CA2324931A1 - Novel nucleic acid transfer agents, compositions containing same and uses - Google Patents

Abstract

The invention concerns novel compounds useful as agents for transferring nucleic acids into cells. Said novel compounds are more particularly related to the lipopolyamine family, and comprise at least a cyclic amidine function. They are useful for transfecting nucleic acids of interest into different cell types, in vitro as well as in vivo or ex vivo.

Description

NOVEL NUCLEIC ACID TRANSFER AGENTS.
COMPOSITIONS CONTAINING THEM AND APPLICATIONS THEREOF
The present invention relates to new compounds useful as agents transfer of nucleic acids into cells. These new compounds are more particularly related to the lipopolyamine family, and include less a cyclic amidine function. They are useful for transfection of acids nucleic acids in different cell types, both in vitro and ex vivo or in vivo.
With the development of biotechnology, the possibility of transferring effectively nucleic acids in 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 any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the realization 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 transfer of genes into cells taken from an organism, with a view to their subsequent re-administration.
Different types of synthetic vectors have been developed to improve the transfer of nucleic acids into cells. Among these vectors, lipids cationic have interesting properties. These vectors are consist of a polar, cationic part, interacting with nucleic acids, and a part lipid, hydrophobic, promoting cell penetration. Examples individuals cationic lipids are in particular monocationic lipids (DOTMA
Lipofectin ~), certain cationic detergents (DDAB), lipopolyamines and especially dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide palmitoylphosphatidylethanolamine (DPPES), the preparation of which has been described for example in patent application EP 394 111. Another family of WO 99/51581 PCT / F'R99 / 00740

2 lipopolyamines is represented by the compounds as described in request WO 97/18185 incorporated herein by reference, and are illustrated in the figure 1.
But so far, the injections into the tissues, and in particular the muscles, were often made with unformulated DNA to facilitate entry in the cells, association with synthetic vectors leading to complexes of size too large to be incorporated into cells.
This is one of the main problems that your present invention proposes to solve. Indeed, the compounds according to the invention have the advantage unexpected from have at least equivalent level of transfection in vivo in muscle to the one obtained with unformulated DNA and in any event a very good level of transfection into other tissues. The association with a compound according to the invention protects DNA from nuclease damage and / or damage to during freeze-drying, which significantly improves stability of nucleolipid formulations. In addition, such an association allows a release slow control of nucleic acids.
Furthermore, the compounds according to the present invention belong to the family cationic lipids and carry an original cationic region which confers to the said compounds with improved properties, including reduced cytotoxicity by report to cationic vectors of the prior art. This cationic part is in effect more precisely represented by one or more particular polyamine (s), wearing one or several cyclic amidine functions which most likely have the effect of "Relocate" the positive charges, making the compound overall less cationic, with the beneficial effects on the plan of toxicity.
Thus, a first subject of the invention relates to new compounds under form D, L, or DL, of general formula (I) CA-Rep R (I) for which

3 ~ O CA represents a cycloamidine group and its mesomeric forms of general formula (II) ,:.
, - ~ I - Rt (II) ~ CHz m \ X / ~ C ~) n for which ~ m, and n are integers independent of each other between 0 and 3 included and such that m + n is greater than or equal to I, ~ R, represents a group of general formula (III) - ~ (CH2) py ~ (*) (III) for which p and q are integers independent of each other understood between 0 and 10 inclusive, Y represents a carbonyl, amino, methylamino group, or else methylene, Y can have different meanings within different [(CH2) PY] groups, and (*) represents either a hydrogen atom or is the place of connection to the Rep group, it being understood that RI can be linked to any atom of the formula general (II), y understood Z, and that there is a single group R1 in formula (II), ~ X represents a group NR2 or CHR2, R2 being either an atom hydrogen either the bond to the group R1 as defined above, Z
~ The grouping - '~ - represents W
* I "case: a grouping of general formula (IV)

4 NH
R'NI 'W' (I ~
for which W 'represents CHR "' or NR"', and R "and R"' represent independently of each other a hydrogen atom, a methyl, or the bond at group Rl as defined above, or else * 2 ""'case: a grouping of general formula (V) NHR ' N ~ W '(V) for which W 'represents CHR "' or NR"', and R' and R "'represent independently of each other a hydrogen atom, a methyl, or the bond at group R ~ as defined above, D Rep is absent or is a dispatcher of general formula (VI) O
- ~ N - (CHy t (VI) Ra R3 whose nitrogen atom is attached to X, V, W, or Z atoms or to the substituent Y
of group R, as the case may be, and ~ t is an integer between 0 and 8 inclusive, ~ r is an integer between 0 and 10 inclusive, r can have meanings different within the different groupings -NR ,, - (CH) T, ~ R3, which can have different meanings within different groupings IVR. ~ - (CH) ~ R3, represents a hydrogen atom, a methyl group, or a grouping of general formula (VII) - ~ - (CH2 ~ --N- ~ g (VII) Rs for which u is an integer between 1 and 10 inclusive, s is an integer between 2 and 8 inclusive may have different meanings within different - (CHZ) S NRs groups, and Rs is a hydrogen atom, a CA group such than

5 defined above, it being understood that the CA groups are independent the from each other and can be different, or a group of formula general (VII) it being understood that the groups of general formula (VII) are independent from each other and can have different meanings, ~ R4 is defined in the same way as R3 or represents a grouping CA
such as defined above, it being understood that the CA groups are independent some from others and may be different, and m R is linked to the carbonyl function of the Rep group of general formula (VI), or if Rep is absent R is linked directly to the group CA, and represented * or a group of formula NR6R ~ for which R6 and R ~ represent independently of each other a hydrogen atom or an aliphatic radical saturated or not, linear or branched, optionally fluorinated, containing 1 to 22 atoms of carbon, with at least one of the two substituents R6 or RT different from hydrogen and the other containing between 10 and 22 carbon atoms, * either a steroid derivative, * or a group of general formula (VIII) - ~ NH (CHz) ~ Q (VIII) for which x is an integer between 1 and 8 inclusive, y is an integer between 1 and 10 inclusive, and either Q represents a group C (O) NR ~ R ~ for which R6 and R ~ are

6 defined as above, or Q represents a group C (O) RS for which Rg represents a group of formula (IX) R

~ N ~ N ~ O ~~ (IX) the ~~ ~ z O
for which z is an integer between 2 and 8 inclusive, and R9 is a aliphatic radical S saturated or not, optionally fluorinated, containing 8 to 22 carbon atoms, or one steroid derivative, and the two R6 substituents are, independently one of the other, defined as above, or Rg represents a group -O-R9 for which R9 is defined as below-above.
According to a variant of the invention, the group R ~ is linked either to Z or to V
on the one hand and to the Rep group on the other hand through Y.
Advantageously, the cycloamidine group CA of formula (II) comprises S, 6, 7 or 8 links.
Furthermore, in another variant of the invention, Rep is a distributor with 1, 2, or 3 "arms". We can for example cite the following distributors its F ~ s HN N ~ HN N
OO
JdN l ld a la Jd 1a NOT, iJ 'Ra''~N'Rn ~ 'N ~ d NO
Ra N v Ra H
I ~ IH
Rs R

7 ~~ "" N
~ a O Rs NO
b N
~ N ~

According to a second variant of the invention, R3 represents a hydrogen atom or a methyl and R4 is as defined above, or else R3 and R. ~ present in the formula (VI) represent hydrogen atoms, or else R ,, is an atom of hydrogen and R3 is a group of formula (VII) in which RS
represents a CA grouping Preferably, in formula (V), p and q are chosen independently one of the other among 2, 3 or 4.
In general, the group R contains at least one segment hydrophobic. For the purposes of the invention, the term "hydrophobic segment" means any lipid type group, promoting cell penetration. In particular, the group R contains at least one aliphatic chain or at least one derivative of steroid.
According to a preferred variant, the group R represents a group of formula NR6R ~, R6 and R ~ being defined as above, or else represents a group of general formula (VIII) in which Q represents a group C (O) NR6R ~, R6 and R ~ being defined as above.
Preferably, R6 and / or R, represent independently of each other a saturated or unsaturated linear aliphatic chain containing 10 to 22 atoms of carbon, preferably in 12, 14, 16, 17, 18, or 19 carbon atoms. II
is by example of groups (CH2) "CH3, (CH2), 3CH ;, (CH2) isCH3, (CH2) mCH3, oleyl etc ...
In a particular embodiment, the groups Rb and R ~ are identical or different and each represents a saturated aliphatic chain or not,

8 linear or branched, possibly fluorinated, containing 10 to 22 atoms of carbon, as defined in the previous paragraph.
When R represents a steroid derivative, this is advantageously selected among cholesterol, cholestanol, 3-a-5-cyclo-5-a-cholestan-6- (3-0l, acid cholic, cholesterylformiate, chotestanylformiate, 3a, 5-cyclo-Sa-cholestan-6 [3-yl formate, cholesterylamine, 6- (I, 5-dimethylhexyl) -3a, 5a-dimethyl-hexadecahydrocyclopenta [aJcyclopropa [2,3 Jcyclopenta [1,2-fJnaphta-lèn-10-ylamine, or cholestanylamine.
These new compounds of general formula (I) can be present under form of non-toxic and pharmaceutically acceptable salts. These non-salts toxic include salts with mineral acids (hydrochloric acids, sulfuric, hydrobromic, phosphoric, nitric) or with organic acids (acids acetic, propionic, succinic, malefic, hydroxymic, benzoic, fumaric, methanesulfonic or oxalic) or with mineral bases (soda, potash, lithine, lime) or with organic bases (tertiary amines such as triethylamine, piperidine, benzylamine).
By way of example illustrating preferred compounds according to the invention, it is possible to cite the compounds of the following formulas not HN "N
~ NH
Compound (1) N-dioctadecylcarbamoylmethyl-2- {3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino) propylamino} -acetamide not . ~~ H- ~, ~ r ''' compound (2) N-ditetradecylcarbamoylmethyl-2- {3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino) propylamino} -acetamide ~~ N ~ Ni ~ N ~ N ~ N ~ N
CN HHHO
H
Compound (3) 2- (3- {4- [3- (4, 5-dihydro-1 H-imidazol-2-ylamino) -propylamino) -butyIamino} -N-ditetradecylcarbamoylmethylacetamide not NYNH
HN
O
~~ N ~ N ~ N ~ N ~ N
~ 'N, HH ~ O
Compound (4I
2- (3- {bis- [3- (4,5-dihydro-1H-imidazol-2-ylamino) -propyl) -amino}
-propylamino) -N-ditétradécylcarbamoylméthyl-acetétamide.

N ~ N ~ N ~ N ~ N ~ N
~ NH H ~ O
Compound (5) N-ditetradecylcarbamoylmethyl-2- {3- [3- (I, 4, 5,6-tetrahydropyrimidin -2-ylamino) -propylamino) propylamino} -acetamide WO 99/51581 PCT / F'R99 / 00740 N ~ N ~ N ~ N ~~~ N
~ NH ~ O
Compound (6) N-dioctadecylcarbamoylmethyl-2-t 3- [3- (1,4,5,6-tetrahydropyrimidin -2-ylamino) -propylamino] propylamino} -acetamide The compounds of the invention can be prepared in various ways. According to a first method, the compounds of the invention can be obtained by synthesis analogous lipopolyamines (i.e. the same structure but without group 5 cycloamidine), the cyclization into cycloamidine groups being carried out in one Secondly. Analogous lipopolyamines can be obtained by any method known to those skilled in the art, and in particular according to the methods described in the application WO 97/18185 or by analogous methods. Cyclization of the heads amidine can for example be carried out by reaction between one and / or more amines 10 primary lipopolyamine and reagents such as hydrogen sulfate do-methylisourea sulfate [J. Med. Chem., 1995, 38 (16), pp. 3053-3061] or the semisulfate of S-methylisothiourea [Int. J. Pept. Prot. Res., 1992, 40, pp. 119-126]. Of preferably, one operates in an aqueous medium in the presence of a hot base [J. Med.
Chem., 1985, pp. 694-698 and J. Med. Chem., 1996, pp. 669-672]. As solvent preferred, mention may be made of water / alcohol mixtures or dimethylformamide. AT
title of base, triethylamine, N-ethyldüsopropylamine, soda, potash etc ... The temperature is preferably between 40 ° C and 60 ° C, and even more preferably, the reaction is carried out at 50 ° C.
Another method consists in carrying out a synthesis of bricks carrying the cycloamidine function which are then grafted) on lipids equipped with distributors. This method has the advantage of accessing a number important to products. For the purposes of the invention, the term “bricks” means any segment functional of the molecule. For example, the cycloamidine group CA as defined in the general formula (II), Rep or even R constitute separate bricks the one of the others within the meaning of the invention.

For example, one can for example proceed as follows 1) Synthesis of the brick R
a) When R represents -NR6R ,, either it is commercially available or he can be synthesized using one of the following methods ~ by alkylative reduction between an amine carrying the group R6 and an aldehyde wearing the group R ~. It is preferably carried out in a chlorinated solvent (for example the dichloromethane, chloroform, dichloro-1,2-ethane etc ... [J. Org. Chem., 1996, pp. 3849-3862]) or in any other organic solvent compatible with the reaction (by example of tetrahydrofuran), in the presence of sodium triacetoxyborohydride, sodium cyanoborohydride or its derivatives (e.g. cyanoborohydride lithium) [J.Am. Chem. Soc., 1971, pp. 2897-2904] and acetic acid.
~ or by substitution of a leaving group carried by R6, by an amine carrying the group R ~. As an example of a leaving group, we can cite the atoms halogen (Br, Cl, I) or the substituents tosyl, mesyl, etc. We preferably operate in presence a basic reagent, for example sodium carbonate, potash, welded triethylamine etc ..., in an alcohol (for example ethanol) at reflux [J. Am.
Chem. Soc., 1996, pp. 8524-8530]
~ or by coupling between a fatty acid (or its derivatives such as chlorides acid fat for example) and a fatty amine. The amide obtained is then reduced by a hydride, for example lithium aluminum hydride or any other hydride known from the man of the trade, in an ether (for example tetrahydrofuran (THF), t-butyl methyl ether (TBME), dimethoxyethane (DME), etc.).
b) When R represents a group of general formula (VIII), the peptide coupling between the group Q and H- [NH- (CH2) X] YCOOH. The coupling peptide is carried out according to conventional methods known to those skilled in the art job (Bodanski M., Principles and Practices of peptide Symhesis, Ed. Springe-Verlag) or by any known analogous method. In particular, the reaction can be carried out in presence of a non-nucleophilic base in suitable aprotic solvents (such as chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane etc ...), at a temperature between 0 and 100 ° C, the pH being adjusted between 9 and 11.
Q is either commercially available or when Q represents a group C (O) R8 with R8 of formula (IX), it can be synthesized by reaction between a commercial chloroformate (e.g. cholesteryl chloroformate) or obtained according to conventional methods known to those skilled in the art from a chloroformate commercial, and a commercial diamine (for example N-ethylenediamine) or obtained according to conventional methods known to those skilled in the art. Of preference, one operates in a chlorinated solvent (for example dichloromethane, chloroform, the 1,2-dichloro-ethane etc ...) or in any other compatible organic solvent with the reaction such as for example dimethylformamide, dimethylsulfoxide, acetonitrile etc ...
The group H- [NH- (CH2) x] y-COOH is a commercial amino acid when y is equal to 1, or is obtained by one or more cyanoethylation reactions according to methods described below in the synthesis of Rep when y is greater than 1.
2) Synthesis of the Rep brick The Rep group is obtained by cyanoethylation or by dicyanoethylation (according to that one wishes to obtain a linear or branched Rep structure) of a amino acid of formula HOOC- (CH2) r NH2 then by reduction of the nitrile functions into amines.
a) mono- or di-cyanoethylation CN
r-1 CN r-1 HOOC- (CH2) r-NH2 + 1 or 2 ~ R ~ H
or CN
R ' r-1 . R'-N
r-CN

Preferably, the operation is carried out in a basic aqueous medium. For example, the reaction is carried out in solvents such as water, alcohols (for example methanol , ethanol etc ...) in the presence of a base such as soda, potash, triethylamine etc ... In the case of monocyanoethylation, we preferably work at cold [J. Am.
Chem. Soc., 1950, pp. 2599-2603]. In the case of dicyanoethylation, we works from preferably hot and with an excess of acrylonitrile [J. Am. Chem. Soc., 1951, pp.
1641-1644]
b) The reduction of the nitrile functions into amines is carried out by hydrogenation catalytic in basic medium or by any other method known to those skilled in the art job. As an example, platinum oxide or alternatively nickel of Raney [J. Org. Chem., 1988, pp. 3 I 08-3 II 1] as a catalyst. Of preferably the chosen solvent is an alcohol (for example methanol, ethanol etc ...) in presence a base, for example soda, potash etc ...
3) Synthesis of the Rep-R brick The Rep-R brick is obtained by peptide coupling between the Rep acid and amine R
obtained in the previous steps.
The peptide coupling is carried out according to the conventional methods known from the man of the profession (Bodanski M., Principles and Practices of peptide Synthesis, Ed.
Springe-Verlag) or by any known analogous method. In particular, the reaction can be carried out in the presence of a non-nucleophilic base in solvents aprotic suitable (such as chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane etc ...), at a temperature between 0 and 100 ° C, the pH being adjusted between 9 and 1 I.
4) Synthesis of the compounds according to the invention CA-Rep-R
The compounds according to the invention are obtained according to several possible methods a) by coupling in basic medium between the terminal amine present on Rep-R
got to the previous step, and CA-S-CH ~, according to the conventional methods known from the man of career. It is preferably carried out in a chlorinated solvent (for example the dichloromethane, chloroform etc ...) or in other organic solvents compatible with the reaction such as for example water, alcohols, dimethylformamide etc ..., in the presence of a base (for example the triethylamine, the soda, potash, N-ethyldüsopropylamine etc ...). and at temperature ambient (20 ° C
about).
The CA-S-CH3 brick is either commercially available (this is the case with example of 2-methylthio-2-imidazoline hydroiodide}, or it can be obtained by action of a carbon disulfide on an appropriate diamine (that is to say chosen in function of the cycloamidine group which it is desired to obtain), followed by methylation.
Through example, the reaction scheme can be illustrated as follows n ~ z CSZ ~ n NH
N / _S
H
n NH MeX ~ n N
N / \ S
HH
Preferably, the reaction process is carried out in an alcohol (for example example ethanol). The methylation step is carried out by the action of a halomethyl, the halogen atom possibly being, for example, an iodine atom [J. Am. Cem. Soc., 1956, pp. 1618-1620 and Bioorg. Med. Chem. Lett., 1994, pp. 351-354].
b) by internal cyclization of the cycloamidine group from the functions amino present on Rep-R, by the action of O-methylisourea hydrogen sulphate or S-methylisothiourea semisulfate. Preferably, one operates in an aqueous medium in presence of a hot base [J. Med. Chem., 1985, pp. 694-698 and J. Med.
Chem., 1996, pp. 669-672). Mention may be made, as preferred solvent, of mixtures water / alcohol or dimethylformamide. As a base, triethylamine can be used, the N-ethyldüsopropylamine, soda, potash etc ... The temperature is included of preferably between 40 ° C and 60 ° C, and even more preferentially, the reaction is set at 50 ° C.

c) by peptide coupling between CA-COOH and Rep-R according to the methods classics known to those skilled in the art, as described above.
CA-COOH brick can be obtained in different ways ~ by the action of a CA-S-CH3 brick on an amino acid or a polyamine acid S according to methods known to those skilled in the art or by any other method similar [J. Am. Chem. Soc., 1956, pp. 1618-1620]. CA-S-CH3 brick is obtained from same way as before, and the amino acid or polyamine is chosen by function of the compound according to the desired invention, or ~ by the action of an S, S-dimethyl-tosyl-iminothiocarbonimidate or one of its 10 derivatives on a polyamino acid according to methods known to those skilled in the art trade or by any other similar method [J. Org. Chem., 1971, pp. 46-48J. Of preferably we operates in an ethanolic medium in the presence of a base (for example sodium hydroxide) and the reflux temperature of the mixture.
As an example of CA-COOH bricks obtainable by one of the 15 methods described above, we can cite the following bricks HH
N ~ HN ll ON ~ N ~
O OH
OH ~ H ~
ab oH c ~ O
NOT
OH
O
Oh of H
N ~ O
fN
O OH
OH

In all the reactions set out above, when the substituents amino present in the different groups can interfere with reactions implemented, it is preferable to protect them beforehand with radicals compatible that can be put in place and removed without affecting the rest of the molecule.
By way of example, the protective radicals can be chosen from Ies radicals described by TW GREENE, Protective Gror ~ ps in Organic Synihesis, J. Wiley-Interscience Publication (1991) or by McOmie, Protective Groups irr Organic Chemistry, Plenum Press (1973).
Another subject of the invention relates to a composition comprising at least a compound of formula (I) as defined above. In particular another object according to the present invention comprises a compound of formula (I) as defined above and a nucleic acid. When a compound according to the invention and an acid nucleic are brought together, they form a complex by interaction between the charges positive present at physiological pH on the compound according to the invention and the charges nucleic acid negatives. This complex is called "complex nucleolipid "
in the following. Preferably, the compound according to the invention and nucleic acid are present in quantities such as the positive charge ratio of the composed on the negative charges of the nucleic acid is between 0.1 and S0, preference between 0.1 and 20. This ratio can be easily adjusted by a person skilled in the art in depending on the compound used, the nucleic acid, and the applications wanted (including the type of cells to be transfected).
For the purposes of the invention, the term “nucleic acid” is understood to mean both an acid deoxyribonucleic than a ribonucleic acid. It can be sequences natural or artificial, and in particular genomic DNA (gDNA), complementary DNA
(CDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA
(RRNA), hybrid sequences or synthetic or semi-sequences synthetic, modified or unmodified oligonucleotides. These nucleic acids can be original human, animal, plant, bacterial, viral, etc. They can be obtained by any technique known to those skilled in the art, and in particular by screening banks, through chemical synthesis, or by mixed methods including modification chemical or enzymatic sequence obtained by screening libraries. Tls can be chemically modified.
Concerning more particularly deoxyribonucleic acids, they can be single or double stranded as well as short oligonuleotides or sequences longer. In particular, the nucleic acids are advantageously made up of plasmids, vectors, episomes, expression cassettes, etc.
These acids deoxyribonucleics may carry an origin of functional replication or not in the target cell, one or more marker genes, sequences regulators of transcription or replication, genes of therapeutic interest, sequences antisense modified or not, regions of binding to other components cellular, etc ...
Preferably, the nucleic acid comprises an expression cassette constituted one or more genes of therapeutic interest under the control of one or many promoters and a transcriptional terminator active in cells targets.
For the purposes of the invention, the expression “gene expression cassette” is intended to mean of interest "a DNA fragment that can be inserted into a vector at sites of specific restrictions. DNA fragment includes an acid sequence nucleic encoding an RNA or a polypeptide of interest and further comprises the sequences necessary for expression (enhancer (s), promoter (s), sequences of polyadenylation etc ...) of said sequence. The cassette and the restriction boxes are designed for ensure insertion of the expression cassette into a reading frame appropriate for transcription and translation.
It is generally a plasmid or an episome carrying one or more genes of therapeutic interest. By way of example, mention may be made of the plasmids described in patent applications WO 96/26270 and WO 97/10343 incorporated herein through reference.
Within the meaning of the invention, the term gene of therapeutic interest in particular any gene encoding a protein product having a therapeutic effect. The product protein thus encoded can be in particular a protein or a peptide. This product protein can be exogenous homologous or endogenous towards the cell target is ie a product that is normally expressed in the target cell when this one does presents no pathology. In this case, the expression of a protein allows through example of compensating for insufficient expression in the cell or expression of a inactive or weakly active protein due to a modification, or of overexpressing said protein. The gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, activity modified, etc ...
The protein product can also be heterologous towards the cell target. In in this case, an expressed protein can for example supplement or provide a activity deficient in the cell, allowing it to fight against a pathology, or stimulate an immune response.
Among the therapeutic products within the meaning of the present invention, one can more particularly mention enzymes, blood derivatives, hormones, the lymphokines: interleukins, interferons, TNF, etc ... (FR 92/03120), factors of neurotransmitters or their precursors or enzymes synthesis, the trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, HARP / pleiotrophin, etc ...) the apolipoproteins (ApoAI, ApoAIV, ApoE, etc ..., FR
93/05125), dystrophin or a minidystrophin (FR 91/11947), the protein CFTR
associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc ..., FR 93/04745), the genes coding for the factors involved in the coagulation (Factors VII, VIII, IX), the genes involved in repair of DNA, suicide genes (thymidine kinase, cytosine deaminase), genes for hemoglobin or other protein transporters, enzymes of the metabolism, catabolism etc ...
The nucleic acid of therapeutic interest can also be a gene or a antisense sequence, the expression of which in the target cell makes it possible to control gene expression or transcription of cellular mRNAs. Such sequences can, for example, be transcribed in the target cell into RNA
complementary of cellular mRNAs and thus block their translation into protein, depending on the technical described in patent EP 140 308. The therapeutic genes include also the sequences encoding ribozymes, which are capable of destroying selectively Target RNA (EP 321 201).
As indicated above, the nucleic acid may also contain one or several genes coding for an antigenic peptide capable of generating in the man or the animal an immune response. In this particular mode of implementation artwork, the invention enables either vaccines or treatments to be carried out immunotherapeutics applied to humans or animals, especially against microorganisms, viruses or cancers. It may especially be peptides specific antigens of the Epstein Ban virus, of the HIV virus, of the hepatitis B
(EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", other viruses or also tumor-specific antigenic peptides (EP 259,212).
Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene encoding for the 1 S antigenic peptide in the desired cell or organ. These can be sequences which are naturally responsible for the expression of the gene considered when these sequences are likely to work in the infected cell. He can also they are sequences of different origin (responsible for the expression other proteins, or even synthetic). In particular, they may be promoter sequences of Genoa eukaryotic or viral. For example, they may be promoter sequences from genome of the cell you want to infect. Likewise, it can be sequences promoters from the genome of a virus. In this regard, we can cite by example the promoters of the E 1 A genes, MLP, CMV, RS V, etc. In addition, these sequences can be modified by adding activation sequences, regulation, etc ... It can also be promoter, inductibie or repressible.
Furthermore, the nucleic acid can also comprise, in particular in upstream of the gene of therapeutic interest, a signal sequence directing the product therapeutic synthesized in secretory pathways of the target cell.
This sequence signal may be the natural signal sequence of the drug, but he can also be any other functional signal sequence, or a signal sequence artificial. The nucleic acid may also have a signal sequence ruler the therapeutic product synthesized to a particular compartment of the cell.
The compositions according to the invention can also comprise one or more 5 adjuvants capable of associating with the complexes formed between the compound according to the invention and the nucleic acid, and to improve the transfecting power thereof.
In another mode of implementation, the present invention therefore relates to compositions comprising a nucleic acid, a compound of formula (I) as defined above before and a or several adjuvants capable of associating with nucleolipid complexes compound 10 (I) / nucleic acid and improve its transfecting power. The presence of that type adjuvants (lipids, peptides or proteins for example) can allow advantageously to increase the transfecting power of the compounds.
With this in mind, the compositions of the invention can comprise as an adjuvant, one or more neutral lipids. Such compositions are 15 particularly advantageous, especially when the charge ratio R
is weak.
The Applicant has indeed shown that the addition of a neutral lipid allows to improve the formation of nucleolipid particles and promote the penetration of the particle in the cell by destabilizing its membrane.
More preferably, the neutral lipids used in the context of present 20 invention are lipids with two fatty chains. So particularly advantageous, natural or synthetic, zwitterionic lipids are used or free of ionic charge under physiological conditions. They can to be more particularly chosen from dioleoylphosphatidylethanolamine (DOPE), oleoylpaimitoylphosphatidylethanolamine (POPE), Ies di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamines and their N-methylated derivatives 1 to 3 times the phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as especially sphingomyelins) or asialogangliosides (such as in particular asialoGMl and GM2).

These different lipids can be obtained either by synthesis or by extraction from organs (example: the brain) or eggs, by techniques classics well known to those skilled in the art. In particular, the extraction of lipids natural can be carried out using organic solvents (see also Lehninger, Biochemistry).
More recently, the Applicant has demonstrated that it is also particularly advantageous to use as an adjuvant, a product intervening or not directly at the level of nucleic acid condensation (WO
96/25508). The presence of such a product, within a composition according to the invention, allows of decrease the amount of compound of formula (I), with the consequences beneficial which toxicologically, without causing any harm to the activity transfectant. By product involved in the condensation of the acid nucleic acid, we mean defining a compacting product, directly or not, acid nucleic acid. More specifically, this product can either act directly at the level of (acid nucleic acid to be transfected or intervene at the level of an additional product which East directly involved in the condensation of this nucleic acid. Of preferably it acts directly at the nucleic acid level. For example, the product precompacting can be any polycation, for example polylysine. According to a mode of preferred embodiment, this product being involved in the condensation of acid nucleic acid derives in whole or in part from a protamine, a histone, or a nucleolin and / or one of their derivatives. Such a product can also be made, in all or part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of patterns which can vary between 2 and 10. In the structure of the compound according to the invention, these patterns can be repeated continuously or not. This is how they can be separated by biochemical links, for example by one or many amino acids, or chemical in nature.
Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant (s) for one equivalent of nucleic acids in moUmol and, more preferably, from 0.5 to 5.

In a particularly advantageous embodiment, the compositions of the present invention further include a targeting element for to guide the transfer of nucleic acid. This targeting element can be an element of extracellular targeting to direct the transfer of DNA to some, cell types or certain desired tissues (tumor cells, cells hepatic, hematopoietic cells ...}. It can also be an element of targeting intracellular to direct the transfer of nucleic acid to some privileged cellular compartments (mitochondria, nucleus etc ...). The element of targeting can be linked to the compound according to the invention or also to the acid nucleic as previously stated.
Among the targeting elements which can be used in the context of the invention, can cite sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives.
Preferably, it these are sugars from peptides or proteins such as antibodies or fragments antibodies, cell receptor ligands or fragments thereof here, receptors or receptor fragments, etc. In particular, it can be ligands growth factor receptors, cytokine receptors, receptors cell lectin type, or ligands with RGD sequence with an affinity for the adhesion protein receptors like integrins. We can also quote them transferrin, HDL and LDL receptors, or the transporter of folate.
The targeting element can also be a sugar making it possible to target lectins such as receptors for asialoglycoproteins or syalydes such as sialyde Lewis X, or an antibody Fab fragment, or a single chain antibody (ScFv}.
The association of targeting elements with nucleolipid complexes of the invention can be carried out by any technique known to a person skilled in the art profession, by example by coupling to a hydrophobic part or to a part which interacts with the nuciéic acid of the compound of general formula (I} according to the invention, or still one group which interacts with the compound of general formula (I) according to the invention or with nucleic acid. The interactions in question can be, according to a fashion preferred, ionic or covalent in nature.

According to another variant, the compositions of the invention can also incorporate at least one nonionic surfactant in sufficient quantity for stabilize the particle size of nucleolipid complexes composed of formula general (I) / nucleic acid. The introduction of non-ionic surfactants prevent the S formation of aggregates, which makes the composition more particularly suitable for in vivo administration. The compositions according to the invention incorporating such agents of surface have an advantage in terms of safety. They present also a added benefit in that they decrease the risk interference with other proteins given the reduction in the overall burden of compositions of nucleolipid complexes.
Surfactants advantageously consist of at least one segment hydrophobic, and at least one hydrophilic segment. Preferably, the segment hydrophobic is chosen from aliphatic chains, polyoxyalkylene, polyester alkylidene, polyethylene glycol with a polyether benryl head and cholesterol, and the hydrophilic segment is advantageously chosen from polyoxyalkylene, alccols polyvinyls, polyvinylpyrrolidones or saccharides. Such agents of surface non-ionic have been described in application WO 98/34648.
A subject of the invention is also the use of the compounds of formula general (I) as defined above for the manufacture of a medicament intended for heal nucleic acid transfer diseases (and more generally polyanions) in primary cells or in established lines. It can be in particular of fibroblast, muscle, nerve cells (neurons, astrcytes, glyal cells), hepatic, of the hematopoietic line (lymphocytes, CD34, dendritics etc ...), epithelial, etc., in differentiated or pluripotent forms (precursors).
Such use is particularly advantageous because the compounds of general formula (I) according to the invention have a reduced cytotoxicity by report cationic lipids of the prior art. The Applicant has in particular implemented evidence that at very high load ratios usually resulting in death of animals following transfection, no apparent cytotoxicity was detected. The compounds according to the invention can be used in particular for the in vitro, ex vivo or in vivo transfection of nucleic acids. For some uses in vivo, for example in therapy or for the study of gene regulation or creation of animal models of pathologies, the compositions according to (invention can to be formulated for topical, cutaneous, oral, rectal administration, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc ... Preferably, the compositions of (invention contain a pharmaceutically acceptable vehicle for a injectable formulation, in particular for direct injection at the level of the organ desired, or for topical administration (on the skin and / or mucosa).
he can in particular, sterile, isotonic solutions, or compositions dry, in particular lyophilized, which, by addition as the case may be of sterilized water or serum physiological, allow the constitution of injectable solutions. The doses acid nucleic acid used for injection as well as the number of administrations can be adapted according to different parameters, and in particular according to the fashion administration used, the pathology concerned, the gene to be expressed, or more the duration of the treatment sought. As regards more particularly the fashion administration, it can either be a direct injection into the tissues, for example at the level of the tumors, or the circulatory pathways, either a treatment of cells in culture followed by their re-implantation in vivo, by injection or graft. The fabrics concerned in the context of the present invention are for example the muscles, the skin, brain, lungs, liver, spleen, bone marrow, thymus, heart, the lymph, blood, bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissues, etc. Advantageously, the transfected tissues are muscles and lungs.
The invention further relates to a method for transferring nucleic acids.
in cells comprising the following steps (1) bringing the nucleic acid into contact with a compound of formula general (I) such as defined above, to form a nucleolipid complex, and (2) bringing the cells into contact with the nucleolipid complex formed in (1).
Contacting the cells with the nucleolipid complex can be performed by incubating the cells with said complex (for in vitro uses or ex vivo), or by injection of the complex into an organism (for uses in 5 vivo). The incubation is preferably carried out in the presence of, for example, 0.01 to 1000 µg of nucleic acid per 106 cells. For in vivo administration, of doses of nucleic acid between 0.01 and 10 mg can for example be used.
In the case where the compositions of the invention additionally contain one or 10 several adjuvants as defined above, the adjuvant (s) are previously mixed with the compound of general formula (I) according to the invention or although a nucleic acid.
The present invention thus provides a particularly advantageous method for the treatment of diseases by administration of a coding nucleic acid for 15 a protein or capable of being transcribed into a nucleic acid capable of correct said disease, said nucleic acid being associated with a compound of general formula (I) such as defined above, under the conditions defined above. More particularly, this method is applicable to diseases resulting from a deficiency in a product protein or nucleic acid, the administered nucleic acid encoding said product 20 protein or being transcribed into a nucleic acid product or constituting said product nucleic acid.
The invention extends to any use of a compound of formula (I) according to the invention for the itt vivo, ex vivo, or in vitro transfection of cells.
In addition to the foregoing arrangements, the present invention also includes 25 other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limit the scope. In particular, the Applicant proposes, without limitation, various protocols operating as well as reaction intermediaries likely to be used in work ... .. .. .. ..
.. ... . ... ... . ..
... . . ... . .. ..
. . . ..
.. ... .. .... .. ..

to prepare the compounds of general formula (I). Of course, he is at the scope of those skilled in the art to draw inspiration from these protocols and / or products intermediaries for develop analogous pigs to lead to others composed of general formula (n according to the invention.
S FIGURES
Figure 1: Structure of synthetic vectors called lipid A, lipid B and lipid C
in the present invention and described in patent application WO 97/18185 incorporated herein by reference.
Wire Schematic representation of the plasmid pXI ~ 774.
hisures 3: Phase diagram of the compound (1) / DNA nucleolipid complexes.
The binding of compound (1) to DNA was determined by following the decrease in the fluorescence (in%, 104 ° lo being the fluorescence of naked DNA) of bromide ethidium (EtBr) (symbol ~, solid line), as described according to Tax located on the right. The cut particles of complexes (in nm) is indicated on Taxe located on the left.
The x-axis represents the transfer agents / DNA charge ratio. The size of complex nucleolipids without co-lipid is represented by the symbol t in line full. The size of nucleolipid complexes containing 25% cholesterol is represented by the symbol ~ in broken line. The size of the complexes nucleolipids containing 40% DOPE is represented by the symbol ~ in broken lines. The method does not allow to determine the size of the particles beyond 3 Eun.
Fisure 4: In vitro gene transfer activity in HeLa cells of complex nucleolipid comprising the compound (1) according to the present invention without co-lipid middle bar in dark gray), with 25% cholesterol (left bar in Grey medium), and with 40% molar DOPE (light gray bar), compared to naked DNA. Only the nucleolipid complexes in which the DNA is completely saturated with the compound according to the invention and whose size East between 100 nm and 300 nm were used.
MODIFIED SHEET

WO 99/51581 PCTlFR99 / 00740 Figure 5: In vitro gene transfer activity of complexes nucleolipids formed from compound (3), in HeLa cells. In ordinate figure the expression of luciferase, expressed in pg per well transfected. On the x-axis, figure the charge ratio between compound (3) and DNA in nmoUpg. The expression was measured each time for formulations without co-lipid (micelles}, with DOPE and with cholesterol.
Figure 6: In vitro gene transfer activity of complexes nucleolipids formed from compound (5), in HeLa cells. In ordinate figure the expression of luciferase, expressed in pg per well transfected. On the x-axis, figure the charge ratio between compound (5) and DNA in nmoUUg. The expression was measured each time for formulations without co-lipid (micelles), with DOPE and with cholesterol.
Fir ~ ure 7: In vitro gene transfer activity of complexes nucleolipids formed from compound (6), in HeLa cells. In ordinate figure the expression of luciferase, expressed in pg per well transfected. On the x-axis, figure the charge ratio between compound (6) and DNA in nmol / ug. The expression has summer measured each time for formulations without co-lipid (micelles), with DOPE and with cholesterol.
FIGURE 8: In vivo gene transfer activity after direct injection into the muscle complexes containing the compound (1) according to the present invention or the compound of formula H2N (CH2) 3NH (CH2) dNH (CH2) 3NHCH2COArgN [(CH2) ~ 7CH3j2 (called lipid A ”in the following) without co-lipid (bar in dark gray), with 25%
of cholesterol (bar in medium gray), and with 40% molar of DOPE (bar in gray clear), compared to naked DNA. Only complexes in which DNA
East completely saturated in lipid and whose size is between 100 nm and 300 nm have been used .
Figure 9: The importance of the invention is illustrated by comparing the activity transfer of gene from two different lipids, the compound (1) according to the invention and the lipid A, and naked DNA via two routes of administration: intravenously (iv) and by way intramuscular (im). Only complexes in which DNA is completely saturated in lipid and whose size is between 100 nm and 300 nm have been used.
Fig-ure 10: Gene transfer activity in vivo 48 hours after injection im some nucleolipid complexes containing compounds (5) or (6) according to the present invention without co-lipid and at 0.25 / 1 charge ratio, compared to naked DNA.
The expression is expressed in pg of luciferase per ml.
Starting from the left, the bars represent: (a) negative control; (b) Naked DNA;
(c) Compound (5) and (d) compound (6).
MATERIAL AND METHODS
A \ MATERIAL
- The starting amino acids, polyamines (or their derivatives) are available commercially. This is the case for example with N- (3-aminopropyl) glycine, N- (2-cyanoethyl) glycine, or 2,4-diaminobutyric acid, or may be synthesized by conventional methods known to those skilled in the art.
- Cyclic isothioureas are also commercial products, such as through example 2-methylthio-2-imidazoline hydroiodide, or may be synthesized by conventional methods known to those skilled in the art.
- Amines substituted by a lipid (s) are commercial or else synthesized at starting from the corresponding amines and aldehydes by alkylative reduction.
- Products such as triethylamine, trifluoroacetic acid, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), dimethylaminopyridine (DMAP), benzyl chloroformate, dicarbonate di-tert-butyl are commercial products. Chloride solutions sodium and sodium carnbonate are saturated. Potassium sulfate solution East concentrated at 0.5 M.

B \ METHODS
1) Physical measures.
Proton NMR spectra were recorded on Bruker spectrometers 400 and 600 MHZ.
Mass spectra were performed on an API-MS / III.
21 Methods of huritication and analysis al Conditions for direct phase chromatography - Thin layer chromatographies (TLC) were performed on plates 0.2 mm thick Merck silica gel.
They are revealed either by UV (254 nm), ninhydrin, by vaporizing (spray light) an ethanolic solution of ninhydrin (40 mg / 100 cm3 of ethanol) to reveal amines or amides by heating to 150 ° C., with fluorescamine, spraying a solution (40 mg / 100 cm3 of acetone) to reveal the primary amines, green of bromocresol, by spraying a solution (0.1% in propanol-2) to reveal the 1 acid S, with vanillin by spraying (light spray) a solution ethanolic vanillin (3%) with 3% sulfuric acid followed by heating to 120 ° C, or to iodine in covering the plate with iodine powder.
- The column chromatographies were carried out on silica gel 60 Merck's grain size 0.063-0.200 mm.
bl HPLC purification conditions (Li ~ high fluid chromatography Performance) preparatory The apparatus is a set for liquid chromatography in fashion gradient allowing UV detection This preparatory chain is composed of following items Pump A: G1LSON model 305 fitted with a 50 SC head.
Pump B: GILSON model 303 fitted with a 50 SC head.
Injection loop. : 5 ml.
Pressure module: GILSON model 806.

Mélan ~ aeur: GII, SON model 811 C equipped with a 23 ml head.
UV detector: GIL, SON model 119 equipped with a preparatory cell.
Fraction collector: GILSON model 202 fitted with racks n ° 21 and from tube to 10 ml glass.
5 Integrator: SHIMADZU model C-R6A.
Column: Column C4 (10 mm) in stainless steel 25 cm long and 2.2cm diameter marketed by VYDAC model 214 TP 1022.
The solution of product to be purified is loaded onto the column by through the injection loop, the eluate is collected in fractions of a tube in 30 seconds. The The detector is set to the wavelengths of 220 nm and 254 nm.
the mobile phases are thus defined Solvent A Solvent B
Demineralized water 2500 cm3 Acetonitrile for HPLC 2500 cm3 Trifluoroacetic acid 2 cm3 Trifluoroacetic acid 2.5 cm3 Gradient Time in minutes% of solvent% of solvent Flow in cm '/ min AB

cl Analog chromatography techniques - HPLC (High Performance Liquid Chromatography) analyzes were carried out 15 on a Merck-Hitachi device fitted with a D 2500 computer integrator HITACHI, an AS-2000A autosampler, an L-6200A intelligent pump, and a detector UV-L-4000 screw with adjustable wavelength set to 220 nm.
Cotton for analytical separations are Browlee columns in steel stainless steel 3 cm long and 0.46 cm in diameter sold by 20 APPLIED BIOSYSTEM.

The stationary phase consists of fAquapore Butyl? micron. The phases mobile are water (with trifluoroacetic acid) and facetonitrile (with acid trifluoroacetic). The injections are 20 μl of a solution of approximately 1 mg / cm3 in a 0.1 cm3 loop valve. The flow rate for analyzes is set between 1 cm '/ min and 4 cm3 / min. The pressure is around 180 bars.
The separation conditions are summarized below Solvent A Solvent B
Demineralized water 2500 cm3 Acetonitrile for HPLC 2500 cm3 Trifluoroacetic acid 2 cm3 Trifluoroacetic acid 2.5 cm3 Gradient Time in minutes% solvent% solvent Flow rate in cm / minute AB

35.1 60 40 4 36.1 60 40 4 3 6.2 60 40 2 EXAMPLES
SYNTHESIS OF COMPOUNDS ACCORDING TO THE INVENTION
EXAMPLE 1 Synthesis of Compound (1) (N-dioctadecylcarbamoylmethyl-2- [3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butyiamino] -propylamino} -acetamide) from of 15 cationic lipid of condensed formula NH2 (CH2) 3NH (CH2) a NH (CH2) 3NHCH2COGIyN [(CH2), ~ CH ~] 2 called “lipid B” in the following (including preparation was described in patent application WO 97/18185 and whose structure is shown in Figure 1).

0.784 mmol of lipid is dissolved in a flask equipped with a magnetic bar B
in 25 cm3 of methanol, and 10.21 mmol of triethylamine are added. Then we sinks slowly (S minutes) on the mixture a solution of O-Methylisourée and acid sulfuric (1.173 mmol) in water (9 cm3). The mixture is kept at 50 ° C in S an oil bath for twenty hours.
Then, the mixture is concentrated to dryness on a rotary evaporator. We solubilize dry extract with a solution of water (4 cm3), ethanol (4 cm3), and acid trifluoroacetic (1 cm3). This solution is injected twice in preparative HPLC.
The interesting fractions (determined by analytical HPLC} are grouped, frozen and freeze-dried. 194 mg (0.163 mmol) of product are thus obtained.
salified.
Yield: 20.8 Analytical HPLC: Rt = 15.99 minutes.
1H NMR spectrum (400 MHz, (CD3) ZSO d6, b in ppm): 0.88 (t, 3 = 6.5 Hz, 6H
CH3 fatty chains); 1.24 (mt, 60H: central CH2 of the fatty chains);
of 1.35 to 1.70 (mt, 4H: 1 CHZ of each fatty chain); 1.57 (mt, 4H: (CHZ) a butyl centrals); 1.88 and 1.96 (2 mts, 2H each: central CHZ of propyl and CH2 central of the cycle); from 2.85 to 3.35 (2 mts, 16H in total: the 8 NCH2);
3.81 (s wide, 2H: NCHZCON); 4.03 (d, J = 5 Hz, ZH: glycyl CONCH2CON); 7.25 and 7.84 (respectively s and s broad, 1H each: the 2 NH of the cycle); 8.61 (t, J =
5.5 Hz, 1H
NHCO); 8.70 and 9.02 (2 mfs, 1H each: 2 NH).
MH + = 846 Example 2 Synthesis of Compound 12) (N-ditetradecylcarbamoylmethyl-2- {3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino] -propyiamino} -acetamide) from of compound of condensed formula NH2 (CH2), NH (CH2) 4 NH (CH2) 3NHCH2COGIyN [(CH2) lzCH3] 2 called “lipid C” in the following (including preparation has been described in patent application WO 97/18185 and whose structure is shown in Figure 1).
1.036 mmol of lipid is dissolved in a flask equipped with a magnetic bar VS
in 30 cm3 of methanol, and 13.13 mmol of triethylamine are added. Then we sinks slowly (5 minutes) on the mixture a solution of O-Methylisourée and acid sulfuric (1.554 mmol) in water (9 cm3). The mixture is kept at 50 ° C in an oil bath for about twenty hours. Then, the mixture is concentrated dry at rotary evaporator. The dry extract is solubilized with a water solution (3 cm3), ethanol S (2 cm3), and trifluoroacetic acid (0.5 cm3). This solution is injected in HPLC
preparatory.
The interesting fractions (determined by analytical HPLC) are grouped, frozen and freeze-dried. Finally, 218 mg (0.2022 mmol) of salified product.
Yield: R = 19.5 Analytical HPLC: Rt = 10.76 minutes.
1H NMR spectrum (400 MHz, (CD3) ZSO d6, 8 in ppm): 0.88 (t, J = 7 Hz, 6H
CH3 fatty chains); from 1.15 to 1.40 (mt, 44H: (CHZ) "central of chains fatty); 1.45 and 1.50 to 1.65 (2 mts, 2H each: 1 CHZ of each chain oily);
1.59 (mt, 4H: the central 2 CH2 of butyl); 1.91 and 1.97 (2 mts, 2H each : CH2 central propyls); from 2.85 to 3.10 (mt, l OH: the 2 NCH2 of butyl - the one of the 2 propyls - and 1 of the 2 NCH2s of the other propyl); 3.23 and 3.30 to 3.50 (2 mts, respectively SH and 1H: the other NCH2 of the other propyl and NCH2 of the chains fatty); 3.79 (mf, 2H: NCHZCON); 4.03 (d, J = S Hz, 2H: CONCH2CON of glycyl); 7.27 and from 8.40 to 9.30 (respectively s broad and mf, 2H and 4H:
NH2 ,.
CF3C00 ', NH + CF3C00- and = NH); 7.88 and 8.61 (respectively s and s broad, 1H
each: respectively NHC = N and CONH).
MH + = 734 Example 3: synthesis of the compound (3) (2- (3- {4- [3- (4,5-dihydro-1H-imidazol-2-ylamino) -propylaminoJ-butylamino} -propylamino) -N-ditétradécylcarbamoylméthyl-acetamide) from lipid C (see Example 2 and Figure 1 for its structure).
In a flask equipped with a bubble counter and a magnetic bar, we dissolve 0.36 mmol of 2-methylmercapto-2-imidazolinium iodide in 0.36 cm3 of sodium hydroxide 1N. To this solution is added 0.36 mmol of lipid C previously dissolved in 1.44 cm3 of 1N sodium hydroxide, 5 cm3 of water, and 4 cm3 of ethanol. The mixture is maintained under agitation until the methyl mercaptan release stops (24 hours). The mixed is then concentrated to dryness on a rotary evaporator. Solubilize the dry extract with a solution of water (4 cm3), ethanol (4 cm3), and trifluoroacetic acid (0.5 cm3).
This solution is injected twice in preparative HPLC.
The interesting fractions (determined by analytical HPLC) are grouped, frozen and freeze-dried. Finally, 213 mg (0.1727 mmol) of salified product.
Yield: R = 48 HPLCa ~ ,, n ~ q "~: Rt = 8.90 minutes.
1 H NMR spectrum (400 MHz, (CD3} ZSO d6 with the addition of a few drops of CD3COOD d4, 8 in ppm): 0.87 (t, J = 7 Hz, 6H: CH3 of the fatty chains); of 1; 15 at 1.40 (mt, 44H: (CH2) 1, central of the fatty chains); 1.45 and 1.55 (2 mts, each: 1 CH2 of each fatty chain); 1.65 (mt, 4H: the 2 central CH2 of the butyl); from 1.80 to 1.95 (mt, 4H: central CH2 of propyls); from 2.80 to 3.05 (mt, lOH
the 2 NCH2 of butyl - the 2 NCHZ of one of the 2 propyls - and 1 of the 2 NCHZ of the other propyl); 3.24 (mt, 6H: the other NCH2 of the other propyl and NCHZ of the chains fat) 3.56 (s, 2H: NCHZCON); 3.62 (s, 4H: NCH2CHZN); 4.02 (d, J = 5 Hz, 2H
CONCH2CON glycyl).
MH + = 777 Example 4: Synthesis of comeose (4) (2- (3- {bis- [3- (4,5-dihydro-1H-imidazol-2-ylamino) -propylJ-amino} -propylamino) -N-ditétradécylcarbamoylméthyl-acetamide) by the method of synthesis of "bricks".
I) SYNTH ~ SE OF BOC-GLY-DITÉTRADÉCYLAMINE (a) The Gly group whose amines are protected by Boc groups (10 mmol) and ditetradecylamine (10 mmol) are introduced into a 250 ml, and 100 cm3 of dichlaromethane are added. The mixture is stirred until dissolution complete. 30 mmol of N-ethyldüsopropylamine (DIEA) and 11 mmol of phosphonium benzotriazol-1-yloxytrisdimethylamine (BOP) are then added. PH is maintained at 10 with DIEA, and the mixture is stirred for 2 hours. When the reaction is complete, (followed by TLC and / or HPLC), the dichloromethane is evaporated and WO 99/51581 PCT / FR99 / 00'140 the solid obtained is taken up in ethyl acetate (300 cm3). The sentence organic is washed with potassium sulphate solution (4 times 100 cm3), carbonate of sodium (4 times 100 cm3), and sodium chloride (4 times 100 cm3).
organic is then dried over magnesium sulfate, filtered and evaporated under vacuum.
S Product (a) is obtained with a yield of 93%.
TLC: Rf = 0.9 (CHCIs / MeOH, 9: 1) MH +: 567 II) SYNTH ~ SE OF [Z-NH (CHZ) 3] aN- (CH2) 3-NH-Boc-CH2-COOH (b) 1) Synthesis of NC- (CH =) Z-NH-Boc-CHi-COOH (c) 10 The amine of N- (cyanoethyl) -glycine (0.1 mol / amine, commercial) is dissolved in 1N sodium hydroxide (200 cm3 / amine) and dioxane (200 cm3). The solution is stirred in an ice bath, then added dropwise solution of O- (t-butoxucarbonyl) Z or p-chlorobenzyloxycarbonyl (C1Z, 0.14 mol / amine) in 200 cm3 of dioxane. The pH is maintained at a value 15 greater than 9. Then the mixture is stirred at about 20 ° C for one night. The dioxane is evaporated under vacuum, then the mixture is acidified to pH 3 using of a potassium sulfate solution. What is insoluble is extracted with acetate ethyl (3 times 100 cm3) then washed with sodium chloride solution (2 times 100 cm3). The organic phase is dried over magnesium sulfate, filtered and 20 evaporated in vacuo. Product (c) of formula NC- (CH2) 2-NH-Boc-CH2-COOH
is obtained with a yield of 98%.
TLC: Rf = 0.66 (CHC13 / MeOH, 8: 2) MH +: 229 2) Synthesis of NHz- (CHZ) 3-NH-Boc-CHI-COOH (d) 25 50 mmol of product (c) is introduced into a 1 liter stainless steel autoclave of formula NC- (CH2) 2-NH-Boc-CH2-COOH. We prepare at the same time in a beak a solution of 10 cm3 of ethanol (95%) and 3.3 g of sodium hydroxide (80 mol).
When the soda is dissolved, 2 cm3 of Raney nickel are introduced onto coal. The autoclave is closed. The initial hydrogenation pressure is 30 52 bar approximately, and it drops to approximately 48.5 bar overnight temperature ambient (20 ° C). The suspension is filtered on paper, the filter is washed with ethanol WO 99! 51581 PCT / FR99 / 00740 (4 times 25 cm '), and the filtrates are concentrated to dryness under vacuum. We obtain the product (d) which is used without further purification in the following.
CÇM: Rf = 0.12 (CHCl3 / MeOH, 6: 4) MH +: 233 3) Synthesis of [NC (CH =) Z] ZN- (CH =) 3-NH-Boc-CH2-COOH (e) In a flask, the product (d) of formula NHz- (CHz) 3-NH-Boc-CHz-COOH
(0.05 mol) and soda (0.1 mol) are dissolved in 150 cm3 of water. The solution is cooled in an ice bath. Under strong agitation, we sink slowly acrylonitrile (0.12 mol) keeping the temperature of the mass below 20 ° C. The reaction mixture is kept overnight at temperature ambient (20 ° C). Then the mixture is maintained at 50 ° C for 2 hours. The solvent is evaporated under vacuum, then the mixture is acidified to pH 3 with a potassium sulfate solution. What is insoluble is extracted with acetate ethyl (3 times 200 cm3), then washed with sodium chloride solution (2 times 100 cm3). The organic phase is dried over magnesium sulfate, then filtered and evaporated in vacuo. The "crude" is possibly purified on a silica column. The product is obtained with a yield of 50%.
TLC: Rf = 0.75 (CHCl3 / MeOH, 6: 4) MH +: 339 4) Synthesis of [Z-NH- (CH2) 3] ZN- (CH2) 3-NH-Boc-CHI-COOH (b) The product (e) of formula is introduced into a 1 liter stainless steel autoclave [NC (CHz) z] zN- (CHz) 3-NH-Boc-CHz-COOH (50 mmol). We prepare at the same time time in a beaker a solution of 10 cm3 of ethanol (95%) and 3.3 g of soda (80 mol). When the soda is dissolved, this solution is introduced in the autoclave. A stream of nitrogen is passed through the autoclave and 2 are introduced.
cm3 Raney Nickel on carbon. The autoclave is closed. Initial pressure of hydrogenation is around 52 bar, and it goes down to around 48.5 bar in a overnight at room temperature (20 ° C). The suspension is filtered on paper, filter is washed with ethanol (4 times 25 cm3), and the filtrates are concentrated to dry under vacuum. We obtain a white solid which is used without others purifications after a CCM analysis.

TLC: Rf = 0.14 (CHC13 / MeOH, 6: 4) The solid obtained above is dissolved in 1N sodium hydroxide (200 cm3 / amine) and dioxane (200 cm3). The solution is stirred in a ice, then a solution of (t-butoxycarbonyl) ZO is added dropwise or of p-chlorobenryloxycarbonyl (0.14 moUamine) in 200 cm3 of dioxane. PH
is maintained at a value greater than 9. Then, the mixture is stirred at room temperature (20 ° C) overnight. The dioxane is evaporated under vacuum, then acidify the mixture to pH 3 with the aid of a sulfate solution potassium. What is insoluble is extracted with ethyl acetate (3 times cm3) then washed with a sodium chloride solution (2 times 100 cm3). The organic phase is dried over magnesium sulfate, filtered and evaporated under empty. The products are analyzed by CCM and / or HPLC.
The crude product is purified on a silica column (dichloromethane / methanol, 8: 2).
Product (b) is obtained with a yield of 66% relative to product (d).
TLC: Rf = 0.42 (CHC13 / MeOH, 6: 4) MH +: 615 III) SYNTI ~ SE OF [Z-NH (CHZ) 3] 2-N- (CH2) 3-NH-Boc-CHZ-COGIyN [(CH2) 13-CHs] 2 (( Product (a) whose amines are protected by Boc groups (1 mmol) East introduced into a balloon equipped with a magnetic bar. 30 cm 'of acid trifluoroacetic at 4 ° C are added, then the solution is stirred during one hour.
When the reaction is complete (monitored by TLC and / or HPLC), the acid trifluoroacetic is evaporated under vacuum then the product is dried by coevaporation with 3 times 30 cm3 ethyl ether.
HPLC: Rt = 12.86 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]). .
The product obtained (Gly-ditétradécylamine, 10 mmol} and the product (b) (10 mmol) are introduced into a 250 cm3 flask, dicloromethane (100 cm3) is added and the mixture is stirred until complete dissolution. 30 mmol of N-ethyldüsopropylamine (DIEA) and 11 mmol of BOP hexafluorophosphate are then added. PH is maintained at 10 with DIEA and the mixture is stirred for two hours.
When the reaction is complete (monitored by TLC and / or HPLC), dichloromethane is evaporated and the solid obtained is taken up in ethyl acetate (300 cm3). The sentence organic is washed with potassium sulphate solution (4 times 100 cm3), carbonate of sodium (4 times 100 cm3), and sodium chloride (4 times 100 cm3). The sentence organic is dried over magnesium sulfate, filtered and evaporated in vacuo.
The products are used without other purifications. The product (f) is obtained with a yield of 75% after purification on a silica column (dichloromethane / methanol, 8: 2).
TLC: Rf = 0.86 (CHC13 / MeOH, 8: 2) HPLC: Rt = 17.44 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
I '~ SYNTH ~ SE OF [NHZ (CH2) 3] 2-N- (CHZ) 3-NH-BOC-CH2-COGIyN [(CHz) j3-CH3] 2 (g) The product (f) whose amines are protected, is introduced into a flask equipped with a magnetic bar and dissolved in 10 cm3 of methanol per gram of product.
Of palladium on carbon (10%, 1 g / g of product) and ammonium formate (1 g / g of product) are added at room temperature. Hydrogenolysis is followed by HPLC.
After two hours, the reaction is complete, the mixture is filtered, and the filter washed with 3 times 10 cc of methanol per gram of product. Bi-distilled water is added and the solution is frozen and lyophilized, or the filtrate is concentrated to dryness and the solid is taken up in ethyl acetate (300 cm3). The organic phase is washed with a sodium carbonate solution (2 times 100 cm '), and a chloride solution of sodium (2 times 100 cm3), then it is dried over magnesium sulfate, filtered and evaporated in vacuo. The products are analyzed by HPLC and are used without other purifications. The product (g) is obtained with a yield of 40% relative to at product (f).
HPLC: Rt = 9.62 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).

WO 99! 51581 PCT / FR99 / 00740 MH +: 795.
V) SYNTHESIS OF THE COMPOUND (4) the product (g) which contains the primary amine to be modified (1 mmoamine) is solubilized in dichloromethane (10 cm3), then 2- hydroiodide is added methylthio S imidazoline (1.2 mmol / amine) and triethylamine (1.3 mmol / amine). The mixture is stirred at room temperature (20 ° C) until the release of sulfide methyl. At the end of the reaction (followed by HPLC), the dichloromethane is evaporated under vacuum.
The product obtained, the amines of which are protected by Boc groups, (1 mmol) is introduced into a balloon equipped with a magnetic bar. 30 cm3 of acid trifluoroacetic at 4 ° C are added, then the solution is stirred during one hour.
When the reaction is complete (monitored by TLC and / or HPLC), the acid trifluoroacetic is evaporated under vacuum then the product is dried by coevaporation with 3 times 30 cm3 ethyl ether.
The product obtained is purified by preparative HPLC and the fractions analyzed.
through HPLC. The compound (4) according to the present invention is thus obtained with a yield 34%.
HPLC: Rt = 10.07 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
1 H NMR spectrum (400 MHz, (CD3) 2S0 d6 at a temperature of 383K, d en ppm): 0.92 (t, J = 7 Hz, 6H: CH3 of the fatty chains); from 1.25 to 1.45 (mt, (CH2), 1 central fatty chains); 1.57 (mt, 4H: 1 CH2 of each chain oily) from 1.70 to 1.90 (mt, 6H: central CHZ of propyls); from 2.50 to 3.40 (mt, 16H:
the 2 NCH2 of propyls and NCHZ of fatty chains); 3.68 (s, 8H: both NCH2CH2N) ; 3.72 (br s, 2H: NCHZCON); 4.06 (s, 2H: glycyl CONCHZCON).
MH +: 831 Example 5: Synthesis of compound (5): (N-ditétradécylcarbamoylméthyl-2- {3- [3-(1,4,5,6-tetrahydropyrimidin-2-ylamino) -propylamino] propylamino} -acetamide) over there method of synthesis of "bricks".

WO 99/51581 PCTlFR99 / 00740 I) SYNTH ~ SE OF BOC-GLY-DITÉTRADÉCYLAMINE (a) We operate in the same way as in the previous example. Product (a) is got with a yield of 93%.
CÇM: Rf = 0.9 (CHC13 / MeOH, 9: 1) 5 MH +: 567 II) SYNTH ~ SE OF Z-NH (CH2) 3-N-Boc- (CHZ) ~ -N-Boc-CH2-COOH (b) 1) Synthesis of NC- (CH2) 2-NH-Boc-CH2-COOH (c) The procedure is the same as above in example 4. The product (c) is obtained with a yield of 98%.
10 CCM: Rf = 0.66 (CHCI3 / MeOH, 8: 2) MH +: 229 2) Synthesis of NHZ- (CHz) 3-NH-Boc-CHI-COOH (d) The product (d) is obtained in the same way as previously in example 4.
TLC: Rf = 0.12 (CHCl3 / MeOH, 6: 4) 15 MH +: 233 3) Synthesis of NC (CHZ) ZN-Boc- (CHZ) 3-NH-Boc-CHZ-COOH (e) In a flask, the product (d) (0.05 mol) and soda (0.1 mol) are solubilized in 150 cm3 of water. The solution is cooled in an ice bath. Sous vive stirring, acrylonitrile (0.05 mol) is poured in slowly, keeping the Mass temperature below 20 ° C. The reaction mixture is maintained overnight at room temperature (20 ° C).
The solvent is evaporated under vacuum, then the mixture is acidified to pH 3 with a potassium sulfate solution. What is insoluble is extracted with (acetate ethyl (3 times 200 cm3), then washed with sodium chloride solution (2 25 ~ times 100 cm3). The organic phase is dried over magnesium sulfate, then filtered and evaporated in vacuo. The product obtained is optionally purified sure a silica column.
The product obtained (0.1 mol / amine) is solubilized in 1N sodium hydroxide (200 cm3 / amine) and dioxane (200 cm '). The solution is stirred in a 30 ice, then a solution of (Boc) 20 or p- is added dropwise chlorobenzyloxycarbonyl (0.14 moUamine) in 200 cm3 of dioxane. PH is maintained at a value greater than 9. Then, the mixture is stirred at temperature room temperature (20 ° C) overnight. The dioxane is evaporated under vacuum, then we acidifies the mixture to pH 3 with a potassium sulphate solution. This who is insoluble is extracted with ethyl acetate (3 times 100 cm3) then washed with a sodium chloride solution (2 times 100 cm3). The organic phase is dried over magnesium sulfate, filtered and evaporated in vacuo. Products are analyzed by CCM and / or HPLC.
The product (e) is thus obtained with a yield is 93%.
TLC: Rf = 0.75 (CHCI ~ / MeOH, 8: 2) MH +: 386 4) Synthesis of Z-NH- (CHZ) 3-N-Boc- (CHZ) 3-N-Boc-CHz-COOH (b) The product (e) (SO mmol) is introduced into a 1 liter stainless steel autoclave.
We at the same time prepare in a beaker a solution of 10 cm3 of ethanol (95%) and 3.3 g of sodium hydroxide (80 mol). When the soda is dissolved, we introduce this solution in (autoclave. A stream of nitrogen is passed through the autoclave and introduces 2 cm3 of Raney Nickel on carbon. The autoclave is closed. The initial hydrogenation pressure is about 52 bar, and it drops to 48.5 bar approximately overnight at room temperature (20 ° C). The suspension is filtered on paper, the filter is washed with ethanol (4 times 25 cm3), and the filtrates are dry concentrated in vacuo. A white solid is obtained which is used without other purifications after TLC analysis.
TLC: Rf = 0.14 (CHC13 / MeOH, 6: 4) The product obtained (0.1 moUamine) is dissolved in 1N sodium hydroxide (200 cm3 / amine) and dioxane (200 cm3). The solution is stirred in a bath of ice, then a p-chlorobenzyloxycarbonyl solution is added dropwise (0.14 mol / amine) in 200 cm3 of dioxane. The pH is maintained at a value greater than 9. Then the mixture is stirred at room temperature (20 ° C) during a night. The dioxane is evaporated in vacuo, then the mixture is acidified to pH 3 at (using potassium sulphate solution. What is insoluble is extracted with ethyl acetate (3 times 100 cm3) then washed with a chloride solution of sodium (2 times 100 cm '). The organic phase is dried over sulphate magnesium filtered and evaporated under vacuum. The product obtained is purified on a silica column (dichloromethane / methanol, 9: 1). Products are analyzed S by CCM and / or HPLC. Product (b) is obtained with a yield of 32 relative to product (d).
TLC: Rf = 0.63 (CHCl3 / MeOH, 9: 1) MH +: 523 III) SYNTH ~ SE OF 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) In a flask with stirring and a stream of nitrogen, 3,4,5,6-tetrahydro-2-pyrimidinethiol (0.0103 mol) is charged, and 5 cm 'of methanol and 0.65 cm' iodide methyl (0.0105 mol) are added. The mixture is brought to reflux for 1 hour then is left to cool to room temperature (20 ° C). The product is precipitated by addition of S cm3 of ethyl ether. The precipitate is filtered and then washed with ether ethyl. The product is then dried overnight under a pressure of 34 mbar.
1.5 g (0.0041 mol) of product (VI) are obtained, ie a yield of 40%.
TLC: Rf = 0.25 (CHCl3 / MeOH, 9: 1) MH +: 131 d) SYNTH ~ SE OF Z-NH (CH2) 3-N-Boc (CH2) 3-N-Boc-CHZ-COGIyN [(CH2) i3-CHa] 2 (g) Product (a) whose amines are protected by Boc groups (1 mmol) East introduced into a balloon equipped with a magnetic bar. 30 cm3 of acid trifluoroacetic at 4 ° C are added, then the solution is stirred during one hour.
When the reaction is complete (monitored by TLC and / or HPLC), the acid trifluoroacetic is evaporated under vacuum and then the product obtained is dried by coevaporation with 3 times cm3 of ethyl ether.
HPLC: Rt = 12.86 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]}.
The product obtained (10 mmol) and the product (b) (10 mmol} are introduced into a ball 30 of 250 cm3. Dichloromethane (100 cm ~) is added and the mixture is stirred until complete dissolution. 30 mmol of DIEA and 11 mmol of BOP are then added.
The pH is maintained at 10 thanks to the DIEA and the mixture is stirred for two hours.
When the reaction is complete (monitored by TLC and / or HPLC), dichloromethane East evaporated and the solid obtained is taken up in ethyl acetate (300 cm3).
The sentence organic is washed with potassium sulfate solution (4 times 100 cm3), of sodium carbonate (4 times 100 cm3), and sodium chloride (4 times 100 cm3). The organic phase is dried over magnesium sulfate, filtered and evaporated under empty.
The products are used without other purifications.
After purification on a silica column (dichloromethane / methanol, 8: 2), get the product (g) with a yield of 85%.
TLC: Rf = 0.9 (CHC13 / MeOH, 9: 1) HPLC: Rt = 19.79in, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
V) SYNTH ~ SE OF NHZ (CHZ) 3] 2-N-Boc- (CHZ) 3-NH-Boc-CH2-COGIyN [(CH2) 13-CH3] 2 (h) The product (g) is introduced into a flask equipped with a magnetic bar and dissolved in 10 cm3 of methanol / g of product. Palladium on carbon (10%, lg / g of product) and ammonium formate (1 g / g product) are added to temperature ambient (20 ° C). The hydrogenolysis is followed by HPLC. After two hours, the reaction is completed, the mixture is filtered, and the filter washed with 3 times 10 cm3 of methanol / g of product. Bi-distilled water is added, and the solution is frozen and freeze-dried, or well the filtrate is concentrated to dryness and the solid is taken up in acetate ethyl (300 cm3). The organic phase is washed with a sodium carbonate solution (2 time 100 cm3), and a sodium chloride solution (2 times 100 cm3), then is dried over magnesium sulfate, filtered and evaporated in vacuo. The products are analyzed by HPLC and are used without further purification. The product (h) is obtained with a yields of 93% compared to the product (g).
TLC: Rf = 0.42 (CHCl3 / MeOH, 6: 4) HPLC: Rt = 14.66 min, (H20 / MeCN: 3 min [40160], 3-20 min [0/100], 35 min [0/100]).

MH +: 838 VI) SYNTHESIS OF THE COMPOUND (5) The product (h) containing the primary amine to be modified (1 mmoamine) is solubilized in dichloromethane (10 cm3), then the product (f) (1.2 mmol / amine) and the triethylamine (I, 3 mmol / amine). The mixture is stirred at room temperature (20 ° C) until the release of methyl sulfide. At the end of the reaction (followed by HPLC}, the dichloromethane is evaporated under vacuum.
The product obtained is purified by preparative HPLC and the fractions analyzed.
through HPLC. Compound (5) is thus obtained with a yield of 38%.
HPLC: Rt = 8.42 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
1H NMR spectrum (400 MHz, (CD3) 2S0 db, 8 in ppm): 0.86 (t, J = 7 Hz, 6H
CH3 fatty chains); from 1.10 to 1.35 (mt, 44H: (CH2), ~ central of chains fatty); 1.44 and 1.53 (2 mts, 2H each: 1 CH2 of each fatty chain);
from 1.80 to 2.00 (mt, 6H: central CHZ of propyls and CH2 of 1,4,5,6-tetrahydro-pyrimidine);
from 2.80 to 3.10 (mt, 10H: NCH2 of propyls and NCH2 of 1,4,5,6-tetrahydro-pyrimidine); 3.15 to 3.45 (mt: 6H corresponding to = NCHZ of 1,4,5,6-tetrahydro-pyrimidine and NCHZ fatty chains); 3.81 (mf, 2H
NCH2CON); 4.04 (d, J = 5 Hz, 2H: glycyl CONCH2CON); 7.89 - 8.62 - 8.75 and

9.01 (4 mfs, 8H in total: the exchangeable and OH of CF ~ COOH}.
MH +: 720 Example 6: Synthesis of the compound (6): (N-dioctadecylcarbamoylmethyl-2- {3- [3-(1,4,5,6-tetrahydropyrimidin-2-ylamino) -propylamino] propylamino} -acetamide) over there method of synthesis of "bricks".
I) SYNTH ~ SE OF BOC-GLY-DITÉTRADÉCYLAMINE (a) We operate in the same way as in the previous example. Product (a) is got with a yield of 93%.
TLC: Rf = 0.9 (CHC13 / MeOH, 9: 1) MH +: 567 II) SYNTH ~ SE OF Z-NH (CH2) 3-N-Boc- (CH2); - N-Boc-CH2-COOH (b) 1) Synthesis of NC- (CH =) 2-NH-Boc-CHI-COOH (c) The procedure is the same as above in example S. The product (c) is obtained with a yield of 98%.
TLC: Rf = 0.66 (CHCI3 / MeOH, 8: 2) 5 MH +: 229 2) Synthesis of NH = - (CH ~) 3-NH-Boc-CHZ-COOH (d) The product (d) is obtained in the same way as previously in Example 5.
TLC: Rf = 0.12 (CHCl3 / MeOH, 6: 4) MH +: 233

10 3) Synthesis of NC (CH2) 2-N-Boc- (CH =) 3-NH-Boc-CHZ-COOH (e) The procedure is the same as above in example 5. The product (e) is thus obtained with a yield is 93%.
TLC: Rf = 0.75 (CHCI ~ / MeOH, 8: 2) MH +: 386 4) Synthesis of Z-NH- (CH2) 3-N-Boc- (CH2) 3-N-Boc-CHi-COOH (b) We operate in the same way as previously in Example 5. We obtain a white solid which is used without further purification after TLC analysis.
TLC: R f = 0.14 (CHCI3 / MeOH, 6: 4) The product obtained is used in the same way as above in order to 20 protect the terminal amine with a benzyloxycarbonyl group. The product (b) is thus obtained with a yield of 32% relative to the product (d).
TLC: Rf = 0.63 (CHCh / MeOH, 9: 1) MH +: 523 III) SYNTH ~ SE OF 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) 25 The operation is carried out in the same way as above in Example 5. We obtain so 1.5 g (0.0041 mol) of product (f), ie a yield of 40%.
TLC: Rf = 0.25 (CHCl3 / MeOH, 9: 1) MH +: 131 IV) SYNTH ~ SE OF Z-NH (CH2) 3-N-Boc (CH2) ~ -N-Boc-CHZ-COGIyN [(CH2), ~ -CH3] z 30 (g) WO 99/51581 PCT / FR99 / 00'140 The procedure is the same as above in example 5. We thus obtain the product (a) from which the Boc groups have been cleaved.
HPLC: Rt = 19.44 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [o / loo]).
This product obtained is used in the same way with the product (b) as previously in Example S. After purification on a silica column (dichloromethane / methanol, 8: 2), product (g) is obtained with a yield of 84%.
ÇCM: Rf = 0.9 (CHCl3 / MeOH, 9: 1) HPLC: Rt = 23.95 min., (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
V) SYNTH ~ SE OF NH2 (CH2); J2-N-Boc- (CHZ) 3-NH-Boc-CH2-COGfyN [(CH2), ~ -(h) The procedure is the same as above with example 5. The product (h) East obtained with a yield of 73% relative to the product (g).
TLC: Rf = 0.28 (CHCl3 / MeOH, 6: 4) HPLC: Rt = 20.59 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
MH +: 83 8 VI) SYNTHESIS OF THE COMPOUND (6) The procedure is the same as above in Example 5. The compound (6) is so obtained with a yield of 68%.
HPLC: Rt = 15.83 min, (H2O / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).
1H NMR spectrum (500 MHz, (CD3) 2S0 d6, b in ppm): 0.88 (t, J = 7 Hz, 6H
CH, fatty chains); from 1.15 to 1.35 (mt, 60H: (CH2), 5 central chains fatty); 1.46 and 1.54 (2 mts, 2H each: 1 CHZ of each fatty chain);
from 1.80 to 2.00 (mt, 6H: central CHZ of propyls and CH2 of 1,4,5,6-tetrahydro-pyrimidine);
2.85 to 3.05 (mt, IOH: NCHZ of propyls and NCH2 of 1,4,5,6-tetrahydro-pyrimidine); 3.15 to 3.45 (mt: 6H corresponding to = NCH2 of 1,4,5,6-tetrahydro-pyrimidine and NCH2 fatty chains); 3.81 (mf, 2H

NCH2CON); 4.04 (d, J = 5 Hz, 2H: glycyl CONCH2CON); 7.88 - 8.61 - 8.74 and 8.99 (4 mfs, 8H in total: exchangeable and OH of CF3COOH).
MH ~: 832 B \ USE OF TRANSFECTING AGENTS ACCORDING TO THE INVENTION
Example 7: preuaration of nucleolinid complexes This example illustrates the preparation of seion nucleolipid complexes the invention.
The compound used in this example is the compound (1) dissolved in chloroform. Amounts of 10 nmoles of compound (1) (i.e. 11.8 ug) per pg DNA
have been used. In some cases, a neutral co-lipid, Cholesterol or DOPE, is previously mixed with the compound. A thin lipid film forms when evaporate the chloroform using a light stream of argon and then it is rehydrated in a mix of 5% dextrose and 20 mM sodium chloride overnight 4 ° C. The samples are then treated with ultrasound for 5 minutes, heated to 65 ° C
for 30 minutes, and finally treated again with ultrasound for 5 minutes. We thus obtains lipid suspensions which are stored at 4 ° C.
until they are used.
The DNA used is the plasmid pXL2774 (FIG. 2) in solution in a mixture 5% dextrose and 20 mM sodium chloride at a concentration of 0.5 mg / ml or 1.0 mg / ml. Plasmid pXL, 2774 has the following characteristics - endotoxin level below 50 EU / mg, - Rate of supercoiled DNA greater than 60%, - RNA content, i.e. AIZNm, tRNA and ribosomal RNA, (determined by HPLC) less than S%, - chromosomal DNA level less than 1%, - protein content less than 1%, - osmolarity lower than IS mosmoles / kg.
The nucleolipid complexes according to the invention are prepared by mixing quickly equal volumes of DNA solution and lipid suspension as described above. The amount of compound complexed with DNA varies from 0.5 nmoles / pg of DNA at 12 nmol / µg of DNA.
Example 8: behavior of complexes formed at different charge rates This example illustrates the behavior of nucleolipid complexes according to the invention at different charge ratios. The impact of adding a co-neutral lipid is also illustrated.
The size of the complexes was first analyzed by measuring the diameter hydrodynamics by dynamic light scattering (Dynamic Laser Light Scattering) using a Coulter N4plus device. the samples are diluted 20 times in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple broadcasts. The effect of the cycloamidine group, composition lipid, and the charge ratio on the size of the complexes nucleolipids according to the invention has thus been studied.
There are three possible phases when increasing the load ratio between the compound (1) according to the invention and the DNA. These three phases determine the therapeutic potential of compound (1). Figure 3 illustrates these 3 phases for the compound (1). The same behavior can be observed for other compounds according to the invention.
At a low charge ratio, the DNA is not saturated with the compound (1). he rest still naked DNA, and the complexes are globally negatively charged.
The particles are small (between 100 and 300 nm). This phase is called "Phase AT ".
The fact that DNA is not completely saturated with compound (1) means than DNA is not completely protected by it. DNA can therefore be subjected to degradations by enrymes (DNAases). Furthermore, the complexes being overall negative, the passage of the cell membrane is difficult.
For these reasons, phase A nucleolipid complexes are effective many less in transfection.

At an intermediate charge ratio, the DNA is completely saturated by the compound (1), and the complexes are generally neutral or slightly positive.
This phase is unstable because the ionic repulsions are minimal and a phenomenon of crosslinking 'can happen. The particle size is well above of the limit detection by dynamic light scattering (much greater than 3 µm).
This unstable phase is called "phase B".
Such a size of complex is not suitable for uses in injection.
However, this does not mean that the complexes are inactive in the phase B, but they are only under a formulation that is not appropriate for their injection for pharmaceutical purposes.
At a relatively high charge ratio, the DNA is over-saturated with the compound (1), and the complexes are generally positive. Due to strong repulsions between the positive charges, this phase is stable. It is referred to as "
phase C ".
Unlike phase A, the nucleolipid complexes are in a form such that DNA is very well protected against enzymes, and their charge overall positive facilitates the passage of the cell membrane of an anionic nature.
The phase C complexes are therefore particularly suitable for use for the transfer of nucleic acids into cells.
In addition to the cycloamidine group of the compound according to the invention, the use of a neutral co-lipid has a strong impact on the stability of complexes, such as that is illustrated in figure 3. The co-lipids added are either DOPE (lipid cationic / DOPE =
3/2), or cholesterol (cationic lipid / cholesterol = 3/1). In general, adding the neutral co-lipid increases the instability of the complexes, resulting in the raise of the amount of compound required to reach phase C. This is very clearly illustrated in Figure 3 when comparing the charge ratio at which the phase It is damage in the presence and absence of co-lipid.
It should be noted that the load ratio values which delimit the three phases A, B and C depend on the compound used. So these values can vary very strongly from one compound to another.

Finally, the affinity of the compound for (DNA as a function of the ratio of load has been studied. For this, the reduction in fluorescence after adding 3 ug of ethidium bromide (EtBr) was measured. Indeed, the substitution of bromide of ethidium from DNA by the compound is an indication of binding to (DNA.
5 The formulation used is diluted 20 times to a final concentration of 25 ug of DNA / ml. The relative fluorescence measured for naked DNA is defined as being 100%. The rate of binding with the compound (1) is represented by the reduction of the relative fluorescence of (sample. Figure 3 shows that the fluorescence decreases when the load ratio increases, which means that a greater number of 10 compound (1) is available to bind to DNA (plus fluorescence decreases, plus one large amount of compound binds to DNA until reaching saturation).
In this way, it has thus been shown that the affinity of the compound (1) according to the invention for DNA is determined by the cycloamidine group, but not through the addition of a co-lipid.
Example 9: In vitro transfection with compound (1) This example illustrates the capacity of the compound (1) according to the invention to transfect (DNA in cells in vitro, compared to unformulated DNA.
24-well microplates are seeded with 60,000 HeLa cells (ATCC) per well, and transfected 24 hours later. Complexes containing 1 ~ tg 20 of DNA are diluted in 0.5 ml of DMEM culture medium (Gibco / BRL) in the absence of serum, then added to each well. The cells are incubated at 37 ° C for 4 hours. The medium containing the complexes is then removed and replaced by a mixture of DMEM and 10% fetal calf serum. Then, the cells are at re-cultured for 24 hours. Finally, the cells are lysed and tested 25 using a luciferase test kit (Promega) and a Dynex luminometer MLX.
The results shown in Figure 4 highlight the difference between the performances of naked DNA compared to the compound (1) / DNA complexes the invention completely saturated: no luciferase activity could be detected (device sensitivity less than 1 pg per well) after transfection in DNA in vitro naked, whereas the gene transfer activity of the complexes according to the invention varies from 200 pgl well at 8000 pg / well.
This example therefore clearly shows the advantageous use of the compound (1) according to (invention for transfection of irr vitro cells.
Example 10: In Vitro Transfection with Compounds (3), (5) and (6) This example illustrates the capacity of compounds (3), (5) and (6) according to the invention to transfect DNA into cells in vitro, compared to non-DNA
formula.
The transfection is carried out according to the protocol of example 9 above, in HeLa cells. The results are illustrated in Figures 5, 6 and 7. We so observe that these 3 compounds have a good level of transfection in vitro.
Example 11: In Vivo Transfection of Compound (1) This example illustrates the capacity of the compound (1) according to the invention to transfect DNA in iu vivo cells, compared to unformulated DNA and lipid A
of condensed formula NHz (CH2) 3NH (CHZ)., NH (CHZ); NHCH2COArgN [(CHZ) "CH3] 2 described in application WO 97/18185 and the structure of which is shown represented at Figure 1.
In vivo gene transfer was performed on Balb / C mice by intramuscular and intravenous administration. The formulations that have been compared are naked DNA formulations, lipid-containing formulations A, or formulations containing the compound (1) according to the invention.
In the case of intramuscular injections, each mouse received 30 μl of formulation containing 15 µg of DNA in the anterior tibia muscle. The fabrics are recovered 7 days after the injection, they are frozen and stored at -80 ° C in waiting to perform the luciferase activity tests. The luciferase activity measurements are made as in Example 8.
In the case of intravenous injections, each mouse received 200 μl of formulation containing SO pg of DNA. Tissues are recovered in this case 24 hours after injection, then frozen and stored in the same way as before.

In vivo gene transfer results are shown in Figure 8 and Figure 9. The ratio between compound (1) and (DNA is 10 nmol / pg of DNA. The ratio between the lipid A and the DNA is 4 nmol / µg of DNA.
FIG. 8 illustrates the activity ij ~ vivo in the muscle of the compound (1) according to (invention compared to naked DNA and lipid A. It can be seen that the levels of luciferase activity are equivalent between naked DNA and compound (1), this latest also having a very improved activity compared to lipid A.
transfer mechanisms involved seem different between (naked DNA and (use of compound (1) according to the present invention. Indeed, the complexes according to the invention used do not contain free DNA (phase C) and moreover, their results in vitro are much superior to those of naked DNA.
FIG. 9 compares the activities of compound (1) according to the invention, of DNA
bare and lipid A, intravenously and intramuscularly.
It is found that the transfection efficiency is roughly equivalent by track intravenously for lipid A and for compound (I). However, by way intramuscular, the transfection efficiency of compound (1) according to the invention is very significantly higher than that of Timid A.
Compared with naked DNA, the compound (1) exhibits transfection by the intravenous, in addition to transfection at least equivalent by route intramuscular.
It therefore appears that the transfer efficiency of (nucleic acid in vivo with the compound (1) according to the invention is generally greater than that with the lipid A which is a known cationic lipid and that of unformulated DNA.
Finally, it appears that the complexes according to the invention have the advantage, through compared to naked DNA transfection, to protect DNA from damage by nucleases, thereby contributing to a significant improvement in stability of formulations. The compounds of the present invention may also be used to protect DNA from damage during freeze-drying, improving there still the stability of the formulations.

Example 12: in vivo transfection of comaose (5) and (6) This example illustrates the capacity of compounds (5) and (6) to transfect from acid nucleic acid in vivo efficiently.
The same protocol as in the previous example is used. Figure 10 shows that the compound (5) and the compound (6), formulated in a ratio of charges 0.25: 1 with DNA without co-lipid, have a level of transfection in vivo greater than or equal to naked DNA 48 hours after im injection The following table gives the results obtained with compounds (S) and (6) in different formulations Report Co-lipidep composition ~ my pg / Poumondadm tration charges com os / DNA

Compos (5) 6/1 DOPE (1: 1) 254.9 1611.3 iv Compos (5) 8/1 DOPE (l: I) 535.2 3558.1 iv Compos (5) 0.5 / 1 Chol. 209.6 1330.5 im (3: 1) Compos (5) 0.5 / 1 DOPE (1: 1) 155.8 974.6 im Compos (6) 6/1 - 175.1 1098.7 iv Compos (6) 5/1 DOPE (1: 1) 407.7 2700.8 iv Compos (6) 0.5 / 1 DOPE (1: 1) 1768, 7 13 005.4 im

Claims (28)

1. Compounds, in D, L or DL form, as well as its salts, of general formula (I):

CA~Rep~R ~ (I) for which:
~ CA represents a cycloamidine group and its mesomeric forms of general formula (II):

for which:
~ m, and n are independent integers between 0 and 3 included and such that m+n is greater than or equal to 1, ~ R1 represents a group of general formula (III):

~(CH2)~Y~(*) ~ (III) for which p and q are independent integers included between 0 and 10 included, Y represents a carbonyl, amino, methylamino group, or else methylene, Y may have different meanings within the different [(CH2)pY] groups, and (*) represents either a hydrogen atom or is the place binding to the Rep group, it being understood that R1 can be bonded to any atom of the formula general (II), y including Z, and there is a single R1 group in formula (II), ~ X represents an NR2 or CHR2 group, R2 being either an atom hydrogen either the bond to the R1 group as defined previously, ~ Grouping represented :
* 1st case: a group of general formula (IV):

for which W' represents CHR''' or NR''', and R'' and R''' represent independently of each other a hydrogen atom, a methyl, or the bond to group R1 as defined above, or else * 2nd case: a group of general formula (V):

for which W' represents CHR''' or NR''', and R' and R''' represent independently of each other a hydrogen atom, a methyl, or the bond to group R1 as defined above, ~ Rep is absent or is a dispatcher of general formula (VI):

whose nitrogen atom is attached to atoms X, V, W, or Z or to the substituent Y
from group R1 depending on the case, and ~ t is an integer between 0 and 8 inclusive, ~ r is an integer between 0 and 10 inclusive, r can have meanings different within the different -NR4-(CH)r- groups, ~ R3, which may have different meanings within different groups NR4-(CH)r R3, represents a hydrogen atom, a methyl group, or a group of general formula (VII) for which u is an integer between 1 and 10 inclusive, s is an integer between 2 and 8 inclusive which may have different meanings within the different -(CH2)s-NR5 groups, and R5 is a hydrogen atom, a CA group such that previously defined, it being understood that the CA groups are independent them from each other and may be different, or else a group of formula general (VII) it being understood that the groups of general formula (VII) are independent from each other and can have different meanings, ~ R4 is defined in the same way as R3 or represents a CA group such as previously defined, it being understood that the CA groups are independent one from others and may be different, and ~ R is linked to the carbonyl function of the Rep group of general formula (VI), or if Rep is absent R is linked directly to the CA group, and represented * or a group of formula NR6R7 for which R6 and R7 represent independently of each other a hydrogen atom or an aliphatic radical saturated or not, linear or branched, optionally fluorinated, containing 1 to 22 atoms of carbon, with at least one of the two substituents R6 or R7 different from hydrogen and the other containing between 10 and 22 carbon atoms, * either a steroid derivative, * or a group of general formula (VIII) for which x is an integer between 1 and 8 inclusive, y is an integer between 1 and 10 inclusive, and either Q represents a C(O)NR6R7 group for which R6 and R7 are defined as above, either Q represents a C(O)R8 group for which R8 represents a group of formula (IX):
for which z is an integer between 2 and 8 inclusive, and R9 is a aliphatic radical saturated or not, optionally fluorinated, containing 8 to 22 carbon atoms, or a steroid derivative, and the two R6 substituents are, independently one of the other, defined as before, or alternatively R8 represents a group -O-R9 for which R9 is defined as below above. 2. Compounds according to claim 1, characterized in that the R1 group is linked either to Z or to V on the one hand and to the Rep group on the other hand by through Y. 3. Compounds according to claim 1 characterized in that the head cycloamidine CA
of formula (II) comprises 5, 6, 7, or 8 members. 4. Compounds according to claim 1, characterized in that R3 represents a atom hydrogen or methyl and R4 is as defined in claim 1, or well R3 and R4 present in formula (VI) represent hydrogen atoms, or well R4 is a hydrogen atom and R3 is a group of formula (VII) in which R5 represents a CA group 5. Compounds according to claim 1 characterized in that, in the formula (V), p and q are chosen independently of each other from 2, 3 or 4. 6. Compounds according to claim 1 characterized in that the R6 groups and R7 are the same or different and each represent aliphatic chains saturated or not, linear or branched, optionally fluorinated, containing 10 to 22 atoms of carbon. 7. Compounds according to claim 1, characterized in that the R6 groups and R7 are the same or different and each represent aliphatic chains saturated or not, linear or branched, optionally fluorinated, and containing 12, 14, 16, 17, 18, or 19 carbon atoms. 8. Compounds according to claim 1 characterized in that when R is a derivative steroid, said steroid derivative is selected from cholesterol, cholesterol, the 3-.alpha.-S-cyclo-5-.alpha.-cholestan-6-.beta.-ol, cholic acid, cholesterol formate, the chotestanylformate, 3.alpha.,5-cyclo-5.alpha.-cholestan-6.beta.-yl formate, cholesterolamine, 6-(1,5-dimethylhexyl)-3.alpha., 5.alpha.-dimethyl-hexadecahydrocyclopenta[a]cyclopropa-[2,3]cyclopenta-[1,2-f]naphtha-len-10-ylamine, or cholestanylamine. 9. Compounds according to claim 1 of formulas 10. Process for the preparation of the compounds according to claims 1 to 9 characterized in what we perform the synthesis of the bricks carrying the function(s) cycloamidine then these bricks are grafted onto lipids equipped with distributors. 11. Process for the preparation of the compounds according to claims 1 to 8 characterized in what one carries out the synthesis of the analogous lipopolyamines then one perform the cyclization to cycloamidine groups. 12. Composition characterized in that it comprises at least one compound of formula general (I). 13. Composition according to claim 12, characterized in that it comprises a compound of general formula (I) and a nucleic acid. 14. Composition according to claims 12 or 13, characterized in that it understand additionally one or more adjuvants. 15. Composition according to claim 14, characterized in that the adjuvants are one or more neutral lipids with two fatty chains. 16. Composition according to claim 15, characterized in that the lipids neutral are natural or synthetic lipids, zwitterionic or devoid of charge ionic under physiological conditions, chosen for example from the dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanol-amine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamines as well than their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galacto-cerebrosides), sphingolipids (such as in particular sphingomyelins) or Again asialogangliosides (such as in particular asialoGM1 and GM2). 17. Composition according to claim 14, characterized in that the adjuvant is a compound intervening directly or indirectly in the condensation of the acid nucleic. 18. Composition according to claim 17, characterized in that said adjuvant drift in whole or in part of a protamine, a histone, or a nucleolin and/or one of their derivatives, or consists, in whole or in part, of peptide units (KTPKKAKKKP) and/or (ATPAKKAA), the number of patterns varying between 2 and 10, and can be repeated continuously or not. 19. Composition according to claims 12 to 18, characterized in that it contains in in addition to one or more non-ionic surfactants) in sufficient quantity for stabilize the particle size of nucleolipid complexes. 20. Composition according to claims 12 to 19, characterized in that it understand a pharmaceutically acceptable carrier for an injectable formulation. 21. Composition according to claims 12 to 19, characterized in that it understand a pharmaceutically acceptable vehicle for application to the skin and/or the mucous membranes. 22. Composition according to claim 13, characterized in that said acid nucleic is deoxyribonucleic acid or ribonucleic acid. 23. Composition according to claim 22, characterized in that said acid nucleic comprises an expression cassette consisting of one or more genes of interest therapy under the control of one or more promoters and a terminator active in target cells. 24. Use of a compound according to one of claims 1 to 9 for make a medicine to cure disease. 25. Use of a compound according to one of claims 1 to 9 for make a drug intended to treat diseases by transfer of nucleic acids in the cells intramuscularly. 26. Method for transferring nucleic acids into cells comprising the steps following:
(1) contacting the nucleic acid with a compound of formula general (I) such as defined above, to form a nucleolipid complex, and (2) bringing the cells into contact with the nucleolipid complex formed in (1). 27. Method of transferring nucleic acids into cells according to the claim 26 characterized in that said nucleic acid and/or said compound are previously mixed with one or more adjuvants. 28. Method of treating diseases by administering a nucleic acid coding for a protein or capable of being transcribed into a nucleic acid capable of to correct said diseases, said nucleic acid being associated with a compound of formula general (I).