MXPA00008970A - 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

NEW TRANSFER AGENTS. OF NUCLEIC ACIDS, COMPOSITIONS THAT CONTAIN THEM AND THEIR APPLICATIONS FIELD OF THE INVENTION The present invention relates to novel compounds useful as nucleic acid transfer agents in cells. These new compounds resemble more closely the family of the 1 ipopoli amine s, and comprise at least one cyclic amidine function. They are useful for the transfection of nucleic acids in different cell types, both in vi tro co or ex vi vo or vi n vi.

BACKGROUND OF THE INVENTION With the development of biotechnologies, the possibility of efficiently transferring nucleic acids in cells is seen as a necessity. It can be about the transfer of nucleic acids in cells i n vi t ro, by RER .: 122206 example, for the production of recombinant proteins, or for laboratory, for the study of the regulation of gene expression, gene cloning, or any other manipulation involving DNA. One can also try to transfer nucleic acids in the cells, for example, for the creation of transgenic animals, the realization of vaccines, market studies or also for therapeutic studies. It may also be the transfer of nucleic acids in ex vivo cells, in bone marrow grafts, in immuno therapies or in other methods involving the transfer of genes in cells taken as a sample from an organism. , in view of its subsequent readministration. Different types of synthetic vectors have been developed to improve the transfer of nucleic acids in cells. Among these vectors, cationic lipids possess interesting properties. These vectors consist of a polar part, cationic, interactive with the nucleic acids, and a hydrophobic lipid part, which favors cell penetration. Particular examples of cationic lipids are mainly the onocationic lipids (DOTMA): Lipo f ect in®), certain cationic detergents (DDAB), the 1-ipopolyamines and in particular the dioctadecyl idogli cil spermine (DOGS) or the 5-carboxiespermi lick of palmitoylphosphidylethanolamine (DPPES), the preparation of which has been described, for example, in patent application EP 394 111. Another family of lipopolyamines is represented by the compounds described in the patent application WO 97/18185 incorporated herein. as reference, and are illustrated in Figure 1. But currently, injections into tissues, and mainly muscles, are often made with unformulated DNA in order to facilitate their entry into cells, the association with synthetic vectors that They lead to complexes of very important size to be incorporated into the cells. It is one of the main problems that the present invention sets out to solve. Indeed, the compounds according to the invention have the intended advantage of presenting a transfection level in vi ve in the muscle at least equivalent to that obtained with the unformulated DNA and in any case a very good level of transfection in the others. tissues. The association with a compound according to the invention protects the DNA from degradations by nucleases and / or deterioration during lipophylation, which contributes to significantly improve the stability of the nuci-olide formulations. In addition, an association allows a slow controlled release of nucleic acids. Therefore, the compounds according to the present invention belong to the family of cationic lipids and carry an original cationic region which gives them improved properties, mainly a reduced cytotoxicity in relation to the cationic vectors of the prior art. In fact, this cationic part is represented more precisely by one or several specific polyamine (s), which support one or more cyclic amidine functions which, very likely to have an effect, "delocalize" the positive charges. , which make the compound less cationic overall, with the beneficial effects that are known on the plane of oxidation.

DESCRIPTION OF THE INVENTION Thus, a first object of the invention refers to new compounds in the form of D, L, or DL, of the general formula (I): CA-Rep-R (I) for 1 to which: (1) CA represents a cycloamidine group and its mesomeric forms of the general formula (II): for which m • m, and n are independent integers between 0 and 3 even and such as m + n is greater than or equal to 1, Ri represents a group of the general formula (III - [• (CH * -? ^ - n (m) for which p and q are integers independent of each other comprised between 0 and 10 even, Y represents a carbonyl, amino, methylamino, or methylene group, and may have different meanings within the different groups _ (CH2) pY], and (* ) represents either a hydrogen atom, or is the locus of the group Rep, it is understood that Ri can be attached to no matter which atom of the general formula (II), and comprises Z, and which has a single group Ri in the formula (II), • X represents a group NR2 or CHR2, R2 is either a hydrogen atom or the link to the group Ri as defined above. The group represents case: a group of the general formula (IV): for which W 'represents CHR "' or NR" ', and R "' independently represents a hydrogen atom, a methyl, or the bond or group Ri as defined above, or 2nd case: a group of the general formula (V): for which W 'represents CHR "' or NR '", and R' and R "'independently represent a hydrogen atom, a methyl, or the bond or group Ri as defined above, (2) Rep is absent or is a distributor of the general formula (VI): whose nitrogen atom is attached to the X, V, W, or Z atoms or a Y substituent of the Ri group as the case may be, and • t is an integer between 0 and 8 even, • r is an integer between 0 and 10 even, r can have different meanings within the different groups -NR - (CH) r-, • R3, which may have different meanings within the different groups NR4- (CH) rR3, represents a hydrogen atom, a methyl group, or a group of the general formula (VII): for which u is an integer comprised between 1 and 10 even, s is an integer between 2 and 8 even that may have different meanings within the different groups - (CH2) s-NRs, and Rs is an atom of hydrogen, a CA group as defined above, it is understood that the CA groups are independent of each other and may be different, or else a group of the general formula (VII) is understood to mean that the groups of the general formula (VII) are independent of each other and may have different meanings, • R is defined in the same way as R3 or represents a CA group as defined above being understood that the CA groups are independent of each other and can be different s, and (3) R is linked to the carbonyl function of the group Rep of the general formula (VI), or if Rep is absent R is directly linked to the group CA, and represents: * or a group of the formula NR6 7 for which Re and R independently represent a hydrogen atom or an aliphatic radical saturated or not, linear or branched, optionally fluorinated, containing 1 to 22 carbon atoms, with at least one two Re or R7 substituents other than hydrogen and another containing between 10 and 22 carbon atoms, * or a steroid derivative or a group of the general formula (VIII) for which x is an integer between 1 and 8 even, and is an integer between 1 and 10 even, and either Q represents a group C (0) NReR7 for which R ß and R7 are defined as above , or Q represents a group C (0) Rs for which Rs represents a group of the formula (IX): for which z is an integer between 2 and 8 even, and R9 is an aliphatic radical saturated or not, optionally fluorinated, containing 8 to 22 carbon atoms, or a steroid derivative, and the two substituents R e are, independently of one another, defined as above, or Rβ represents a group -O-R 9 for which R 9 is defined as previously. According to a variant of the invention, the group Ri is linked either to Z or V on the one hand and to the group Rep on the other hand by the intermediary of Y. Advantageously, the cycloamidine group CA of the formula (II) comprises , 6, 7 or 8 links. On the other hand, in another variant of the invention, Rep is a dealer in 1, 2, or 3 <; < branches > > . For example, the following distributors can be mentioned: According to a second variant of the invention, R3 represents a hydrogen atom or a methyl and R4 is as defined above, or R3 and R present in the formula (VI) represent the hydrogen atoms, or R4 is a hydrogen atom and R3 is a group of the formula (VII) in which R5 represents a CA group.

Preferably, in formula (V), p and q are independently chosen from each other, between 2, 3 or 4. In general, the R group contains at least one hydrophobic segment. It is understood in the sense of the invention by < < hydrophobic segment > > any group of lipid type, which favors cell penetration. In particular, the group R contains at least one aliphatic chain or at least one derivative of t -roid. According to a preferred variant, the group R represents a group of the formula NRßR7, Re and R7 are defined as above, or represent a group of the general formula (VIII) in which Q represents a group C (0) NRdR7 , Re and R are defined as above. Preferably, Rβ and / or R7 independently represent a saturated or unsaturated linear aliphatic chain containing 10 to 22 carbon atoms, preferably 12, 14, 16, 17, 18 or 19 carbon atoms. These are, for example, groups (CH2.11CH3. (CH2)? 3CH3, (CH2)? 5CH3, (CH2)? 7CH3, oleyl, etc. In a particular mode of implementation, groups R6 and R7 they are identical or different and each represents a saturated or non-saturated linear or branched aliphatic chain, optionally fluorinated, containing 10 to 22 carbon atoms, as defined in the preceding paragraph.When R represents a steroid derivative, it is chosen advantageously between cholesterol, cholestanol, 3-a-5-cyc or 5-a-choles t an-6-β-ol, cholic acid, cholesteryl formate, cotes tani 1 formi ato, 3a-5-cyclo-5-colé s tan-6ß-yl formate, cholesteryl amine, 6- (1, 5-dimethyl-1-hexy-1) -3 a, 5a-dimethyl-hexadecahydro-cyclopenta [a] cyclopropa [2, 3] cyclopenta [1, 2-f] na phthalenol-10-alamine, or the colé s tani lamina These new compounds of the general formula (I) can be presented in the form of non-toxic salts and pharmaceutically These non-toxic salts comprise the salts with the mineral acids (hydrochloric, sulfuric, bromohydric, phosphoric, nitric acids) or with the organic acids (acetic, propionic, succinic, maleic, hydroxylamine, benzoic, fumaric, methanesulfonic or oxalic) or with the mineral bases (soda, potash,. litina, lime) or also with organic bases (tertiary amines such as triethylamine, piperidine, benzylamine). As an illustrative example of the preferred compounds according to the invention, mention may be made of the compounds of the following formulas: Compound (1) N-dioctade cilcarbamoi lmethyl-2-. { 3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -but i] amino] propyl amino} -acetamide Compound (2) N-ditetradecylcarbamoylmethyl-2-. { 3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino] propylamino} -acetamide Compound (3) 2- (3- { 4- [3- (4, 5-dihydr o-lH-imidazol-2-ylamino) propylamino] -butylamino.} - N -tetradecylcarbamoylmethyl-acetamide Compound (4) 2- (3 - { Is - [3- (4, 5-dihydro-lH-imidazol-2-ylamino propyl] -amino}. -propylamino) -N-di tetradec i lcarbamoylmet i 1 -acetamide Compound (5) N-ditetradecylcarbamoylmethyl-2-. { 3- [3 - (1,4,5,6-te tr ahydr-opirimidin-2-ylamino) -propylamino] -ropylamino} -acetamide Compound (6) N-dioctadecylcarbamoylmethyl-2-. { 3- [3- (1, 4, 5, 6 -tetrahydropyrimidin-2-ylamino) -propylamino] propylamino} -acetamide The compounds of the invention can be prepared in different ways. According to a first method, the compounds of the invention can be obtained by synthesis of 1 ipopolyamines analogous (ie, the same structure but without cycloamidine group), cyclization in cycloamidine groups is carried out in a second time. The analogous 1 ipopo 1 i amine can be obtained by any method known to the person skilled in the art., and mainly according to the methods described in the application WO 97/18185 or by analogous methods. The cyclization of amidine heads can be effected, for example, by the reaction between one and / or several primary amines of the lipopolyamine and the reactants such as the acid sulfate of O-me t i li s ourea sulfate [J. Med. Chem., 1995, 38 (16), pp. 3053-3061] or the semisulfate of S-me t i 1 i sot iourea [Int. J. Pept. Prot. Res., 1992, 40, p. 119-126]. Preferably, the operation is carried out in the 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 the preferred solvent, water / alcohol or dimethylformamide mixtures may be mentioned. As a base, triethylamine, N-e tildi is opropi sheet, soda, potash, etc. can be used. The temperature is preferably between 40 ° C and 60 ° C, and more preferably, the reaction is carried out at 50 ° C. Another method is to carry out a synthesis of blocks that carry the cycloamidine function, which is then grafted onto the lipids equipped with distributors. This method has the advantage of accessing a significant number of products. In the sense of the invention, it is meant by <; < blocks > > any functional segment of the molecule. For example, the cycloamidine group CA as defined in the general formula (II), Rep or even R constitute the different blocks from each other, in the sense of the invention. As an example, you can proceed, for example, in the following way: 1) Synthesis of the R block: a) When R represents -NReR7, or is commercially available, or can be synthesized according to one of the following methods: • by alkylating reduction between an amine bearing the group R β and an aldehyde carrying the group R 7. It is preferably operated in a chlorinated solvent (for example, dichloromethane, chloroform, dic loro-1,2-ethane, etc. [J. Org. Chem., 1996, pp. 3849-3862] or in any other organic solvent compatible with the reaction (for example, tetrahydrofuran), in the presence of sodium triacetoxyborohydride, sodium cyanoborohydride or its derivatives (for example, lithium cyanoborohydride) [J. Am. Chem., 1971, pp. 2897 -2904] and acetic acid • either by substitution of a starting group borne by Rβ, by an amine bearing the group R.sup.7 As an example of the starting group, the halogen atoms (Br, Cl, I) or the substituents tosyl, mesyl, etc. It is preferably used in the presence of a basic reagent, for example, sodium carbonate, potassium carbonate, soda, triethylamine, etc., in an alcohol (eg, ethanol) with reflux [J. Am. Chem. Soc., 1996, pp. 8524-8530] • or by coupling between a fatty acid (or its derivatives such as fatty acid chlorides 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 to the person skilled in the art, in an ether (for example, tetrahydrofuran (THF), t-butylmethyl lé ter (TBME), dimethoxyethane (DME), etc.). b) When R represents a group of the general formula (VIII), the peptide coupling between the group Q and H- [NH- (CH2) x] and COOH is effected. The peptide coupling is carried out according to the classical methods known to the person skilled in the art (Bodanski M., Prin cipi es and Pra c t i ce s of pep t i de Syn th e s i s, Ed. Spr inge-See lag) or by any other known analogue method. Primarily, the reaction can be carried out in the presence of a non-nucleophilic base in suitable aprotic solvents (such as chloroform, dimethylformamide, methylmercury, acetonitrile, dichloromethane, etc.), at a temperature between 0 and 100 ° C, the pH is adjusted between 9 and 11. Q is available either commercially, or when Q represents a group C (0) R8 with R8 of the formula (IX), it can be synthesized by the reaction between a commercial chloroformate (for example, the cholesteryl chloroformate) or obtained according to the classical methods known per se. the person skilled in the art from a commercial chloroformate, and a commercial diamine (for example, Ne ti lendiamine) or obtained according to the classical methods known to the person skilled in the art. Preferably, it is operated in a chlorinated solvent (for example, dichloromethane, chloroform, dichloro-1,2-ethane, etc.) or in any other organic solvent compatible with the reaction such as, for example, dimethylformamide, dimethisulfoxide , acetonitrile, etc. The group H - [NH - (CH2) x] y - COOH is a commercial amino acid when y is equal to 1, or it is obtained by one or more cyanide reactions according to the methods described above. in Rep's synthesis when and is superior to l. 2) Synthesis of the Rep block: The Rep group is obtained by cyanoe ti lation or by di cyanoe tilation (depending on whether you want to obtain a linear or branched Rep structure) of an amino acid of the formula HOOC- (CH2) r-NH2 then by reduction of nitrile functions in amines. a) mono- or di -cianoe ti lación: Preferably, it is operated 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 monoethoxylation, preference is given to working cold [J. Am. Chem. Soc., 1950, pp 2599-2603]. In the case of diction, preference is given to working hot and with an excess of acrylonitrile [J. Am. Chem. Soc., 1951, pp 1641-1644] b) The reduction of the nitrile functions in amines is effected by catalytic hydrogenation in basic medium or by any other method known to the person skilled in the art. As an example, platinum oxide or even Raney nickel [J. Org. Chem., 1988, pp 3108-3111] as a catalyst. Preferably, the chosen solvent is an alcohol (for example, methanol, ethanol, etc.) in the presence of a base, for example, soda, potash, etc. 3) Synthesis of the Rep-R block: The Rep-R block is obtained by peptide coupling between the Rep acid and the amine R obtained in the preceding steps. The peptide coupling is carried out in accordance with the classical methods known to the person skilled in the art (Bodanski M., Pr inc ipi es and Pr actices of pep ti do Syn th es is, Ed. Springe-Verlag) or by any other known analog method. Primarily, the reaction can be carried out in the presence of a non-nucleophilic base in suitable aprotic solvents (such as chloroform, dimethylformamide, idyl ether, acetonitrile, dichloromethane, etc.), at a temperature between 0 and 100 ° C, the pH is adjusted between 9 and 11. 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 obtained in the preceding step, and CA-S-CH3, according to the classical methods known to the person skilled in the art. It is preferably operated in a chlorinated solvent (for example, 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, triethylamine, soda, potash, N-ethyldiisopropylamine, etc.), and at room temperature (approximately 20 ° C). The block CA-S-CH3 is available either commercially (as is the case, for example, from the acid iodide of 2-methyl-11-io-2 -imido zolon), or it can be obtained by the action of a disulfide of carbon on an appropriate diamine (ie, chosen according to the cycloamidine group that is desired), followed by methylation. For example, the reaction scheme can be illustrated in the following way: Preferably, the reaction process is carried out in an alcohol (for example, ethanol). The methylation step is carried out by the action of a halogenomethyl, the halogen atom can be, 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 amino functions present on Rep-R, by the action of acid sulfate of O-me t ilis ourea or of semisulfate of S-me tili s or thiourea. Preferably, the operation is carried out in the aqueous medium in the presence of a base in heat [J. Med. Chem., 1985, pp. 694-698 and J. Med.

Chem., 1996, pp. 669-672]. As the preferred solvent, water / alcohol mixtures or dimethylformamide can be mentioned. As a base, it is possible to use triethylamine, N-ethyl sopropylamine, soda, potash, etc. The temperature is preferably between 40 ° C and 60 ° C, and also more preferably, the reaction is carried out at 50 ° C. c) by peptide coupling between CA-COOH and Rep-R according to the classical methods known to the person skilled in the art, as described above. The CA-COOH block can be obtained in different ways: • by the action of a block CA-S-CH3 on an amino acid or a polyamine acid according to the methods known to the person skilled in the art or by any other analogous method [J. Am. Chem. Soc., 1956, pp. 1618-1620]. The block CA-S-CH3 is obtained in the same manner as above, and the amino acid or polyamine is chosen depending on the compound according to the desired invention, or • by the action of an S, S-dimethylol 1-iminothiocarbonylidene of one of its derivatives on a polyamine acid according to methods known to the person skilled in the art or by any other analogous method [J. Org. Chem., 1971, pp. 46-48]. Preferably, the operation is carried out in the ethanolic medium in the presence of a base (for example, soda) and at the reflux temperature of the mixture. As an example of CA-COOH blocks that can be obtained by one of the methods described above, the following blocks can be cited: In any of the reactions discussed above, when the amino substituents present in the different groups can interfere with the reactions implemented, it is preferable to protect them previously by the compatible radicals that can be put in place and eliminated without touching the rest of the molecule. As an example, the protecting radicals can be chosen from the radicals described by T. W. GREENE, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991) or by McOmie, Protective Groups in Organic Chemistry, Plenum Press (1973). Another object of the invention relates to a composition comprising at least one compound of the formula (I) as defined above. In particular, another object according to the present invention comprises a compound of the formula (I) as defined above and a nucleic acid. When a compound according to the invention and a nucleic acid are mixed, they form a complex by interaction between the positive charges present at physiological pH on the compound according to the invention and the negative charges of the nucleic acid. This complex is called < < nucleolipi di co complex "in the following. Preferably, the compound according to the invention and the nucleic acid are present in amounts such as the ratio of the positive charges of the compound on the negative charges of the nucleic acid or comprised between 0.1 and 50, preferably between 0.1 and 20. This relationship it can be easily adjusted by the person skilled in the art depending on the compound used, the nucleic acid, and the applications sought (mainly of the type of cells to be transfected). It is understood in the sense of the invention by < < nucleic acid > > also a deoxyribonucleic acid as a ribonucleic acid. It can be natural or artificial sequences, and mainly of genomic DNA (GDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or synthetic sequences or emi s int e ti ca s, oligonucleotides modified or not. These nucleic acids may be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to the person skilled in the art, and mainly by screening or sieving of banks, by chemical synthesis, or also by mixed methods that include the chemical or enzymatic modification of sequences obtained by screening banks. They can be modified chemically. With reference more particularly to the soxir ribonucleic acid acids, they can be single or double strands as well as short oligonucleotides or longer oligonucleotides. In particular, nucleic acids are advantageously constituted by plasmids, vectors, episomes, expression cassettes, etc. These deoxyribonucleic acids can carry a functional origin of replication or not in the target cell, one or several marker genes, transcriptional or replication regulatory sequences, genes of therapeutic interest, modified or non-modified antisense sequences, regions of link to other cellular compounds, etc.

Preferably, the nucleic acid comprises an expression cassette consisting of one or several genes of therapeutic interest under the control of one or more promoters and of a transcriptional terminator active in the target cells. In the meaning of the invention, the term "expression cassette of a gene of interest" means a fragment of DNA which can be inserted into a vector of specific restriction sites. The DNA fragment comprises a nucleic acid sequence encoding an RNA or a polypeptide of interest and further comprising the sequences necessary in the expression (primer (s), pr omot or (s)), polyadenylation sequences, etc.) of the sequence. The cassette and restriction sites are known to ensure an insertion of the expression cassette into an appropriate reading frame for transcription and translation. Generally it is a plasmid or an episome that carries one or several genes of therapeutic interest. As an example, mention may be made of the plasmids described in patent applications WO 96/26270 and WO 97/10343 incorporated herein by reference. In the sense of the invention, the gene of therapeutic interest is understood to be, in particular, any gene coding for a protein product that has a therapeutic effect. The protein product thus encoded may be primarily a protein or a peptide. This protein product can also be exogenous homologous or endogenous to the target cell, ie a product which is normally expressed in the target cell when it does not present any pathology. In this case, the expression of a protein makes it possible, for example, to mitigate an insufficient expression in the cell or the expression of an inactive or weakly active protein as a result of a modification, or else to express the protein. The gene of therapeutic interest can thus be encoded for a mutant of a cellular protein having an increased stability, a modified activity, etc. The protein product can also be heterologous to the target cell. In this case, an expressed protein can, for example, complete or provide a deficient activity in the cell, which allows it to fight against a pathology, or stimulate an immune response. Among the therapeutic products in the sense of the present invention, enzymes, blood derivatives, hormones, lymphokines, interleukins, interferons, TNF, etc. can be mentioned more particularly. (FR 92/03120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pl eyo t róf ina, etc.) apol ipopr o t ei na s (ApoAI, ApoAIV, ApoE, etc., FR 93/05125), dystrophin or a mini-syndrome (FR 91/11947), the CFTR protein associated with mucovi s, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc., FR 93/04745), the genes coding for the factors involved in coagulation (Factors VII, VIII, IX), the genes involved in the repair of DNA, the genes suicides (thymidine kinase, cytosine deaminase), the genes of hemoglobin or other protein transporters, metabolism enzymes, catabolism etc.

The nucleic acid of therapeutic interest can also be a gene or an antisense sequence, whose expression in the target cell allows controlling the expression of genes or the transcription of cellular mRNAs. Such sequences can be transcribed, for example, in the target or target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in EP 140 308. The therapeutic genes also comprise the sequences coding for ribozymes, which are capable of selectively destroying target RNA (EP 321 201). As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide, capable of generating an immune response in man or animal. In this particular mode of implementation, the invention allows the implementation of vaccines or immunotherapy treatments applied to man or animal, mainly against microorganisms, viruses or cancers. It can be mainly antigenic peptides specific to Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, "syncitia forming virus" (ie virus formator). of syncytium), of other viruses or also of tumor-specific antigenic peptides (EP 259 212). Preferably, the nucleic acid also comprises the sequences that allow the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the cell or the desired organ. These may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be about sequences of different origin (responsible for the expression of other proteins, or also synthetic). Primarily, it can be promoter sequences of eukaryotic or viral genes. For example, it can be promoter sequences from the genome of the cell to be infected. Similarly, it can be promoter sequences from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the EIA, MLP, C.MV, RSV, etc. genes. In addition, these expression sequences can be modified by the addition of activation, regulation, etc. sequences. It can also be the promoter, which can be induced or can be recovered. On the other hand, the nucleic acid can also comprise, in particular in the amount of the gene of therapeutic interest, a sequence signal that directs the therapeutic product synthesized in the secretion pathways of the target cell. This sequence signal can be the natural sequence signal of the therapeutic product, but it can also be any other functional sequence signal, or an artificial sequence signal. The nucleic acid may also comprise a sequence signal which directs the synthesized therapeutic product into a particular compartment of the cell. The compositions according to the invention may further comprise one or more adjuvants capable of associating the complexes formed between the compound according to the invention and the nucleic acid, and of improving the transfection power. In a mode of implementation, the present invention relates to compositions comprising a nucleic acid, a compound of formula (I) as defined above and one or more adjuvants capable of associating nucleolipid complexes (I) / nucleic acid complex and in improving the power of transfection. The presence of this type of adjuvants (lipids, peptides or proteins, for example) can advantageously make it possible to increase the transfection power of the compounds. In this view, the compositions of the invention may comprise one or more neutral lipids as an adjuvant. Such compositions are particularly advantageous, especially when the ratio or proportion of charges R is weak. Indeed, the Applicant has shown that the addition of a neutral lipid makes it possible to improve the formation of null particles and to favor the penetration of the particle in the cell by destabilizing its membrane. More preferably, the neutral lipids used in the context of the present invention are lipids of two fatty chains. In a particularly advantageous manner, natural or synthetic, zwitterionic or ion-deprived lipids are used under physiological conditions. In particular, dioleoylphosphatidylethanolamine (DOPE), oleoylpalmyl toylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl fos fat idile tanolamines as well as their N-methyl derivatives 1 to 3 times, phosphatidylglycerols- , the di acylglycerols, the glycosyl diacyl glycerol, the gone brains, (such as mainly the galact ocer ebro s ida s), the spingol ipidos (such as mainly the ones that are ingested) or the as ialoganglios gone (such as mainly asialoGMl and GM2). These different lipids can be obtained either by synthesis, or by extraction from organs (example: the brain) or from eggs, by the classical techniques well known to the person skilled in the art. In particular, the extraction of natural lipids can be carried out by means of organic solvents (see also Lehninger, Biochemistry).

More recently, the Applicant has shown that it is also particularly advantageous to use, as an adjuvant, a product that directly intervenes or not at the level of nucleic acid condensation (WO 96/25508). The presence of a product, within a composition according to the invention, makes it possible to reduce the amount of the compound of the formula (I), with the beneficial consequences that result on the t oxy-logical plane, without prejudging the transfection activity. By product that intervenes at the level of the nucleic acid condensation, it is understood to define a compaction product, directly or not, the nucleic acid. More precisely, this product can be reacted directly either at the level of the nucleic acid to transfect or intervene at the level of an appended product which is directly involved in the condensation of this nucleic acid. Preferably, it reacts directly to the level of the nucleic acid. For example, the precompactant product can be no matter what pollination, for example, polylysine. According to a preferred embodiment, this product acts at the level of condensation of the nucleic acid derived in all or part of a protamine, a histone, or a nucleolin and / or one of its derivatives. A product can also be constituted, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of the motifs that can vary between 2 and 10. In the structure of the compound according to the invention, These reasons can be repeated continuously or not. This is how they can be separated by ligatures or bonds of a biochemical nature, for example, by one or several amino acids, or of a chemical nature. Preferably, the compositions of the invention comprise 0.01 to 20 equivalents of adjuvant (s) for one nucleic acid equivalent in mol / mol and, more preferably 0.5 to 5. In a particularly advantageous embodiment, the Compositions of the present invention further comprise a target element that allows targeting the transfer of the nucleic acid. This element of target can be an element of extracellular target that allows orienting the transfer of DNA towards certain cell types or certain desired tissues (tumorigenic cells, liver cells, hematopoietic cells.). It can also be an element of intracellular target that allows to guide the transfer of the nucleic acid towards certain privileged cell compartments (my cells, nucleus, etc.). The target element can be linked to the compound according to the invention or also to the nucleic acid as that has been specified above. Among the target elements that can be used in the context of the invention, mention may be made of sugars, peptides, proteins, oligonucleotides, lipids, neurometers, hormones, vitamins or their derivatives. Preferably, these are sugars of peptides or proteins such as antibodies or fragments of antibodies, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc. In particular, it can be ligands of growth factor receptors, cytokine receptors, cell-type lectin receptors, or ligands in the RGD sequence with an affinity for adhesion protein receptors such as integrins. The receptors for transferrin, HDL and LDL, or the folate transporter can also be mentioned. The target element can also be a sugar that allows lectins to turn white, such as the receptors to the acetyl coprotinate as well as the sialides such as the Lewis X sialide, or also a Fab fragment of antibodies, or a chain antibody. simple (ScFv). The association of the target elements with the complexes of the invention, can be carried out by any technique known to the person skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the compound of the general formula (I) according to the invention, or else to a group which interacts with the compound of the general formula (I) according to the invention or with the nucleic acid. The interactions in question can be, according to a preferred mode, ionic or covalent in nature. According to another variant, the compositions of the invention can also incorporate at least one nonionic surface agent in an amount sufficient to stabilize the size of the particles of nucleic complexes composed of the formula (I) / nucleic acid. The introduction of non-ionic surface agents prevents the formation of aggregates, which makes the composition more particularly adapted to an in vivo administration. The compositions according to the invention incorporating such surface agents have an advantage over the safety plane. They also have an additional advantage in this regard, that they reduce the risk of interference with other proteins, taking into account the reduction of the overall load of the complexes of core 1-ipic complexes. The surface agents are advantageously composed of at least one hydrophobic segment, and of at least one hydrophilic segment. Preferably, the hydrophobic segment is chosen from aliphatic chains, polyoxyalkylene, alkylidene polyesters, polyethylene glycols of benzylic polyether head and collesterol, and the hydrophilic segment is advantageously chosen from polyoxyalkylene, polyalkylene alcohols, polyvinyl lpyrrolidones or saccharides. Such non-ionic surface agents have been described in the application WO 98/34648. Another subject of the invention is the use of the compounds of the general formula (I) as defined above for the manufacture of a medicament for the treatment of diseases by transfer of nucleic acids (and more generally polyanions) in primary cells. or in the established lines. In particular, it can be treated with cells that are oblate, muscular, nervous (neurons, astreites, glial cells), hepatic, hema t opoyé ti ca (lymphocytes, CD34, dendritic, etc.), epithelial cells, etc. ., under the differentiated form or pluripotent (precursors). Such use is particularly advantageous because the compounds of the general formula (I) according to the invention have reduced cytotoxicity with respect to the cationic lipids of the prior art. The Applicant has mainly shown that in the very high load ratios that commonly cause the death of animals consecutively to transfection, no apparent cytotoxicity has decreased. The compounds according to the invention can be used mainly for transfection in vi t ro, ex vi ve or i n vi t ro of nucleic acids. For in vivo uses, for example, in therapy or for the study of gene regulation or the creation of animal models of pathologies, the compositions according to the invention can be formulated for the purpose of topical administration, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intrat racheal, intraperitoneal, etc. Preferably, the compositions of the invention contain a pharmaceutically acceptable carrier for an injectable formulation, primarily for direct injection at the level of the desired organ, or for topical administration (on the skin and / or mucosa). It can be, in particular, sterile, isotonic, or dried compositions, in particular lyophilized, which, by addition according to the case of sterilized water or physiological saline solution, allow the constitution of injectable solutes. The doses of nucleic acid used for the injection as well as the number of administrations can be adapted according to different parameters, and mainly depending on the mode of administration used, the aforementioned pathology, the gene to be expressed, or also the duration of the sought treatment. As regards more particularly the mode of administration, it can be treated either by direct injection into the tissues, for example, at the level of the tumors, or the circulatory routes, or by a treatment of cells in culture followed by its reimplantation in vi, by injection or graft. The tissues referred to in the context of the present invention are, for example, the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilages, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissues, etc. Advantageously, the transfected tissues are the muscles and the lungs. The invention further relates to a method of transferring nucleic acids in cells comprising the following steps: (1) contacting the nucleic acid with a compound of the general formula (I) as defined above, to form a nucleolipi di complex, and (2) contacting the cells with the nucleic acid complex. ipidi co formed in (1). The cells can be contacted with the nucleic acid complex by incubation of the cells with the complex (for in vivo or ex vivo use), or by injection of the complex into an organism (for in-vitro use). alive ) . Incubation is preferably carried out in the presence of, for example, 0.01 to 1000 μg of nucleic acid per 10 6 cells. For in vivo administration, nucleic acid doses between 0.01 and 10 mg can be used, for example.

In the case where the compositions of the invention also contain one or more adjuvants as defined above, the adjuvant (s) are previously mixed with the compound of the general formula (I) according to the invention or with the acid nucleic. The present invention thus provides a particularly advantageous method for the treatment of diseases by the administration of a nucleic acid encoding a protein or that can be transcribed into a nucleic acid capable of correcting the disease, the nucleic acid is associated with a compound of the general formula (I) as defined above, under the conditions defined above. More particularly, this method is applicable to diseases that result from a deficiency in a protein or nucleic product, the nucleic acid administered coding for the protein product or that is transcribed in a nucleic product or also that constitutes the nucleic product. The invention is understood in any use of a compound of the formula (I) according to the invention for the transfection i n vi, ex vi ve, or in vi t ro of cells. In addition to the foregoing devices, the present invention also comprises other features and advantages that result from the examples and figures that follow, and which should be considered as illustrative of the invention without limiting the scope. Mainly, the Applicant proposes in non-limiting quality various operative protocols as well as the reaction intermediates susceptible to be put into practice to prepare the compounds of the general formula (I). Of course, it is within the scope of the person skilled in the art to be inspired by these protocols and / or intermediates to bring them to the point of analogous processes in order to lead to other compounds of the general formula (I) according to the invention.

FIGURES Figure 1: Structure of synthetic vectors termed lipid A, lipid B, lipid c and lipid D in the present invention and described in patent application WO 97/18185 incorporated herein by reference.

Figure 2: Schematic representation of plasmid pXL2774.

Figure 3: Phase diagram of the compound nucleolipid complexes (1) / DNA. The binding of compound (1) to DNA has been determined by following the decrease in fluorescence (in%, 100% is the fluorescence of naked DNA) of ethidium bromide (EtBr) (symbol •, solid line), as described by the axis and located on the right. The particle size of the complexes (in nm) is indicated on the y-axis on the left. The x-axis represents the charge ratio of DNA / transference agents. The size of the nucleolipid complexes without co-lipid is represented by the symbol B in solid line. The size of the nucleolipid complexes containing 25% cholesterol is represented by the symbol D in broken line. The size of the ipolic nucleol complexes containing 40% DOPE is represented by the symbol - dashed line. The method does not allow to determine the size of the particles beyond 3 μm.

Figure 4: Transfer activity of the viral gene in the HeLa cells of the nucleolipid complexes containing the compound (1) according to the present invention without co-lipid (middle bar in dark gray), with 25% cholesterol (left bar in medium gray), and 40% molar DOPE (right bar in light gray), compared to naked DNA. Only the nucleolipid complexes in which the DNA is completely saturated in the compound according to the invention, and whose size is between 100 nm and 300 nm, have been used.

Figure 5: Transfer activity of the viral gene of the nucleolipid complexes formed from the compound (3), in the HeLa cells. In the ordered figure, the expression of luciferase, expressed in pg per refined cavity. In abscissa, the charge ratio between compound (3) and DNA in nmol / μg is shown. The expression has been measured at each time for the formulations without colipid (micelles), with DOPE and with cholesterol.

Figure 6: Transfer activity of the viral gene of the complexes nuci eolipí di eos formed from the compound (5), in HeLa cells. In orderly figure the expression of luciferase, expressed in pg per cavity t r ans f e c t ada. In abscissa, the charge ratio between compound (5) and DNA in nmol / μg is shown. The expression has been measured each time for the formulations without colipid (micelles), with DOPE and with cholesterol.

Figure 7: Transfer activity of the viral gene of the nuclear complexes formed by the compound (5), in the HeLa cells. In orderly figure the expression of luciferase, expressed in pg per cavity t r ans f ected. In abscissa, the charge ratio between compound (6) and DNA in nmol / μg is shown. The expression has been measured each time for the formulations without colipid (micelles), with DOPE and with cholesterol.

Figure 8: Transfer activity of the in vi ve gene after direct injection into the muscle of the complexes containing the compound (1) 'according to the present invention or the compound of the formula H 2 N (CH 2) 3 NH (CH 2) 4NH (CH2) 3NHCH2COArgN [(CH2) 17CH3] 2 (called < < < Lipid A > > in all the following) without co-lipid (dark gray bar), with 25% cholesterol (medium gray bar) ), and with 40% molar of DOPE (light gray bar), comparatively with naked DNA. Only the complexes have been used, in which the DNA is completely saturated in lipid and of which the size is between 100 nm and 300 nm.

Figure 9: The importance of the invention is illustrated by comparing the gene transfer activity of two different lipids, the compound (1) according to the invention and the lipid A, and the DNA aligned via two administration routes: intravenously (iv) and intramuscularly (im). Only the complexes in which the DNA is completely saturated in lipid and of which the size is between 100 nm and 300 nm, have been used.

Figure 10: Transfer activity of the gene in vi 48 hours after the im injection, of the nucleolipid complexes containing the compounds (5) or (6) according to the present invention without co-lipid and with charge ratio 0.25 / 1, comparatively with 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).

MATTER!. AND METHODS A \ MATERIAL The starting amino acids, polyamines (or their derivatives) are commercially available. This is the case, for example, of N- (3-aminopr opyl) glycine, N- (2-cyanoe tyl) glycine, or also of 2-diaminobutyric acid, or they can be synthesized by methods classics known to the person skilled in the art. The cyclic isothioureas are also commercial products, such as, for example, the acid iodide of 2-methyl t-thio-2-imidazoline, or they can be synthesized by the classical methods known to the person skilled in the art. The amines substituted by the / a lipid (s) are marketed or synthesized from the corresponding amines and aldehydes by alkylating reduction. Products such as triethylamine, trifluoroacetic acid, hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium (BOP), dimethylaminopyridine (DMAP), benzyl chloroformate, di-tert-butyl dicarbonate are commercial products. The solutions of sodium chloride and sodium carbonate are saturated. The potassium sulfate solution is concentrated at 0.5 M.

B \ METHODS Physical Measures The Proton NMR spectra were recorded on the Bruker 400 and 600 MHz spectrometers.

The mass spectra were performed on an API-MS / III. 2) Methods of purification and analysis a) Direct phase chromatography conditions - Thin layer chromatographies (MLC) were performed on 0.2 mm thick Merck silica gel plates. They are disclosed by U.V. (254 nm), by ninhydrin, vaporizing (light mist) an ethanolic solution of ninhydrin (40 mg / 100 cm of ethanol) to reveal the amines or amides by heating at 150 ° C, with the fluorescamine, vaporizing a solution (40 mg / 100 cm3 of acetone) to reveal the primary amines, when pouring bromocresol, vaporizing a solution (0.1% in 2-propanol) to reveal the acids, with vanillin vaporizing (light dew) an ethanolic solution of vanillin (3 %) with 3% sulfuric acid followed by heating at 120 ° C, or with iodine coating the iodine powder plate.

Column chromatographies were carried out on Merck silica gel 60 granulometry at 0.063-0.200 mm.

Purification conditions CLHP (Preparative High Performance Liquid Chromatography) The apparatus is a set for liquid phase chromatography in the gradient mode that allows a U.V detection. This preparatory chain is composed of the following elements: Pump A: GILSON model 305 equipped with a head 50 SC Pump B: GILSON model 303 equipped with a head 50 SC. Injection circuit: 5 ml. Pressure module: GILSON model 806. Me z clador: GILSON model 811 C equipped with a 23 ml head. UV detector: GILSON model 119 equipped with a preparative cell.

GILSON model 202 action collector equipped with slide n ° 21 and glass tube 10 ml. Integrator: SHIMADZU model C-R6A. Column: Column C4 (10 mm) of stainless steel 25 cm long and 2.2 cm in diameter marketed by VYDAC model 214 TP 1022. The solution of the product to be purified is loaded onto the column by the intermediary of the injection circuit, the eluent it is recovered by fractions of a tube in 30 seconds. The detector is regulated by the wavelengths of 220 nm and 254 nm. The mobile phases are thus defined: Sun A Sol see you B Water Acetonitrile for demineralized 2500 ero, "HPLC 2500 cm3 Acid trifluoroacetic acid 2 cm" Trifluoroacetic 2.5 cm ' Gradient c) Analytical chromatography techniques - The CLHP analysis (Crorna t ogra fiaida High Performance) have been carried out on a Mer ck-Hi t achí apparatus equipped with a D 2500 HITACHI calculator integrator, of an aut omos tr ador AS-2000A , of an intelligent L-6200A pump, and of a UV-vis detector L-4000 with adjustable wavelength set at 220 nm. The columns for the analytical separations are the Browlee columns in stainless steel, 3 cm long and 0.46 cm in diameter, marketed by APPLIED BIOSYSTEM. The stationary phase is constituted by the Aquapore Butyl 7 microns. The mobile phases are water (with trifluoroacetic acid) and acetonitrile (with trifluoroacetic acid).

The injections are 20 μl of a solution of approximately 1 mg / cm3 in a 0.1 cm3 loop funnel. The performance for the analyzes is regulated between 1 cm3 / min and 4 cm3 / min. The pressure is about 180 bars. The conditions of separation are summarized below: Solvent A Solvent B Water Acetonitrile for Demineralized 2500 cm "HPLC 2500 crtr Acid Trifluoroacetic acid 2 cm" Trifluoroacetic 2.5 cm " Gradient EXAMPLES AXSINTESIS OF THE COMPOUNDS ACCORDING TO THE INVENTION Example 1: Synthesis of compound (I) (N-dioctadecylcarbamoylmethyl-2 { 3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino] -propylamino.}. -acet amide) from the cationic lipid of the condensed formula NH2 (CH2) 3NH (CH2) 4NH (CH2) 3NHCH2COGlyN [(CH2) i7CH3] 2 called <; < lipid B > > in the following (the preparation of which has been described in patent application WO 97/18185 and whose structure is shown in figure 1). In a ball flask equipped with a magnetic bar, 0.784 mmol of lipid B are dissolved in 25 cm3 of methanol, and 10.21 mmol of triethylamine are added. Then a solution of O-Methylisourea and sulfuric acid (1173 mmol) in the water (9 cm3) is poured slowly (5 minutes) into the mixture. The mixture is maintained at 50 ° C in an oil bath for twenty hours. The mixture is then concentrated to dryness in the evaporator. The dried extract is solubilized with a solution of water (4 cm), ethanol (4 cm3), and trifluoroacetic acid (1 cm3). This solution is injected twice into preparative CHLP. The interesting fractions (determined by analytical CLHP) are regrouped, frozen and lyophilized. This gives 194 mg (0.163 mmol) of salified product.

Performance: 20.8% CLHPanaütico: Rt = 15.99 minutes. XR NMR spectrum (400 MHz, (CD3) 2SO d6, d in ppm): 0.88 (t, J = 6.5 Hz, 6H: CH3 of the fatty chains): 1.24 (mt, 60H: CH2 central fatty chains); from 1.35 to 1.70 (mt, 4H: 1 CH2 of each fat chain, 1.57 (mt, 4H: (CH2) 2 central butyl), 1.88 and 1.96 (2 meters, 2H each: central CH2 of propyl and CH2 central of cycle), from 2.85 to 3.35 (2 meters, 16H in total: 8 NCH2), 3.81 (s broad, 2H: NCH2C0N), 4.03 (d, J = 5 Hz, 2H: glycine CONCH2CON), 7.25 and 7.84 ( respectively sys 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: the 2 NH), MH + = 846 Example 2 : Synthesis of the compound (2) (N-ditetradecylcarbamoylmethyl-2- { 3- [4- (2-imino-tetrahydro-pyrimidin-1-yl) -butylamino] -propylamino.} - acetamide) from the compound of the condensed formula NH2 (CH2) 3NH (CH2) 4 NH (CH2) 3NHCH2COGlyN [(CH2) 13CH3] 2 called "lipid C" in the following (the preparation of which has been described in patent application WO 97/18185 and which structure is represented in figure 1.) In a ball flask equipped with a magnetic rod dissolves n 1036 mmol of lipid C in 30 cm3 of methanol, and 13.13 mmol of triethylamine are added. Then a solution of O-Methylisourea and sulfuric acid (1554 mmol) in the water (9 cm3) is poured slowly (5 minutes) into the mixture. The mixture is kept at 50 ° C in an oil bath for about twenty hours. Then, the mixture is concentrated to dryness in the water bath. The dried extract is solubilized with a solution of water (3 cm 3), ethanol (2 cm 3), and trifluoroacetic acid (0.5 cm). This solution was injected into preparative CLHP. Interesting fractions (determined by analytical CLHP) were regrouped, frozen and lyophilized. Finally, 218 mg (0.2022 mmol) of the salified product are obtained. Performance: R = 19.5% CLHPanaiitica: Rt = 10.76 minutes. NMR spectrum 1H NMR (400 MHz, (CD3) 2 SO d6, d in ppm): 0.88 (t, J = 7 Hz, 6H: CH3 of fatty chains); from 1.15 to 1.40 (mt, 44H: (CH2) n central fatty chains); 1.45 and from 1.50 to 1.65 (2 meters, 2H each: 1 CH2 of each fatty chain); 1.59 (mt, 4H: the 2 central CH2 butyl); 1.91 and 1.97 (2 meters, 2H each: central CH2 of propyl); from 2.85 to 3.10 (mt, 10H: the 2 NCH2 of the butyl - the 2 NCH2 of one of the 2 propyl - and 1 of 2 NCH2 of the other propyl); 3.23 and 3.30 to 3.50 (2 meters, respectively 5H and 1H: the other NCH2 of the other propyl and NCH2 of fatty chains); 3.79 (mf, 2H: NCH2CON); 4.03 (d, J = 5 Hz, 2H: CONCH2CON glycyl); 7.27 and from 8.40 to 9.30 (respectively broad s and mf, 2H and 4H: NH2 + CF3COO; NH + CF3COO "y = NH); 7.88 and 8.61 (respectively sys broad, 1H each: respectively NHC = N and CONH) .MH 734 Example 3: Synthesis of compound (3) (2- (3- { 4- [3- (4,5-dihydro-lH-imi-zol-2-ylamino) -propylamino] -buti-1-amino} -propi lamino) -N-ditetradecylcarbamoylmethyl-acetamide) from lipid C (see example 2 and figure 1 for its structure) In a ball flask equipped with a bubble counter and a magnetic bar, they dissolve 0.36 mmol of the 2-met ilmer capto-2-imidazolinium iodide in 0.36 cm3 of sodium hydroxide 0. To this solution 0.36 mmol of the lipid C previously dissolved in 1.44 cm3 of sodium hydroxide, 5 cm3 of water, and 4 cm of water were added. The mixture was kept under stirring until the release of the methyl mercaptan was stopped (24 hours), and the mixture was then concentrated to dryness in the evaporator. n a solution of water (4 cm), ethanol (4 cm), and trifluoroacetic acid (0.5 cm3). This solution is injected twice in preparative CLHP. Interesting fractions (determined by analytical CLHP) are regrouped, frozen and lyophilized. Finally, 213 mg (0.1727 mmol) of the salified product are obtained.

Performance: R = 48% CLHPanaiitica: Rt = 8.90 minutes. NMR NMR spectrum (400 MHz, (CD3) 2SO d6 with the addition of any CD3COOD drops d4, d in ppm): 0.87 (t, J = 7 Hz, 6H: CH3 of fatty chains); from 1.15 to 1.40 (mt, 44H: (CH2) n central fatty chains); 1.45 and 1.55 (2 meters, 2H each, 1 CH2 of each fatty chain); 1.65 (mt, 4H: the 2 central CH2 butyl); from 1.80 to 1.95 (mt, 4H: central CH2 of the propyl); from 2.80 to 3.05 (mt, 10H: the 2 NCH2 of the butyl - the 2 NCH2 of one of the 2 propyl - and 1 of 2 NCH2 of the other propyl); 3.24 (mt, 6H: the other NCH2 of the other propyl and NCH2 of the fatty chains); 3.56 (s, 2H: NCH C0N); 3.62 (s, 4H: NCH2CH2N); 4.02 (d, J = 5 Hz, 2H: CONCH2CON of the gyicyl).

MH 777 Example 4: Synthesis of compound (4) (2- (3. {Bis- [3- (4, 5-dihydro-lH-imidazol-2-ylamino) -propyl] -amino}. -propylamino) - N-di tradecil carbamoi lmet il-acetamida) by the method of the synthesis of «blocks».

SYNTHESIS OF BOC - GL Y- DI TETRADE CI LAM IA (a The Gly group from which the amines are protected by Boc groups (10 mmol) and ditetradecylamine (10 mmol) are placed in a 250 ml ball flask, and 100 cm 3 of dichloromethane are added The mixture is stirred until complete dissolution, then 30 mmol of Ne-tildiisopropylamine (DIEA) and 11 mmol of benzothiazol-1-yloxy trisdimethylamine phosphonium (BOP) are added. to 10 thanks to the DIEA, and the mixture is stirred for 2 hours.When the reaction is achieved, (followed by CCM and / or CHLP), the dichloromethane is evaporated and the solid obtained is recovered in ethyl acetate (300 cm) The organic phase is washed with a solution of potassium sulphate (4 times 100 cm3), sodium carbonate (4 times 100 cm3), and sodium chloride (4 times 100 cm3), then the organic phase is dried over sulphate. of magnesium, filtered and evaporated under vacuum.The product (a) is obtained with a yield of 93%. %.

CCM Rf = 0.9 (CHCl3 / MeOH, 9: 1 II) SYNTHESIS OF [Z -NH (CH2) 3] 2-N- (CH2) 3-NH-B0C-CH2-COOH (b) 1) Synthesis of NC- (CH2) 2-NH-Boc-CH2-COOH (c) The amine of N- (cyanoe ti 1) -gli ciña (0.1 mol / amine, commercial) is solubilized in the soda ÍN (200 cm / amine) and dioxane (200 cm3). The solution is stirred in an ice bath, then a solution of O- (t-but-oxycarbonyl) 2 or of p-chlorobenzyloxycarbonyl (C1Z, 0.14 mol / amine) in 200 cm of dioxane is added dropwise. The pH is maintained at a value greater than 9. The mixture is then stirred at about 20 ° C overnight. The dioxane is evaporated under vacuum, then the mixture is acidified to pH 3 with the help of a potassium sulfate solution. What 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 under vacuum. The product (c) of the formula NC- (CH2) 2-NH-B0C-CH2-COOH is obtained in a yield of 98%.

CCM: Rf = 0.66 (CHCl3MeOH, 8: 2) MH +: 229 2) Synthesis of NH2- (CH2) 3-NH-Boc-CH2-COOH (d) Into a 1 liter stainless steel autoclave, 50 mmol of the product are introduced (c) of the formula NC- (CH 2) 2-NH-Boc-CH 2 -COOH. A solution of 10 cm3 of ethanol (95%) and 3.3 g of soda (80 mol) is prepared in a container at the same time. When the soda dissolves, they are introduced 2 cm3 of raney nickel on carbon. The autoclave closes. The initial hydrogenation pressure is about 52 bar, and it drops to about 48.5 bar in one night at ambient temperature (20 ° C). The suspension is filtered on paper, the filtrate is washed with ethanol (4 times 25 cm 3), and the filtrates are concentrated to dryness under vacuum. The product (d) is obtained which is used without further purification as it is.

CCM Rf = 0.12 (CHCls / MeOH, 6: 4 MH 233 3) Synthesis of [NC (CH2) 2] 2-N- (CH2) 3-NH- Boc-CH2-COOH (e) In a ball flask, the product (d) of the formula NH2- (CH2) 3-NH-B0C-CH2-COOH (0.05 mol) and soda (0.1 mol) are solubilized in 150 cm3 of water. The solution is cooled in an ice bath. Under agile agitation, it is poured slowly acrylonitrile (0.12 .mol) keeping the temperature of the mass below 20 ° C. The reaction mixture is kept overnight at room temperature (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 solution of potassium sulfate. What is insoluble is extracted with ethyl acetate (3 times 200 cm), then washed with a sodium chloride solution (2 times 100 cm). The organic phase is dried over magnesium sulfate, then filtered and evaporated under vacuum. The product "unrefined" is eventually purified on a silica column. The product (e) is obtained with a 50% yield.

CCM: Rf = 0.75 (CHCl3 / MeOH, 6.4) MH 399 4) Syntheses i s of [Z -NH - (CH2) 3] 2 -N- (CH2) 3 -NH - Boc- CH2 -COOH (b) In a stainless steel autoclave of 1 liter, the product (e) of the formula [NC (CH2) 2] 2-N- (CH2) 3-NH-Boc-CH2-COOH (50 mmol) is introduced. A solution of 10 cm3 of ethanol (95%) and 3.3 g of soda is prepared in a container at the same time. (80 mol). When the soda dissolves, this solution is introduced into the autoclave. A stream of nitrogen is passed to the autoclave and 2 cm of Raney Nickel is introduced on carbon. The autoclave closes. The initial hydrogenation pressure is about 52 bar, and it drops to about 48.5 bar in one night at room temperature (20 ° C). The suspension is filtered on paper, the filtrate is washed with ethanol (4 times 25 cm3), and the filtrates are concentrated dryness under vacuum. A white solid is obtained which is used in other purifications after the CCM analysis.

CCM Rf = 0.14 (CHCl3 / MeOH, 6: 4) The solid obtained above is solubilized in the sodium hydroxide (200 cm 3 / amine) and dioxane (200 cm 3). The solution is stirred in an ice bath, then a solution of (t-but oxycarboni 1) 20 or p-20 chlorobenzyloxy carbonyl or (0.14 mol / amine) in 200 cm dioxane is added dropwise. The 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 the mixture is acidified to pH 3 with the help of a solution of potassium sulfate. What 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 under vacuum. The products are analyzed by CCM and / or CLHP.

The crude or unrefined product is purified on a silica column (dichloromethane / methanol, 8: 2).

The product (b) is obtained with a yield of 66% in relation to the product (d).

CCM: Rf = 0.42 (CHCl3 / MeOH, 6: 4) MH: 615 III) SYNTHESIS OF [Z-NH (CH2) 3] 2-N- (CH2) 3-NH-Boc • CH2-COGlyN [(CH2) 13-CH3] 2 (f) The product (a) whose amines are protected by the Boc groups (1 mmol) is introduced into a ball flask equipped with a magnetic bar. 30 cm3 of trifluoroacetic acid at 4 ° C are added, then the solution is stirred for one hour. When the reaction is achieved (followed by CCM and / or CLHP), the trifluoroacetic acid is evaporated under vacuum then the product is dried by coevaporation with 3 times 30 cm3 of ethyl ether.

CLHP: Rt = 12.86 min, (H20 / MeCN 3 min [40/60], 3-20 min [0/100], 35 min [0/100] The product obtained (Gly-ditetr adecylamine, 10 mmol) and product (b) (10 mmol) are placed in a 250 cm3 flask, dichloromethane (100 cm) is added and the mixture is stirred until complete dissolution. . 30 mmol of N- and t ildii s opropylamine (DIEA) and then 11 mmol of BOP hexafluorophosphate are added. The pH is maintained at 10 thanks to the DIEA and the mixture is stirred for two hours. When the reaction is achieved (followed by CCM and / or CLHP), the dichloromethane is evaporated and the solid obtained is recovered with ethyl acetate (300 cm). The organic phase is washed with a solution of potassium sulfate (4 times 100 cm), sodium carbonate (4 times 100 cm), and sodium chloride (4 times 100 cm 3). The organic phase is dried over magnesium sulfate, filtered and evaporated under vacuum. The products are used in other purifications. The product (f) is obtained with a yield of 75% after purification on a silica column (dichloromethane / methanol, 8: 2).

CCM: Rf = 0.86 (CHCl3 / MeOH, 8: 2) CLHP: Rt = 17.44 min, (H20 / MeCN 3 min [40/60] 3-20 min [0/100], 35 min [0/100] IV) SYNTHESIS OF [NH2 (CH2) 3] 2 ~ N- (CH2) 3-NH -B0C-CH2-COGlyN [(CH2) 13-CH3] 2 (g) The product (f) whose amines are protected, is introduced into a ball flask equipped with a magnetic bar and dissolved in 10 cm3 of methanol per gram of product. Palladium on carbon (10%, lg / g of the product) and ammonium formate (1 g / g of the product are added at room temperature.) Hydrogenolysis is continued by CLHP After two hours, the reaction is reached, the mixture is filtered, and the filtrate is filtered. Wash 3 times with 10 cm of methanol per gram of the product The bidistilled water is added and the solution is frozen and lyophilized, or the filtrate is concentrated to dryness and the solid is recovered in ethyl acetate (300 cm3). The organic phase is washed with a sodium carbonate solution (2 times 100 cm.sup.3), and a sodium chloride solution (2 times 100 cm.sup.3), then dried over magnesium sulfate, filtered and evaporated under vacuum. The products are analyzed by CLHP and are used without further purification The product (g) is obtained with a yield of 40% with respect to the product (f).

CLHP 9.62 min, (H20 / MeCN 3 min [40/60] 3-20 min [0/100], 35 min [0/100] MH 795 V) SYNTHESIS OF THE COMPOUND (4) The product (g) which contains the primary amine to be modified (1 mmol / amine) is solubilized in dichloromethane (10 cm), then the acid iodide of 2-methylthioimidazoline (1.2 mmol / amine) and triethylamine ( 1.3 mmol / amine). The mixture is stirred at room temperature (20 ° C) until the decoupling of the methyl sulfide is stopped. At the end of the reaction (followed by CLHP), the dichloromethane is evaporated under vacuum.

The product obtained, of which the amines are protected by the Boc groups, (1 mmol) is introduced into a ball flask equipped with a magnetic bar. 30 cm3 of trifluoroacetic acid at 4 ° C are added, then the solution is stirred for one hour. When the reaction is reached (followed by CCM and / or CLHP), the trifluoroacetic acid is evaporated under vacuum then the product is dried by coevaporation with 3 times 30 cm of diethyl ether.

The product obtained is purified by preparative HPLC and the fractions are analyzed by CLHP. The compound (4) according to the present invention is thus obtained in a yield of 34%.

CLHP: 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) 2 SO d6 at a temperature of 383K, d in ppm): 0.92 (t, J = 7 Hz, 6H: CH3 of fatty chains); from 1.25 to 1.45 (mt, 44H: (CH2) n central fatty chains); 1.57 (mt, 4H: 1 CH2 of each fatty chain); from 1.70 to 1.90 (mt, 6H: propyl CH2 central); from 2.50 to 3.40 (mt, 16H: the 2 NCH2 of propyl and the NCH2 of fatty chains); 3.68 (s, 8H: the 2 NCH2CH2N); 3.72 (broad s, 2H: NCH2CON); 4.06 (s, 2H: CONCH2CON glycyl).

MH: 831 Example 5: Synthesis of the compound (5): (N-ditetradecylcarbamoylmethyl-2- { 3- [3- (1, 4, 5, 6-tet rahi dropi rimidi n- 2 -i 1 ami no) -propyl amino ] propi lamino.} - ace t amide by the synthesis method of «blocks».

I) SYNTHESIS OF BOC-GLY- DI TETRADECI LAMÍ NA (a) It is operated in the same way as in the previous example. The product (a) is obtained with a yield of 93%.

CCM Rf = 0.9 (CHCl2 / MeOH, 9: 1) MH 567 II) SYNTHESIS OF Z-NH (CH2) 3-N-B0C- (CH2) 3-N-Boc-CH2-COOH (b) 1) Synthesis of NC- (CH2) 2-NH-Boc-CH2-COOH (c) It is operated in the same way as above in Example 4. The product (c) is obtained with a yield of 98%.

CCM: Rf = 0.66 (CHCl3 / MeOH, 8: 2) MH +: 229 2) Synthesis of NH2- (CH2) 3-NH-Boc-CH2-COOH (d) The product (d) is obtained in the same manner as above in Example 4.

CCM: Rf = 0.12 (CHCl3 / MeOH, 6.4) MH +: 233 3) Synthesis of NC (CH2) 2-N-Boc- (CH2) 3-NH- Boc-CH2-COOH (e) In a ball flask, the product (d) (0.05 mol) and the soda (0.1 mol) are solubilized in 150 cm3 of water. The solution is cooled in an ice bath. Under agile agitation, the acrylonitrile (0.05 mol) is slowly poured keeping the temperature of the mass below 20 ° C. The reaction mixture is kept overnight at room temperature (20 ° C).

The solvent is evaporated under vacuum, then the mixture is acidified to pH 3 with a solution of potassium sulfate.

What is insoluble is extracted with ethyl acetate (3 times 200 cm 3), then washed with a sodium chloride solution (2 times 100 cm 3). The organic phase is dried over sodium sulfate magnesium, then filtered and evaporated under vacuum. The product obtained is optionally purified on a silica column.

The product obtained (0.1 mol / amine) is solubilized in the sodium hydroxide (200 cm / amine) and dioxane (200 cm) The solution is stirred in an ice bath, then a solution of (Boc) 20 is added dropwise. of p-chlorobenzyloxycarbonyl (0.14 mol / amine) in 200 cm3 of dioxane.The pH is maintained at a value greater than 9. Then, the mixture Stir at room temperature (20 ° C) overnight. The dioxane is evaporated under vacuum, then the mixture is acidified to pH 3 with the aid of a potassium sulfate solution. What 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. It is filtered and evaporates under vacuum. The products are analyzed by CCM and / or CLHP.

The product (e) is thus obtained with a yield of 93%.

CCM: Rf = 0.75 (CHCl3 / MeOH, 8: 2) MH 386 4) Synthesis of Z-NH- (CH2) 3-N-Boc- (CH2) 3-N- Boc-CH2-COOH (b) Into a 1 liter stainless steel autoclave, the product (e) (50 mmol) is introduced. A solution of 10 cm3 of ethanol (95%) and 3.3 g of soda (80 mol) is prepared in a container at the same time. When the soda is Dissolve, this solution is introduced into the autoclave. A stream of nitrogen is passed in the autoclave and 2 cm3 of Raney Nickel on carbon are introduced. The autoclave closes. The initial pressure hydrogenation is about 52 bar, and the same drops to about 48.5 bar in one night at room temperature (20 ° C). The suspension is filtered on paper, the The filtrate is washed with ethanol (4 times 25 cm3), and the filtrates are concentrated to dryness under vacuum. A white solid is obtained which is used without further purifications after a CCM analysis.

CCM Rf = 0.14 (CHCl3 / MeOH, 6: 4 The product obtained (0.1 mol / amine) is solubilized in the soda IN (200 cm 3 / amine) and dioxane (200 cm 3). The solution is stirred in an ice bath, then a solution of p-chlor obencil oxycarboni (0.14) is added dropwise. mo / amine) in 200 cm dioxane. The pH is maintained at a higher value of 9. Then, the mixture is stirred at room temperature (20 ° C) overnight. The dioxane is evaporated under vacuum, then acidifies the mixture to pH 3 with the help of a solution of potassium sulfate. What is insoluble is extracted with ethyl acetate (3 times 100 cm3) then washed with a sodium chloride solution (2 times 100 cm). The organic phase is dried over magnesium sulfate, filtered and evaporated under vacuum. The product obtained is purified on a silica column (dichloromethane / methanol, 9: 1). The products are analyzed by CCM and / or CLHP. The product (b) is obtained with a yield of 32% in relation to the product (d).

CCM: Rf = 0.63 (CHCl / MeOH, 9: 1) MH 523 III) SYNTHESIS OF 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) In a shake flask with nitrogen stream, 3, 4, 5, 6-tet rahydro-2-pyrimidine thiol (0.0103 mol) is charged, and 5 cm3 of methanol and 0.65 cm of methyl iodide (0.0105 mol) ) are added. The mixture is refluxed for 1 hour then allowed to cool to room temperature (20 ° C). The product is precipitated by the addition of 5 cm of ethyl ether. The precipitate is filtered, then washed with ethyl ether. The product is then dried under a pressure of 34 mbar. 1.5 g (0.0041 mol) of the product (VI) are obtained, either a yield of 40%.

CCM Rf = 0.25 (CHCls / MeOH, 9: 1) MH 131 d) SYNTHESIS OF Z-NH (CH2) 3-N-Boc (CH2) 3-N-Bo c-CH2-COGlyN [(CH2) 13-CH3] 2 (g) The product (a) from which the amines are protected by Boc groups (1 mmol) is introduced into a ball flask equipped with a magnetic bar. 30 cm of trifluoroacetic acid at 4 ° C are added, then the solution is stirred for one hour. When the reaction is reached (followed by CCM and / or CLHP), the trifluoroacetic acid is evaporated under vacuum then the product obtained is dried by coevaporation with 3 times 30 cm of ethyl ether.

CLHP: Rf = 12.86 min, (H20 / 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 placed in a 250 cm3 ball flask. Dichloromethane (100 cm3) is added and the mixture is stirred until complete dissolution. Then 30 mmol of DIEA and 11 mmol of BOP are added. The pH is maintained at 10 thanks to the DIEA and the mixture is stirred for two hours. When the reaction is achieved (followed by CCM and / or CLHP), the dichloromethane is evaporated and the solid obtained is recovered in ethyl acetate (300 cm). The organic phase is washed with a solution of potassium sulphate (4 times 100 cm3), 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 vacuum. The products are used without other purifications.

After purification on a silica column (dichloromethane / methanol, 8: 2), the product (g) is obtained in a yield of 85%.

CCM: Rf = 0.9 (CHCl3 / MeOH, 9: 1 CLHP Rt = 19.79 in, (H20 / MeCN 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).

V) SYNTHESIS OF NH2 (CH2) 3] 2 -NB or c- (CH2) 3-NH -Boc -CH2 -COGlyN [(CH2) i3-CH3] 2 (h) The product (g) is introduced into a flask of ball equipped with a magnetic bar and dissolved in 10 cm3 of methanol / g of product. Palladium on charcoal (10%, 1 g / g of product) and ammonium formate (1 g / g of product) are added at room temperature (20 ° C). Hydrogenolysis is continued by CLHP. After two hours, the reaction is reached, the mixture is filtered, and the filtrate is washed 3 times with 10 cm of methanol / g of the product. The bidistilled water is added, and the solution is frozen and lyophilized, or the filtrate is concentrated to dryness and the solid is recovered in ethyl acetate (300 cm) The organic phase is washed with a sodium carbonate solution (2 times 100 cm), and a sodium chloride solution (2 times 100 cm3), then dried over magnesium sulfate, filtered and evaporated under vacuum.The products are analyzed by CLHP and used without further purifications. h) is obtained with yields of 93% in relation to the product (g).

CCM Rf 0.42 (CHCl3 / MeOH, 6: 4 CLHP: Rf = 14.66 min, (H20 / MeCN: 3 min [40/60], 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 mmol / amine) is solubilized in dichloromethane (10 cm3), then the product (f) (1.2 mmol / amine) and triethylamine (1.3 mmol / amine) are added. . The mixture is stirred at room temperature (20 ° C) until the stoppage of the methyl sulfide release. At the end of the reaction (followed by CLHP), the dichloromethane is evaporated under vacuum.

The product obtained is purified by preparative HPLC and the fractions analyzed by CLHP. The compound (5) is thus obtained with a yield of 38%.

CLHP: Rt = 8.42 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).

H NMR spectra (400 MHz, (CD3) 2SO, d in ppm): 0.86 (t, J = 7 Hz, 6H: CH3 of fatty chains); from 1.10 to 1.35 (mt, 44H: (CH2) n central fatty chains); 1.44 and 1.53 (2 meters, 2H each: 1 CH2 of each fatty chain); from 1.80 to 2.00 (mt, 6H: central CH2 of propyl and CH2 of 1,4,5,6-tetrahydro-pyrimidine); from 2.80 to 3.10 (mt, 10H: NCH2 of propyl and NCH2 of 1, 4, 5, 6-tet rahydro-pyrimidine); from 3.15 to 3.45 (mt: the 6H corresponding to the = NCH2 of the 1, 4, 5, 6-tetrahydro-pyrimidine and the NCH2 of the fatty chains); 3.81 (mf, 2H: NCH2CON); 4.04 (d, J = 5 Hz, 2H: CONCH2CON glycyl); 7.89-8.62-8.75 and 9.01 (4 mfs, 8H in total: the interchangeable and OH of CF3COOH).

MH: 720 Example 6; Synthesis of compound (6): (N-Dioctadecylcarbamoylmethyl-2- {3- [3- (1, 4, 5, 6-te-trahydropyrimidin-2-ylamino) -propi-1-amino] -propyl-amino}. -acet t amide) by the method of synthesis of "blocks".

SYNTHESIS OF BOC-GLY- DI TETRADEC I LAM INA (a It is operated in the same way as in the previous example. The product (a) is obtained with a yield of 93%.

CCM: Rf = 0.9 (CHCl3 / MeOH, 9: 1 MH '567 II) SYNTHESIS OF Z-NH (CH2) 3-N-Boc- (CH2) 3-N-B0C-CH2-COOH (b) 1) Synthesis of NC- (CH2) 2-NH-Boc-CH2-COOH (c) It is operated in the same way as above in Example 5. The product (c) is obtained with a yield of 98%.

CCM Rf = 0.66 (CHCl3 / eOH, 8: 2) MH +: 229 2) Synthesis of NH2- (CH2) 3-NH-Boc-CH2-COOH (d) The product (d) is obtained in the same way as previously in Example 5.

CCM: Rf = 0.12 (CHCl3 / MeOH, 6: 4) MH *: 233 3) Synthesis of NC (CH2) 2-N-Boc- (CH2) 3-NH-Boc-CH2-COOH (e) Operate in the same manner as above in Example 5. The product (e) is thus obtained with a yield is 93%.

CCM: Rf = 0.75 (CHCl3 / MeOH, 8: 2) MH 386 4) Synthesis of Z-NH- (CH2) 3-N-Boc- (CH2) 3- NH-Boc-CH2-COOH (b) It is operated in the same way as above in Example 5. A white solid is obtained which is used without further purifications after a CCM analysis.

CCM Rf = 0.14 (CHCl3 / MeOH, 6: 4) The product obtained is used in the same manner as above in order to protect the terminal amine by a benzyloxycarbonyl group. The product (b) is thus obtained with a yield of 32% in relation to the product (d).

CCM: Rf = 0.63 (CHCl3 / MeOH, 9: 1) MH "523 III) SYNTHESIS OF 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) It is operated in the same way as in the previous example 5. It is thus obtained 1. 5 g (0.0041 mol) of the product (f), or a yield of 40%.

CCM: Rf = 0.25 (CHCl3 / MeOH, 9: 1) MH: 131 IV) SYNTHESIS OF Z-NH (CH2) 3-N-BOC (CH2) 3-N-BOC-CH2-COGlyN [(CH2) 17-CH3] 2 (g) The operation is carried out in the same manner as in Example 5. The product (a) whose Boc groups have been separated or is sold is thus obtained.

CLHP: Rt = 19.44 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 [0/100]).

This product obtained is used in the same way with the product (b) as above in Example 5. After purification on a silica column (dichloromethane / methanol, 8: 2), the product (g) is obtained in a yield 84% CCM Rf = 0.9 (CHCls / MeOH, 9: 1) CLHP: Rt = 23.95 min, (H20 / MeCN: 3 min [40/60], 3-20 min [0/100], 35 min [0/100]).

V) SYNTHESIS OF NH2 (CH) 3] 2-N-Boc- (CH2) 3-NH-Boc-CH2-COGlyN [(CH2) 17-CH3] 2 (h) The operation is carried out in the same manner as above with Example 5. The product (h) is obtained with a yield of 73% with respect to the product (g).

CCM: Rf = 0.28 (CHC / MeOH, 6: 4) CLHP: Rt = 20.59 min, (H20 / MeCN: 3 min. [40/60], 3-20 min [0/100], 35 min [0/100]) MH +: 838 VI) SYNTHESIS OF THE COMPOUND (6) It is operated in the same way as above in Example 5. Compound (6) is thus obtained with a yield of 68%.

CLHP ": Rt = 15.83 min, (H20 / MeCN: .3 min [40/60], 3-20 min [0/100], 35 min [0/100]).

H NMR NMR spectrum (500 MHz, (CD3) 2SO, d in ppm): 0.88 (t, J = 7 Hz, 6H: CH3 of fatty chains); from 1.15 to 1.35 (mt, 60H: (CH2)? 5 central fatty chains); 1.46 and 1.54 (2 meters, 2H each: 1 CH2 of each fatty chain); from 1.80 to 2.00 (mt, 6H: central CH2 of propyl and CH2 of 1, 4, 5, 6- te t r ahidro-pir imidina); from 2.85 to 3.05 (mt, 10H: NCH2 of propyl and NCH2 of 1, 4, 5, 6- t e rahi dro-pir imidine); from 3.15 to 3.45 (mt: the 6H corresponding to the = NCH2 of the 1, 4, 5, 6- te rahydro-pir imidine and 'to the NCH2 of fatty chains); 3.81 (mf, 2H: NCH2CON); 4.04 (d, J = 5 Hz, 2H: C0NCH2C0N glycyl); 7.88-8.61 - 8.74 and 8.99 (4 mfs, 8H in total: the interchangeable and OH of CF3COOH).

MH +: 832 BXUTILIZATION OF TRANSFECTION AGENTS IN COMPLIANCE WITH THE INVENTION Example 7: Preparation of nucleolipid complexes This example illustrates the preparation of the nucleolipid complexes according to the invention.

The compound used in this example is the compound (1) in solution in the chloroform. Quantities of 10 mmol of compound (1) (either 11.8 μg) per μg of DNA has been used. In certain cases, a neutral colipid, Cholesterol or DOPE, is previously mixed with the compound. A final lipid film is formed when the chloroform is evaporated with the help of a light flow of argon, then rehydrated in a mixture of 5% dextrose and 20 mM sodium chloride, overnight, at 4 ° C. The samples are then treated with ultrasound for 5 minutes, heated at 65 ° C for 30 minutes, and finally treated again with ultrasound for 5 minutes, heated at 65 ° C for 30 minutes, and finally treated again with ultrasound for 5 minutes. Lipid suspensions are thus obtained which are stored at 4 ° C until use.

The DNA used is the plasmid pXL2774 (FIG. 2) in solution in a mixture of 5% dextrose and 20 mM sodium chloride at a concentration of 0.5 mg / ml or 1.0 mg / ml. Plasmid pXL2774 has the following characteristics: lower endotoxin level 50 EU / mg, - Superior supercoiled DNA rate 60% - RNA content, ie mRNA, tRNA and ribosomal RNA, (determined by HPLC) less than 5%, chromosomal DNA rate lower than 1%, protein content less than 1%, lower osmolarity 15 mosmoles / kg The nucleolipid complexes according to the invention are prepared by rapidly mixing equal volumes of DNA solution and lipid suspension as described above. The amount of the compound complexed with the DNA varies from 0.5 nmoles / μg of DNA to 12 nmoles / μg of DNA.

Example 8: behavior of the complexes formed at different load ratios This example illustrates the behavior of the iplicic nucleol complexes according to the invention at different charge ratios. The impact of the addition of a neutral colipid is also illustrated.

The size of the complexes was first analyzed by measuring the hydrodynamic diameter by dynamic diffusion of the light (Dynamic Laser Light Scattering) with the help of a Coulter N4plus device, the samples are diluted 20 times in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple diffusions. The effect of the cycloamidine group, the lipid composition, and the ratio or proportion of charge on the size of the nucleolipid complexes according to the invention has been studied.

Three possible phases are distinguished when the ratio or load ratio between the compound (1) according to the invention and the DNA is increased. These three phases determine the therapeutic potential of the compound (1). Figure 3 illustrates these 3 phases for compound (1). The same behavior can be observed for other compounds according to the invention.

Having a low charge percentage, the DNA is not saturated by the compound (1). In addition the rest of the naked DNA, and the complexes are globally charged in a negative way. The particles are smaller (between 100 and 300 nm). This phase is called "Phase A".

The fact that the DNA is not completely saturated by the compound (1) means that the DNA is not completely protected by it. DNA can be subjected to degradation by enzymes (DNAases). On the other hand, the complexes are totally negative, the passage of the cell membrane is difficult. For these reasons, the nucleolipid complexes of phase A are of lesser efficiency in transfection.

Having the percentage of intermediate charge, the DNA is completely saturated by the compound (1), and the complexes are globally neutral or slightly positive. This phase is unstable because the ionic repulsions are minimal and a phenomenon of "crosslinking" can occur. The size of the particles is well below the limit of detection for the dynamic diffusion of light (much higher than 3 μm). This unstable phase is called "phase B".

A size of complexes is not adapted for the uses in injection. However, this does not mean that the complexes are inactive in phase B, but they are only under a formulation that is not suitable for injection into a pharmaceutical object.

Having a relatively high percentage charge, the DNA is overstuffed by the compound (1), and the complexes are globally positive. In fact the forces of repulsion between the positive charges, this phase is stable. It is designated under the name "phase C". Contrary to phase A, the nucleolipid complexes are in such a form that the DNA is very well protected against enzymes, and their overall positive charge facilitates the passage of the cell membrane of anionic nature. The complexes of phase C are particularly adapted to a use for the transfer of nucleic acids in cells.

In addition to the cycloamidine group of the compound according to the invention, the use of a neutral colipide has a strong impact on the stability of the complexes, as illustrated in Figure 3. The added colipids are either DOPE (lipid cation ion / DOPE = 3/2), or cholesterol (lipid cat ion ion co / cabbage terol = 3/1). In general, the addition of the neutral colipid increases the instability of the complexes, which leads to an increase in the amount of the compound required to reach phase C. This is very clearly illustrated in figure 3 when comparing the ratio or percentage of load to which phase C is reached in the presence and absence of the colipid.

It is pointed out that the values of the relation or percentage of load that delimit the three phases A, B and C depend on the compound used.

Thus, these values can vary very strongly from one compound to another.

Finally, the affinity of the compound against DNA as a function of the ratio or percentage of charge has been studied. For this, the reduction of fluorescence after the addition of 3 μg of ethidium bromide (EtBr) has been measured. In effect, the substitution of ethidium bromide in the DNA for the compound is an indication of DNA binding.

The formulation used is diluted 20 times to a final concentration of 25 μg DNA / ml. The relative fluorescence measured for naked DNA is defined as being 100%. The rate of binding to compound (1) is represented by the reduction of fluorescence relative to the sample. Figure 3 shows that the fluorescence decreases when the ratio or percentage of charge increases, which means that a greater amount of the compound (1) is available to bind to the DNA (plus the decreasing fluorescence, plus a large amount of the compound binds to the DNA until saturation is achieved).

In this way, it has thus been shown that the affinity of the compound (1) according to the invention for the DNA is determined by the cycloamidine group, but it goes through the addition of a lipid.

Example 9: In vitro transfection with the compound (1) This example illustrates the ability of the compound (1) according to the invention to transfect the DNA in the cells visually, in comparison with the unformulated DNA.

The 24-cavity microplates are seeded with 60,000 HeLa cells (ATCC) per well or cavity, and transfected 24 hours later. Complexes containing 1 μg of DNA are diluted in 0.5 ml of culture medium DMEM (Gibco / BRL) in the absence of serum, then added in each well or cavity. The cells are incubated at 37 ° C for 4 hours. The medium containing the complexes below is cleaned and replaced by a mixture of DMEM and 10% fetal bovine serum. Then, the cells are put back into culture for 24 hours. Finally, the cells are lysed and tested using a luciferase test suite (Promega) and a Dynex MLX luminometer.

The results indicated in figure 4 indicate the difference between the performances of the naked DNA in relation to the compound (1) / DNA of the invention fully saturated: no luciferase activity can be detected (sensitivity of the device less than 1 pg per cavity) ) after transfection in naked DNA, when the gene transfer activity of the complexes according to the invention varies from 200 pg / well to 8000 pg / well.

This example clearly shows the advantageous use of the compound (1) according to the invention for the transfer of in vi ve cells.

Example 10: In vitro transfection with compounds (3), (5) and (6) This example illustrates the ability of the compounds (3), (5) and (6) according to the invention to transfect the DNA in the cells in vi, ro compared to the unformulated DNA.

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. It is thus observed that these 3 compounds present a good level of transfection i n vi tro.

Example 11: live transfection of the compound (1) This example illustrates the ability of the compound (1) according to the invention to transfect the DNA in the cells in vi, comparatively with the unformulated DNA and the lipid A of the condensed formula NH2 (CH2) 3NH (CH2) 4NH ( CH2) 3NHCH2COArgN [(CH2)? 7CH3] 2 described in the application WO 97/18185 and of which the structure is represented in figure 1.

The transfer of the in vi ve gene has been effected on Balb / C mice by intramuscular and intravenous administration. The formulations which have been compared are naked DNA formulations, the formulations containing the lipid A, or the 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 muscle of the tibia. The tissues are recovered 7 days after the injection, they are frozen and stored at -80 ° C until the luciferase activity tests are performed. The measurements of luciferase activity are made as in example 8.

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

The results of transfer of the in vi ve gene are present in Figure 8 and Figure 9. The ratio or percentage between the compound (1) and the DNA is 10 nmoles / μg of DNA. The ratio or proportion between lipid A and DNA is 4 nmoles / μg of DNA.

Figure 8 illustrates the activity in vi ve in the muscle of the compound (1) according to the invention comparatively with the naked DNA and with the lipid A. It is found that the levels of luciferase activity are equivalent between the naked DNA and the compound (1), the latter also having a much improved activity in relation to lipid A. The transfer mechanisms involved seem different between naked DNA and the use of compound (1) according to the present invention. In fact, the complexes according to the invention used do not contain free DNA (phase C) and, moreover, their results in vi t r o are much higher than those of the naked DNA.

Figure 9 compares the activity of compound (1) according to the invention, naked DNA and lipid A, intravenously and intramuscularly.

It is found that the efficiency of the transfection is little close to the intravenous equivalent for lipid A and for compound (1). On the other hand, by intramuscular route, the efficiency of the transfection of the compound (1) according to the invention is clearly superior to that of the limitation A.

In relation to the naked DNA, the compound (1) presents an intravenous transfection, in addition to at least equivalent transfection intramuscularly.

It thus appears that the efficiency of the nucleic acid transfer with the compound (1) according to the invention is overall superior to that with the lipid A which is a known cationic lipid and this one of the unformulated DNA.

Finally, it seems that the complexes according to the invention have the advantage, with respect to the transfection of naked DNA, of protecting the DNA from degradations by the nucleases, which thus contribute to a significant improvement of the stability of the formulations. The compounds of the present invention can also be used to protect the DNA from deterioration during lyophilization, thereby improving the stability of the formulations.

Example 12: transfection of the compounds (5) and (6) This example illustrates the ability of the compounds (5) and (6) to be transfected from the nucleic acid in an effective manner.

The same protocol as in the preceding example is put into practice. Figure 10 shows that the compound (5) and the compound (6), formulated in a ratio or percentage of charges 0.25: 1 with the DNA without a colipid, have a level of transfection in vi ve or equal to the naked DNA 48 hours after the injection in im The following table gives the results obtained with the compounds (5) and (6) in different formulations: It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (28)

1. Compounds in the form of D, L, or DL, as well as their salts, of the general formula (I): CA-Rep-R (I) characterized in that: (i) CA represents a cycloamidine group and its mesomeric forms of the general formula (II): for which • m, and n are integers independent of each other between 0 and 3 even and such that m + n is greater than or equal to 1, • Ri represents a group of the general formula (III): for which p and q are integers independent of each other comprised between 0 and 10 even, Y represents a carbonyl, amino, methylamino, or methylene group, and may have different meanings within the different groups [(CH2) PY] and (*) represents either a hydrogen atom, or is the locus of the group Rep, it is understood that Ri may be attached to no matter which atom of the general formula (II), and comprises Z, and which has a single group Ri in the formula (II), • X represents a group NR2 or CHR2, R2 is either a hydrogen atom or the link to the group Ri as defined above, • The group represents: case: a group of the general formula (IV) for which W 'represents CHR' "or NR" ', and R "' independently represents a hydrogen atom, a methyl, or the bond or group Ri as defined above, or case: a group of the general formula (V for which W 'represents CHR "' or NR" ', and R' and R "'independently represent a hydrogen atom, a methyl, or the bond or group Ri as defined above, (ii) Rep is absent or is a distributor of the general formula (VI): whose nitrogen atom is attached to the X, V, W, or Z atoms or a Y substituent of the Ri group as the case may be, and t is an integer between 0 and even, • r is an integer between 0 and 10 even, r can have different meanings within different groups -NR - (CH) r-. • R3, which may have different meanings within the different groups NR4- (CH) rR3, represents a hydrogen atom, a methyl group, or a group of the general formula (VII): for which u is an integer between 1 and 10 even, s is an integer between 2 and 8 even that may have different meanings within the different groups - (CH2) s-NRs, and Rs is a hydrogen atom, a CA group as defined above, it is understood that the CA groups are independent of each other and may be different, or else a group of the general formula (VII) is understood to mean that the groups of the general formula (VII ) are independent of each other and may have different meanings, • R4 is defined in the same way as R3 or represents a CA group as defined above, it being understood that the CA groups are independent of each other and can be different, and (iii) R is linked to the carbonyl function of the group Rep of the general formula (VI), or if Rep is absent R is directly linked to the group CA, and represents: * or a group of the formula NR6R7 for which Re and R7 independently represent a hydrogen atom or a saturated or non-saturated linear or branched aliphatic radical, optionally fluorinated, containing 1 to 22 carbon atoms, with at least two Re or R7 substituents other than hydrogen and another containing between 10 and 22 carbon atoms, a steroid derivative, a group of the general formula (VIII) - [NH (CHafcL-Q (VIII) for which x is an integer between 1 and 8 even, and is an integer between 1 and 10 even, and either Q represents a group C (0) NR6R7 for which R ß and R7 are defined as above , or Q represents a group C (0) Rs for which Rs represents a group of the formula (IX): for which z is an integer comprised between 2 and 8 even, and Rg is an aliphatic radical saturated or not, optionally fluorinated, containing 8 to 22 carbon atoms, or a steroid derivative, and the two Rβ substituents are, independently each other, defined as above, or Re represents a group -O-R9 for which R9 is defined as above.

2. The compounds according to claim 1, characterized in that the group Ri is linked either to Z or to V on the one hand and to the Rep group on the other hand by the intermediary of Y .

3. The compounds according to claim 1, characterized in that the CA cycloamidine head of the formula (II) comprises 5, 6, 7 or 8 bonds.

4. The compounds according to claim 1, characterized in that R3 represents a hydrogen atom or a methyl and R is as defined in claim 1, or R3 and R present in the formula (VI) represent the hydrogen atoms, or R4 is a hydrogen atom and R3 is a group of the formula (VII) in which R5 represents a CA group.

5. The compounds according to claim 1, characterized in that, in formula (V), p and q are independently chosen from each other, between 2, 3 or 4.

6. The compounds according to claim 1, characterized in that the groups R6 and R7 are identical or different and each represents saturated or non-saturated, linear or branched aliphatic chains, optionally fluorinated, containing 10 to 22 carbon atoms.

7. The compounds according to claim 1, characterized in that the groups Rβ and R7 are identical or different and each represents aliphatic chains saturated or not, linear or branched, optionally fluorinated, and containing 12, 14, 16, 17, 18 or 19 carbon atoms.

8. The compounds according to claim 1, characterized in that R is a steroid derivative, the steroid derivative is chosen from cholesterol, cholestanol, 3-a-5-cyclo-5-ales-6-coles ß-ol, cholic acid, cholesteryl formate, cot is tanyl formate, 3a-5-cyc-5a-col is t an- 6ß-il formate, colé s ter amine, 6- ( 1,5-dimethylhexy-1) -3 a, 5a-dimethyl-hexadecahydro-cyclopenta [a] cyclopropa [2, 3] cyclopenta [1, 2-f] naphthalen-10-ylamine, or the coles tani lami na.

9. The compounds according to claim 1, characterized in that they are of the following formulas: Compound (2) Compound (3) Compound (4) Compound (5) Compound (6)

10. The process for preparing the compounds according to claims 1 to 9, characterized in that the synthesis of blocks carrying the cycloamidine functions is carried out, and then these blocks are grafted onto the lipids equipped with distributors.

11. The process for preparing the compounds according to claims 1 to 8, characterized in that the synthesis of the analogous lipopolyamines is carried out, followed by the cyclization of cycloamidine groups.

12. The composition characterized in that it comprises at least one compound of the general formula (I) •

13. The composition according to claim 12, characterized in that it comprises a compound of the general formula (I) and a nucleic acid.

14. The composition according to claims 12 or 13, characterized in that it also comprises one or more adjuvants.

15. The composition according to claim 14, characterized in that the one or more adjuvants are one or several neutral lipids of two fatty chains.

16. The composition according to claim 15, characterized in that the neutral lipids are natural or synthetic lipids, zwitterionic or free of ionic charge under physiological conditions, chosen for example between dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), the di- stearoyl, -palmitoyl, -myristoyl phosphatidylethanolamines as well as their N-methyl derivatives 1 to 3 times, the phosphatidylglycerols, the diacylglycerols, the glycosyldiacylglycerols, the cerebrosides (such as mainly the galactocerebrosides), the fingolipids (such as mainly the fingomyelins) or also asialogangliosides (such as mainly asialoGMl and GM2).

17. The composition according to claim 14, characterized in that the adjuvant is a compound that intervenes directly or not at the level of nucleic acid condensation.

18. The composition according to claim 17, characterized in that the adjuvant is derived in all or part of a protamine, a histone, or a nucleolin and / or one of its derivatives, or is constituted, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of motifs that can vary between 2 and 10, and that can be repeated continuously or not.

19. The composition according to claims 12 to 18, characterized in that it also contains one or more non-ionic surface agents (s) in an amount sufficient to establish the particle size of the nucleolipid complexes.

20. The composition according to claims 12 to 19, characterized in that it comprises a pharmaceutically acceptable carrier for an injectable formulation.

21. The composition according to claims 12 to 19, characterized in that it comprises a pharmaceutically acceptable carrier for an application on the skin and / or mucous membranes.

22. The composition according to claim 13, characterized in that the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid.

23. The composition according to claim 22, characterized in that the nucleic acid comprises an expression cassette consisting of one or several genes of therapeutic interest under the control of one or more promoters and of an active transcriptional terminator in the target cells.

24. The use of a compound according to one of claims 1 to 9 for manufacturing a medicament for treating diseases.

25. The use of a compound according to one of claims 1 to 9, for manufacturing a medicament for treating diseases by transfer of nucleic acids into cells intramuscularly.

26. The method of nucleic acid transfer in cells, characterized in that it comprises the following etap: (i) mixing the nucleic acid with a compound of the general formula (I) as defined above, to form a nucleolipidic complex, and (ii) contacting the cells with the nucleolipid complex formed in (i).

27. The method of transferring nucleic acids in cells according to claim 26, characterized in that the nucleic acid and / or the compound are previously mixed with one or more adjuvants.

28. The method of treating diseases by administering a nucleic acid encoding a protein or that can be transcribed into a nucleic acid capable of correcting the diseases, the nucleic acid is associated with a compound of the general formula (I)