. More preferably, x and y independently of each other have a value of 14, 15, 16, 17 or 18. When x is 0 or 1, y is preferably 30 to 50, or 40 to 50. More preferably, y is 44 to 50.

For the purposes of the present invention, the term "oligonucleotide" refers to at least one nucleotide, deoxynucleotide, ribo, which is a monomeric unit which differs by the presence of a base which may be selected from adenine, guanine, cytosine, thymidine or uracil. Nucleotides and / or deoxyribonucleotides. See Lehninger Biochimie, Flammarion Medicine Sciences, 2nd edition, p. 305-329]. Because of their base pairing properties, oligonucleotides are widely used in molecular biology, for example as linkers (adherent molecules) or probes.

In addition, oligonucleotides may be used in the form of conjugates, ie, in the form of coupling with one or more other molecules having distinctive properties. For example, the coupling of oligonucleotides with reactive chemical groups, fluorescent or chemiluminescent groups, or with groups capable of enhancing intermolecular interactions to facilitate entry into cells. Such conjugates are 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, for example, the ability to enhance the influx of complexes into cells, to reduce the degree of degradation by nucleases, to increase the stability of the complexes, to monitor the fate of oligonucleotides in an organism, and the like. There is an advantage. Thus, oligonucleotides, when grafted to a delivery agent according to the present invention, allow the delivery agent to provide additional properties (eg, targeting, labeling, etc.).

Oligonucleotides can be obtained according to conventional methods known to those of skill in the art and include 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 Tetrahedron, Beaucage et al., The Synthesis of Modified Oligonucleotides by the Phosphoramiditeb Approach and Their Application, Vol. 49, no. 28, pp. Modified oligonucleotides may also be synthesized according to the methods described in 6123-6194, 1993.

For the purposes of the present invention, the term "peptide" refers to a chain containing one or more amino acids that are linked together via attachment of a peptide species [Lehninger Biochimie, Flammarion Medicine Sciences, 2nd edition]. These are 20 "traditional" amino acids, i.e. those commonly found in protein compositions (alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, aspartic acid, glutamine, lysine, arginine, histidine , Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamic acid), or they may also be "rare" amino acids such as 4-hydroxyproline, desmocin, 5-hydroxylysine, N-methyllysine, 3 -Methylhistidine, isodesmocin, etc. Finally, they also appear in free or bound form in various cells or in various tissues, and are generally expressed in α-amino acids (e.g., β-alanine, γ- Aminobutyric acid, homocysteine, ornithine, cannavanin, zenkalic acid, β-cyanoalanine, etc. Such peptides may be, for example, In this context, for example, RGD or NLS peptides may be used, and they may also be used for analysis techniques such as, for example, fluorescence spectroscopy, infrared spectroscopy, nuclear magnetic resonance (NMR), etc. Peptide sequences having labeling properties, ie, allowing identification, in this regard, for example, Arg-Gly-Asp (arginine-glycine-) of primary and / or secondary receptors for integrin-type adhesion proteins. Straight chain or cyclic peptide or pseudopeptide sequences containing aspartic acid) recognition epitopes.

The peptides according to the invention can also be substituted for one or more of the functional groups, for example in the αcarboxyl, in the αamine functional groups and / or in the functional groups of the respective amino acid side chains. Straight, branched or cyclic saturated or unsaturated aliphatic groups of 1 to 24 carbon atoms, such as, for example, cholesteryl, arachidyl or retinoyl radicals, or for example substituted or unsubstituted benzyloxycarbonyl, benzyl esters, Or substitution by mono or polyaromatic groups such as rhodaminyl derivatives. The advantage of such substitutions is in modifying the chemical and / or biological properties of the peptides, for example labeling them.

When peptides are used as hydrophilic substituents, they are selected from hydrophilic peptides, ie peptides consisting solely of hydrophilic amino acids, or those consisting of partially hydrophilic amino acids and whose composition makes them totally hydrophilic.

According to a preferred alternative of the invention, all Z groups represent a hydrogen atom.

According to a more specific advantageous aspect of the present invention, the delivery agent is represented by the formula (3).

In the above formula,

R represents a polycation,

x and y independently of each other represent an integer from 10 to 22, X and Y independently of one another represent a hydrogen atom or a sugar, at least one of the X and Y substituents represents a sugar, or

Or x is 0 or 1 and y is an integer from 20 to 50, X is a hydrogen atom and Y is a sugar.

For the purposes of the present invention, the polycations, sugars and x and y in formula (3) are as defined above for formula (1).

More particularly preferred delivery agents are of formula (3), wherein x and y independently of each other represent an integer from 10 to 22, one of X and Y represents a hydrogen atom and the other a sugar. According to another advantageous alternative, the delivery agent according to the invention is of formula (3), x is 0, y is an integer from 40 to 50, X represents a hydrogen atom and Y is a sugar.

The present invention also relates to isomers of the product of formula (1), if present, and also mixtures or salts thereof.

In particular, the compounds of the present invention may be in the form of non-toxic pharmaceutically acceptable salts. Such non-toxic salts include inorganic acids (e.g. hydrochloric acid, sulfuric acid, bromic acid, phosphoric acid and nitric acid), organic acids (acetic acid, propionic acid, succinic acid, maleic acid, hydroxymaleic acid, benzoic acid, fumaric acid, methanesulfonic acid or oxalic acid), Salts with inorganic bases (sodium hydroxide, potassium hydroxide, lithium hydroxide, lime) or organic bases (tertiary amines such as triethylamine, piperidine, benzylamine).

According to the invention, the preparation of the product of formula 1 is carried out using the following steps:

1) First, an alkyl chain of carbon number x containing hydroxyl functional groups and ester functional groups (where x is defined above) is prepared by ring opening of the corresponding lactone. The reaction is generally carried out in alcohol, at a basic pH and at temperatures between −10 ° C. and room temperature. For example, the alcohol can be methanol or ethanol.

2) Then, the X group is attached to the difunctional alkyl chain obtained in the previous step. If X represents a sugar, condensation is carried out at a temperature of -5 to 10 ° C., in the presence of Lewis acid, in a chlorinated solvent such as, for example, dichloromethane or chloroform. Lewis acids can be selected, for example, from tin chloride, iron chloride, p-toluenesulfonic acid (tsOH), trimethylsilyltrifluoromethanesulfonic acid (TMStf), boron trifluoride etherate and the like [Kazunobu Toshima et al. , Recent Progress in O-glycosilation Methods and its Application to Natural Products Synthesis, Chem. Rev, 1993, Vol. 93, pp. 1503-1531.

When X represents a hydrophilic or non-hydrophilic peptide group, peptide coupling can be carried out according to conventional methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or using analogous methods known to those skilled in the art. Is performed. In particular, the reaction is generally carried out at a temperature of 0 to 100 ° C. in a suitable aprotic solvent in the presence of a non-nucleophilic base and the pH is adjusted to 9 to 11. For example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as the solvent. The non-nucleophilic base used is preferably a tertiary amine, calcium carbonate or sodium dicarbonate. Even more preferably, the base used is a tertiary amine, for example triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out at 0 to 50 ° C, preferably at 10 to 30 ° C.

If X wishes to represent a hydroxyl group, this step is not carried out.

When X represents an amino group, the reaction is carried out by nucleophilic substitution according to conventional methods known to those skilled in the art, allowing the amine to be obtained from an alcohol.

When X represents an -OAlk group, alkylation of the alcohol functionality is carried out according to conventional methods known to those skilled in the art or according to analogous methods. For example, diazo compounds of the formula Alk-N ₂ may optionally be reacted in the presence of a catalyst such as HBF ₄ or silica gel. In basic media it is also possible to work under Williamson reaction conditions in which Hal reacts a chain with an alcohol functional group to an Alk-Hal compound of formula wherein halogen represents a halogen atom such as chlorine, bromine or iodine.

The same Williamson type reaction can also be used when X wants to represent a polyol.

Finally, when X represents an oligonucleotide, the latter is coupled to the bifunctional chain according to known conventional methods for covalent grafting of oligonucleotides. For example, oligonucleotides can be grafted through a suitable linker (attachment molecule).

3) Third, the ester functional groups present in the bifunctional chain are hydrolyzed with acid functional groups according to known methods. For example, the procedure can be carried out at a reflux temperature of 50 to the reaction mixture, in a basic medium in a high boiling alcohol.

4) Subsequently, substituted or unsubstituted alkyl of formula (4) according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or using any analogous method known to those skilled in the art. The amine chain is coupled with the compound obtained in the previous step.

H ₂ N- (CH ₂ ) y Y

In the above formula,

y and Y are as defined above.

In particular, the reaction is generally carried out at a temperature of 0 to 100 ° C. in a suitable aprotic solvent in the presence of a non-nucleophilic base and the pH is adjusted to 9 to 11. For example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as the solvent. The non-nucleophilic base used is preferably a tertiary amine, calcium carbonate or sodium dicarbonate. Even more preferably, the base used is a tertiary amine, for example triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out at 0 to 50 ° C, preferably at 10 to 30 ° C.

Groups of formula (4) are commercially available or can be obtained by condensation of Y to the corresponding unsubstituted alkylamine according to methods analogous to those described above in 2).

5) Then, the amide obtained in the previous step is reduced to amine. The procedure for this is carried out according to conventional methods known to those skilled in the art. For example, the procedure is carried out by reacting lithium aluminum hydride LiAlH ₄ in anhydrous organic solvent such as anhydrous tetrahydrofuran. Other reducing agents that can be used are, for example, borane, borane in dimethyl sulfide (BH ₃ SMe ₂ ), sodium borohydride / titanium tetrachloride (NaBH ₄ , TiCl ₄ ), phosphorus oxychloride (POCl ₃ / Zn) on zinc ), Phosphorus pentasulphide on Raney nickel (P ₄ S ₁₀ ) [Richard C. Larock, Comprehensive Organic Transformations, VCH Publishers Inc., 1989]. The procedure can also be performed by catalytic hydrogenation. Advantageously, the reduction is carried out by reacting lithium aluminum hydride LiAlH ₄ in anhydrous tetrahydrofuran at the reflux temperature of the mixture.

Thus, the compound of formula 5 is obtained.

In the above formula,

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

6) Finally, in the last step, according to the conventional peptide coupling method (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or using any similar method known to those skilled in the art, The acid derivative corresponding to the polycation R is coupled with the compound of formula 4 obtained in the previous step.

In particular, the reaction is generally carried out at a temperature of 0 to 100 ° C. in a suitable aprotic solvent in the presence of a non-nucleophilic base and the pH is adjusted to 9 to 11. For example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as the solvent. The non-nucleophilic base used is preferably a tertiary amine, calcium carbonate or sodium dicarbonate. Even more preferably, the base used is a tertiary amine, for example triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out at 0 to 50 ° C, preferably at 10 to 30 ° C.

Acid derivatives corresponding to polycations are commercially available.

According to another alternative, the transfection agent according to the invention can be prepared by carrying out the following steps:

1) First, an alkyl chain of carbon number x containing hydroxyl functional groups and ester functional groups (where x is defined above) is prepared by ring opening of the corresponding lactone. The reaction is generally carried out in alcohol, at a basic pH and at temperatures between −10 ° C. and room temperature. For example, the alcohol can be methanol or ethanol.

2) Then, the substituted or unsubstituted alkylamine chain of formula 4 is coupled to this bifunctional alkyl chain.

Formula 4

H ₂ N- (CH ₂ ) y Y

In the above formula,

y and Y are as defined above.

The reaction is carried out at temperatures above the melting point of each product, possibly under vacuum. The reaction may also be carried out in the presence of an alcoholic solvent, at reflux temperature. For example, the solvent may be methanol or ethanol. The procedure is carried out, for example, at a temperature of 45 to 60 ° C.

The reaction can also be carried out at the reflux temperature of the mixture, in the presence of an alcohol such as methanol as a solvent. Another alternative is to directly couple the compound of formula 4 with lactone (in this case, the first step of ring-opening the lactone is not performed).

Groups of formula (4) are commercially available or can be obtained by condensing Y to the corresponding unsubstituted alkylamine according to methods analogous to those described above.

3) Then, the difunctional bicatenary amide obtained in the previous step is reduced to amine. The procedure for this is carried out according to a conventional method. For example, the procedure is carried out by reacting lithium aluminum hydride (LiAlH ₄ ) in anhydrous organic solvent such as anhydrous tetrahydrofuran. Other reducing agents that may be used are, for example, borane, boron hydride dimethyl sulfide (BH ₃ -SMe ₂ ), sodium borohydride / titanium tetrachloride (NaBH ₄ , TiCl ₄ ), phosphorus oxychloride (POCl ₃ on zinc) / Zn), phosphorus pentasulphide on Raney nickel (P ₄ S ₁₀ ) and the like (Richard C. Larock, Comprehensive Organic Transformations, VCH Publishers Inc., 1989). The procedure can also be carried out by catalytic hydrogenation.

Advantageously, the reduction is carried out by reacting lithium aluminum hydride LiAlH ₄ in anhydrous tetrahydrofuran at the reflux temperature of the mixture.

Thus, the compound of formula 6 is obtained.

In the above formula,

Y, x and y are as defined above.

4) Then, the X group is condensed to the amine of formula 6 obtained in the previous step. Condensation is performed according to a method similar to that described above for the first synthetic route.

5) Finally, in the last step, according to the conventional peptide coupling method (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or using any similar method known to those skilled in the art, The acid derivative corresponding to the polycation R is coupled with the compound of formula 6 obtained in the previous step.

In particular, the reaction is generally carried out at a temperature of 0 to 100 ° C. in a suitable aprotic solvent in the presence of a non-nucleophilic base and the pH is adjusted to 9 to 11. For example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene can be used as the solvent. The non-nucleophilic base used is preferably a tertiary amine, calcium carbonate or sodium dicarbonate. Even more preferably, the base used is a tertiary amine, for example triethylamine (TEA) or N-ethyldiisopropylamine. Advantageously, the peptide coupling is carried out at 0 to 50 ° C, preferably at 10 to 30 ° C.

Acid derivatives corresponding to polycations are commercially available.

In addition, when X, Y and / or polycation substituents can interfere with the reaction, it is desirable to protect them in advance with radicals that are compatible and can be introduced and removed without affecting the rest of the molecule. The procedure for this is in accordance with conventional methods known to those skilled in the art, in particular T.W. GREENE, Protective Groups in Organic Synthesis, 2nd Edition, Wiley-Interscience, McOMIE, Protective Groups in Organic Chemistry, Plenum Press (1973), or in accordance with the methods described in Philip J Kocienski, Protecting Groups, Thieme.

In addition, after each step of the preparation process, separation and purification of the obtained compound may be continued according to a method known to those skilled in the art.

Examples of advantageous nucleic acid delivery agents include, in accordance with the present invention, the following compounds.

Another subject of the invention relates to a nucleic acid delivery agent as described above, and a composition comprising a nucleic acid. The amount of each component can be easily adjusted by one skilled in the art as a function of the delivery agent used, the nucleic acid and the desired application (especially the cell type to be transfected).

For the purposes of the present invention, the term "nucleic acid" means both deoxyribonucleic acid and ribonucleic acid. These may be natural or artificial sequences, 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, Or oligonucleotides that are unmodified or unmodified. Such nucleic acids may be of human, animal, plant, bacterial, viral, or the like origin. They can be obtained using any technique known to those skilled in the art, in particular by library screening, by chemical synthesis, or by using mixing methods including chemical or enzymatic modification of the sequences obtained by library screening. They may be chemically modified.

Especially in the case of deoxyribonucleic acids, they can be single-chain or double-stranded, and short oligonucleotides or long sequences. In particular, nucleic acids typically consist of plasmids, vectors, episomes, expression cassettes and the like. Such deoxyribonucleic acids may be present in the origin of replication, one or more marker genes, transcriptional or replication regulatory sequences, corresponding therapeutic genes, antisense sequences that may or may not be modified, other cellular components that may or may not function in target cells. And an area for joining.

Preferably, the nucleic acid comprises a regulatory sequence, for example one or more promoters active in the target cell and one or more corresponding therapeutic genes under the control of the transcription terminator.

For the purposes of the present invention, the term "therapeutic genes" in particular means any gene encoding a therapeutic protein product. The protein product thus encoded may in particular be a protein or peptide. Such protein products may be products that are exogenous, homologous or endogenous to the target cell, ie normally expressed in the target cell without pathological symptoms. In this case, the expression of the protein allows to overcome the expression of the inactive or weakly active protein or overexpress the protein, for example due to insufficient expression or modification in the cell. The therapeutic genes of interest may also encode mutants of cellular proteins with increased stability, modified activity, and the like. Protein products may also be heterologous to target cells. In this case, the expressing protein may, for example, compensate for or provide for insufficient activity in the cell, thereby combating pathological symptoms or stimulating an immune response.

Therapeutic products for the purposes of the present invention are more specifically enzymes, blood derivatives, hormones, lymphokines; Interleukin, interferon, TNF, etc. (FR 92/03120), growth factor, neurotransmitter or precursor thereof or synthetase thereof, tropic factor (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / Pleurotropin, etc.), apolipoproteins (ApoAI, ApoAIV, ApoE et al., FR 93/05125), dystrophin or minidistopins (FR 91/11947), CFTR proteins associated with cystic fibrosis, tumor suppressor genes (p53, Rb , Rap1A, DCC, k-rev et al., FR 93/04745), genes encoding coagulation related factors (factors VII, VIII and IX), DNA repair related genes, suicide genes (thymidine kinase, cytosine deaminase) , Hemoglobin or other carrier protein genes, metabolic genes, catabolic enzymes, and the like.

The therapeutic nucleic acid of interest may also be an antisense sequence or gene, the expression of which in the target cell makes it possible to regulate the expression of the gene or the transcription of cellular mRNA. Such sequences can be transcribed into RNA which, for example, in target cells, is complementary to cellular mRNA and thus blocks translation into proteins, according to the techniques described in patent EP 140 308. The therapeutic gene also includes a sequence encoding a ribozyme capable of selectively destroying target RNA (EP 321 201).

As noted above, nucleic acids may also contain one or more genes that encode antigenic peptides that can generate an immune response in humans or animals. In this particular embodiment, the invention allows the production of a vaccine or the execution of an immunotherapy treatment applied to humans or animals, in particular against microorganisms, viruses or cancer. These may be, in particular, antigenic peptides specific for Epstein-Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo rabies virus, conjugate-forming virus or other virus, or antigenic peptides specific for tumors. have.

Preferably, the nucleic acid also includes sequences that allow expression of the therapeutic gene and / or antigenic peptide encoding genes of interest in the cell or organ of interest. These may be sequences that are naturally responsible for the expression of the gene of interest when the sequence can function in an infected cell. These may be sequences of different origins (which are also responsible for the expression of other proteins, or even synthetic proteins). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell to be infected. Likewise, they can be promoter sequences derived from the viral genome. In this regard, for example, gene promoters such as E1A, MLP, CMV, RSV, and the like can be given. In addition, these expression sequences can be modified by adding an activation sequence, a regulatory sequence and the like. Promoters can also be inducible or inhibitory.

The nucleic acid may also contain a signal sequence, particularly upstream of the therapeutic gene, that directs the synthesized therapeutic product into the secretory pathway of the target cell. Such signal sequences may be natural signal sequences of the therapeutic product, but may also be other functional signal sequences or artificial signal sequences. The nucleic acid may also contain signal sequences that direct the synthesized therapeutic product towards a particular compartment of the cell.

The composition according to the present invention may also contain one or more adjuvants which can bind to and enhance its transfection capacity with the delivery agent / nucleic acid complex. In another aspect, the present invention therefore relates to a composition comprising at least one adjuvant capable of binding to and enhancing the transfection ability of the nucleic acid, the aforementioned nucleic acid delivery agent and the delivery agent / nucleic acid complex. The presence of this type of adjuvant (eg lipid, peptide or protein) advantageously allows to increase the transfection ability of the compound. In this regard, the compositions of the present invention may comprise one or more neutral lipids as an adjuvant.

More preferably, the neutral lipids used in the context of the present invention are lipids with two fatty chains. Particularly advantageously, natural or synthetic lipids are used which are zwitterionic or lack ionic charge in physiological conditions. They are more specifically dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidyl-ethanolamine and also 1-3 Derivatives thereof that are once N-methylated, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (e.g. galactocerebrosides), sphingolipids (e.g. sphingomy in particular) Elin) or asialogangliosides (for example asialo GM1 and GM2).

These various lipids can be obtained by synthesis or extraction from eggs or organs (eg, the brain), using conventional techniques known to those skilled in the art. In particular, extraction of natural lipids can be performed with organic solvents (see Lehninger, Biochemistry).

More recently, the Applicant has also shown that it is particularly advantageous to use a compound as an adjuvant directly involved in the condensation of the nucleic acid as described in patent application WO 96/25508. The presence of such compounds in the compositions according to the invention makes it possible to reduce the amount of transfection agent, with beneficial results therefrom from a toxicological point of view, without damaging effects on transfection activity. The expression "compound involved in the condensation of nucleic acid" is defined as a compound that directly or indirectly condenses nucleic acids. More precisely, such compounds may act directly on the nucleic acid to be transfected or may be involved at the level of additional compounds directly involved in the condensation of such nucleic acids. Preferably, it acts directly on the nucleic acid. In particular, the predense agent may be a polycation, for example polylysine. According to a preferred embodiment, the agent involved in condensation of the nucleic acid is derived, in whole or in part, from protamine, from histones or nucleolines, and / or from derivatives thereof. Such agents may also consist of all or part of peptide units (KTPKKAKKP) and / or (ATPAKKAA) and the number of units may range from 2 to 10. In the structure of the compounds according to the invention, these units can be repeated continuously or discontinuously. Thus, they can be separated by binding of biochemical species, for example one or more amino acids, or chemical species.

Preferably, the composition of the present invention comprises from 0.01 to 20 equivalents, more preferably from 0.5 to 5, of adjuvant per nucleic acid equivalent in moles / molar equivalents.

In a particularly advantageous embodiment, the composition according to the invention also comprises a targeting element which enables the delivery of the nucleic acid. Such targeting elements can be extracellular targeting elements that can direct the delivery of DNA towards a particular desired cell type or tissue (tumor cells, liver cells, hematopoietic cells, etc.). Such targeting elements may also be intracellular targeting elements that can direct the delivery of nucleic acids towards certain desired cell compartments (mitochondria, nucleus, etc.). The target element may bind to the nucleic acid delivery agent according to the invention or to a nucleic acid as described above. When the targeting element is bound to the nucleic acid delivery agent of Formula 1, this element preferably constitutes one of the X or Y substituents.

Targeting elements that can be used in the context of the present invention include sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof. Preferably, they are sugars, peptides or proteins such as antibodies or antibody fragments, ligands or fragments thereof of cellular receptors, fragments of receptors or receptors and the like. In particular, they are ligands of growth factor receptors, cytokine receptors or cellular lectin type receptors, or RGD sequence-containing ligands that are affinity for receptors for adhesive proteins such as integrins. Receptors for transferrin, HDL and LDL, or folate transporters. The targeting element may also be a sugar, or Fab antibody fragment or single-chain antibody (ScFv), which enables targeting of sialic glycoproteins or lectins such as receptors for sialylated species such as for example sialyl Lewis X. .

The association of the nucleolipid complex with the targeting element can be carried out using techniques known to those skilled in the art, for example in the hydrophobic moiety or in the moiety that interacts with the nucleic acid of the delivery agent according to the invention, or with the delivery agent or nucleic acid according to the invention. It can be formed by coupling to a group. The interaction may be ionic or covalent in nature, depending on the desired mode.

Subject of the invention is also the use of a compound as defined above for the delivery of polynucleotides (and more generally polyanions) into cells in vitro, in vivo or ex vivo. More precisely, the subject of the present invention is the use of a compound as defined above for the manufacture of a medicament for the treatment of a disease, in particular a disease resulting from a lack of a protein or nucleic acid product. Polynucleotides contained in a pharmaceutical product constitute a nucleic acid product that encodes a protein or nucleic acid product or that can correct a disease in vivo or ex vivo.

In vivo, for example for treatment or for use in gene control studies or in the generation of animal models of pathological conditions, the compositions according to the invention may be used topically, skin, oral, rectal, vaginal, parenteral, intranasal, intravenous, muscle It can be formulated for administration via the intra, subcutaneous, intraocular, transdermal, intrabronchial, intraperitoneal route, and the like. Preferably, the compositions of the present invention contain a pharmaceutically acceptable vehicle for injectable formulations, in particular for direct injection into the target organ, or for administration via the topical route (on the skin and / or mucosa). They may in particular be isotonic sterile solutions or, in some cases, dry, in particular lyophilized compositions which allow the injectable solute to be configured upon addition of sterile water or physiological saline. The dosage of nucleic acid used for injection, and also the frequency of administration, can be adjusted as a function of various parameters, in particular as a function of the dosage form used, the corresponding physiological symptoms, the gene to be expressed or the desired duration of treatment. More specifically, the dosage form may involve the treatment of cells in culture that are injected directly into tissues, such as tumors, or into the circulatory system, or which are followed by in vivo re transplantation by injection or transplantation. Suitable tissues in the context of the present invention include, for example, muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testes, Ovary, rectum, nervous system, eyes, glands and connective tissue.

Another subject of the invention is

(1) the nucleic acid is contacted with a delivery agent as defined above to form a complex,

(2) a method of treating a human or animal, comprising contacting a human or animal cell with the complex formed in (1).

The present invention also provides

(1) the nucleic acid is contacted with a delivery agent as defined above to form a complex,

(2) a method of delivering a nucleic acid into a cell, comprising contacting the cell with the complex formed in (1).

The cells can be contacted with the complex by culturing it with the complex (for in vitro or ex vivo use) or by injecting the complex into the organism (for in vivo use). Cultivation is preferably carried out, for example, in the presence of 0.01 to 1000 μg of nucleic acid per 10 ⁶ cells. For in vivo administration, nucleic acid dosages, for example in the range of 0.01 to 10 mg can be used.

When the composition of the invention also contains one or more of the above defined adjuvants, the adjuvants are previously mixed with the delivery agent and / or the nucleic acid according to the invention.

Accordingly, the present invention encompasses in vivo or in vitro administration of nucleic acids capable of correcting a disease, encoding a protein, or possibly transcribed into a nucleic acid, wherein the nucleic acid has a compound of formula 1 under the conditions defined above. Provided are methods of associating, in particular, particularly advantageous in vivo nucleic acids for the treatment of diseases.

Nucleic acid delivery agents of the invention can be used, in particular, to deliver nucleic acids to primary cells or to chemotactic cell lines. These may be fibroblasts, myocytes, neurons (neurons, astrocytes, glia), hepatocytes, hematopoietic cells (lymphocytes, CD34, dendritic cells, etc.), epithelial cells, etc., in differentiated or multipotent forms (precursors).

In addition to those listed above, the present invention also includes other features and advantages that will appear in the embodiments and drawings that will be described below and that the present invention should be considered to be illustrative of the scope of the invention without limitation. In particular, the Applicant provides, without limitation, reaction intermediates that can be used in the preparation of various operating protocols and also of the delivery agents of formula (I). Of course, it is within the skill of one in the art to use these protocols or intermediates as a basis for developing similar processes in terms of producing these same compounds. It is also within the capacity of those skilled in the art to use the synthesis processes described in the various patent applications described above as the basis for synthesizing the polycation R included in Formula 1 (WO 96/17823, WO 97/18185, WO 97/31935, etc.). .

A. Materials and Methods

a) material

Starting material polyamines such as spermidine, spermine, tris- (2-aminoethyl) amine, phenylenediamine, diaminoalkane and the like are commercially available or they are prepared using conventional methods (e.g. To give branched amines).

Many compounds are also commercially available, for example triethylamine, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzyl chloroformate, 11-bromodecanol and the like. Product.

Amberlite IR 120 is a commercial ion exchange resin (BDH catalog).

Dimethyl sulfoxide (DMSO), previously treated with potassium hydroxide, is distilled on calcium hydride and stored on 4 mm molecular sieves.

Dichloromethane is distilled over phosphorus pentoxide and then stored on a 4 mm molecular sieve.

Tetrahydrofuran (THF) is distilled on sodium in the presence of benzophenone.

For reactions requiring anhydrous conditions, all glassware is flame dried under a stream of nitrogen.

b) method

Spectroscopic analysis

Nuclear magnetic resonance (NMR) spectra are recorded on a Brucker MSL 30 spectrometer at a frequency of 300 MHz for protons and 75 MHz for carbon. All chemical shifts are reported in ppm relative to the frequency of tetramethylsilane (TMS) or solvent. Spectra are recorded using residual signal of TMS or solvent as internal control. The multiplicity of the signal is indicated using the following abbreviations: s (single), d (double), t (triple), q (quad) and m (multiple)

Chromatography Technology

Reaction kinetics are monitored by thin layer chromatography (TLC) using silica gel (Merck Silicagel 60 F254) containing a fluorescent indicator as a support. The chromatogram is developed by spraying an alcohol solution of anisealdehyde.

All column chromatography is carried out under compressed air pressure with silica gel 60 (0.05-0.02 mm) as stationary phase. The mobile phase used depends on the type of synthesis (medium pressure chromatography).

HPLC (High Performance Liquid Chromatography) analysis at 220 nm on a Waters LC 4000 instrument equipped with a C4 analytical column (stainless steel "Brownlee column", 3 cm long, 0.46 cm in diameter) and "Waters 486" detector sold by Applied Biosystem. Do this. The stationary phase is 7 micron butyl aquapore and the mobile phase is acetonitrile (2500 cm ³ ) supplemented with deionized water (2500 cm ³ ) or trifluoroacetic acid (2.5 cm ³ ). The flow rate is 1 ml per minute.

B. Synthesis of Transfection Agent

Example 1 Synthesis of (3- [4- (3-aminopropylamino) butylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecyl α-D-mannopyranoside (Compound 1)

a) Synthesis of 3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] acetic acid (FRM 375)

Sodium cyanoborohydride NaBH ₃ CN (0.548 g; 8.74 mmol) is added to a solution of spermine (5 g; 24.96 mmol) in methanol (125 ml). The solution is then vigorously stirred. A solution of glyoxylic acid (2.34 g; 25.46 mmol) in methanol (80 ml) is added over a 100 minute period using a pressure dropping funnel. After overnight, di-tert-butyl dicarbonate (27.67 g; 129.79 mmol) solubilized in triethylamine (3.86 ml; 27.71 mmol) and tetrahydrofuran (55 ml) was added to the mixture. After overnight, the mixture is concentrated on a rotary evaporator and dissolved with ethyl acetate (63 ml) and washed with potassium hydrogen sulphate and brine. The mixture is then dried over magnesium sulphate and concentrated. The resulting product is purified by chromatography (CH ₂ Cl ₂ / MeOH 9: 1). Yield 30%.

¹ H NMR (CDCl 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 methyl 15-hydroxypentadecanoate

6.66 cm ³ (13.31 mmol) of 2N sodium methylate is added to 10 g (41.60 mmol) of pentadecaractone in 41.60 cm ³ of methanol at 0 ° C.

After 9 hours, 9.24 cm ³ of acetic acid was added and reacted for 15 minutes. The solution is then evaporated to dryness in vacuo, then the residue is dissolved in dichloromethane and the mixture is washed with sodium bicarbonate. The organic phase obtained is dried using magnesium sulfate and the solvent is evaporated in a rotary evaporator. Purify in 6: 4 hexane / ethyl acetate mixture. Methyl 1-ol-pentadecanoate is obtained in 80% yield.

¹ H NMR (CDCl 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, H-1), 3.67 (s, 3H, H-16).

c) Synthesis of methyl pentadecanoate 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside

5.26 cm ³ (44.94 mmol) of tin chloride is added to 8.72 g (22.47 mmol) of pentaacetylated mannose in 56 cm ³ of dichloromethane at 0 ° C. over 30 minutes. Subsequently, 7.34 g (26.96 mmol) of methyl 1-ol-pentadecanoate obtained in advance in a) 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 phase is extracted with diethyl ether and the organic phase is washed successively with sodium carbonate solution, and brine and then dried over magnesium sulfate. The product obtained after evaporation to dryness in vacuo is purified by medium pressure chromatography in a 7: 3 heptane / ethyl acetate mixture. Yield 53%.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.26 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 2.01, 2.05, 2.12 and 2.17 (s , 3H, OCO CH ₃ ), 2.29 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 7.89 Hz, OCH _a CH ₂ ), 3.66 (m, 1H, J = 7.89 Hz, OCH _b CH ₂ ), 3.67 (s, 3H, COO CH ₃ ), 4.05 (ddd, 1H, J = 9.56 Hz and 5.57 Hz, H-5), 4.1 (dd, 1H, J = 5.57 Hz and 12.32 Hz, H-6a), 4.29 (dd, 1H, J = 5.57 Hz and 12.32 Hz, H-6b), 4.8 (d, 1H, J = 1.85 Hz, H-1), 5.23 (dd, 1H, J = 1.85 Hz and 3.23 Hz, H-2), 5.27 (dd, 1H, J = 9.97 Hz and 9.56 Hz, H-4), 5.35 (dd, 1H, J = 9.97 Hz and 3.23 Hz, H-3).

d) Synthesis of methyl pentadecanoate α-D-mannopyranoside

3.63 g (6.01 mmol) of the product obtained in the previous step dissolved in 12 cm ³ of methanol are treated with 3 cm ³ (6.01 mmol) of 2N sodium methylate. Upon completion of the reaction, the latter was neutralized with Amberlite IR120 (1 weight / volume), filtered and evaporated to dryness in vacuo.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 2.34 (t, 2H, J = 7.62 Hz, H-14), 3.41 (m, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.74 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.67 (s, 3H, CH ₃ OCO), 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, H-1).

e) Synthesis of methyl pentadecanoate 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside

4.54 g (27.36 mmol) of potassium iodide, 1.09 g (27.36 mmol) of 60% sodium hydride and 3.25 cm ³ (27.36 mmol) of benzyl bromide were dissolved in the preceding step d) in 20 cm ³ of dimethylformamide (DMF). To 2 g (4.56 mmol) of the obtained product was added successively. After 12 hours, 18.24 cm ³ saturated ammonium chloride solution was added and reacted for 10 minutes. The mixture is then diluted with water and the organic phase is extracted with ethyl acetate. This organic phase is then washed with water and brine and finally dried over magnesium sulfate. Further washing with sodium thiosulfate saturated solution to remove iodide ions. The mixture is evaporated in vacuo and the resulting oil is purified in a 9: 1 heptane / ethyl acetate mixture. The product is obtained in 60% yield.

¹ H NMR (CDCl ₃ ): δ (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, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.63 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.67 (s, 3H, CH ₃ OCO), 3.75 (m, 1H, J = 8.97 Hz and 6.21 Hz, H-5), 3.78 (s, 2H, CH ₂ Ph ₂ ), 3.90 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6a), 3.97 (dd, 1H, 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 ₂ Phe), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 ), 4.74 (1H, J = 2.52 Hz, H-1), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4), 7.35 (m, 20H, Phe).

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

4.68 cm ³ of 25% sodium hydroxide solution is added to 0.50 g (0.73 mmol) of the product obtained in the previous step e) dissolved in 7 cm ³ of methanol. The reaction mixture is heated at reflux for 30 minutes. The mixture is then neutralized with 5% hydrochloric acid solution at low temperature. The organic phase is extracted with ethyl acetate and evaporated to dryness in vacuo. Purify in 4: 6 heptane / ethyl acetate mixture. The product is obtained in 62% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ ), 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, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.63 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.75 (m, 1H, 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, 1H, 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 ₂ Phe), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2), 4.74 (1H, J = 2.52 Hz, H-1), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4), 7.35 (m, 20H, Phe).

g) Synthesis of N-octadecyl-15-carbamoylpentadecanyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside

0.29 g of a product of the product obtained in the preceding step f) in which 0.23 g (0.52 mmol) of BOP, 0.21 cm ³ (1.48 mmol) of diisopropylethylamine and 0.12 g (0.44 mmol) of octadecylamine were dissolved in 5 cm ³ of chloroform. (0.37 mmol) was added successively. When the reaction is complete, the mixture is diluted with dichloromethane and washed with water. It is then dried using magnesium sulfate and then evaporated to dryness in vacuo. The product obtained is purified by medium pressure chromatography in a 6: 4 heptane / ethyl acetate mixture. The product is obtained in 98% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 50H, (CH ₂ ) ₂₅ ), 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, 1H , J = 6.71 Hz, O CH _a CH ₂ ), 3.63 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.75 (m, 1H, 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, 1H, 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 ₂ Phe), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2), 4.74 (1H, J = 2.52 Hz, H-1), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4), 5.37 (band, 1H, HN CO), 7.35 (m, 20H, Phe).

h) Synthesis of 15-octadecylaminopentadedecanyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside

0.056 g (1.50 mmol) of lithium aluminum hydride AlLiH ₄ are added to 0.77 g (0.75 mmol) of the product obtained in the preceding step g) in 15 cm ³ of anhydrous tetrahydrofuran (THF). The mixture is heated at reflux for 10 hours. The reaction mixture is then cooled in an ice bath and 56 μl of water is added, after 10 minutes 112 μl of 2N sodium hydroxide is added and finally 56 μl of water is added after 10 minutes. The mixture is filtered and evaporated to dryness in vacuo. The product obtained is purified in a 9: 2: 0.5 dichloromethane / methanol / 28% ammonia mixture. The product is obtained in 86% yield.

¹ H NMR (CDCl 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 ₂ CH ₂ , H-17, H-14, H-17 and NH), 2.57 (t, 4H, J = 7.92 Hz, H-15 and H-16), 3.34 (m, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.63 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.75 (m, 1H, 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, 1H, 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 ₂ Phe), 4.69 (dd , 1H, J = 2.52 Hz and 2.91 Hz, H-2), 4.74 (1H, J = 2.52 Hz, H-1), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4), 7.35 (m, 20H, Phe).

i) (3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16 Synthesis of -octadecyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside

0.38 g (0.85 mmol) of BOP, 0.425 cm ³ (2.44 mmol) of diisopropylethylamine and 3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonyl obtained in step a) 0.48 g (0.73 mmol) of amino) butyl-tert-butoxycarbonylamino] acetic acid (FRM 375) are added successively to 0.63 g (0.61 mmol) of the solution of the product previously obtained in step h) in 10 cm ³ of chloroform. . After 4 hours, the mixture is diluted with dichloromethane and washed with water. Dry using magnesium sulfate and evaporate to dryness in vacuo. The product obtained is purified by medium pressure chromatography in a 6: 4 heptane / ethyl acetate mixture. The product is obtained in 80% yield.

¹ H NMR (CDCl 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, 17H, OCH ₂ CH ₂ , 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, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.63 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.75 (m, 1H, 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, 1H, 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 ₂ Phe), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2), 4.74 (1H, J = 2.52 Hz, H-1), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz , H-4), 7.35 (m, 18H, Phe).

j) (3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16 Synthesis of -octadecyl α-D-mannopyranoside

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

¹ H NMR (CDCl 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, 17H, OCH ₂ CH ₂ , 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, 1H, J = 6.71 Hz, O CH 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, O CH b CH} 2), 4.72 (1H, J = 2.52 Hz, H-1).

k) Synthesis of (3- [4- (3-aminopropylamino) butylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecyl α-D-mannopyranoside (Compound 1)

21.50 cm ³ of distilled tetrahydrofuran (TFA) is added to 0.37 g (0.28 mmol) of the product obtained in the preceding step j). After 1 hour the reaction mixture is concentrated at low temperature and then lyophilized. The purity of the product dissolved in methanol is demonstrated by HPLC as described in the "Materials and Methods" section.

¹ H NMR (CDCl ₃ ): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH ₂ ) ₂₅ ), 1.4-1.6 (m, 17H, OCH ₂ CH ₂ , 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, 1H, _{J = 6.71 Hz, O CH 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 _b CH ₂ ), 4.72 (1H, J = 2.02 Hz, H-1).

Example 2: (3- [4- (3-aminopropylamino) butylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecyl 6-deoxy-α-L-mannopyranoside (compound 2) Synthesis

a) Synthesis of 3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] acetic acid (FRM 375)

Sodium cyanoborohydride NaBH ₃ CN (0.548 g; 8.74 mmol) is added to a solution of spermine (5 g; 24.96 mmol) in methanol (125 ml). The solution is then vigorously stirred. A solution of glyoxylic acid (2.34 g; 25.46 mmol) in methanol (80 ml) is added over a 100 minute period using a pressure dropping funnel. After overnight, di-tert-butyl dicarbonate (27.67 g; 129.79 mmol) solubilized in triethylamine (3.86 ml; 27.71 mmol) and tetrahydrofuran (55 ml) was added to the mixture. After overnight, the mixture is concentrated on a rotary evaporator and dissolved with ethyl acetate (63 ml) and washed with potassium hydrogen sulphate and brine. The mixture is then dried over magnesium sulphate and concentrated. The resulting product is purified by chromatography (CH ₂ Cl ₂ / MeOH 9: 1). Yield 30%.

¹ H NMR (CDCl 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 methyl 15-hydroxypentadecanoate

6.66 cm ³ (13.31 mmol) of 2N sodium methylate is added to 10 g (41.60 mmol) of pentadecaractone in 41.60 cm ³ of methanol at 0 ° C. After 9 hours, 9.24 cm ³ of acetic acid was added and reacted for 15 minutes. The solution is then evaporated to dryness in vacuo, then the residue is dissolved in dichloromethane and the mixture is washed with sodium bicarbonate. The organic phase obtained is dried using magnesium sulfate and the solvent is evaporated in a rotary evaporator. Purify in 6: 4 hexane / ethyl acetate mixture. Methyl 1-ol-pentadecanoate is obtained in 80% yield.

¹ H NMR (CDCl 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, H-1), 3.67 (s, 3H, H-16).

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

2.49 cm ³ (21.30 mmol) of tin chloride is added to 3.55 g (10.65 mmol) of tetraacetylated rhamnose in 27 cm ³ of dichloromethane at 0 ° C. over 30 minutes. Then, 3.48 g (12.78 mmol) of methyl 1-ol-pentadecanoate obtained above are added. After 2 hours the reaction mixture is diluted with ethyl ether and poured into a solution of sodium phosphate (Na ₂ PO ₄ ). The aqueous phase is extracted with diethyl ether and the organic phase is washed successively with sodium carbonate solution, and brine and then dried using magnesium sulfate. After evaporation to dryness in vacuo the product is purified by medium pressure chromatography in a 7: 3 heptane / ethyl acetate mixture. The product is obtained in 60% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.20 (d, 3H, J = 6.45 Hz, H-6), 1.26 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 1.98, 2.04 and 2.16 (s, 3H, OCO CH ₃ ), 2.29 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.66 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.67 (s, 3H, COO CH ₃ ), 3.88 (m, 1H, J = 6.45 Hz and 9.97 Hz, H -5), 4.70 (d, 1H, J = 1.72 Hz, H-1), 5.06 (dd, 1H, J = 9.97 Hz and 9.97 Hz, H-4), 5.22 (dd, 1H, J = 1.72 Hz and 3.52 Hz, H-2), 5.30 (dd, 1H, J = 3.52 Hz and 9.97 Hz, H-3).

d) Synthesis of methyl pentadecanoate α-deoxy-L-6-mannopyranoside

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

¹ H NMR (CDCl ₃ ): δ (ppm) 1.20 (d, 3H, J = 6.45 Hz, H-6), 1.26 (m, 20H, (CH ₂ ) ₁₀ ), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 2.29 (t , 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, O CH b CH 2), 3.66 (m, 1H, J = 6.71 Hz, OCH _b CH ₂ ), 3.67 (s, 3H, CH ₃ OCO), 3.6-3.9 (m, 4H, H-2, H-3, H-4 and H-5), 4.70 (d, 1H , J = 1.72 Hz, H-1).

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

3.32 g (20.00 mmol) of potassium iodide, 0.80 g (20.00 mmol) of 60% sodium hydride and 2.38 cm ³ (20.00 mmol) of benzyl bromide were obtained in the preceding step d) in 30 cm ³ of anhydrous dimethylformamide (DMF). To 2.09 g (5.00 mmol) of one product is added successively. After 12 hours, 20 cm ³ of saturated ammonium chloride solution was added and reacted for 10 minutes. The mixture is then diluted with water and the organic phase is extracted with ethyl acetate. This organic phase is then washed with water and brine and finally dried over magnesium sulfate.

Further washing with sodium thiosulfate saturated solution to remove iodide ions. The mixture is evaporated to dryness in vacuo and the resulting oil is purified in a 9: 1 heptane / ethyl acetate mixture. The product is obtained in 60% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ , 1.33 (d, 3H, J = 6.21 Hz, H-6), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 2.31 (t , 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, O CH a CH 2), 3.61 (dd, 1H, J = 8.96 Hz And 9.5 Hz, H-4), 3.66 (m, 1H, J = 6.71 Hz, O CH _b CH ₂ ), 3.67 (s, 3H, CH ₃ OCO), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, 1H, 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 (1H, J = 2.01 Hz, H-1), 7.35 (m, 15H, Phe).

f) Synthesis of pentadecanoic acid 2,3,4-tri-O-benzyl-6-deoxy-α-L-mannopyranoside

4.68 ml of 25% sodium hydroxide is added to 0.50 g (0.73 mmol) of the solution of the product obtained in the preceding step e) in 7 cm ³ of methanol. The reaction mixture is heated at reflux for 30 minutes. The mixture is then neutralized with 5% hydrochloric acid solution at low temperature. The organic phase is extracted with ethyl acetate and evaporated to dryness in vacuo. Purify in 4: 6 heptane / ethyl acetate mixture. The product is obtained in 72% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.28 (m, 20H, (CH ₂ ) ₁₀ ), 1.33 (d, 3H, J = 6.21 Hz, H-6), 1.59 (m, 4H, OCH ₂ CH ₂ and H-13), 2.34 (t , 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, O CH a CH 2), 3.61 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4), 3.66 (m, 1H, J = 6.71 Hz, O CH b CH 2), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd , 1H, 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 (1H, J = 2.01 Hz, H-1), 7.35 (m, 20H, Phe).

g) Synthesis of N-octadecyl-15-carbamoylpentadedecanyl 2,3,4-tri-O-benzyl-6-deoxy-α-L-mannopyranoside

0.60 g (1.56 mmol) of BOP, 0.72 cm ³ (4.16 mmol) of diisopropylethylamine and 0.34 g (1.25 mmol) of octadecylamine were added to 0.70 g of a solution of the product previously obtained in step f) in 13 cm ³ of chloroform. 1.04 mmol) in succession. When the reaction is complete, the mixture is diluted with dichloromethane, washed with water, dried over magnesium sulfate and then evaporated to dryness in vacuo. The product obtained is purified by medium pressure chromatography in a 6: 4 heptane / ethyl acetate mixture. The product is obtained in 84% yield.

¹ H NMR (CDCl 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, 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), 3.40 (m, 1H, J = 6.71 Hz, O CH a CH 2), 3.61 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4), 3.66 ( m, 1H, J = 6.71 Hz , O CH b CH 2), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, 1H, 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 (1H, J = 2.01 Hz, H-1), 5.37 (band, 1H, HN CO), 7.35 (m, 15H, Phe).

h) Synthesis of 15-octadecylaminopentadedecanyl 2,3,4-tri-O-benzyl-6-deoxy-α-L-mannopyranoside

0.065 g (1.72 mmol) of lithium aluminum hydride AlLiH ₄ are added to 0.81 g (0.86 mmol) of the product obtained in the preceding step g) in 15 cm ³ of anhydrous tetrahydrofuran (THF) and the mixture is heated at reflux for 10 h. The reaction mixture is then cooled in an ice bath and 65 μl of water is added, after 10 minutes 130 μl of 2N sodium hydroxide is added, and finally after additional 10 minutes additional 65 μl of water. The mixture is filtered and evaporated to dryness in vacuo. 9: 2: 0.5 Purify in dichloromethane / methanol / 28% ammonia mixture. The product is obtained in 93% yield.

¹ H NMR (CDCl 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, 9H, OCH ₂ CH ₂ , H-17, H-14, H-17 and NH), 2.57 (t, 4H, J = 7.92 Hz, H-15 and H-16), 3.40 (m , 1H, J = 6.71 Hz, O CH a CH 2), 3.61 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4), 3.66 (m, 1H, J = _{6.71 Hz, O CH b CH 2} ), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5), 3.75 (dd, 1H, 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 (1H, J = 2.01 Hz, H-1), 7.35 (m, 15H, Phe).

i) (3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16 Synthesis of -octadecyl 2,3,4 -tri-O-benzyl-6-deoxy-α-L-mannopyranoside

0.53 g (1.20 mmol) of BOP, 0.30 cm ³ (1.72 mmol) of diisopropylethylamine and 0.62 g (0.95 mmol) of FRM 375 obtained in step a) were obtained in the preceding step h) dissolved in 7 cm ³ of chloroform. To 0.78 g (0.86 mmol) of the solution of the product is added continuously. After 4 hours, the mixture is diluted with dichloromethane, washed with water, dried over magnesium sulphate and evaporated to dryness in vacuo. The resulting product is purified by "flash" chromatography in a 6: 4 heptane / ethyl acetate mixture. The product is obtained in 72% yield.

¹ H NMR (CDCl 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, OCH ₂ CH ₂ , 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, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.65 (s, 2H, CH ₂ Phe), 3.66 (m, 1H, J = 6.71 Hz, OCH _b CH ₂ ), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5), 3.99 (s, 2H, CH ₂ Phe), 4.02 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4), 4.32 (s, 2H, CH ₂ Phe), 4.57 (dd, 1H, J = 2.01 Hz and 3.02 Hz, H-2), 4.73 (1H, J = 2.01 Hz, H-1 ), 4.82 (dd, J = 3.02 Hz and 8.96 Hz, H-3), 7.35 (m, 18H, Phe).

j) (3- [4- (3-tert-butoxycarbonylaminopropyl-tert-butoxycarbonylamino) butyl-tert-butoxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16 Synthesis of -octadecyl 6 - deoxy - α-L-mannopyranoside

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

¹ H NMR (CDCl ₃ ): 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 CH ₂ , 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, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.66 (m, 1H, J = 6.71 Hz, O CH _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) (3- [4- (3-aminopropylamino) butylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecyl 6-deoxy-α-L-mannopyranoside (Compound 2) Synthesis of

24 cm ³ of distilled tetrahydrofuran (TFA) are added to 0.40 g (0.31 mmol) of the product obtained in the preceding step j). After 1 hour the reaction mixture is evaporated to dryness under vacuum at low temperature and then lyophilized. The purity of the product dissolved in methanol is verified by HPLC.

¹ H NMR (CDCl ₃ ): 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 CH ₂ , 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), 3.40 (m, 1H, J = 6.71 Hz, O CH _a CH ₂ ), 3.66 (m, 1H, J = 6.71 Hz, O CH _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).

Example 3: 1-[-(3- [4- (3-aminopropylamino) butylaminopropylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecanyl 6-deoxy-β-L Synthesis of Galactopyranoside (Compound 3)

a) Synthesis of {3- [4- (3-benzyloxycarbonylaminopropyl-benzyloxycarbonylamino) butylbenzyloxy carbonylamino] propylamino} acetic acid

Sodium cyanoborohydride NaBH ₃ CN (1.10 g; 17.47 mmol) is added to a solution of spermine (10 g; 49.91 mmol) in methanol (200 ml). The solution is then vigorously stirred. A solution of glyoxylic acid (4.59 g; 49.91 mmol) in methanol (120 ml) is added over a 100 minute period using a pressure dropping funnel. 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 added successively in 10 portions. Subsequently, stirring is actively carried out while maintaining the temperature of the bath at 5 to 10 ° C. After 2 hours at room temperature the mixture is extracted with ether and neutralized with 5N hydrochloric acid solution. The organic phase is then dried over magnesium sulphate and then concentrated on a rotary evaporator. The resulting product is purified by chromatography (100% CH ₂ Cl ₂ , then 9: 1 CH ₂ Cl ₂ / MeOH). Yield 52%.

¹ H NMR (CDCl 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 ₂ COO), 5.07 (s, 8H, CH ₂ ), 7.27 (m, 20H, Phe).

b) Synthesis of methyl 15-hydroxypentadecanoate

Pentadecaractone (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 were added and reacted for 15 minutes. The solution is then concentrated, then the resulting oil is dissolved in dichloromethane and washed with sodium bicarbonate. After decantation, the organic phase is dried over magnesium sulphate and evaporated. Purification in a 6: 4 hexanes / ethyl acetate (AcOEt) mixture affords methyl 15-hydroxypentadecanoate in 80% yield.

¹ H NMR (CDCl ₃ ): δ (ppm) 1.29 (m, 20H, (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, H-1), 3.67 (s, 3H, H-16).

c) Synthesis of N-octadecyl-15-hydroxypentadecanamide

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

¹ H NMR (CDCl ₃ ): δ (ppm) 0.88 (t, 3H, J = 6.96 Hz, H-33), 1.26 (m, 54H, (CH ₂ ) 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, H-16), 3.64 (t, 2H, J = 5.84 Hz, H-1), 5.39 (band NHCO).

¹³ C NMR (CDCl 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 (C-1), 174.31 (CO)

d) Synthesis of 15-octadecylaminopentadecanol

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

¹ H NMR (CDCl 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 band NH), 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, H-1).

¹³ C NMR (CDCl 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 (C-1).

e) Synthesis of N- [benzyloxycarbonyl] -15-octadecylaminopentadecanol

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

¹ H NMR (CDCl 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, H-14, H-17), 3.20-3.22 (m, 4H, H-15 and H-16), 3.64 (t, 2H, J = 5.84 Hz, H-1), 5.12 (s, 2H , OCH ₂ Phe), 7.34 (m, 5H, Phe).

¹³ C NMR (CDCl ₃ ): δ (ppm) 14.48 (C-33), 25.8, 26.9 and 31.94 (C-2, C-14 and C-17), 29.72 ((CH ₂ ) ₂₇ ), 47.26- 48.04 (C-15 and C-16), 63.08 (C-1), 66.79 (OCH ₂ ), 128.40 (Phe).

f) Synthesis of 15- [N- (benzyloxycarbonyl) octadecylamino] pentadecanyl 2,3,4-tri-O-acetyl-6-deoxy-β-L-galactopyranoside

1.5 g (4.52 mmol) of tetraacetylated fucose is reacted with 0.634 ml (5.42 mmol) of tin tetrachloride in dry acetonitrile (50 ml) for 30 minutes. Then, 3.132 g (4.97 mmol) of N- [benzyloxycarbonyl] -15-octadecylaminopentadecanol obtained in the previous step e) are added. After 5 hours, the reaction is extracted and the product obtained is chromatographed (6: 4 heptane / ethyl acetate). Yield 69%.

¹ H NMR (CDCl ₃ ): 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 CH ₂ , H-14 and H-17), 1.95, 2.05 and 2.15 (s, 3H, OCO CH ₃ ), 3.14-3.25 (m, 4H, H-15 and H-16), 3.44 (m, 1H, O CH _a CH ₂ ), 3.63 (m, 1H, O CH _b CH ₂ ), 3.79 (m, 1H, H-5), 4.41 (d, 1H, J = 7.98 Hz, H-1), 4.99 (dd, 1H, J = 3.52 Hz and 10.46 Hz, H-3), 5.09 (s, 2H, OCH ₂ Phe), 5.16 (dd, 1H, J = 7.98 Hz and 10.46 Hz, H-2), 5.23 (dd, J = 3.52 Hz and 3.31 Hz, H-4), 7.32 (m, 5H, Phe).

¹³ C NMR (CDCl ₃ ): δ (ppm) 14.68 (C-33), 17.31 (C-2), 20.75 ( CH ₃ COO), 27.29 (C-6), 29.72 ((CH ₂ ) ₂₇ )), 25.89-31.98 (OCH ₂ CH ₂ , C-14, C-17), 47.25-48.04 (C-15 and C-16), 66.91 ( CH ₂ Phe), 69.63 (O CH ₂ CH ₂ ), 69.45 (C -2), 70.57 (C-5), 70.85 (C-4), 71-44 (C-3), 96.25 (C-1), 128.43 (Phe), 156.21 and 171.30 (CO).

g) Synthesis of 15-octadecylaminopentadedecanyl 2,3,4-tri-O-acetyl-6-deoxy-β-L-galactopyranoside

15- [N- (benzyloxycarbonyl) octadecylamino] pentadecanyl 2,3,4- from 10% (0.5 g) palladium on activated carbon under hydrogen pressure obtained in the preceding step f) in methanol (100 ml) To a solution of tri-O-acetyl-6-deoxy-β-L-galactopyranoside (2.72 g; 4.23 mmol). The reaction is quantitative.

¹ H NMR (CDCl ₃ ): 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 CH ₂ , H-14 and H-17), 1.88-1.93 (band NH), 1.95, 2.05 and 2.15 (s, 3H, OCO CH ₃ ), 2.64 (m, 4H, H-15 and H-16), 3.46 (m, 1H, O CH _a CH ₂ ), 3.63 (m, 1H, O CH _b CH ₂ ), 3.79 (m, 1H, H-5 ), 4.41 (d, 1H, J = 7.98 Hz, H-1), 4.99 (dd, 1H, J = 3.52 Hz and 10.46 Hz, H-3), 5.16 (dd, 1H, J = 7.98 Hz and 10.46 Hz , H-2), 5.23 (dd, J = 3.52 Hz and 3.31 Hz, H-4).

¹³ C NMR (CDCl ₃ ): δ (ppm) 14.68 (C-33), 17.31 (C-2), 20.75 ( CH ₃ COO), 27.29 (C-6), 29.72 ((CH ₂ ) ₂₇ )), 25.89-31.98 (OCH ₂ CH ₂ , C-14, C-17), 47.75-48.04 (C-15 and C-16), 69.63 (O CH ₂ CH ₂ ), 69.45 (C-2), 70.57 (C -5), 70.85 (C-4), 71.44 (C-3), 96.25 (C-1), 171.30 (CO).

h) (3- [4- (3-aminopropylamino) butylaminopropylbenzyloxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecanyl 2,3,4-tri-O- Synthesis of Acetyl-6-deoxy-β-L-galactopyranoside

Diisopropylethylamine (0.491 ml; 2.82 mmol), BOP (0.457 g; 1.03 mmol) and {3- [4- (3-benzyloxycarbonylaminopropylbenzyloxycarbonylamino) butyl obtained in step a) Benzyloxycarbonylamino] propylamino} acetic acid (0.748 g; 0.94 mmol) is added successively to a solution of 0.60 g (0.94 mmol) of the compound obtained in the preceding step g) in chloroform (15 ml). The resulting oil is purified by chromatography (4: 6 heptanes / ethyl acetate). (3- [4- (3-aminopropylamino) butylaminopropylbenzyloxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecanyl 2,3,4-tri-O-acetyl- 6-deoxy-β-L-galactopyranoside is obtained in 45% yield.

¹ H NMR (CDCl ₃ ): 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 ), 1.39-1.67 (m, 15H, OCH ₂ CH ₂ , H-14, H-17, NH, CH ₂ ), 1.95, 2.05 and 2.15 (s, 3H, OCOCH ₃ ), 3.05- 3.35 (m, 18H, H-15, H-16 and CH ₂ N), 3.43 (m, 1H, O CH _a CH ₂ ), 3.67 (m, 1H, J = 6.74 Hz, O CH _b CH ₂ ), 3.79 (m, 1H, H-5), 4.41 (d, 1H, J = 7.98 Hz, H-1), 4.99 (dd, 1H, J = 3.52 Hz and 10.46 Hz, H-3), 5.05 (s, 8H, CH ₂ Phe), 5.16 (dd, 1H, J = 7.98 Hz and 10.46 Hz, H-2), 5.23 (dd, J = 3.52 Hz and 3.31 Hz, H-4), 5.47 (band CONH, 1H) , 7.32 (m, 20H, Phe).

¹³ C NMR (CDCl ₃ ): δ (ppm) 14.84 (C-33), 20.75 ( CH ₃ COO), 27.29 (C-6 ′), 29.72 ((CH ₂ ) 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 (O CH ₂ CH ₂ ), 69.45 (C- 2), 70.57 (C-5), 70.85 (C-4), 71.44 (C-3), 96.25 (C-1), 128.31 (Phe), 157.01 and 171.30 (CO).

i) 1-[-(3- [4- (3-aminopropylamino) butylaminopropylbenzyloxycarbonylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecanyl 6-deoxy-β Synthesis of -L-galactopyranoside

A saturated methanol solution of ammonia (1 ml) is added to a methanol solution (3 ml) (0.60 g; 0.94 mmol) containing the product obtained in the preceding step h). After 1 hour the mixture is concentrated.

¹ H NMR (CDCl ₃ ): 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 ₂ ), 3.05-3.35 (m, 18H, H-15, H-16 and CH ₂ N), 3.4-3.7 (m, 6H, O CH ₂ CH ₂ , H-3, H-4, H-5, H-2), 4.73 (d, 1H, J = 7.98 Hz, H-1 ), 5.05 (s, 8H, CH ₂ Phe), 5.47 (band CONH, 1H), 7.32 (m, 20H, Phe).

j) 1-[-(3- [4- (3-aminopropylamino) butylaminopropylamino] methylenecarbamoyl) -15-pentadecanyl-16-octadecanyl 6-deoxy-β-L-gal Synthesis of Lactopyranoside (Compound 3)

10% (0.032 g) palladium on charcoal in methanol is added to a solution of the product (0.072 g; 0.05 mmol) obtained in the preceding step i). After overnight the mixture is filtered through fiberglass paper and then concentrated on a rotary evaporator. The product is then HPLC purified on a C-4 preliminary column.

¹ H NMR (CDCl ₃ ): 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, CH ₂ ), 2.92-3.19 (m, 18H, H-15, H-16 and CH ₂ N), 3.4-3.7 (m, 6H , O CH 2 CH 2, H-3, H-4, H-5, H-2), 4.73 (d, 1H, J = 7.98 Hz, H-1).

C. Use of the delivery agent according to the invention

Example 4 Preparation of Transfer Agent / Nucleic Acid Complex with Compound 2 and Measurement of Its Size

This example illustrates the preparation of a complex between a delivery agent and a nucleic acid according to the invention, the size of which is then measured.

The glycolipid used in this example and the following example is Compound 2 dissolved in chloroform at a concentration of 10 mg / ml. In some cases, the neutral co-lipid cholesterol or DOPE is premixed with compound 2.

The lipid solution is prepared in the following way: the desired amount of sample is collected and the solvent is evaporated on an argon stream and left to dry for 1 hour. The lipid is then rehydrated overnight at 4 ° C. with a solution containing 5% dextrose and 10 mM sodium chloride. The next day the lipid solution is heated at 60 ° C. for 5 minutes and then sonicated for 1 minute. The operation is repeated until the lipid particles have stabilized in size.

The DNA used is plasmid pXL3031 (FIG. 1) dissolved 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 luc gene, which encodes luciferase under the control of the P / E CMV promoter of cytomegalovirus. The length is 3671 bp. A diagram of this plasmid is shown in FIG. Plasmid pXL3031 is purified according to the method described in patent application WO 97/35002.

Compound 2 / DNA complexes are prepared by rapidly mixing a solution of plasmid DNA with a suitable volume of compound 2 (depending on the desired charge ratio) at room temperature. The amount of transfection agent is 0.25-12 nmol per μg DNA.

The size of the complex is analyzed by measuring the hydrodynamic diameter by dynamic laser light scattering using a Coulter N4Plus machine. Samples are diluted 20-fold in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple dispersions.

At a ratio of 3 nmol of lipid per μg DNA, the following results were obtained;

Size (nm) Michelle 130 nm Formulation by Cholesterol 153 nm Formulation by DOPE 137 nm

The term "micelle" indicates that Compound 2 is used alone, ie without the addition of neutral co-lipids, thus forming a micelle solution.

The table above shows that the complexes obtained have a size of approximately 130 to 150 nm, which size is particularly suitable for pharmaceutical use by injection.

Example 5: Behavior of Complexes Formed from Compound 2 at Various Charge Ratios

This example illustrates the behavior of the delivery agent / nucleic acid complex according to the invention when varying the charge ratio. The effect of adding co-lipids (cholesterol or DOPE) is also described.

Typically, three physicochemical phases are identified when increasing the delivery agent / DNA charge ratio (B. Pitard et al., Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer, PNAS, Vol. 94, pp. 14412-14417, 1997). These three phases determine the therapeutic ability of the delivery agent.

At low charge ratios, the DNA is not saturated with the transfer agent. DNA that does not form a complex remains, and the complex is negatively charged and small in size. This stable phase is referred to as "phase A".

The fact that DNA is not fully saturated with delivery agents means that DNA is not fully protected. Thus, DNA can be exposed to degradation by nucleases. In addition, since the complex is negatively charged as a whole, it is difficult to cross cell membranes. For this reason, the nuclear lipid complex of phase A is relatively inert.

At moderate charge ratios, the DNA is fully saturated with the delivery agent, and the complexes are generally neutral or slightly positive.

This phase is unstable because the ion repulsion is minimal and aggregation may occur. The particle size far exceeds the detection limit by dynamic laser light scattering (much larger than 3 μm). This unstable phase is called "B phase". These complex sizes are not suitable for injection, but do not mean that the complexes are inert on B: they are only formulations that are not suitable for their injection for pharmaceutical purposes.

At higher charge ratios, DNA is supersaturated with the transfer agent and the complex is totally positive. Because of the strong repulsion between the positive charges, this phase is stable. This is referred to as "phase C". Unlike phase A, the complexes obtained are in a form in which DNA is very well protected against nucleases, and the overall positive charge of these complexes promotes adhesion to cell membranes that are anionic and crosses the membrane. Thus, the C phase complex is particularly suitable for the delivery of nucleic acids into cells.

These three zones A, B and C are also updated with compound 2 according to the invention as delivery agent.

Charge ratio 0.25 0.5 0.75 One 1.5 2 3 4 6 8 10 12 Michelle A A B B B B C C C C C C + Cholesterol A A B B B C C C C C C C + DOPE A A B B B C C C C C C C

As shown in the table above, the B region, which is an unstable region, is particularly small and occurs at very low charge ratios. Region C starts with 2 nmol of lipid per μg of DNA when Compound 2 is used with co-lipids (cholesterol or DOPE), and 3 nmol of lipid per μg of DNA when compound alone is used. As defined above, this is a particularly advantageous area for pharmaceutical use.

As a result, when one of the cationic lipids described in patent WO / 18185, the C region began to form at a charge ratio of at least 2 depending on the sodium chloride concentration of the solution (B. Pitard et al., PNAS USA, 94, See FIG. 3A in pp. 14412-14417, 1997).

Thus, Compound 2 is a particularly advantageous delivery agent because it is stable at low charge ratios, allowing the formation of stable complexes with small amounts of glycolipids, with beneficial results in terms of toxicity.

Example 6: Use of Compound 2 for In Vitro DNA Delivery

This example describes the DNA transfection ability of the delivery agents according to the invention in cells, in vitro, in the presence and absence of neutral co-lipids (cholesterol or DOPE) at various charge ratios.

Seeds are propagated overnight by seeding 60,000 HeLa cells per well in 24-well microplates. After overnight, and therefore at the time of transfection, the cell number is 100,000 cells per well.

Each well is formed with Compound 2 and contacted with a complex containing 1 μg of plasmid DNA in 0.5 ml of serum-free DMEM medium (Gibco / BRL). Cells are incubated at 37 ° C. for 5 hours. The medium containing the complex is then removed and replaced with DMEM culture medium containing 10% fetal calf serum. The cells are then incubated for 24 hours. Finally, the cells are lysed and analyzed using the Luciferase Assay Kit (Promega) and Dynex MLX Photometer.

The results obtained are shown in the histogram of FIG. Delivery efficiency is expressed in pg / well by luciferase expression. It is noted that the maximum transfection is at approximately 500 pg / well.

In conclusion, this example clearly shows that Compound 2 according to the present invention can be used to form complexes that can facilitate DNA delivery into cells in vitro.

Example 7; Use of Compound 2 for In Vivo DNA Delivery

This example demonstrates the DNA transfection ability of a delivery agent according to the invention into cells in vivo.

In vivo gene transfer is performed by intrabronchial, intravenous and intramuscular administration in Balb / C mice.

For intramuscular injection, each mouse is administered 30 μl of the formulation containing 15 μg of plasmid DNA to the anterior muscle of the tibia. Tissues are harvested 7 days after injection and stored at −80 ° C. while waiting to perform luciferase activity assays.

For intravenous injection, each mouse receives 200 μl of the formulation containing 50 μg of plasmid DNA. Tissues are recovered 24 hours after injection, then frozen and stored in the same manner as above.

FIG. 3 illustrates the activity of complexes formed with Compound 2 on in vivo gene delivery by intramuscular injection. These results clearly show that the complex formation of Compound 2 with DNA according to the present invention can promote the delivery of DNA into cells in vivo.

Likewise, delivery agents as defined herein can be used to facilitate DNA delivery into cells of other tissues.

Claims (33)

A nucleic acid delivery agent, characterized in that it comprises a hydrophobic spacer that is chemically bonded to a polycion first and to at least one hydrophilic substituent second. 2. The hydrophobic spacer of claim 1, wherein the hydrophobic spacers consist of two or three hydrocarbon-based straight chain fatty chains containing from 10 to 20 carbon atoms per chain, and each chain may be different in length, or the hydrophobic spacers are from 20 to A nucleic acid delivery agent characterized by a very long hydrocarbon-based straight chain fatty chain containing 50 carbon atoms. The nucleic acid delivery agent of claim 1 wherein the hydrophilic substituent is selected from hydroxyl or amino substituents, polyols, sugars or hydrophilic peptides. 4. The nucleic acid delivery agent of claim 1 or 3, wherein at least one of the hydrophilic substituents is a sugar. The nucleic acid delivery agent of claim 1 which is optionally an isoform of Formula 1 in isomeric form, and also mixtures or salts thereof, if present. Formula 1 In the above formula, R represents a polycation, Z represents a hydrogen atom or a fluorine atom, the various Z are mutually independent, x and y independently of each other represent an integer of 10 to 22, X and Y independently of each other a hydrogen atom, an -OAlk group (wherein Alk represents a straight or branched chain alkyl of 1 to 4 carbon atoms), hydroxyl Group, amino group, polyol, sugar, hydrophilic or non-hydrophilic peptide, or oligonucleotide, at least one of the X and Y substituents represents a hydrophilic group selected from hydroxyl group, amino group, polyol, sugar or hydrophilic peptide , Or x is 0 or 1, y is an integer from 20 to 50, X is a hydrogen atom or an -OAlk group, where Alk represents straight or branched chain alkyl of 1 to 4 carbon atoms, and Y is hydroxyl Hydrophilic group selected from the group, amino group, polyol, sugar or hydrophilic peptide. 6. A nucleic acid delivery agent according to claim 1 or 5, which is optionally an isoform of Formula 3 in isomeric form, and also mixtures or salts thereof, if present. Formula 3 In the above formula, R represents a polycation, x and y independently of each other represent an integer from 10 to 22, X and Y independently of one another represent a hydrogen atom or a sugar, at least one of the X and Y substituents represents a sugar, or Or x is 0 or 1 and y is an integer from 20 to 50, X is a hydrogen atom and Y is a sugar. 7. The nucleic acid delivery agent of claim 6, wherein x and y independently represent an integer of 10 to 22, one of X and Y represents a hydrogen atom, and the other represents a sugar. 8. The nucleic acid delivery agent according to any one of claims 1 and 5 to 7, wherein the polycation is a straight or branched polyamine and each amino group is separated by one or more methylene groups. 9. The nucleic acid delivery agent according to claim 8, wherein the polycation is represented by the formula (2). Formula 2 In the above formula, R ₁ , R ₂ and R ₃ independently of one another represent a hydrogen atom or a (CH ₂ ) _q NR′R ″ group where q can range from 1 to 6, which may vary from R ₁ , R ₂ and Independent of the R ₃ groups, at least one of R ₁ , R ₂ and R ₃ is not a hydrogen atom, R ′ and R ″ independently of one another represent a (CH ₂ ) _q NH ₂ group in which a hydrogen atom or q is as defined above, m represents an integer from 1 to 6, n and p independently represent mutually an integer of 0 to 6, when n is 2 or more, m may have a different value and R ₃ has a different meaning in formula (2), and when n is 0, R ₁ and At least one of the R ₂ substituents is not a hydrogen atom. The polycation according to any one of claims 1 and 5 to 7, wherein the polycation is selected from spermine, spermidine, cadaverine, putrescine, hexamethylenetetramine (hexamine), methacrylamidopropyltrimethylammonium chloride ( AMBTAC), 3-acrylamido-3-methylbutyltrimethylammonium chloride (AMBTAC), polyvinylamine, polyethyleneimine, or ionene. 8. The nucleic acid delivery agent according to any one of claims 3 to 7, wherein the sugar is a mono-, oligo- or polysaccharide molecule. 12. The sugar composition according to claim 11, wherein the sugar is glucose, mannose, rhamnose, galactose, fructose, maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarabose, genthiobiose, sophorose , Melibiose, dextran, α-amylose, amylopectin, fructan, mannan, xylan, and arabinan. 6. The method of claim 5, wherein the oligonucleotide is any chain containing one or more nucleotides, deoxynucleotides, ribonucleotides and / or deoxyribonucleotides, optionally coupled with one or more molecules having distinctive properties. Nucleic acid delivery agents. 6. The peptide according to claim 5, wherein the peptide is any chain containing one or more amino acids which are mutually linked via the binding of the peptide species and which are optionally substituted with one or more aliphatic groups which may be saturated or unsaturated, and straight, branched or cyclic. A nucleic acid delivery agent characterized by. The delivery agent of claim 1, wherein Chemical formula The delivery agent of claim 1, wherein Chemical formula The delivery agent of claim 1, wherein Chemical formula 18. A composition comprising a nucleic acid delivery agent as defined in any one of claims 1 to 17, and a nucleic acid. 19. The composition of claim 18, wherein the nucleic acid is deoxyribonucleic acid or ribonucleic acid. 20. The composition of claim 18 or 19, wherein the nucleic acid comprises one or more therapeutic genes of interest under the control of regulatory sequences. 21. The composition of any one of claims 18 to 20, wherein the nucleic acid is an antisense sequence or gene. 19. The composition of claim 18, which also contains one or more adjuvants. The composition of claim 22, wherein the adjuvant is one or more neutral lipids. The composition of claim 23, wherein the neutral lipid is a lipid containing two fatty chains. 25. The method of claim 23 or 24, wherein the neutral lipid is for example dioleoylphosphatidylethanolamine (DOPE), oleyl palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl. Phosphatidyl-ethanolamine and derivatives thereof which are also N-methylated one to three times, phosphatidylglycerol, diacylglycerol, glycosyldiacylglycerol, cerebroside (e.g. galactocerebroside), sphingolipid ( For example, it is a natural or synthetic lipid which is zwitterionic or deficient in ionic charge under physiological conditions, in particular selected from sphingomyelin or asialogangliosides (for example asialoGM1 and GM2). Composition. 23. The composition of claim 22, wherein the adjuvant is a compound that directly or indirectly participates in the condensation of the nucleic acid. The adjuvant of claim 26, wherein the adjuvant is derived, in whole or in part, from protamine, from histones or nucleolins, and / or derivatives thereof, or consists entirely or part of peptide units (KTPKKAKKP) and / or (ATPAKKAA), The number of units can range from 2 to 10 and can be repeated continuously or discontinuously. 28. The composition of any one of claims 18 to 27, comprising a pharmaceutically acceptable vehicle in the injection formulation. 28. The composition of any one of claims 18 to 27, comprising a pharmaceutically acceptable vehicle for skin and / or mucosal application. Use of a delivery agent according to any one of claims 1 to 17 for the manufacture of a medicament for the treatment of a disease. (1) contacting the nucleic acid with the delivery agent according to any one of items 1 to 17 to form a complex, (2) contacting a human or animal cell with the complex formed in (1). (1) the nucleic acid is contacted with a delivery agent as defined above to form a complex, (2) contacting the cell with the complex formed in (1). 33. The method of claim 31 or 32, wherein the delivery agent and / or nucleic acid is premixed with one or more adjuvants as defined in any one of claims 22 to 27.