CA2446951C - Polythiourea lipid derivatives - Google Patents

Abstract

The invention relates to novel compounds that are used for the transfer of nucleic acids into cells. More specifically, said novel compounds are polythiourea lipid derivatives and are used for the in vitro, ex vivo or in vivo transfection of nucleic acids into different cell types.

Description

LIPID DERIVATIVES OF POLYTHIORA

The present invention relates to novel compounds which allow the transfer of nucleic acids into cells. More specifically, these new Compounds are lipid derivatives of polythiourea. They are useful for the in vitro, ex vivo or in vivo transfection of nucleic acids into different Types cell.

With the development of biotechnologies, the possibility of transferring effectively nucleic acids in the cells has become a need. he can This is the transfer of nucleic acids into cells in vitro, by example for the production of recombinant proteins, or in the laboratory, for the study of regulation of gene expression, gene cloning, or other manipulation involving DNA. It can also be the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines tagging studies or also therapeutic approaches. he can again the transfer of nucleic acids into cells ex vivo, in approaches to bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells taken from an organism with a view to their subsequent readministration.

Today, several methods are proposed for delivery intracellular exogenous genetic material. One of them, in particular, based on the use of non-viral vectors which is a very advantageous to viral methods which are not completely devoid of risks.
These synthetic vectors have two main functions: complexing and compact the nucleic acid to be transfected, and promote its passage through the membrane plasma and possibly through the nuclear envelope.

Several families of synthetic vectors have thus been developed, such as for example polymers or biochemical vectors (made up of a cationic protein associated with a cellular receptor ligand), but a progress

2 important has been accomplished with the development of lipofectants, and more particularly cationic lipids. It has thus been highlighted that lipids cationic, because of their overall positive charge, interfered with spontaneously with globally negative DNA, forming nucleolipid complexes capable of the protect the DNA against nucleases and focus on membranes for intracellular release of DNA.

Different kinds of cationic lipids have been synthesized to date:
lipids comprising a quaternary ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE ...), lipopolyamines such as DOGS, DC-Chol or the lipopolyamines disclosed in the patent application WO 97/18185, lipids combining at the same time an ammonium group quaternary and a polyamine such as DOSPA, or lipids with various other cationic entities, especially amidinium groups (for example ADPDE, ADODE or the lipids of the patent application WO 97/31935).

However, the use of these cationic lipids as a transfection still poses many problems, and their effectiveness remains to be improved.
Especially, it has been observed that to obtain effective nucleolipid complexes and stable it is generally necessary for these complexes to be highly cationic.
However, it would be desirable to have vectors that are not not cationic agents so as to form particles with the nucleic acid overall neutral or negative. Indeed, it has been observed that the complexes globally formed between the nucleic acid and the cationic lipids have tendency to be captured by the reticuloendothelial system which induces their elimination. In addition, plasma proteins tend to adsorb to their surface because of the charge overall positive result of the complexes formed, and this results in a loss of power of transfection. Moreover, in a context of local injection, the presence of a charge positive overall effect prevents diffusion of acid complexes nucleic outside the administration site, because the complexes are adsorbed on the matrices extracellular; the complexes can no longer reach the cells targets, this which, as a result, leads to a decrease in the effectiveness of transfer by

3 relative to the amount of complexes injected. Finally, it was also found at many times that cationic lipids have killed the inflammatory effect.

The object of the present invention is precisely to propose new transfectant compounds, originated by their polythiourea function, and are likely to be used effectively for in vitro transfection, ex vivo or in vivo nucleic acids. These new compounds are particularly interesting because :
- the absence of positive charges in their structure makes it possible to solve numerous problems raised by the use of cationic vectors discussed below.
before, - just like cationic lipids, they are able to complex and compact nucleic acids and promote their transfection.
SUMMARY OF THE INVENTION
The present invention thus has for its first object transfecting compounds characterized in that they consist of a polythiourea portion connected to a lipid via a spacer.

In particular, the subject of the present invention is transfectant compounds of general formula (I):

X 1 - N] YL I
HH (HMI 1 () RR

in which :

= -1 is an integer chosen from 0 and 1 = n is an integer chosen from 1, 2, 3, 4, 5 and 6, = ni is an integer chosen from 2, 3 and 4, and can not take values different to within the different groups - [NH-CS-NH- (CH (R)) m] -,

4 R 'represents a group of general formula (II):

S
4 CH N 'j ~ NX (II) (z) p HH
q wherein q is an integer selected from 1, 2, 3, 4, 5 and 6, and p is a chosen whole among 2, 3 and 4, p being able to take different values within the different groups - [(CH2) p NH-CS-NH] -, R represents either a hydrogen atom or a group of general formula (II) such as defined above, it being understood that when n is 1 and -e is 0, then at least one group R is of formula (II) = X, in formulas (I) and (II), represents a linear aliphatic group or cyclic, saturated or unsaturated, and comprising 1 to 8 carbon atoms, a group mercaptomethyl (-CH2SH), or a hydrophilic chain chosen from groups:
- (CH2) X (CHOH) ù H with x an integer from 0 to 10, and u an integer selected among 1, 2, 3, 4, 5 and 6, or - (OCH2CH2O), - H with v an integer chosen from 1, 2 and 3, with the proviso that not more than one substituent X, both in the formulas * (I) and (II), represents a hydrophilic chain, = Y represents a spacer = and L represents:
- or a group -N (RI) R2 with RI and R2 which represent, independently of one another, a hydrogen atom or a fatty aliphatic chain, or a group of formula - (CH2) t-OZ with t represents an integer selected from 11, 12, 13, 14 or 15 and Z represents a sugar, a polyol or a PEG, it being understood that at least one of R1 and R2 is different from hydrogen, or a group -OR3, with R3 representing a steroid derivative, if appropriate in their isomeric forms or mixtures thereof.
The present invention also relates to compositions characterized in that that they comprise a transfecting compound as described above, and a nucleic acid.
The present invention also relates to a method for transferring acids nucleic cells in the cells characterized in that it comprises the steps following:
(1) contacting the nucleic acid with a transfectant compound such than described above, to form a complex, and (2) contacting the cells with the complex formed in (1).
The present invention also relates to the use of a transfectant compound such described in this application for treatment or prevention infections viral or bacterial, or cancerous states.
The present invention also relates to a method of transferring acids nucleic cells in cells characterized in that it comprises the steps following:
(1) contacting the nucleic acid with a transfectant compound such than described in the present application, to form a complex, and (2) contacting the cells in vitro with the complex formed in (1).
DESCRIPTION OF THE INVENTION

According to the present invention, spacer means any chemical group allowing both the liaison between the polythiourea party and the part lipid of the molecule, and to move these two parts apart in order to reduce any unwanted interaction between them. Preferred spacers may for example to be 5a consisting of one or more chemical functions chosen from among the alkyls having 1 to 6 carbon atoms, the functions ketone, ester, ether, amide, amidine, carbamate, thiocarbamate, glycerol, drawn, thiourea, or cycles aromatics. By for example, the spacer Y may be chosen from groups of formula:
-NH-C (O) -CH2-CH2-or: - (CH2 -), - W- (CH2) j-where i and j are integers selected from 1 to 6 inclusive and W is a group selected from ketone, ester, ether, amide, amidine, carbamate, thiocarbamate, glycerol, urea, thiourea, or even cycles aromatic, For the purposes of the present invention, the term aliphatic chains fatty acids containing from 10 to 22 carbon atoms, saturated or unsaturated and possibly containing one or more heteroatoms, provided that said fatty aliphatic chains have lipid properties. Of preferably they are linear or branched alkyl groups containing 10 to 22 atoms of carbon and 1, 2 or 3 unsaturations. Preferably, said alkyl groups include 10, 12, 14, 16, 18, 20 or 22 carbon atoms. We can mention more especially the aliphatic groups - (CH 2) 1 1 CH 3, - (CH 2) 13 CH 3, (CH2) 15CH3 and - (CH2) 17CH3.

For the purposes of the invention, the term "sugar" means any molecule consisting of or several saccharides. By way of example, mention may be made of sugars such as the pyranoses and furanoses, for example glucose, mannose, rhamnose, the galactose, fructose, or maltose, lactose, sucrose, sucrose, the fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorosis, the melibiose etc ... Preferably, the sugar or sugars are chosen from glucose, the mannose, rhamnose, galactose, fructose, lactose, sucrose and the cellobiose. In addition, it can also be called complex sugars, that is to say of several sugars covalently coupled to each other, each sugar being of preference chosen from the list cited above. As polysaccharides suitable are dextran, α-amylose, amylopectin, fructans, the mannans, xylans and arabinans. Some of the preferred sugars can outraged interact with cellular receptors, like some types of of lectins.

According to the invention, the term "polyol" is also understood to mean any molecule hydrocarbon linear, branched or cyclic comprising at least two hydroxyl functions. We can mention by way of example glycerol, ethylene glycol, propylene glycol, Tetritols, pentitols, cyclic pentitols (or quercitols), hexitols such as mannitol, the sorbitol, dulcitols, cyclic hexitols or inositols etc ... (Stanek and al., The Monosaccharides Academic Press, pp. 621-655 and pp. 778-855). According to one aspect preferred, the polyols are chosen from alcohols of general formula:

HO s OH
OH
for which s is selected from 2, 3, 4, 5 and 6.

When the compounds of general formula (I) according to the invention contain a polyethylene glycol (PEG) group, this usually comprises between 2 and 120 reasons -OCH 2 CH 2 O-, and preferably between 2 and 80 -OCH 2 CH 2 O- units. It could be of PEG simple, that is to say whose end of the chain is terminated by a group hydroxyl, or PEG whose end group is chosen from among the alkyls, by example methyl.

For the purposes of the present invention, the term "steroid derivatives" means polycyclic compounds of the cholestane type. These compounds can be natural or no and are more preferably selected from cholesterol, cholestanol, 3-a-

5-cyclo-5-a-cholestan-6- (3-ol, cholic acid, cholesterylformate, chotestanylformate, 3a, 5-cyclo-5a-cholestan-6p-yl formate, the cholesterylamine, 6- (1,5-dimethylhexyl) -3a, 5α-diméthylhexadécahydrocyclopenta [a] cyclopropa [2,3] -cyclopenta [1,2-f] naphthalen-10-ylamine, or cholestanylamine.

According to a preferred variant of the invention, the transfecting compounds have for general formula (III):

N N- CH Y 'j ~ N ~ HH (2nR (III) wherein X, m, n, and Y are as previously defined in the formula general (I), with the exception of n which is different from 1, and RI and R2 represent independently of one another, a hydrogen atom or a chain fatty aliphatic, it being understood that at least one of R1 and R2 is different of hydrogen.

Even more preferentially, the transfecting compounds of the invention have for general formula (IV):

(IV) NNN- (2 CH YN 1 HH mn R2 wherein m, n, and Y are as previously defined in the formula General (I), except for n which is different from 1, and R1 and R2 represent, independently from each other, a hydrogen atom or a fatty aliphatic chain, being understood that at least one of R1 and R2 is different from hydrogen.

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

The preparation of the compounds of general formula (I) according to the present invention is carried out by implementing the following steps, in the order presented or according to any other known and equally adapted variant, using the techniques of classical organic synthesis, in solution or on solid supports, although known from the skilled person:

1) Obtaining the Lipid Part L

When the lipid portion L of the compounds of general formula (I) is represented by a group N (R1) R2 with R1 and / or R2 which represent a fatty aliphatic chain, the amine of formula HN (R1) R2 is first formed.
Said amine can be obtained by condensation of a carboxylic acid and a amine containing one the substituent R1 and the other the substituent R2 for form the amide corresponding, followed by the reduction of said amide thus obtained.

The formation of the amide is advantageously carried out by mixing the constituents and melting by heating at a temperature above temperature of fusion of the substances involved, usually between 20 C and 200 C, then elimination of water produced by dehydration of the environment; or more advantageously presence a desiccant such as, for example, phosphorus pentoxide or any other other substance that can absorb water. The formation of this intermediate amide can also be done using a variant of this method or any other amide formation method (such as, for example, type coupling peptide) involving carboxylic acids or their derivatives, conditions and reagents [Larock RC, Comprehensive Organic Transformations, VCH Editors] and well known to those skilled in the art.

The reduction of the amide previously obtained in amine of formula HN (R1) R2 may be carried out for example using a reducing agent such as hydride double lithium and aluminum, or any other effective hydride or reductant in this case. The procedure is preferably carried out in a non-protic solvent (for example tetrahydrofuran or ethers), at a temperature below temperature boiling solvent and under dry atmosphere and / or inert.

According to another variant, the lipid portion designated as HN (Rn) R2 may be available commercially.

When Rn and / or R2 represents (nt) a group of formula - (CH2) t-OZ, proceeds as described above to form the alkyl part, and then a simple coupling with a sugar, a polyol or a commercial PEG according to the techniques classics known to those skilled in the art.

When the lipid portion L of the compounds of general formula (I) is represented by a group -OR3, this group is preferably chosen from the commercially available products.

2) Grafting the spacer Y

The spacer Y is then attached to the lipid portion L obtained in step preceding according to conventional techniques known to those skilled in the art. according to a preferred variant, an amide bond is made by N-acylation of the part lipid In a suitable solvent such as dichloromethane, chloroform, tetrahydrofuran, or any other ether, at a temperature below temperature boiling solvent, and under dry atmosphere and / or inert. This reaction himself preferably unrolled in the presence of an amino base such as N, N-dimethylaminopyridine, or in the presence of this base mixed with bases non-nucleophilic amino acids such as triethylamine or ethyldiisopropylamine.
Pyridine can also be used alone or in combination with another based, diluted by one of the solvents mentioned or used itself as a solvent.

3) Formation of the polythiourea chain The third part of the synthesis of the compounds of general formula (I) consists of the successive introduction of the thiourea motifs. This one will be realized in a sequence of reactions that can be repeated as many times as necessary to get the desired polythiourea part. According to a preferred method, the operation is carried out way next :

A) First graft to the YC (O) -L group obtained in step previous, the first part of the pattern as a link -HN-(CHR) ,,, -.
For this, it is advantageous to operate from a diamine ring of formula (CHR) m NH 2 in the presence of a coupling agent, for example the hexafluorophosphore 1-benzotriazolyloxytris (pyrrolidino) phosphonium (PyBOP), hexafluoro-phosphorus of 1-benzotriazolyloxytris (dimethylamino) phosphonium (BOP), the hexafluorophosphorus or O- (1H-benzotriazol-1-yl) -N, N, N ', N'-N'-tetrafluoroborate Tetramethyluronium (HBTU or TBTU), dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), or 1- (3-trimethylaminonopropyl) -3-ethylcarbodiimide iodide, supported or no.
This coupling is carried out in a suitable solvent, for example the dichloromethane, chloroform, tetrahydrofuran, or any other ether, temperature below the boiling point of the solvent, and under atmosphere dry and / or inert. It is also carried out in the presence of a non-amino base nucleophile, for example ethyldiisopropylamine, triethylamine or the triisopropylamine. If the nature of the lipid part and the spacer is compatible, a sequence of the SCN- (CHR) m type, or a precursor, can be grafted, allowing thus to continue the synthesis by a step such as that described hereinafter in C), B) The product obtained in the previous step is then converted, according to a preferred technique, isothiocyanate by treatment with carbon disulfide (CS2) or by any other reagent known to those skilled in the art to obtain such a [H. Ulrich, Chemistry and Technology of Isocyanates, Wiley (1996).
The Chemistry of Cyanates and their Thio Derivatives, S. Patai Ed., Wiley (1977). S.
Ozaki, Recent Advances in Isocyanate Chemistry, Chem. Rev. 72, 457 (1972).].
The The reaction is advantageously carried out in a solvent such as, for example, tetrahydrofuran, or any other compatible etheric solvent, at a temperature variant between the refrigerant mixtures and about 20 C. It also operates in presence of an agent capable of promoting the reaction and / or trapping hydrogen sulphide released during the course of the reaction, for example the dicyclohexylcarbodiimide (DCC).

C) Then the thiourea unit is formed from the isothiocyanate obtained at step previous to allow for the introduction of another segment of formula - (CHR) m. Advantageously, a diamine of formula H2N- (CHR) m -NH 2, possibly protected, is reacted, in its neutral form or in the form salt of acid, with the isothiocyanate obtained in the previous step. This reaction is done optionally in the presence of a non-nucleophilic amino base, for example the triethylamine, ethyldiisopropylarine, triisopropylamine or 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). It is preferably carried out in a solvent such as dichloromethane, chloroform, tetrahydrofuran, or all other ether or compatible solvent, at a temperature that may be between that of refrigerant mixtures and the reflux temperature of the solvent.

Then we repeat sequentially and in the required order the steps B) and C) described above, until the target structure is obtained, so that introduce the desired pattern in n copies. To get connected structures, we proceeds from analogous way by introducing at the appropriate moment the molecule (s) Required to obtain a substitution R as described by formula (II).

4) Termination of the polythiourea part by introduction of the substituent X

The last step allowing the termination of the chain (s) of type polythiourea consists of the introduction of the substituent X. For this, one uses the conventional methods of grafting known to those skilled in the art chosen from function of the nature of the substituent X. For example, when X represents an alkyl, operates in reacting an alkylisothiocyanate, when present necessary, non-nucleophilic amine base, for example triethylamine, ethyldiisopropylamine, triisopropylamine or 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). The reaction is conducted in a solvent suitable, for example dichloromethane, chloroform, tetrahydrofuran, or any other compatible ether, at a temperature between of the refrigerant mixtures and the reflux temperature of the solvent.

Naturally, when the different substituents can interfere with the reaction, it is better to protect them with radicals compatible and can be put in place and eliminated without touching the rest of the molecule.
We operates for this according to conventional methods known to those skilled in the art, and especially according to the methods described in TW Greene, Protective Croups in Organic Synthesis, Wiley-Interscience, in McOmie, Protective Groups in Organic Chernistry, Plenum Press, or in PJ Kocienski, Protecting Groups, Thieme.

Moreover, each step of the preparation process can be followed, if appropriate, steps of separation and purification of the compound obtained according to all method known to those skilled in the art.
The preferred compounds according to the present invention are 3- (2- [3- [2- (3- [2- (3- (diterradecylcarbamoyl) propionylamino] ethyl thioureido) ethyl] thioureido-ethyl-1-methylthiourea or DT-3TU, which comprises three thioureas and corresponds to formula (I) wherein X is -CH3, m is equal to 2, n is 3, R is hydrogen, f is 0, L = -N (R1) R2 where R1 = R2 =
C14H29, and Y = NH-CO-CH2-CH2.

3- (2- {3- [2- (3- {2- [3- (2- {3- [ditertradecylcarbamoyl] propionylamino} -ethyl) -thioureido] -ethyl} -thioureido) -ethyl] -thioureido} -ethyl) -1-methylthiourea or DT-4TU which contains four thiourea groups and responds to the general formula (I), wherein X is -CH3, m is 2, n is equal 4, R is a hydrogen, f is 0, L = -N (R1) R2 where R1 = R2 = C14H29, and Y = NH-CO-CH2.

- The compound 2- (3- ~ 2- [3- (diterradecylcarbamoyl) propionylamino] ethyl ~ -thioureido) ethyl] propan-1,2-diol or DT-2TU diol corresponds to the general formula (I) in which X = HO OH

m = 2; R = H; n = 2; C = 0; L = -N (R1) R2 where R1 = R2 = C14H29, and Y = NH-CO-CH2-CH2.
The compound 2- (3- {2- [3- (2- {3- [2- (3- (diterradecyl) carbamoyl) propionylamino} ethyl] -thioui ido} -ethyl} thioureido] -ethyl} thioureido) -ethyl] -propane-1,2-diol or DT-3TU diol corresponds to formula (I) in which X = HO OH

m2;
R = H;
n = 3;

-e = 0;
L = -N (RI) R2 where R1 = R2 = C14H29, and Y = NH-CO-CH2-CH2.

Another subject of the invention relates to compositions comprising a transfecting compound according to the invention and a nucleic acid. Quantities respective components of each component can be easily adjusted by the according to the transfecting compound used, the nucleic acid, and of the applications sought (including the type of cells to be transfected).

For the purposes of the invention, the term "nucleic acid" is understood to mean an acid deoxyribonucleic acid as a ribonucleic acid. It can be sequences Natural artificial, and in particular genomic DNA (gDNA), complementary DNA
(CDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA
(RRNA), hybrid sequences such as DNA / RNA chimeroplasts or synthetic or semi-synthetic sequences, modified oligonucleotides or no.
These nucleic acids can be of human, animal, plant origin, bacterial, viral, etc ... They can be obtained by any technique known to man of profession, and in particular by screening of banks, by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by screening banks. They can be chemically modified. In general, they contain at least 10, 20, 50 or 100 consecutive nucleotides, and of preferably at least 200 consecutive nucleotides. Even more preferentially, they contain at least 500 consecutive nucleotides.

With particular regard to deoxyribonucleic acids, they can be single or double strand, as well as short oligonucleotides or longer sequences. In particular, the nucleic acids are advantageously consisting of plasmids, vectors, episomes, cassettes expression, etc. These deoxyribonucleic acids can carry an origin of replication prokaryotic or eukaryotic functional or not in the target cell, one or many marker genes, regulatory sequences of transcription or replication, genes of therapeutic interest, modified or unmodified antisense sequences, of the regions of binding to other cellular components, etc.

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

For the purposes of the invention, the term gene of therapeutic interest includes any gene encoding a protein product having a therapeutic effect. The product protein thus coded may be in particular a protein or a peptide. This product Protein can be exogenous homologous or endogenous towards the cell target, that is, a product that is normally expressed in the target cell when it presents no pathology. In this case, the expression of a protein allows for example to overcome an insufficient expression in the cell or the expression of an inactive or weakly active protein due to modification, or overexpressing said protein. The gene of therapeutic interest can as well code for a mutant of a cellular protein, having increased stability, an activity modified, etc ... The protein product can also be heterologous vis-à-screw of the target cell. In this case, an expressed protein may for example supplement or bring a deficient activity in the cell, allowing him to fight against a pathology, or stimulate an immune response.

Among the therapeutic products within the meaning of the present invention, it is possible to quote especially enzymes, blood derivatives, hormones, lymphokines and cytokines and their inhibitors or antagonists: interleukins, interferons, TNF antagonists of interleukin-1, the soluble receptors of interleukin 1 or TNFa, etc., growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF-B, NT3, NT5, HARP / pleiotrophin, etc.) apolipoproteins (ApoAI, ApoAIV, ApoE, etc.), dystrophin or a minidystrophin, CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc ...), genes coding for factors involved in coagulation (Factors VII, VIII, IX), the genes involved in the repair of DNA, the genes suicides (thymidine kinase, cytosine deaminase), the genes for hemoglobin or other carriers proteins, metabolic enzymes, catabolism etc ...
The nucleic acid of therapeutic interest can also be a gene or Lure antisense sequence or a DNA coding for a ribozyme-functional RNA, whose the expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, to be transcribed in the target cell into RNA complementary to cellular mRNAs and block their translation into protein, according to the technique described in EP patent 140 308. Therapeutic genes also include coding sequences for ribozymes, which are capable of selectively killing target RNAs (EP

201).

As indicated above, the nucleic acid may also include one or several genes encoding an antigenic peptide, capable of generating the man or the animal an imminent response. In this particular mode of setting artwork, the invention allows the realization of either vaccines or treatments immunotherapeutic agents applied to humans or animals, in particular for treating or prevent infections, for example viral or bacterial, or cancerous.

It may be in particular antigenic peptides specific for the virus of Epstein Barr, HIV, hepatitis B virus (EP 185 573), the virus of the pseudo-rabies, "syncitia-forming virus", other viruses or peptides tumor-specific antigenic agents (EP 259,212).

Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or the coding gene for the antigenic peptide in the desired cell or organ. These may be sequences that are naturally responsible for the expression of the gene considered when these sequences are likely to work in the infected cell. he can also sequences of different origin (responsible for the expression other proteins, or even synthetic). In particular, it can be sequences promoters of eukaryotic or viral genes. For example, it may be sequences promoters from the genome of the cell that we want to infect. Similarly, he can these are promoter sequences derived from the genome of a virus. In this respect, can mention, for example, the promoters of the genes EIA, MLP, CMV, RSV, etc.
outraged, these expression sequences can be modified by addition of sequences activation, regulation, etc ... It can also be a promoter, inducible or repressible.

On the other hand, the nucleic acid may also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the product therapeutically synthesized in the secretory pathways of the target cell.
This sequence signal may be the natural signal sequence of the therapeutic product, but he can also be any other functional signal sequence, or a sequence artificial signal. The nucleic acid may also include a sequence signal directing the synthesized therapeutic product to a particular compartment of the cell.

The compositions according to the invention may also comprise one or more adjuvants capable of associating with transfectant / acid complex complexes nucleic and to improve the transfecting power. In another mode of implementation work, the The present invention thus relates to compositions comprising an acid nucleic acid, a transfecting compound as defined above and at least one adjuvant able to to associate with the transfectant / nucleic acid complexes and to improve the transfectant capacity. The presence of this type of adjuvant (lipids, peptides, protein or polymers for example) can advantageously make it possible to increase the power transfecting compounds. In this context, the compositions of the invention can include as an adjuvant, one or more neutral lipids, which have in particular the property of forming lipid aggregates. The term aggregate lipid is a generic term that includes liposomes of all types that time unilamellar and multilamellar cells) as well as micelles or aggregates more amorphous.

More preferentially, the neutral lipids used in the context of the present The invention is lipids with two fatty chains. In particular advantageous, natural or synthetic zwitterionic lipids are used or without ionic charge under physiological conditions. They can to be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), distearoyl, -palmitoyl, -mirystoylphosphatidylethanolamines and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as galactocerebrosides), sphingolipids (such sphingomyelins) or asialogangliosides (such as especially asialoGM1 and GM2). Advantageously, the adjuvants lipid used in the context of the present invention are chosen from DOPE, DOPC
where the cholesterol.

These different lipids can be obtained either by synthesis or by extraction from organs (eg the brain) or eggs, by techniques classics well known to those skilled in the art. In particular, the extraction of lipids can be achieved by means of organic solvents (see also Lehninger, Biochemistry).

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

According to another alternative, the adjuvants mentioned above improve the transfecting power of the compositions according to the present invention, especially peptides, proteins or certain polymers such as polyethylene glycol, can be conjugated to the transfecting compounds according to the invention, and not simply mixed. In this case, they are covalently linked either to the substituent X in the general formula (I), ie at the end of the alkyl chain (s) Ri and / or R2 when these are fatty aliphatic chains. he is also advantageous to use as adjuvant, polyethylene glycol so bound covalently cholesterol (chol-PEG). Indeed, when the latter is conjugated to the compounds transfectants according to the present invention, it makes it possible to obtain particles of sizes reduced, and thus avoid their aggregation and increase the half-time life of particles in the bloodstream. The amount of transfectant example DT-3TU used according to the present invention is such that the particles have sizes less than 500nm. Preferably, the amount of transfectant, such as DT-used is at least 40nmol of DT-3TU lipids / g of DNA (see Examples 11, 13, and 14, below).

According to a particularly advantageous embodiment, the compositions according to the present invention further include a targeting element allowing to guide the transfer of the nucleic acid. This targeting element can be a extracellular targeting element to guide the transfer of acid nucleic to certain cell types or desired tissues (cells tumors, hepatic cells, hematopoietic cells ...). he can also an intracellular targeting element to guide the transfer of acid nucleic acid towards certain privileged cellular compartments (mitochondria, core etc ...). The targeting element can be mixed with the transfectant compounds according to the invention and the nucleic acids, and in this case the targeting element is of covalently bonded to a fatty alkyl chain (at least 10 carbon atoms).

carbon) or polyethylene glycol. According to another alternative, the element of targeting is covalently bound to the transfecting compound according to the invention, either at level of the substituent X, either on the spacer Y, or else on the end of R1 and / or when these represent fatty aliphatic chains. Finally, the element of targeting can also be linked to the nucleic acid as has been specified previously.

Among the targeting elements that can be used in the context of the invention, can include sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives.
Preferably, he These are sugars, peptides, vitamins or proteins such as example of antibodies or antibody fragments, cellular receptor ligands or some fragments thereof, receptors or fragments of receptors.
By for example, they may be growth factor receptor ligands, receptors for cytokines, cell-type lectins, receivers folate, or RGD-sequence ligands with affinity for adhesion proteins like integrins. We can also mention the receivers transferrin, HDL and LDL, or the folate transporter. The element of targeting can also be a sugar to target lectins such that asialoglycoprotein or syalide receptors such as Sialyl Lewis X, or still an antibody Fab fragment, or a single chain antibody (ScFv).

The subject of the invention is also the use of the transfectant compounds such as defined above for the transfer of nucleic acids into cells in vitro, in vivo or ex vivo. More specifically, the subject of the present invention is the use of transfecting compounds according to the invention for the preparation of a medicament destined to treat diseases, especially diseases that result from deficiency in one protein or nucleic product. The polynucleotide contained in said drug code said protein or nucleic product, or constitutes said product nucleic, able to correct said diseases in vivo or ex vivo.

For in vivo uses, for example in therapy or for the study of gene regulation or the creation of animal models of pathologies, the compositions according to the invention may be formulated for administration by topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, Intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneally, etc. Preferably, the compositions of the invention contain a a pharmaceutically acceptable carrier for an injectable formulation, especially for direct injection into the desired organ, or for a administration by topical route (on skin and / or mucosa). It may be particularly solutions Sterile, isotonic, or dry compositions, especially freeze-dried, who by addition of sterilized water or physiological saline as appropriate the constitution of injectable solutions. The doses of nucleic acids used for injection and the number of administrations can be adapted function of different parameters, especially depending on the mode of administration used, The pathology, the gene to be expressed, or the duration of the treatment sought. With regard more particularly to the mode of administration, he can be a direct injection into the tissues, for example at the level of tumors, either an injection into the circulatory pathways or a treatment of cells in culture followed by their reimplantation in vivo, by injection or graft. The tissues In the context of the present invention are, for example, muscles, the skin, brain, lungs, liver, spleen, bone marrow, thymus, the heart, lymph, blood, bones, cartilage, pancreas, kidneys, bladder, the stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissues, etc ...

Another subject of the present invention concerns a transfer method nucleic acids in the cells comprising the steps of:
(1) contacting the nucleic acid with a transfecting compound according to the the present invention to form a complex, and (2) contacting the cells with the complex formed in (1).

The invention further relates to a method of treating the human body or animal comprising the following steps:

(1) contacting the nucleic acid with a transfecting compound according to the the present invention to form a complex, and (2) bringing the cells of the human or animal body into contact with the complex form in 1).

The contacting of the cells with the complex can be carried out by incubation of the cells with said complex (for in vitro or ex vivo), injection of the complex into an organism (for vivo). On the one in general, the amount of nucleic acid to be administered depend on very many factors such as, for example, the disease to be treated or prevent, nature of the nucleic acid, promoter strength, biological activity of product expressed by the nucleic acid, the physical condition of the individual or the animal (weight, age, etc.), the method of administration and the type of formulation.
In In general, the incubation is preferably carried out in the presence of, for example, 0.01 to 1000 g of nucleic acid for 106 cells. For in vivo administration, of the nucleic acid doses ranging from 0.01 to 50 mg may for example be used.
The administration can be carried out in single dose or repeated by interval.

In the case where the compositions of the invention additionally contain one or several adjuvants as defined above, the adjuvant (s) are previously mixed with the transfecting compound according to the invention and / or acid nucleic. Alternatively, the adjuvant (s) may be administered prior to administration of the nucleolipid complexes.

According to another advantageous alternative, the tissues can be subjected to a chemical or physical treatment to improve transfection. In the case physical treatment, it can use electrical impulses as in the case of electrotransfer, or mechanical fears as in the case of the sodoporation.

The present invention thus provides a particularly advantageous method for the transfer of nucleic acids in vivo, in particular for the treatment of diseases, including the in vivo or in vitro administration of a nucleic acid encoding for a protein or can be transcribed into a nucleic acid capable of correct said disease, said nucleic acid being associated with a transfectant compound according to the invention under the conditions defined above.

The transfectant compounds of the invention are particularly useful for the transfer of nucleic acids into primary cells or into lines established. he may be fibroblastic, muscle, nerve cells (neurons, astrocytes, glial cells), hepatic, hematopoietic (lymphocytes, CD34, dendritic, etc ...), epithelial etc ..., in differentiated or pluripotent form (Precursors).

Another object of the present invention is also to provide kits for which comprise one or more transfectant compounds according to the invention and / or their mixtures. Such kits may be in the form of a packaging compartmented to receive different containers such as for example of the bulbs or tubes. Each of these containers includes the different items necessary to perform the transfection, individually or mixed: by example a transfectant compound (s) according to the invention, one or more acid (s) nucleic (s), adjuvant (s), cells, etc.

In addition to the foregoing, the present invention also includes other features and benefits that will emerge from the examples and figures that follow, and which must be considered as illustrating the invention without limit the scope. In particular, the applicant proposes a non-limiting protocol procedure as well as reaction intermediates that could be implemented to prepare the transfecting compounds according to the invention. Of course, he is at the the scope of the art to draw inspiration from this protocol or products intermediate to develop similar processes to lead to these same compounds.

ABBREVIATIONS USED

BET: ethidium bromide DCC: dicyclohexylcarbodiimide DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DTTU: 3- (2- [3- [2- (3- [2- (3- (diterradecylcarbamoyl) propionylamino] ethyl-thio]

ureido) ethyl] thioureido-ethyl-1-methylthiourea.

In the case where the DTTU molecule contains 3 thioureas, it will be designated DT-3TU, 4 thiourea DT-4TU, and so on.
EPC: La-phosphatidylcholine 95% (egg) PyBOP: benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate TBE: tris-borate-EDTA
TFA: trifluoroacetic acid THF: tetrahydrofulan LEGENDS OF FIGURES

Figure 1: Evolution of the fluorescence rate (in%) according to the quantity of EPC / DT-3TU mixture (in nmoles) per g of nucleic acid and depending on the amount of EPC alone (in nmoles) per g of nucleic acid (control mixture).

Figure 2: Evolution of the fluorescence rate (in%) according to the quantity of DPPC / DT-3TU mixture (in nmoles) per g of nucleic acid and depending on the quantity of DPPC alone (in nmoles) per gg of nucleic acid (control mixture).

Figure 3: Agarose gel (0.8% / TBE) showing compaction of the plasmid pXL3031 (g) as a function of the amount of EPC / DT-3TU liposome (in nmoles) used.

Figure 4: Zeta potential (in mV) corresponding to the surface potential of liposomes DPPC / DT-3TU-DNA as a function of the amount of lipid (nmoles) per g of acid nucleic.

Figure 5: Efficacy of in vitro transfection of HeLa cells by complex formed between DNA and DT-3TU / DPPC liposomes (1: 2) at different ratios lipid / DNA in nmoles / g, with or without serum.

The y-axis represents the expression of luciferase in RLU / g of protein.
The x-axis indicates the amount of DT-3TU (in mnoles) per g of DNA.
Figure 6: Protein level (in absorbance) of untreated HeLa cells or treated with EPC + DT-3TU / DNA liposomes at different lipid / DNA ratios n voles / g.

Figure 7: Agarose gel (0.8% / TBE) showing the compaction of the plasmid pXL3031 (pg) as a function of the amount of DT-3TU / DPPC nanoemulsion (in nmoles) used.
Figure 8: Agarose gel (0.8% / TBE) showing the compaction of the plasmid pXL3031 (pg) as a function of the amount of nanoemulsion DT-3TU / DPPC / Chol-PEG (in nmoles) used.
Figure 9: Evolution of the percentage of compacted DNA according to the quantity of DT-4TU / DPPC mixture (in nmoles) per g of nucleic acid compared with different amounts of the DT-3TU / DPPC mixture (in nmoles) per μg of acid nucleic.
Figure 10: Agarose gel (0.8% / TBE) showing the protection of the plasmid pXL3031 (pg) by the mixture of DT-3TU / DPPC lipids vis-à-vis DNases. The control DNA
in presence of DNases was deposited in well 2, DNA in the presence of 30, 40 nmol of lipid / μg DNA and 40nmol / μg + 6% chol-PEG treated with DNase were summer deposited in wells 3,4 and 5 respectively.
Figure 11: Agarose gel (0.8% / TBE) showing the protection of the plasmid pXL3031 (pg) by the mixture of DT-3TU / DPPC lipids vis-à-vis the serum. Well 1 corresponds to DNA alone, wells 2, and 3 to DNA alone and in the presence of 40nmol /. boy Wut of nanoemulsions of DT-3TU / DPPC in 150mM NaCl and then in 20% serum (well 4 and 5) and in 100% serum (wells 6 and 7).
Figure 12: In Vivo Transfection Efficiency in Muscle by Complexes trained between DNA and liposomes DT-3TU / DPPC, at different lipid / DNA ratios nmoles / μg with and without electrotransfer (e- / e +).

Figure 13: In Vivo Biodistribution in Mice of DT3TU / DPPC / DOPE-complexes Rh / DNA after 30 min, 1h and 6h in the blood, lungs and system RES.
This figure shows the stealthiness of these particles: 50% of complex have been found after 30 minutes in the bloodstream.

EXAMPLES
The usual reagents and catalysts such as triethylamine, acid trifluoroacetic acid, p-toluene sulphonic acid, hexafluorophosphate benzotriazol-1-yloxytripyrrolidinophosphonium (PyBOP), dicylclohexylcarbodiimide (DCC), carbon disulfide, tetradecylamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine or diisopropylethylamine are available commercially.

Proton Nuclear Magnetic Resonance spectra (1H NMR) have been recorded on 300, 400 and 600 MHz Bruker spectrometers. Travel are expressed in ppm (parts per million) and the multiplicities per the usual abbreviations.

In all the following, the nucleic acid used is the plasmid pXL3031 described 20 in Gene Therapy (1999) 6, pp. 1482-1488, which contains the gene read coding for luciferase under the control of the P / E CMV promoter of cytomegalovirus.
This plasmid is shown in FIG. 7. Its size is 3671 bp. The solution of used plasmid is diluted to 1.262 g / l in water for preparation injectable.

EXAMPLE 1 Synthesis of the DT-3TU

3- (2- [3- [2- (3- {2- [3- (diterradecylcarbamoyl) propionylamino] ethyl ureido) ethyl] thioureido ~ ethyl) -1-methylthiourea or DT-3TU is according to the formula (I), in which:
X = -CH3;

m = 2;
HR;
n = 3;
.e = 0;

Y = NH-CO-CH2-CH2, and L = -N (RI) R2 where R1 = R2 = C14H29 =
a) Synthesis of ditetradecylamide (1) In a flask equipped with a magnetic stirrer connected to a receiving flask containing a drying agent (P205) are mixed with 131.6 mmol of acid tetradecanoic acid (30 g) and 140.5 mmol of tetradecylamine (30 g). The mixture The reaction is then heated for 4 hours at 170 ° C. under reduced pressure.
(50 min Hg). The crude is then solubilized in THF (700 ml;
for help to solubilize) then add 4 equivalents of Amberlyst-15TM resin (8 boy Wut) to fix the excess amine. After stirring for 20 minutes, the solution is filtered then the filtrate is concentrated to obtain 55.11 g of a white solid (yield:
99%).
1 H NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.5Hz, -CH3), 1.25 (n, 42H, -CH2-), 1.47 (m, 2H, CO-CH 2 -CH 2), 1.60 (m, 2H, N-CH 2 -CH 2), 2.15 (t, 2H, J = 7.5 Hz, CO 2 CH1), 3.23 (dt, 2H, J = 7 Hz, N-CH2), 5.50 (s, 1H, NH).
13C NMR (CDCl3): δ (ppm) 14.09 (C14 + C'13), 22.71 (C13 + C'12), 25.90 (C'2), 26.99 ( C3), 29.69 (-CH2-), 31.96 (C12 + C11), 36.97 (C'1), 39.56 (C1), 160 (CO).

b) Synthesis of ditetradecylamine (2) In a flask equipped with a condenser and a desiccant tube, dissolve 47 mmol of ditetradecylamide (20 g) in 700 ml of anhydrous THF under nitrogen. We the mixture is cooled to 0 ° C. and then 89 mmol of lithium hydride is added aluminum LiAIH4 (3.4 g). After addition, the mixture is then heated to reflux during 5 hours under vigorous agitation. When the reaction is complete, the mixture is cooled to 0 C to perform the hydrolysis by successive addition of 3.4 ml of water, 6.8 ml 2N sodium hydroxide and 3.4 ml of water. After an hour of agitation at room temperature, the crude reaction product is filtered on BuchnerTM and filtrate is concentrated. The product obtained is then purified on 1.5 equivalents of resin (15 g) in 300 ml of THF with stirring for 30 minutes. The resin is filtered to be redissolved in 300 ml of THF with addition of 2 equivalents of triethylamine (19.2m1). After stirring for 30 minutes, the solution is filtered and the filtrate is concentrated to obtain 17.72 g of a white solid (yield: 92%).

1H NMR (CDCl3): δ (ppm) 0.87 (t, 6H, J = 6.5Hz, -CH3), 1.25 (m, 44H, -CII2-), 1.46 (in, 4H, N-CH 2 -CH 2), 2.58 (t, 4H, J = 7 Hz, N-CH 2).

13C NMR (CDCl3): δ (ppm) 14.06 (C14), 22.69 (C13), 27.48 (C3), 28.29 (C2), 29,69 (C4-C1i), 31.96 (C12), 50.15 (C1).

c) Synthesis of N, N-ditetradecylsuccinamic Acid (3) In a flask are added successively in 125 ml of dichloromethane, 12.65 mmol of succinic anhydride (1.266 g), 12.65 nunol of 4-dimethyl-aminopyridine (1.546 g) and 10.75 nunol of ditetradecylamine (4.407 g). The mixed The reaction mixture is stirred for 18 hours at room temperature. The reaction completed extraction is carried out with dichloromethane and hydrochloric acid (1N).
The organic phase is then washed with brine and dried over sodium sulfate.
magnesium, filtered and concentrated to give 4.21 g of product (3) (yield:
66%).

H NMR (CDCl3): δ (ppm) 0.85 (t, 6H, J = 6.3 Hz, -CH3), 1.23 (m, 44H, -CH2-), 1.48 (m, 4H, N-CH 2 -CH 2), 2.64 (s, 4H, CO-CH 2 -CH 2 -CO), 3.22 (br, 4H, N-CH 2).
13C NMR (CDCl3): δ (pprn) 14.08 (C14), 22.69 (C13), 27.74 (C3) 1 28.10 (CO-CH2) CH2-CO), 28.92 (C2), 29.67 (C4-C11), 30.07 (CO-CH2-CH2-CO), 31.95 (C12), 46.21 and 47.98 (C1), 4171.46 (CO-NH (C14H29) 2), 173.14 (NH-CO).

27a d) Synthesis of tert-butyl ester of 2-aminoethylcarbamic acid (4) 18.6 nu-nols of di-tert-butyl dicarbonate (4 g) are added dropwise to 102.83 mmol of ethylene diamine (6.17 g) in solution in chloroform (20 ml) under nitrogen. The reaction mixture is then stirred for 18 hours at temperature i room. Once the reaction is complete, the solution is concentrated. oil resulting dissolved in dichloromethane is washed with a saturated aqueous solution in sodium carbonate. Then the organic phase is dried over sodium sulphate magnesium, filtered and concentrated. The crude product is purified by flash chromatography (dichloromethane / methanol 9: 1). 2.38 g of product are thus obtained (4) (yield:
80%).

1H NMR (CDCl3): δ (ppm) 1.40 (s, 9H, (CH3) 3), 1.52 (s, 2H, NH2), 2.59 (t, 2H, J = 5.9 Hz, N-CH 2), 3.12 (q, 2H, 4J = 5.4 Hz, NHBoc-CH 2), 5.1 (s, 1H, NHBoc) 13C NMR (CDCl3): δ (ppm) 28.15 (CH3) 3, 41.67 (CH2-NHBoc), 43.46 (CH2-NH2), 78.31 (C- (CH 3) 3, 156.21 (C = O).

e) Synthesis of the tert-butyl ester of 2- [3- (diterradecylcarbamoyl) -propionylamino] ethylcarbamic (5) 8.841 nmol of PyBOP (4.601 g), 9.72 mmol of the amine (4) obtained in the previous step (1.558 g) and 24.31 mmol of diisopropylethylamine (4.24 ml) to a solution of 8.84 mmol of the acid (3) got previously (4.5 g) in 88 ml of dichloromethane. The solution is then agitated for 4 hours at room temperature. At the end of the reaction, the mixture reaction is filtered and the product is purified by flash chromatography (Heptane / ethyl acetate of ethyl 5: 5 then heptane / ethyl acetate 2: 8). 3.79 g of the ester (5) (yield: 66%).

1H NMR (CDCl3): δ (ppm) 0.87 (t, 6H, J = 6.6Hz, -CH3), 1.25 (m, 44H, -CH2-), 1.43 (s, 9H, (CH 3) 3) 1.48 (m, 4H, N-CH 2 -CH 2), 2.56 (t, 2H, J = 6.7 Hz, CH 2 O);
2.69 (t, 2H, J = 6.2 Hz, CH24), 3.28 (m, 4H, N-CH2), 3.3 (m, 4H, CH21 + CH2).
13C NMR (CDCl3): δ (ppm) 14.07 (C14), 22.56 (C13), 26.99 (CH3) 3, 27.73.
(C-3), 28.29 (C'2), 28.59 (C-2), 29.27 (C-4-C-11), 29.55 (C-3), 31.41 (C C "12) 39.85 (C2), 40.39 (Ci), 46.21 and 47.98 (C-1), 78.77 (C (IV) -Boc), 156.33 (CO-Boc), 171.46 (CO-NH (C14H29) 2), 173.14 (C'1).

f) Synthesis of 2- [3- (diterradecylcarbamoyl) propionylamino] ethylamine (6) To 2.44 mmol of the ester (5) obtained in the previous step (1.59 g) is added 12.2 mmol of distilled TFA (0.94 ml). After two hours, the reaction is total.
The product is co-evaporated twice with cyclohexane on a rotary evaporator.
cold.
The yield is quantitative.

1 H NMR (CDCl3): δ (ppm) 0.91 (t, 6H, J = 6.6Hz, -CH3), 1.29 (m, 44H, -CH2-), 1.51 (m, 4H, N-CH 2 -CH 2), 2.59 (t, 2H, J = 6.7 Hz, H 3), 2.71 (t, 2H, J = 6.2 Hz, H'3) 3.29 (m, 4H, N-CH 2), 3.31 (m, 4H, H 1, H 2).
13C NMR (CDCl3): δ (ppm) 14.00 (C-14), 22.67 (C-13), 27.35 (C-3), 27.95.
(C'2), 28.53 (C2), 29.65 (C-4-C-11), 30.70 (C-3), 31.94 (C-12), 37.83 (C2), 40.08.
(C2), 47.85 and 49.42 (C-1), 171.72 (CO-NH (C14H29) 2), 173.26 (C'1).

g) Synthesis of ((1,1-dimethylethoxycarbonyl) amino) ethylisothiocyanate (7) In a flask, 43.69 minol of DCC (9.015 g) are successively added, 297.9 mmol of carbon disulfide (17.9 ml) in 27.5 ml of THF. The mixture is cooled to -7 C with a bath of ice and ammonium chloride NH4Cl (4/1).
We the previously obtained amine (4) (43.69 mmol) is then added dropwise boy Wut) dissolved in 20.5 ml of anhydrous THF for 30 minutes. We leave the mixture return to room temperature and stir for 21 hours. After evaporation, diethyl ether is added to precipitate the dicyclohexylurea formed. The solution is filtered, the filtrate is concentrated and then purified by chromatography flash (heptane / ethyl acetate 80:20) to give 6.357 g of product (7) wish (yield: 72%).

1H NMR (CDCl3): δ (ppm) 1.38 (s, 9H, (CH3) 3), 3.31 (q, 2H, 4J = 5.8 Hz, NHBoc-CH2), 3.59 (t, 2H, J = 5.6 Hz, S = C N-CH2), 5.16 (s, 1H, NHBoc) 13C NMR (CDCl3): δ (ppm) 28.54 ((CH3) 3), 41.03 (CH2-NHBoc), 45.53 (CH2-N = C = S), 79.71 (C- (CH3) 3, 132.72 (C = S), 156.21 (C = O).

h) Synthesis of the tert-butyl ester of 2- (3- ~ 2- [3- (diterradecyl) carbamoyl) propionylamino] ethyl 4hioureido) ethyl carbamic acid (8) To 2.44 mmol of the amine (6) previously obtained (1.62 g) is added directly 9.76 mmol triethylamine (1.36 ml) and allowed to shake for minutes. 24.4 ml of dichloromethane and 2.92 mmol of the isothiocyanate (7) obtained in the previous step (0.59 g). The mixture reaction is Stirred at room temperature for 12 hours. The mixture is then evaporated then purified by flash chromatography (ethyl acetate / heptane 6: 4 and then 100%
acetate ethyl). 1.343 g of the desired ester (8) are thus obtained (yield: 73 %).

1H NMR (CDCl3): δ (ppm) 0.67 (t, 6H, J = 6.4Hz, -CH3), 1.05 (m, 44H, -CH2-), 1.26 (s, 9H, CH3) 3), 1.35 (m, 4H, N-CH2-CH2), 2.31 (m, 2H, H3'3), 2.49 (m, 2H, H3'3), 3.06 (m, 4H, N-CH2), 3.11 (m, 4H, H1, H2), 3.47 (m, 4H, H2, H-1), 7.14 (2H, H thiourea).
13C NMR (CDCl3): δ (ppm) 13.95 (C414), 22.57 (C4-13), 26.92 ((CH3) 3), 27.07 (4'C3), 27.78 (C3'2), 28.39 (C4'2), 28.82 (C3'3), 29.55 (C4'4-C4 '11), 31.83 (C4 '12), 39.45 (C-2), 43.63 (C2 and C-1), 46.39 and 48.16 (C4'1), 79.24 (C (IV) -Boc), 156.53 (CO-Boc), 171.72 (CO-NH (C14H29) 2), 173.71 (C3'1), 182.97 (C = S).

i) Synthesis of 2- (3- [2- [3- (diterradecylcarbamoyl) propionylamino] ethyl ~ -thioureido) ethyl amine (9) 1.98 mmol of the product (8) obtained in the previous step (1.5 g) is added 9.86 mmol of distilled TFA (0.76 ml). After 3 hours, the reaction is total. The The product is co-evaporated twice with cyclohexane on a rotary evaporator.
cold. The yield is quantitative.

1H NMR (CDCl3): δ (ppm) 0.67 (t, 6H, J = 6.4Hz, -CH3), 1.05 (m, 44H, -CH2-), 1.26 (s, 9H, CH3) 3), 1.31 (m, 4H, N-CH2-CH2), 2.31 (m, 2H, H3'3), 2.49 (m, 2H, H3'3), 3.06 (m, 4H, N-CH2), 3.11 (m, 4H, H1, H-2), 3.44 (m, 4H, H2, H-1), 7.10, (2H, thiourea).

13C NMR (CDCl3): δ (ppm) 13.85 (C4'14), 22.49 (C4'13), 27.01 (C4'3), 27.72 (C3'2) 28.29 (C4'2), 28.79 (C3'3), 29.49 (C4'4-C4'11), 31.77 (C4'12), 39.42 (C-2), 43.75 (C2 and C "1), 46.29 and 48.09 (C4 '1), 171.72 (CO-NH (C14H29) 2), 173.71 (C3' 1), 182.97 (C = S).

j) Synthesis of the tert-butyl ester of 2- ~ 3- [2- (3- ~ 2- [3-(ditétradécyl-carbamoyl) propionylamino] ethyl ~ thioureido) ethyl] thioureido Whyl carbamic acid (10) To 1.98 inmol of the amine (9) obtained in the previous step (1.47 g) is added directly 7.92 mmol of triethylamine (1.1 ml) and allowed to shake for minutes. 19.8 ml of dichloromethane and 2.38 mmol of the previously obtained isothiocyanate (7) (0.461 g) and allowed to react to temperature stirring for 12 hours. The mixture is then evaporated and purified by flash chromatography (ethyl acetate / heptane 6: 4 then acetate ethyl / methanol 98: 2). 1.136 g of the desired product (10) is obtained (yield:
67%).

1H NMR (CDCl3): δ (ppm) 0.74 (t, 6H, J = 6.6Hz, -CH3), 1.12 (m, 44H, -CH2-) 1 1.30 (s, 9H, CH3) 3), 1.45 (m, 4H, N-CH2-CH2), 2.41 (m, 2H, H5'2), 2.58 (m, 2H, H5'2), 3.12 (m, 4H, N-CH2), 3.25 (m, 4H, H1, H4'2), 3.56 (m, 8H, H2, H "1, H" 2) , H4 '1), 7.14 (4H, thiourea).

13C NMR (CDCl3): δ (ppm) 14.06 (C6'14), 22.57 (C6'13), 27.11 (C6'3), 26.93 ((CH3) 3), 27.79 (C5'2), 28.38 (C6'2), 28.81 (C5'3), 29.56 (C6'4-C6'11), 31.83 (C6'12), 39.55 (C4'2), 43.66 (C2, C-1, C-2, C4'1), 46.49 and 48.23 (C6'1), 79.28 (C (IV) -Boc), 156.61 (CO-Boc), 171.96 (CO-NH (C14H29) 2), 173.72 (C5 '1), 182.93 (C = S).

k) Synthesis of 2- [3- [2- (3- [2- (3- (diterradecylcarbamoyl) propionylamino] -ethyl ~ thioureido) ethyl] thioureidoethylamine (11) To 1.15 mmol of the product (10) obtained in the previous step (1 g) is added 5.84 mmol of distilled TFA (0.45 ml). After 3 hours, the reaction is total. The The product is co-evaporated twice with cyclohexane on a rotary evaporator.
cold. The yield is quantitative.

1H NMR (CDCl3): δ (ppm) 0.85 (t, 6H, J = 6.6Hz, -CH3), 1.25 (m, 40H, -CH2-), 1.48 (m, 4H, N-CH2-CH2), 1.52 (m, 4H, N-CH2-CH2-CH2), 2.65 (m, 2H, H5'2), 2.77 (m, 2H, H5'3), 3.26 (m, 4H, N-CH2), 3.43 (m, 4H, H1, H4'2), 3.85 (m, 8H, H2, H "1, H₂, H4₁), 7.44 (4H, thiourea).

13C NMR (CDCl3): b (ppm) 14.05 (C6 14), 22.68 (C6'13), 27.05 (C6'3), 27.58 (C5'2) 28.71 (C6'2), 29.36 (C5'3), 29.67 (C6 4-C6'11), 31.94 (C6'12), 40.49 (C5'1), 43.49 (C has of C = S), 47.40 and 49.07 (C6'1), 172.16 (CO-NH (C14H29) 2), 174.00 (NH-CO), 182.73 (C = S).

1) Synthesis of the DT-3TU (12) 0.28 mmol of the amine (11) obtained in the previous step (0.24 g) is added directly 1.12 mmol of triethylamine (0.16 ml) and allowed to shake for minutes. 2.8 ml of dichloromethane and 0.34 mmol of methylisothiocyanate (0.024 g) and allowed to react at room temperature under stirring for 12 hours. The mixture is then evaporated and then purified by HPLC
(High Performance Liquid Chromatography) on a C4 column with gradient following: initially a mixture water / methanol 95: 5 until 100% of methanol. The obtained product is again purified on a small column of silica (acetate 80:20 ethyl / heptane and then 100% ethyl acetate). We thus obtain 118 mg of DTTU (yield: 51%).

1H NMR (CDCl3): δ (ppm) 0.86 (t, 6H, J = 6.7Hz, -CH3), 1.24 (m, 40H, -CH2-), 1.44 (m, 4H, N-CH 2 -CH 2), 1.54 (m, 4H, N-CH 2 -CH 2 -CH 2), 2.52 (m, 2H, H 6 '2), 2.67 (m, 2H, H6'2), 3.05 (m, 3H, -CH3 terminal), 3.21 (m, 4H, N-CH2), 3.32 (m, 2H, H5'2) 3.75 (m, 10H, H '1, H 2, H 3' 1, H 3 '2, H 5' 1), 7.14 (6H, thiourea H).

13C NMR (CDCl3): δ (ppm) 14.09 (C7'14), 22.69 (C7'13), 27.24 (C7'3), 27.89 (C6'2) 28.87 (C7'2), 29.38 (C6'3), 29.67 (C7'4-C7'11), 31.26 (terminal CH3), 31.94 (C7'12) 39.71 (C5'2), 43.63 (C'1, C'2, C3'1, C3'2, C-1), 46.67 and 48.40 (C7'1), 172.16 (CO-NH (C14H29) 2), 174.00 (C6'1), 182.73 (C = S).

EXAMPLE 2 Synthesis of the DT-4TU
DT-4TU or 3- (2- {3- [2- (3- {2- [3- (2- {3- [diterradecylcarbamoyl]]
propionylamino} -ethyl) -thioureido] -ethyl} -thioureido) -ethyl] -thioureido } -ethyl) -1-methylthiourea. The DT-4TU is according to the general formula (I), in which:

X = -CH3; m = 2; R = H; n = 4; -8 = 0; Y = NH-CO-CH2-CH2; and L = -N (RI) R2 where Ri =
R2 = C14H29 =
To synthesize the DT-4TU, the same will be done from the amine (11).
a) Synthesis of the t-butyl ester of 2 {3- {2- [3 e - {3- [2- (3-(ditétradécyl-carbamoyl) propionylamino) -ethyl] thioureido} -ethyl) -thioureido] -ethyl}
thioureido) -ethyl carbamic (13) sso , Al / N \ j4 ^ 3 3 / I {1 '\ N
\ 1 /% 4 ~~} IN 1 ~ N 1 N, - N
17 II 15 17 I 71 II 9 rl T rl 5 II -0 s 0 To the amine (11) (0.8 g, 92 mmol) was added triethylamine (0.643 ml, 4.6 mmol) and allowed to stir for 15 minutes. Then CH2Cl2 (9.2 mL) is added. We then add the isothiocyanate (7) (0.224 g, 1.104 mmol) and allow react to stirring for 20h. The mixture is then evaporated and then purified by chromatography (ethyl acetate / heptane 8: 2 then ethyl acetate / methanol 90: 10); 252 mg of the desired product are obtained (yield: 46%).

1H NMR (CDCl3): δ (ppm) 0.86 (t, 6H, J = 6Hz, H-14 '), 1.25 (m, 44H, H-4'-H-11 '), 1.42 (s, 9H, CH3) 3), 1.45 (m, 4H, H-2'), 2.55 (m, 2H, H-2), 2.69 (m, 2H, H-3), 3.22 (m, 4H, H-1 '), 3.31 (1n, 4H, H-5 and H-15), 3.74 (m, 12H, H-6, H-8, H-9). , H-H-12, H-14), 7.31 (6H, thiourea).
13C NMR (CDCl3): δ (ppm) 14.06 (C-14 '), 22.66 (C-13'), 27.21 (C-3 '), 27.88 (C-2) 28.50 ((CH 3) 3), 28.87 (C-2 '), 29.64 (C-4'-C-11'), 31.27 (C-3), 31.92 (C 12 '), 39.639 (C-5), 40.44 (C-13), 43.73 (C-6, C-8, C-9, C-11, C-12, C-14), 46, 64 and 48, 38 (C-l '), 79.10 (C-17), 156.63 (C-16), 172.35 (C-1), 174.04 (C-4), 182.65 (C-7, C-10, C-13).

b) Synthesis of 2- (3- {2- [3- (2- {3- [2- (3- (diterradecyl-carbamoyl)) propionylamino) ethyl] -thioureido} ethyl) -thioureido] ethyl} -thioureido) -ethyl amine (14) s H Fi s O
CF3000- H, N '' ~ HH 1119'N ~~ HH 6 N 4 az r 'N
s O

To the amine boc (13) obtained in the previous step (0.22 g, 0.23 mmol) is added distilled TFA (0.142 ml, 1.84 mmol). After 6 hours, the reaction is total. The The product is co-evaporated twice with cyclohexane on a rotary evaporator.
cold and put in dessicator on soda tablets for the night. The yield is quantitative.

1H NMR (CDCl3): δ (ppm) 0.74 (t, 6H, J = 6.6Hz, H-14 '), 1.12 (m, 44H, H-4'-H).
11 '), 1.45 (m, 4H, H-2'), 2.41 (m, 2H, H-2), 2.58 (m, 2H, H-3), 3.12 (m, 4H, H-l '), 3.25 (m, 4H, H-5 and H-15), 3.56 (m, 12H, H-6, H-8, H-9, H-11, H-12, H-14), 7.14 (6H, thiourea H).
13C NMR (CDCl3): δ (ppm) 14.06 (C-14 '), 22.57 (C-13'), 27.11 (C-3 '), 27.79 (C-2) 28.38 (C-2 '), 28.81 (C-4'-C-1), 29.56 (C-3), 31.83 (C-12'), 39.55 (C), -5, C-9), 43.66 (C-6, C-8, C-9, C-11, C-12, C-14), 46.49 and 48.23 (C-1 '), 171.96 (C-1), 173 72 (C-4), 182.93 (C-7, C-10, C-13).

c) Synthesis of 3- (2- {3- [2- (3- {2- [3- (2- {3- [ditertradecylcarbamoyl]) propionylamino} ethyl) -thioureido] ethyl} -thioureido) ethyl]} -thioureido -ethyl) -1-methylthiourea (DT-4TU) (15) n '14' 1ZN 10 5 N 4 3 17 15 HH 11 s HH 5 z ss O
To 0.23 mmol of the amine (14) obtained in the previous step (0.224 g) is added directly 1.38 mmol triethylamine (0.19 ml) and allowed to shake for minutes. 2.3 ml of dichloromethane and 0.46 mmol of methylisothiocyanate (0.034 g) and allowed to react at room temperature under stirring for 12 hours. The mixture is then evaporated and then purified by flash chromatography (100% ethyl acetate and then ethyl acetate / methanol 95: 5).
This gives 109 mg of DT4TU (yield: 51%).

1H NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.3Hz, H-14 '), 1.26 (m, 44H, H-4'-H-11 '), 1.45 1.45 (m, 4H, H-2'), 1.58 (1n, 4H, H-3 '), 2.57 (m, 2H, H-2), 2, 73 (m, 2H, H-3), 3.03 (m, 3H, H-17), 3.23 (1n, 4H, H-1 '), 3.31 (m, 2H, H-5), 3.73 (m, 14H). , H-6, H-8, H-9, H-11, H-12, H-14, H-15), 7.14 (8H, thiourea).
13C NMR (CDCl3): b (ppm) 14.09 (C-14 '), 22.69 (C-13'), 27.24 (C-3 '), 27.89 (C-2), 28.87 (C-2 '), 29.38 (C-3), 29.67 (C-4'-C-11'), 31.26 (C-17), 31.94 (C-12) '), 39.71 (C-5), 43.63 (C-6, C-8, C-9, C-11, C-12, C-14, C-15), 46.67 and 48.40 (C-1) '), 172.16 (C-1), 174.00 (C-4), 182.73 (C-7, C-10, C-13, C-16).

EXAMPLE 3 Synthesis of DT-2TU Diol The compound 2- (3- ~ 2- [3- (diterradecylcarbaloyl) propionylamino] ethyl thioureido) ethyl] propan-1,2-diol or DT-2TU corresponds to the general formula (I) in which x = HO OH

M = 2;
R = H;
n = 2;
1 = 0;
Y = NH-CO-CH2-CH2; and L = -N (R1) R2 where R1 = R2 = C14H29.

a) Synthesis of 4-isothiocyanatomethyl-2,2-dimethyl- [1,3] dioxalane (16) H_ N - S
'3 H

o ~

In a flask is added successively the DCC (3.146 g, 15.25 mmol), the disulfide of carbon (6.253 mL, 104.005 mmol) in THF (9.6075 mL). The mixture is Cool to -7 C with ice / NH4Cl (4/1). Then add the 2,2-dimethyl-1,2-dioxalane-4-methanamine (2 g, 15.25 mmol) dissolved in THF
anhydrous (7.1675 mL) dropwise for 30 minutes. We leave the mixture return to room temperature and let stir for 21H. After evaporation, one add diethyl ether. We filter, we evaporate and the mixture is chromatography.
1H NMR (CDCl3): δ (ppm) 1.33 and 1.44 (s, 3H, H-5, H-6), 3.57 (dd, 1H, J = 4.8 Hz, J = 14.4 Hz, H-1), 3.69 (dd, 1H, J = 4.9 Hz, J = 14.4 Hz, H-1 '), 3.80 (dd, 1H, J = 5.4 Hz and J = 8.7 Hz, H-3), 4.09 (dd, 1H, J = 6.3 Hz, and J = 8.7 Hz, H-3), 4.28 (m, 1H, H-2).

13C NMR (CDCl3): δ (ppm) 25.17, 26.77 (C-5, C-6), 47.49 (C-1), 66.55 (C-3), 73.70 (C-2), 110.29 (C-4), 132.76 (N = C = S).

b) Synthesis of 2- (3- ~ 2- [3- (diterradecylcarbamoyl) propionylamino]
ethyl-thioureido) ethyl-4-ylmethyl-2,2-dimethyl- [1,3] dioxalane (17) O
13 11 HuH
NN ~ 6 \ 6 3 2 O 1Z II NN ^ 5 NN
1np s HH 4p To 0.19 mmol of the amine (9) obtained in the previous step (0.146 g) is added directly 0.95 mmol of diisopropylethylamine (0.165 ml) and allowed to shake for 15 minutes. 1.9 ml of dichloromethane and 0.209 are then added.
mmol isothiocyanate (16) (0.027 g) and allowed to react at room temperature under stirring for 12 hours. The mixture is then evaporated and then purified on C8 reverse phase cartridge with a 100% water gradient up 100% acetonitrile.
The product (17) is obtained with a yield of 49%.

11H NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.3Hz, H-14 '), 1.25 (m, 44H, H-4'-H) 11 '), 1.34 and 1.43 (s, 3H, H-15, H-16), 1.48 (m, 4H, H-2'), 1.56 (m, 4H, H-3 '), 2.52 (m, 2H, H-2), 2.68 (m, 2H, H-3), 3.23 (m, 4H, H-1 '), 3.37 (m, 2H, H-5), 3.73 (m, 8H, H-6, H-8, H-9, H-11), 4.05 (m, 2H, H-13), 4.33 (m, 1H, H-12), 7.14 (4H, H).
thiourea).
13C NMR (CDCl3): δ (ppm) 14.06 (C-14 '), 22.67 (C-13'), 25.32 (C-3 '), 27.00 and 27.17 (C-15, C-16), 27.80 (C-2), 28.56 (C-2 '), 28.84 (C-3), 29.66 (C-4'), VS-11 '), 31.25 (C-17), 31.92 (C-12 '), 39.78 (C-5), 43.66 (C-6, C-8, C-9), 44.53 (C -11) 46.73 and 47.13 (C-1 '), 66.87 (C-136), 74.62 (C-12), 109.05 (C-14), 172.21 (C-1), 174.14 (C-4), 183.32 (C-7, C-10, C-13, C-16).

c) Synthesis of [2- (3- [2- [3- (diterradecylcarbamoyl) propionylamino] ethyl -thioureido) ethyl] propan-1,2-diol (DT-2TUdiol) (18) H s O
; s ~ z il N`1o / N --- '-NN ^ 5 N 4 3 z N
HO OH s HHO

The protected diol (17) (0.05 g, 0.5 mmol) is dissolved in 1 mL of HCl solution 1N / THF (1/1) at t; the mixture is stirred for 18h. the crude is then extracted with with dichloromethane (2 x 5 ml). The organic phases are collected then neutralized with sodium hydrogen carbonate solution. The phases aqueous are extracted with dichloromethane. The organic phases are collected then dried over magnesium sulphate and then evaporated. The crude is chromatographed sure reverse phase C8 with a 100% water gradient up to 100% acetonitrile. The product (18) is obtained with a yield of 49%.

1111 NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.3Hz, H-14 '), 1.26 (m, 44H, H-4'-H) 11 '), 1.43 (m, 4H, H-2'), 1.59 (m, 4H, H-3 '), 1.79 (s, 2H, OH), 2.50 (m, 2H); , H-2) 2.70 (m, 2H, H-3), 3.24 (m, 4H, H-1 '), 3.39 (m, 2H, H-5), 3.72 (m, 6H, H- 6, H-8, H-9), 3.9 (m, 2H, H-11), 4.22 (m, 2H, H-13), 4.58 (m, 1H, H-12), 7.14 (4H, H);
thiourea).
13C NMR (CDCl3): b (ppm) 14.06 (C-14 '), 22.67 (C-13'), 25.32 (C-3 '), 27.80 (C-2) 28.56 (C-2 '), 28.84 (C-3), 29.66 (C-4'-C-11'), 31.25 (C-17), 31.92 (C-12) '), 39.78 (C-5), 43.66 (C-6, C-8, C-9), 45.86 (C-11), 46.73 and 47.13 (C-1 '), 63.54 ( VS-13), 70.97 (C-12), 172.21 (C-1), 174.14 (C-4), 183.32 (C-7, C-10).

EXAMPLE 4 Synthesis of DT-3TU Diol The compound 2- (3- {2- [3 {2- {3- [2 {3 (difechadecyl-carbamoyl) propionylamino) -ethyl]
thioureido} -ethyl) thioui ido] -ethyl} thioureido) -ethyl] -propane-1,2-diol or DT-3TU diol answer to the formula (I) in which x = ~ - <-HO OH
m = 2;
HR;
n-- 3;
$ = 0;
Y = NH-CO-CH2-CH2; and L = -N (RI) R2 where R1 = R2 = C14H29, To synthesize the DT-3TU diol, the same will be done from the amine (11).

a) Synthesis of 2 (3- {2- [3 {2- {3- [2- (3- (diterradecyl) carbamoyl) propionylamino) -ethyl] -thioureido} -ethyl} thioureido] -ethyl} thioureido) -ethyl-4-ylmethyl-2,2-dimethyl [1,3] dioxalane (19) s H 0 NN 1 ~ to N ~~~ NN ^ / N aa 1 N
H0H HHH sz 0 s 190 To 0.19 mmol of the amine (11) obtained in the previous step (0.165 g) is added directly 0.95 mmol of diisopropylethylamine (0.165 ml) and allowed to shake for 15 minutes. 1.9 ml of dichloromethane and 0.209 are then added.
mmol isothiocyanate (16) (0.027 g) and allowed to react at room temperature under stirring for 12 hours. The mixture is then evaporated and then purified on C8 reverse phase cartridge with a 100% water gradient up to 1 00% acetonitrile.
The product (19) is obtained with a yield of 49%.

1H NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.3Hz, H-14 '), 1.25 (m, 44H, H-4'-H-11 '), 1.34 and 1.43 (s, 3H, H-18, H-19), 1.48 (m, 4H, H-2'), 1.56 (m, 4H, H-3 '), 2.52 (m, 2H, H-2), 2.68 (m, 2H, H-3), 3.23 (in, 4H, H-1 '), 3.37 (m, 2H, H-5), 3.73 (M, 12H, H-6, H-8, H-9, H-11, H-12, H-14), 4.05 (m, 2H, H-16), 4.33 (m, 1H, H- 15) 7.14 (6H, thiourea).
13C NMR (CDCl3): δ (ppm) 14.06 (C-14 '), 22.67 (C-13'), 25.32 (C-3 '), 27.00 and 27.17 (C-18, C-19), 27.80 (C-2), 28.56 (C-2 '), 28.84 (C-3), 29.66 (C-4'), VS-1l), 31.25 (C-17), 31.92 (C-12 '), 39.78 (C-5), 43.66 (C-6, C-8, C-9, C-11), 44 53 (C-14), 46.73 and 47.13 (C-1 '), 66.87 (C-16), 74.62 (C-15), 109.05 (C-17), 172.21 (C-1), 174.14 (C-4), 183.32 (C-7, C-10, C-13, C-16).
b) Synthesis of 2 (3- {2- [3- (2- {3- [2- (3 (ditertraderyl) trbamoyl) propionylamino) -ethyl] -thioureido} -ethyl) -thioureido] -ethyl} -thioureido) -ethyl] -propane-1,2 diol (DT-3Tudiol) (20) sso 12 HH e II H6 H 3 jj 1 ~
em N 9 HH s 2 H
Ho The protected diol (19) (0.05 g, 0.5 mmol) is dissolved in 1 ml of solution HCl 1N / THF (1/1) at rt; the mixture is stirred for 18h. the crude is then extract with dichloromethane (2x5 ml). The organic phases are collected then neutralized with sodium hydrogen carbonate solution. The phases aqueous are extracted with dichloromethane. The organic phases are collected then dried over magnesium sulphate and then evaporated. The crude is chromatographed sure reverse phase C8 with a 100% water gradient up to 100% acetonitrile. The product (20) is obtained with a yield of 55%.

1H NMR (CDCl3): δ (ppm) 0.88 (t, 6H, J = 6.3Hz, H-14 '), 1.26 (m, 44H, H-4'-H-11 '), 1.43 (m, 4H, H-2'), 1.59 (m, 4H, H-3 '), 1.79 (s, 2H, OH), 2.50 (m, 2H); , H-2) 2.70 (m, 2H, H-3), 3.24 (m, 4H, H-1 '), 3.39 (m, 2H, H-5), 3.72 (m, 1H, 6, H-H-9, H-11, H-12), 3.9 (m, 2H, H-14), 4.22 (m, 2H, H-16), 4.58 (m, 1H, H-15). ) 7.14 (6H, thiourea H).
13C NMR (CDCl3): δ (ppm) 14.06 (C-14 '), 22.67 (C-13'), 25.32 (C-3 '), 27.80 (C
2) 28.56 (C-2 '), 28.84 (C-3), 29.66 (C-4'-C-1'), 31.25 (C-17), 31.92 (C); -12 '), 39.78 (C-5), 43.66 (C-6, C-8, C-9, C-11), 44.53 (C-14), 46.73 and 47.13 (C-1 '), 63.54 (C-16), 70.97 (C-15), 109.05 (C-17), 172.21 (C-1), 174.14 (C-4), 183.32 (C-7, C-10, C-C-16).

EXAMPLE 5 Compaction of the nucleic acid in the presence of DT-3TU (12) The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to associate with the nucleic acids.

This can easily be demonstrated by a bromide fluorescence test of ethidium: the absence of fluorescence testifies to the absence of nucleic acid free Which means that the nucleic acid is compacted by the compound transfectant.

The nucleic acid is brought into the presence of increasing amounts of DT-3TU
(12), by equivolumetric mixing of lipid solutions of different titles in nucleic acid solutions. 800 l samples are thus prepared.
of nucleic acid complexes of concentration 0.01 g / ml in a solution of 150 mM sodium chloride with increasing amounts of DT-3TU (12).

In the same way, a control was carried out by putting the nucleic acid into presence increasing amounts of EPC (see Figure 1) or DPPC (see Figure 2), by equivolumetric mixture of lipid solutions of different nucleic acid solutions. 800 l samples of complex Nucleic acid concentration of 0.01 g / ml in a chloride solution of 150 mM sodium with increasing amounts of EPC or DPPC (FIGS.

respectively).

Ethidium bromide fluorescence is measured over time (measured at C) using a FluoroMax-2 (Jobin Yvon-Spex), with wavelengths excitation and emission of 260 nm and 590 nm respectively. The widths of Slots for excitation and emission are set at 5 nm. The value of fluorescence is recorded after adding 3 g of ethidium bromide to 1 g / l per ml of solution DNA / lipid (at 0.01 mg DNA / ml).

The results are summarized in Figures 1 and 2.

In Figure 1, the curve including squares shows that the addition of a increasing amount of DT-3TU / EPC lipid mixture (0.75 to 20 nmol 3TU) relative to a fixed amount of nucleic acid (8 g) induces a decrease of fluorescence linked to the reduction of the insertion of ethidium bromide between the base pairs of DNA. This indicates that the association between DT liposomes 3TU / EPC and the DNA is strong enough to exclude ethidium bromide from complex. We were able to obtain up to 90% exclusion from fluorescence or 90% DNA-lipid association DT-3TU / EPC. In order to show the active role of lipid DT-3TU in this lipid / DNA combination, a control was performed.
he to observe the interaction between EPC lipid and DNA, this is represented by the curve containing diamonds. When the EPC is brought into contact with DNA
under conditions identical to those used for the study of the complexes DT
3TU / EPC-DNA, only a slight decrease in fluorescence is observed (approximately 5%), which can be attributed to the increased turbidity of the mixture. This control therefore reflects the lack of association of EPC alone with DNA in terms experimental studies.

This example thus illustrates the ability of lipid DT-3TU to associate with the nucleic acid.

In the same way, in Figure 2, the curve including squares shows that the addition of an increasing amount of DT-3TU / DPPC lipid mixture (0.75 to nmoles of DT-3TU) relative to a fixed amount of nucleic acid (8 g) induced a decrease in fluorescence when an identical amount of bromide ethidium is added to the different samples. This indicates that the association between the DT-3TU / DPPC liposomes and the DNA is strong enough to exclude the ethidium bromide complexes. We were able to obtain up to 90%
fluorescence exclusion is 90% DNA-DT-3TU / DPPC association. In order to show the active role of lipid DT-3TU in this lipid / DNA combination, a control has been achieved. It consists in observing the interaction between the lipid DPPC and DNA, this is represented by the curve containing lozenges. When the DPPC is brought into contact with DNA under conditions identical to those used for the study of the DT-3TU / DPPC-DNA complexes, only a slight decrease in fluorescence is observed (about 5%). This control therefore reflects the absence association of DPPC alone with DNA under experimental conditions pre-cited.

This example illustrates the ability of lipid DT-3TU to associate with the nucleic acid.

EXAMPLE 6 Compaction of DNA with DT-3TU / EPC Complexes The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to compact the nucleic acids.

This can easily be demonstrated by an electrophoretic delay test on DNA agarose gel evidenced by use of ethidium bromide (BET): the absence of migration of the nucleic acid on the gel testifies to the compaction of the nucleic acid. Free nucleic acid meanwhile is not subject to a delay on gel.

On an agarose gel (0.8% agarose in 1N TBE) were deposited different DNA / DT-3TU samples with increasing amounts of lipid DT-3TU compared to DNA. The gel was subjected to an electric current of one hour and half at 70V and 70 mA to migrate the DNA by electrophoresis. The bands have been revealed with BET and by absorption under a UV lamp.
results are represented in Figure 3.

The gel shows the electrophoretic migration of DNA when it is not associated with lipids (well 1), then its retention difference when it is associated with lipids. Wells 2 to 6 represent the DNA (0.01 g / l) associated with quantities increasing liposomes DT-3TU / EPC: 0.75 then 5 then 10 then 15 and finally 20 lipid ninoles DT-3TU. The comparison between well 1 and other wells indicated that the more one increases the amount of lipid DT-3TU, the more the DNA is retained on gel to be totally delayed from 3 nmol of DT-3TU / g of DNA, zone aggregation of complexes. Wells 8 to 13 correspond respectively to DNA
alone (0.1 g / l, 1 g for the gel), the lipoplexes formed at the concentration of 0.1 g / 1 of DNA with lipid / DNA ratios: 0.75 or 5 or 10 or 15 and finally 20 nmol / g DNA. In the same way, we can observe that at this concentration of DNA
compatible with in vivo experiments, the DNA is compacted from the report 5 nmol lipid / g of DNA.

This example thus illustrates the capacity of the lipid DT-3TU to be compacted the nucleic acid.

EXAMPLE 7 Measurement of the Zeta Potential of the DT-3TU / DNA Compositions The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to compact the nucleic acids while maintaining a structure globally anionic, neutral, or very weakly cationic.

This can be demonstrated by a measure of Zeta potential: the given measure in mV testifies to the surface charge of the particle relative to mobility electrophoretic sample.

The nucleic acid is brought into the presence of increasing amounts of mixture DT-3TU / EPC lipid by equivolumetric mixing of lipid solutions of different titers in nucleic acid solutions. We thus prepare samples of 2 ml of nucleic acid complexes of concentration 0.01g / l in tune solution of 20 mlvi sodium chloride with increasing amounts of DT-3TU.
Zeta potential measurement (mV) is performed using a zetasizerTM 3000 hsa (Malvern). The value of the potential is carried out 3 times in succession on 2 ml sample DT-3TU / EPC-DNA. The results are summarized in Figure 4.
DT-3TU / EPC liposomes are added to the DNA in an area ranging from 0.75 to 20 μmol of lipids per g of DNA. In this zone of variation of the amount of lipid, the Zeta potential varies from -35 mV to +15 mV. The part negative corresponds to what is shown in Figures 1, 2 and 3, namely that the Zeta potential is negative when the DNA is not fully compacted. More we add lipid, the more the DNA is compacted and the more the Zeta potential approaches zero, the lipoplexes then have a virtually zero surface potential. The Zeta potential bECOMES
then slightly positive to 8 nunoles of lipid / g of DNA. The relativity of this measurement must take into account the comparison of the different samples during a same experience. It is therefore important to note the evolution of the Zeta potential in function of increasing the amount of lipid up to a value weakly positive.

This example thus confirms the compaction of the DNA by the compounds transfectants according to the present invention, in particular the DT-3TU, and shows that the formed lipoplexes have a surface potential close to neutrality.

EXAMPLE 8 In Vitro Transfection of DT-3TU / DNA Compositions The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to transfect cells in vitro.
This study was conducted for lipoplexes comprising different amounts DT-3TU: 1.5 or 5 or 10 or 15 or 20 μl of DT-3TU / g of DNA. Each of these conditions was tested with and without fetal calf serum (+ Serum or -Serum) Cell culture: HeLa cells (American type Culture Collection (ATCC) Rockville, MD, USA) derived from cervical epithelial carcinoma human, are cultured in the presence of a MEM type medium (minimum essential meditun) with addition of 2 mM L-glutamine, 50 units / ml of penicillin, and 50 units / ml of streptomycin. Medium and additives derived from Gibco / BRL Life Technologies (Gaithersburg, MD, USA). The cells are cultivated in flasks at 37 C and 5% carbon dioxide in incubator.

Transfection: one day before transfection, HeLa cells are transferred in 24-well plates with tm number of cells from 30000 to 50000 per well. These Dilutions represent approximately 80% confluency after 24 hours.
For transfection, the cells are washed twice and incubated at 37 ° C.
with 500 l medium with serum (10% FCS v / v) or serum free.
50 l of complexes containing 0.5 g of plastidic DNA are added in each wells (the complexes are prepared at least 30 minutes before adding in well).
After two hours at 37 [deg.] C. the plates without serum are supplemented with 10%
(V / v) of SVF (Fetal Calf Serum).
All the plates are placed 24 hours at 37 ° C. and 5% of carbon.
Determination of luciferase activity: Briefly, transfected cells are washed twice with 500 μl of PBS (phosphate buffer) and then lysed with 250 μl reagent (PromegaTM cell culture lysis reagent, Luciferase kit Assay System).
A 10 μl aliquot of the centrifuged lysate supernatant (12000 xg) 4 C is measured with WallacTM Victor2 luminometer (1420 Multilabel listen).
Luciferase activity is measured by light emission in the presence of luciferin, from coenzyme A and ATP for 10 seconds and reported to 2000 treated cells.
The luciferase activity is thus expressed in Relative Light Unit (RLU
:
Relative light unit) and normalized with the protein concentration of the sample obtained using a Pierce BCA kit (Rockford, IL, USA).

The results summarized in Figure 5 show an efficiency of optimal transfection for lipoplexes comprising 5 or 10 nmoles of DT-3TU

per μg of DNA. The presence of serum induces a weak inhibition of transfection in all cases.

EXAMPLE 9 Determination of the Toxicity of DT-3TU / DNA Lipoplexes with Respect to cells The purpose of this example is to illustrate the absence of toxicity of the compounds transfectants according to the invention.

The level of protein is measured after transfection. The protocol of transfection is identical to that described for Example 8.

Protein level determination: Briefly, transfected cells are washed twice with 500 l of PBS (phosphate buffer) and then lysed with 250 l reagent (Promega cell culture lysis reagent, Luciferase Assay kit System).
A 50 l aliquot of centrifuged lysate supernatant (12000 xg) is transferred to a tube in the presence of 50 l of 0.1 M iodoacetamide, 0.1 M hydrochloric acid at pH 8.2 and left for 1 hour at 37 ° C.
solutions previous ones are deposited in a 96-well plate and 200 l of reagent BCA
protein assay (Pierce, Montluson, France) are added. The plate is then centrifuged at 2500 rpm. then incubated at 37 ° C. for 30 minutes. In parallel, a range of bovine serum albumin (BSA) is performed to correlate the value absorbent obtained for the samples at a present amount of protein in the sample.

The results summarized in Figure 6 show a level of protein similar regardless of the condition used, including lipoplexes 0.75 or 5 or 10 or 15 or 20 nmol of DT-3TU per g of DNA. The presence of lipid DT-3TU
therefore does not alter the cell and no toxicity has been observed in the terms used.

This example illustrates one of the major advantages of the compounds transfectants according to the invention, namely their very low toxicity probably linked the absence of positive charges in their structure.

EXAMPLE 10 Compaction of the Nucleic Acid by DT Nanoemulsions The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to associate with the nucleic acids.

This can easily be demonstrated by an electrophoretic delay test on DNA agarose gel evidenced by use of ethidium bromide (BET): the absence of migration of the nucleic acid on the gel testifies to the compaction of the nucleic acid. Free nucleic acid meanwhile is not subject to a delay on gel.

On an agarose gel (0.8% agarose in TBE 1N) are deposited different DNA / DT-3TU samples with different formulations of lipid DT-3TU compared to DNA. The gel is subjected to an electric current of one hour and half at 70V and 40 mA to migrate the DNA by electrophoresis. The bands have been revealed with BET and by absorption under a UV lamp.
results are shown in Figure 7.
In the same way, the ability of the DT-3TU diol compound to compact DNA
is shown by an agarose gel (0.8% agarose in 1N TBE) on which are deposited different DNA / DT-3TUdiol samples with different DT-3TUdiol lipid formulations relative to DNA. The gel is subjected to a electrical current of one hour and a half at 70V and 40 mA in order to migrate DNA
by electrophoresis. The bands are revealed with BET and by absorption under a UV lamp.

The gel shows the electrophoretic migration of DNA when it is not associated with lipids (well 1), then its retention difference when it is associated with lipids. Wells 2 to 5 represent DNA (0.01g / l) associated with nanoemulsions of DT-3TU / DPPC (60nmol DT-3TU / g DNA) containing or not calcium and ethanol. Well 2 represents 60 nmol / g of DNA without Cal, without EtOH, in the well 3 was added 2% EtOH, in well 4, 60eq Cal + / PO-DNA, in the well 5, 2% EtOH and 60eq of Cal +. The comparison between well 1 and other wells indicated that the different DT-3TU formulations studied are delaying the migration of DNA
on the gel, which was also obtained after dialysis of Ca2 + constituents and EtOH.

This example thus illustrates the capacity of lipid DT-3TU incorporated into different formulations to compact the nucleic acid.

EXAMPLE 11 Compaction of the Nucleic Acid by Stabilized Complexes of The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to associate with the nucleic acids.

This can easily be demonstrated by an electrophoretic delay test on DNA agarose gel evidenced by use of ethidium bromide (BET): the absence of migration of the nucleic acid on the gel testifies to the compaction of the nucleic acid. Free nucleic acid meanwhile is not subject to a delay on gel.

On an agarose gel (0.8% agarose in 1N TBE) were deposited different DNA / DT-3TU / DPPC and DNA / DT-3TUdiol / DPPC samples comprising increasing amounts of DT-3TU lipid relative to the associated DNA or not to PEG-cholesterol. The gel was subjected to an electric current of one hour and half to 70V and 40 mA in order to migrate the DNA by electrophoresis. The bands were revealed with BET and absorption under a UV lamp The results are shown in Figure 8.

The advantage of cholesterol-PEG insertion in DT-formulations 3TU / DPPC comes from the possibility of reducing particle size to a quantity of lipid which would lead to aggregation without insertion of lipid-PEG.
The interest is to optimize the quantities of transfectants injected in vivo. Indeed, sizes of particles required to have stealth objects vis-à-vis proteins serum so an increased half-life time in the bloodstream should to be mostly less than 500m-n. Gold to get sizes of particles of this order, it is necessary to use at least 40mnol of lipid DT-3TU / g of DNA.
The insertion of lipid-PEG in the lipid formulations DT-3TU makes it possible to reduce the amount of DT-3TU required for DNA compaction and training of particles mostly less than 500 nm.

The gel shows the electrophoretic migration of DNA when it is not associated with lipids (well 1), then its retention difference when it is associated with lipids. Wells 2 to 5 represent the DNA (0.01g / t) associated with quantities nanoemulsions of DT-3TU / DPPC with or without cholesterol PEG (20 ethylene glycol units) as a particle stabilizer.
Well 2 represents 20 nmol / g of DNA + 15% chol-PEG, well 3 comprises 20 nmol / ELg of DNA + 20% chol-PEG, well 4 represents 30 nmol / g of DNA + 15% chol-PEG and well 5 represents 20 nmol / g of DNA + 20% chol-PEG. The comparison between the Well 1 and the other wells indicate that the different formulations of DT-3TU
investigated delay the migration of DNA on the gel, indicating the possibility to incorporate into these formulations polymers of polyethylene glycol without release the DNA of the complexes, so without destabilizing them.

EXAMPLE 12 Compaction of the Nucleic Acid in the Presence of DT-4TU (15) The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to associate with the nucleic acids.

This can easily be demonstrated by a bromide fluorescence test of ethidium: the absence of fluorescence testifies to the absence of nucleic acid free which means that the nucleic acid is compacted by the compound transfectant.

The nucleic acid is brought into the presence of increasing amounts of DT-4TU, by equivolumetric mixing of lipid solutions of different the nucleic acid solutions. Samples of 800 ffl complex nucleic acid concentration of 0.01 g / ml in a solution of sodium at 150 nM with increasing amounts of DT-4TU (15).
In the same way, a control was carried out by putting the nucleic acid into presence of increasing amounts of DT-3TU (12) to compare efficiencies of complexion of a lipid with 3 thioureas (see Figure 2) with respect to a lipid carrying 4 thioureas, by equivolumetric mixing of lipid solutions of different titers in nucleic acid solutions. We thus prepare samples of 800 l of nucleic acid complexes of concentration 0.01 g / ml in a 5% glucose solution with increasing amounts of DPPC.

Ethidium bromide fluorescence is measured using a FluoroMax-2 (Jobin Yvon-Spex), with wavelengths of excitation and emission values of 260 nm and 590 nm respectively. Slit widths for excitation and emission are set at 5 iun. The fluorescence value is recorded after addition of 3 l of ethidium bromide (lg / l) per ml of solution DNA / lipid (0.01 g / l of DNA). The results are summarized in Figure 9.
The curve including squares shows that the addition of a growing amount of lipid mixture DT-3TU / DPPC (0.75 to 30 nmol of DT-3TU) relative to a fixed amount of nucleic acid (8 g) induces a decrease in fluorescence linked to reducing the insertion of ethidium bromide between base pairs of DNA.
This indicates that the association between DT-3TU / DPPC liposomes and DNA is strong enough to exclude ethidium bromide from the complexes. We have thus obtained 70% compaction of the DNA using 30nmol of lipids DT-3TU / DPPC per μg of DNA. The active role of lipid DT-3TU in this association Lipids / DNA has been shown in Figures 1 and 2. In the same way, quantities LD-4TU / DPPC lipid mixture (0.75 to 30 nmol of DT-3TU) was added to a fixed amount of nucleic acid (8 g). This association induces a decreased fluorescence due to reduction of bromide insertion ethidium between the base pairs of the DNA. This indicates that the association between liposomes DT-4TU / DPPC and the DNA is strong enough to exclude bromide of éthidiiun complexes. We were able to obtain 60% compaction of DNA for 30 nmol of lipid / μg of DNA (circles, FIG. 9), which is comparable with the complexing efficiency of the DT-3TU under the same conditions.
This example thus illustrates the ability of lipid DT-4TU to associate with the acid nucleic.

EXAMPLE 13 DNA Protection Against DNAses by Complexes The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to protect the nucleic acids of enzymatic hydrolyses whose DNases.

This can easily be demonstrated by an electrophoretic delay test on DNA agarose gel evidenced by use of ethidium bromide (BET): the free DNA or complexed with the lipid is treated with a quantity appropriate of DNase. The DNA, extracted from the enzymatic digestion medium, is deposited on gel and its integrity is verified by comparing its migration with that of acid untreated nucleic acid.

On an agarose gel (0.8% agarose in 1N TBE) were deposited different samples of extracted DNA that had previously been treated with 2.10-4 M of DNase (Sigma). The DNase treatment was performed on free DNA and on DNA

51a complexed with increasing amounts of DT-3TU lipid relative to DNA.
The gel was subjected to an electric current of one hour and a half at 70V and 70 mA
in order to to migrate the DNA by electrophoresis. The strips were revealed with BET
and by absorption under a UV lamp. The results are represented in the figure 10.

The gel shows the electrophoretic migration of untreated DNA by the DNase (well 1), then its difference in retention when treated with DNase. The Well 2 represents the same amount of DNA (3 g) treated with 2.10 -4 M DNase.
Following this treatment (2.10-4 M DNase, 30 min, 37 C), the band corresponding to the DNA is not revealed which indicates a degradation of the DNA. The acid nucleic acid, complexed with 30 and 40 nmol of DT-3TU lipid per g of DNA and 40 nmol of lipid DT-3TU + 6% Chol-PEG was treated with 2.10-4 M DNase. After extraction DNA with a phenol / chloroform mixture and precipitation of the latter, acid Nucleic acid was deposited on this gel in wells 3, 4 and 5 respectively.
The DNA
migrates in a similar way to DNA not treated with DNase, which indicates that DNA
is intact, it has not been degraded by the DNase treatment, so it has been protected by lipid DT-3TU. The DNA in the lipid complexes DT-3TU is therefore not accessible to enzymatic hydrolysis, it is protected from the hydrolysis of DNase.
This example thus illustrates the ability of lipid DT-3TU to protect the acid nucleic enzymatic hydrolysis.

EXAMPLE 14: DNA protection against serum by DT-complexes The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to protect the nucleic acids from degradation in a middle serum.

This can easily be demonstrated by an electrophoretic delay test on DNA agarose gel evidenced by use of ethidium bromide (BET): the DNA free or complexed with the lipid is incubated in different proportions of serum at 37 ° C. The DNA, extracted from serum medium, is deposited on gel and its integrity is verified by comparing its migration with that of acid untreated nucleic acid.

On an agarose gel (0.8% agarose in 1N TBE) were deposited different samples of extracted DNA that was previously incubated in 150 mM
NaCl, 20% serum and 100% serum. Saline and serum treatments have been made on free DNA and on DNA complexed with increasing amounts of lipid DT-compared to the DNA. The gel was subjected to an electric current of one hour and a half at 70V and 40 mA in order to migrate the DNA by electrophoresis. The bands have summer revealed with BET and absorption under a UV lamp. The results are shown in Figure 11.
The gel shows the electrophoretic migration of the control DNA (well 1), then its difference in retention when treated, uncomplexed, in medium saline (15OmM
NaCl) (well 2), when complexed with 40 mnol of lipid DT-3TU / g of DNA
(well 3). The migration of the DNA extracted from the saline medium is comparable for the 2 well indicating that the DNA remains intact under these conditions. The wells following show in the same order the DNA (wells 4 and 6) and the DNA + 40mnol of lipid DT
3TU (wells 5 and 7) in two different serum conditions: 20% serum for the wells 4 and 5 and 100% serum for wells 6 and 7. When the DNA is free, it is completely degraded in the two serum conditions studied after 30 minutes at 37 C (wells 4 and 6). In contrast, the complexion of DNA with nanoemulsions consisting of DT-3TU / DPPC leads to the protection of nucleic acid since the migration band corresponding to the DNA is revealed (wells 5 and 7). The DNA
in the lipid complexes DT-3TU is therefore protected from degradation in medium serum compared to naked DNA.
This example thus illustrates the ability of lipid DT-3TU to protect the acid nucleic acid degradation in the serum.

EXAMPLE 15 In Vivo Transfection of the DT-3TU / DNA Compositions The purpose of this example is to illustrate the key compound transfectant capacity according to the invention to transfect biological tissues in vivo.

This can be demonstrated by intramuscular injection of DNA complexes coding for luciferase. The muscle samples are then taken 96h after the injection and the level of luciferase expression is measured in using a lurninometer (wallac).
Complexes with increasing amounts of lipid DT-3TU per g of DNA were injected into both cranial tibial muscles of mice, which were applied or not electrical pulsations (Bureau, M et al, BBA 2000, 1474 (3): 353-9).

Complexes with increasing amounts 40 and 60 runol of lipid DT-3TU per μg of DNA are injected under a volume of 30 L at a rate of 3 g of DNA per animal in both cranial tibial muscles of C57b1 / 6 mice anesthetized with a mixture of Ketamin / Xylazine. This injection was followed or not the application of transcutaneous electrical pulsations applied by intermediate electrodes located on either side of the muscle (Bureau, M et al, BBA 2000, 1474 (3): 353-9).

After euthanasia of the mice 96 hours post-injection, the muscles are removed and crushed in 1ml of lysis buffer. After centrifugation (10 min, 12000 rpm, 4 C), 10 l of supernatant are removed and deposited in 96 well plates for reading from luciferase after addition of 50 1 of luciferase substrate. The arbitrary level of luminescence is read into the supernatant using a luuninometer (Wallac, Victor).
The values obtained are shown in Figure 12. They represent the levels of expression relating to the dose of lipid associated with the nucleic acid, 20 and 40 nmol of lipid DT-3TU / g of DNA. The expression levels obtained are significant, and greater than the background noise represented by the muscle taken as control that corresponds to 5.104. DNA complexed by different amounts of Lipid DT-3TU is therefore able to transfect muscle tissue with a level significant transfection.

This example illustrates the capacity of the transfecting compounds according to the invention at transfect tissues in vivo.

EXAMPLE 16 In Vivo Biodistribution of DT-3TU / DPPC / DNA Complexes The purpose of this example is to illustrate the ability of transfectant compounds according to the invention to circulate a long time in the circulation in vivo because of their neutral character.

This can be demonstrated by injecting DNA complexes containing a Fluorescent lipid in the bloodstream of mice. Samples blood are then performed at different times after the injection and the level of fluorescence in the circulation is measured using a FluoroMax-2 TM (Jobin Yvon-Spex).
Complexes comprising 40 nmol of DT-3TU lipid per g of DNA, 1 mole equivalent of DPPC / DT-3TU lipid and 0.7% lipid-rhodamine per report total lipids were injected in a volume of 200. l at the rate of 11 pg of DNA
per animal in the caudal vein of C57b1 / 6 mice anesthetized with a mix of Ketamin / Xylazine.
At 30 minutes, lh and 6h post-injection, the blood is collected by puncture intracardiac on anesthetized mouse. After euthanasia of the mice, the liver, the spleen and The lungs are immediately removed, weighed and homogenized in PBS
(5 1 mg of tissue). Lipids are extracted from 100 l of blood and homogenates organs with 3 ml of a 1: 1 mixture of chloroform and methanol agitation vigorous for 30 min then by centrifugation. Fluorescence is read in the supernatant using a FluoroMax-21M (Jobin Yvon-Spex), with lengths excitation and emission waveform of 550 nm and 590 nm respectively. The slit widths for excitation and emission are set at 5 nm.
The values obtained are shown in Figure 13. They represent the 55a percentage of the injected dose obtained in the blood, lungs and system reticuloendothelial (cumulative liver and spleen) 30 minutes, 1 hour and 6 hours after injection. The Fluorescence measurement in the blood after 30 minutes represents 50% of the dose injected which is far superior to what can be achieved with surfactants cationic type DNA. After Ili, 17% of the injected dose could be found this which again represents a remarkable improvement over complex Cationic. The neutral character of these lipid / DNA complexes (zeta very weakly positive: figure 4) thus has a real advantage for obtaining particles stealth serum proteins. It should also limit their interactions with the macrophages and kupffer cells of the spleen and liver which can explain the amount of liposome found at 30 min and 1 h in the blood.

The amount of lipoplex found in the lung is low compared to to that found in the case of cationic lipoplexes. The neutrality of these liposomes should also decrease non-specific interactions with the negative endothelium of the lungs.

This example illustrates the capacity of the transfectant compounds according to the invention to be stealthy vis-à-vis serum proteins.

Claims (20)

1. The transfecting compounds of general formula:
in which:
.cndot. ~ is an integer chosen from 0 and 1 .cndot. n is an integer chosen from 1, 2, 3, 4, 5 and 6, .cndot. m is an integer chosen from 2, 3 and 4, m being able to take values different within the different groups -[NH-CS-NH-(CH(R))m]-, .cndot. R' represents a group of general formula (II):
where q is an integer selected from 1, 2, 3, 4, 5 and 6, and p is a chosen integer among 2, 3 and 4, p being able to take different values within the different -[(CH2)p-NH-CS-NH]- groups, .cndot. R represents either a hydrogen atom or a group of formula general (II) as defined above, it being understood that when n is 1 and ~ is 0, then at least one R group is of formula (II), .cndot. X, in formulas (I) and (II), represents an aliphatic group linear or cyclic, saturated or unsaturated, and comprising 1 to 8 carbon atoms, a group mercaptomethyl (-CH2SH), or else a hydrophilic chain chosen from groups:
-(CH2)x-(CHOH)uH where x is an integer chosen between 0 and 10, and u an integer selected among 1, 2, 3, 4, 5 and 6, or else -(OCH2CH2O)vH with v an integer chosen from 1, 2 and 3, it being understood that not more than one substituent X, at a time in formulas (I) and (II), ne represents a hydrophilic chain, .cndot. Y represents -NH-CO-CH2-CH2-, .cndot. and L represents -N(R1)R2 with R1 and R2 representing C14H29, where appropriate in their isomeric forms or their mixtures. 2. The transfecting compounds according to claim 1, of general formula:
wherein X, m, n, R1, R2 and Y are as defined in claim 1, to except for n which is different from 1, where appropriate in their isomeric forms or their mixtures. 3. The transfecting compounds according to claim 1 or 2, of formula general wherein m, n, R1, R2 and Y are as defined in claim 1, at except for n which is different from 1, where appropriate in their forms isomers or their mixtures. 4. Compositions characterized in that they comprise a compound transfectant according to any one of claims 1 to 3, and an acid nucleic. 5. Compositions according to claim 4, characterized in that the acid nucleic acid is deoxyribonucleic acid or ribonucleic acid. 6. Compositions according to any one of claims 4 to 5, characterized in that they further comprise one or more adjuvants. 7. Compositions according to claim 6, characterized in that said adjuvant is polyethylene glycol, polyethylene glycol covalently bonded to a cholesterol molecule, or a natural or synthetic neutral lipid, switterionic or devoid of ionic charge under physiological conditions. 8. Compositions according to claim 7, characterized in that said lipid neutral is chosen from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmito-ylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphati-dylethanolamines and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides, sphingolipids or asialogangliosides. 9. Compositions according to claim 8, characterized in that the cerebrosides are galactocerebrosides, in that the sphingolipids are of the sphingomyelins, and in that the asialogangliosides are asialo GM1 or GM2. 10. Compositions according to any one of claims 8 to 9, characterized in that they further comprise a targeting element chosen from the sugars, peptides, proteins, oligonucleotides, lipids, neurotransmitters, hormones, vitamins or their derivatives. 11. Compositions according to claim 10, characterized in that said element targeting agent defined in claim 10 is covalently linked either to the compound transfectant according to one of Claims 1 to 3, or to the nucleic acid. 12. Compositions according to any one of claims 4 to 11, characterized in that they are injectable and further comprise a vehicle pharmaceutically acceptable for an injectable composition. 13. Compositions according to any one of claims 4 to 11, characterized in that they are topical compositions and further comprise a vehicle pharmaceutically acceptable for administration to the skin and/or mucous membranes. 14. Use of a transfecting compound according to any one of claims 1 to 3 for the transfer of nucleic acids within a cell. 15. Use of a transfecting compound according to any one of claims 1 to 3 for the manufacture of a medicament intended to treat or prevent viral or bacterial infections, or cancerous conditions. 16. Use of a transfecting compound according to any one of claims 1 to 3 for the treatment or prevention of viral infections Where bacterial, or cancerous states. 17. Method for transferring nucleic acids into cells characterized by this that it includes the following steps:
(1) bringing the nucleic acid into contact with a transfecting compound such that defined according to any one of claims 1 to 3, to form a complex, and (2) bringing the cells into contact in vitro with the complex formed in (1). 18. Method for transferring nucleic acids into cells according to the claim 17, characterized in that said transfecting compound and/or said acid nucleic acid are mixed beforehand with one or more adjuvant(s) and/or with a targeting element as defined in any one of claims 10 at 11. 19. Method for transferring nucleic acids into cells according to the claim 17, characterized in that one or more adjuvant(s) chosen among polyethylene glycol, polyethylene glycol covalently bonded to a molecule of cholesterol and a natural or synthetic neutral lipid, zwitterionic or lacking of ionic charge under physiological conditions and is (are) previously administered to the cells. 20. Method for transferring nucleic acids into cells according to one any one of claims 17 to 19, characterized in that the cells are in in addition to being subjected to physical treatment intended to improve transfection, said physical treatment being electrical impulses or mechanical forces.