JP2002513543A - Novel nucleic acid transfer agents, compositions containing them and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel compound useful as a substance for transferring a nucleic acid into a cell. Such new compounds are more particularly related to the lipopolyamine family and contain at least one cyclic amidine function. They are useful for transferring nucleic acids of interest to various cell types in vitro as well as in vivo or ex vivo.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel compound useful as a substance for transferring a nucleic acid into a cell. These novel compounds, in particular with respect to the lipopolyamine family, contain at least one cyclic amidine function. They are useful for transferring nucleic acids to various cell types in vitro, ex vivo or in vivo.

With the development of biotechnology, it has become necessary to be able to transfer nucleic acids into cells efficiently. This involves transferring nucleic acids into cells in vitro, e.g., to produce recombinant proteins or to study gene expression regulation, gene cloning, or any other manipulation involving DNA in the laboratory. May be included. It may also include transferring the nucleic acid into cells in vivo, for example, for creating a transgenic animal, producing a vaccine, performing a labeling test or therapeutic approach. Furthermore, ex vivo transfer of nucleic acids to cells as an approach for bone marrow transplantation, immunotherapy, or other methods, including transferring genes to cells harvested from the organism for subsequent re-administration, has been described. May be included.

[0003] Various types of synthetic vectors have been developed to improve the transfer of nucleic acids into cells. Among these vectors, cationic lipids have advantageous properties. These vectors consist of a cationic polar moiety that interacts with nucleic acids and a hydrophobic lipid moiety that facilitates cell penetration. Specific examples of cationic lipids are, in particular, monocationic lipids (DOTMA, Lipofectin®), some cationic detergents (DDAB), lipopolyamines, and especially dioctadecylamidoglycylspermine (DOGS) or palmitoyl phosphatidyl Ethanolamine 5-carboxyspermylamide (DPPES), the preparation of which is described, for example, in patent application EP 394,111. Another lipopolyamine family is described in patent application WO 97/18185, which is incorporated herein by reference.
The compounds are represented by the compounds described in No. 1 and are illustrated in FIG.

[0004] However, up to now, injection into tissues, especially muscle, has been by DNA, which is often not formulated to facilitate cell entry. Combinations with synthetic vectors result in complexes that are too large in size to integrate into cells.

[0005] That is one of the main problems that the present invention proposes to solve. In fact, the compounds according to the invention show at least the same level of in vivo transfer to muscle as obtained for non-formulated DNA, and in each case very good levels of transfer to other tissues It has an unexpected advantage. The combination with the compounds according to the invention protects the DNA from degradation by nucleases and / or degradation during freeze-drying and contributes to significantly improving the stability of the nucleolipid preparation. Further, such a combination allows for a slow, controlled release of the nucleic acid.

[0006] Furthermore, the compounds according to the invention belong to the family of cationic lipids, which provide novel cationic regions which confer improved properties on the compounds, in particular low cytotoxicity compared to the prior art cationic vectors. Carry. This cationic moiety may, in effect, more positively charge the positive charge, possibly reducing the cationicity of the compound as a whole.
Expressed as one or more specific polyamines bearing one or more cyclic amidine functionalities that have a "delocalizing" effect, thereby resulting in a beneficial effect known from a toxicological standpoint.

A first object of the present invention relates to novel compounds in D, L or DL form of the general formula (I): CA-Rep-R (I) wherein CA is a cycloamidine of the general formula (II) Represents the group and its mesomeric form:

[0008]

Embedded image Wherein m and n are, independently of one another, an integer from 0 to 3 inclusive, such that m + n is 1 or more; R ₁ represents a radical of the general formula (III) :

[0009]

Embedded imageIn the formula, p and q are each independently an integer from 0 to 10 including both ends, and Y is
A carbonyl, amino, methylamino or methylene group, wherein Y is a different group [(
CH₂)_p-Y] can have different meanings,^*) Is hydrogen field
Is a moiety that represents a bond or binds to a Rep group;₁Is a general formula including Z
May be bonded to any atom of formula (II), and formula (II) has one R ₁ It is understood that the group is present, where X is NR₂Or CHR₂R represents a group₂Is a hydrogen atom or as defined above
Una R₁A bond to a group,

[0010]

Embedded image Represents one of the following: ^* First case: a group of general formula (IV):

[0011]

Embedded image Wherein, W 'represents a CHR "or NR", R "and R", independently of one another, a hydrogen atom, or represents a bond to the R ₁ group as defined methyl or above, or ^* a second Case: group of the general formula (V):

[0012]

Embedded image Wherein W ′ represents CHR ″ or NR ″, R ′ and R ″ independently of one another represent a hydrogen atom, methyl or a bond to an R ₁ group as defined above; Or a spacer of general formula (VI):

[0013]

Embedded image Wherein the nitrogen atom is an X, V, W or Z atom or optionally a substituent Y of the R ₁ group
Is bound to have, and · t in an integer of from 0 inclusive to 8, · r is an integer from 0 inclusive to 10, group r is different -NR 4 - _{(C
H) r} - is also possible to have different meanings in, · _{R 3} are different groups _NR 4 - _(CH) obtained has a different meaning in the r _{R 3,} a hydrogen atom, a methyl group or formula Represents a group of (VII):

[0014]

Embedded imageWherein u is an integer from 1 to 10 inclusive, and s is a different group-(CH₂ )_s-NR₅Can have different meanings within and is an integer from 2 to 8 inclusive
, R₅Is a hydrogen atom, a CA group as defined above, provided that the CA groups are independently of each other
It is understood that they can be different or are groups of the general formula (VII)
It is understood that the groups of formula (VII) are independent of one another and may have different meanings.
・ R₄Is R₃Or as defined above, or a CA group as defined above
With the proviso that the CA groups are independent of each other and may differ.
R is attached to the carbonyl function of the Rep group of general formula (VI), or
If no Rep group is present, R is directly bonded to the CA group and one of the following
Forget:^*NR₆R₇A group having the formula₆And R₇Are, independently of one another, a hydrogen atom or 1
Optionally fluorinated linear or branched, saturated or unsaturated, containing from 22 to 22 carbon atoms
A sum aliphatic radical, wherein at least one of the two substituents R₆Or R ₇ Is not hydrogen, the other contains 10 to 22 carbon atoms,^*Or a steroid derivative,^*Alternatively, groups of general formula (VIII):

[0015]

Embedded imageWherein x is an integer from 1 to 8 including both ends, and y is from 1 to 10 including both ends.
And Q is R₆And R₇Is as defined above for C (O) NR₆R ₇ Or Q represents R₈Represents a group of formula (IX): C (O) R₈Of
Express:

[0016]

Embedded image Wherein z is an integer from 2 to 8 inclusive and R ₉ is an optionally fluorinated saturated or unsaturated aliphatic radical or steroid derivative containing from 8 to 22 carbon atoms; substituents, R _6, independently of one another, either as defined above, or R ₈ represents a group -O-R ₉ R ₉ is as defined above.

According to one variant of the invention, the R ₁ group is linked on the one hand to either Z or V and on the other hand to the Rep group via Y.

Advantageously, the cycloamidine group CA of formula (II) comprises 5, 6, 7 or 8 members.

Further, in another variant of the invention, Rep is a spacer with one, two or three “arms”. Examples include the following spacers:

[0020]

Embedded image

According to a second variant of the invention, R ₃ represents a hydrogen atom or methyl and R ₄ is as defined above, or R ₃ and R present in formula (VI) ₄ represents a hydrogen atom, or R ₄ is a hydrogen atom, and R ₃ is a group of the formula (VII) wherein R ₅ represents a CA group.

Preferably, in formula (V), p and q are independently selected from 2, 3 or 4.

Generally, the R group has at least one hydrophobic segment. For the present invention,
"Hydrophobic segment" is understood to mean a lipid-type group that promotes cell penetration. In particular, the R group comprises at least one aliphatic chain or at least one steroid derivative.

According to a preferred variant, the R group represents a group of the formula NR ₆ R ₇ , wherein R ₆ and R ₇ are as defined above, or a group of the general formula (VIII), Medium Q is C
(O) represents the group NR ₆ R ₇ , wherein R ₆ and R ₇ are as defined above.

Preferably, R₆And / or R₇Are independently of each other 10 to 22 charcoal
Containing elemental atoms, preferably 12, 14, 16, 17, 18 or 19 carbon atoms
Represents a saturated or unsaturated linear aliphatic chain. They are, for example, (CH₂)₁₁CH ₃ , (CH₂)₁₃CH₃, (CH₂)_FifteenCH₃, (CH₂)₁₇CH₃Or
Oleyl group and the like.

In certain embodiments, R ₆ and R ₇ are the same or different and are each optionally fluorinated containing 10 to 22 carbon atoms as defined in the section above. Represents a straight or branched, saturated or unsaturated aliphatic chain.

When R represents a steroid derivative, such steroid derivative is advantageously cholesterol, cholestanol, 3-α-5-cyclo-5-α-cholestan-6-β-ol, cholic acid, cholesteryl formate, Cotestanyl formate, formic acid 3α
, 5-cyclo-5α-cholestan-6β-yl, cholesterylamine, 6- (1
, 5-Dimethylhexyl) -3a, 5a-dimethylhexadecahydrocyclopenta [a] cycloprop [2,3] cyclopenta [1,2-f] -naphthalene-1
Selected from 0-ylamine or cholestanylamine.

These novel compounds of the general formula (I) can be provided in the form of non-toxic pharmaceutically acceptable salts. These non-toxic salts include salts with inorganic acids (hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid or nitric acid) or organic acids (acetic acid, propionic acid, succinic acid, maleic acid, hydroxymaleic acid, benzoic acid) , Fumaric acid, methanesulfonic acid or oxalic acid), or a salt with an inorganic base (sodium hydroxide, potassium hydroxide, lithium hydroxide or calcium hydroxide), or an organic base (triethylamine, piperidine or benzylamine) Tertiary amines).

Examples showing preferred compounds according to the invention include compounds of the formula:

[0030]

Embedded image Compound (1) N-dioctadecylcarbamoylmethyl-2- {3- [4- (2-iminotetrahydropyrimidin-1-yl) butylamino] propylamino} acetamide

[0031]

Embedded image Compound (2) N-ditetradecylcarbamoylmethyl-2- {3- [4- (2-iminotetrahydropyrimidin-1-yl) butylamino] propylamino} acetamide

[0032]

Embedded image Compound (3) 2- (3- {4- [3- (4,5-dihydro-1H-imidazol-2-ylamino) -propylamino] butylamino} -N-ditetradecylcarbamoylmethylacetamide

[0033]

Embedded image Compound (4) 2- (3- {bis [3- (4,5-dihydro-1H-imidazol-2-ylamino) propyl] amino} propylamino) -N-ditetradecylcarbamoylmethylacetamide

[0034]

Embedded image Compound (5) N-ditetradecylcarbamoylmethyl-2- {3- [3- (1,4,5,6-
Tetrahydropyrimidin-2-ylamino) propylamino] propylamino
Acetamide

[0035]

Embedded image Compound (6) N-dioctadecylcarbamoylmethyl-2- {3- [3- (1,4,5,6-
Tetrahydropyrimidin-2-ylamino) propylamino] propylamino
Acetamide

The compounds of the present invention can be prepared in various ways. According to the first method,
Similar lipopolyamines (ie the same structure except without the cycloamidine group)
And then cyclizing to a cycloamidine group to give the compounds of the invention. Similar lipopolyamine groups can be prepared by any method known to those of skill in the art.
In particular, it can be produced according to the method described in patent application WO 97/18185 or by a similar method. The cyclization of the amidine head can be carried out, for example, by reacting one and / or more primary amines of lipopolyamine with O-methylisourea hydrogen sulfate [J. Med. Chem. , 1995, 38 (16), p. 305
3-3061] or S-methylisothiourea hemisulfate [Int. J. pept.
Prot. Res. , 1992, 40, p. 119-126]. The procedure is preferably carried out in an aqueous medium at elevated temperature in the presence of a base [J. Med. Chem. 1985, p. 694-698 and J
. Med. Chem. , 1996, p. 669-672]. Preferred solvents include water / alcohol mixtures or dimethylformamide. As the base, triethylamine, N-ethyldiisopropylamine, sodium hydroxide, potassium hydroxide and the like can be used. The temperature is preferably between 40 ° C and 60 ° C,
More preferably, the reaction is carried out at 50 ° C.

Another method involves synthesizing a building block bearing a cycloamidine function, and then grafting it to a lipid comprising a spacer. This method has the advantage of providing access to a large number of products. For the purposes of the present invention, "block" is understood to mean any functional segment of a molecule. For example,
A cycloamidine group CA, Rep or R as defined in general formula (II)
Constitute different blocks for the present invention.

As an example, the procedure can be carried out, for example, as follows: 1) Synthesis of building block R: a) When R represents —NR ₆ R ₇ , either obtain it commercially or use the following method By alkylating reduction between an amine having R ₆ and an aldehyde having R ₇ group. Such a procedure is preferably performed using a chlorinated solvent (eg, dichloromethane, chloroform, 1,2-dichloroethane, etc. [J. Org. Chem., 1996, p. 384].
9-3862]) or sodium triacetoxyborohydride, sodium cyanoborohydride or a derivative thereof (eg lithium cyanoborohydride) in another organic solvent compatible with the reaction (eg tetrahydrofuran) [J. Am. Che
m. Soc. , 1971, p. 2897-2904] and acetic acid.

Alternatively, by replacement of the leaving group carried by R ₆ with an amine carrying the R ₇ group. Examples of leaving groups include halogen atoms (Br, Cl, I) or tosyl or mesyl substituents. Such a procedure is preferably carried out under reflux [J. Am.
Chem. Soc. , 1996, p. 8524-8530] in an alcohol (eg ethanol) in the presence of a basic reagent such as sodium carbonate, potassium hydroxide, sodium hydroxide, triethylamine and the like.

• Alternatively, by a bond between a fatty acid (or a derivative thereof such as a fatty acid chloride) and a fatty amine. Next, the obtained amide is converted into an ether (for example, tetrahydrofuran (THF), t-butyl methyl ether (TBME), dimethoxyethane (DM
E) etc.) in which reduction is carried out with hydrides, for example lithium aluminum hydride or other hydrides known to the person skilled in the art.

B) When R represents a group of the general formula (VIII), the group Q and H- [NH- (C
H _{2) x]} implementing the peptide bond between the _y COOH. Peptide binding is accomplished by conventional methods known to those skilled in the art (Bodanski M., Principles and practice of peptide synthesis,
(Pringe-Verlag compilation) or by a similar method known in the art. In particular, in a suitable aprotic solvent (such as chloroform, dimethylformamide, methylpyrrolidine, acetonitrile, dichloromethane, etc.)
The reaction can be carried out in the presence of a non-nucleophilic base at a temperature of 0 to 100 ° C. and adjusting the pH to 9 to 11.

Q is commercially available, or Q is a C (O) with R ₈ of formula (IX)
) When representing the R ₈ group, it is either commercially available (for example cholesteryl chloroformate) or obtained from commercially available chloroformate according to conventional methods known to those skilled in the art, or commercially available (for example N- Ethylenediamine)
Alternatively, it can be synthesized by reaction with a diamine obtained according to conventional methods known to those skilled in the art. Preferably, such a procedure comprises a chlorinated solvent (eg, dichloromethane,
(Chloroform, 1,2-dichloroethane, etc.) or other organic solvents that are compatible with the reaction, such as dimethylformamide, dimethylsulfoxide, acetonitrile and the like.

The group H- [NH- (CH ₂ ) x] y-COOH is a commercially available amino acid when y is equal to 1, or the method described in the synthesis of Rep below when y is greater than 1. According to one or more cyanoethylation reactions.

2) Synthesis of the building block Rep: The Rep group is used to cyanoethylate or dicyanoethylate amino acids of the formula HOOC- (CH ₂ ) r-NH ₂ (to obtain either linear or branched Rep structures). (If desired) and then by reducing the nitrile function to an amine.

A) Mono- or dicyanoethylation

[0046]

Embedded image

Preferably, such a procedure is performed in a basic aqueous medium. For example, in the presence of a base such as sodium hydroxide, potassium hydroxide, triethylamine, etc., water,
The reaction is performed in a solvent such as an alcohol (eg, methanol, ethanol, etc.). In the case of monocyanoethylation, the operation is preferably carried out at low temperatures [J. A
m. Chem. Soc. , 1950, p. 2599-2603]. In the case of dicyanoethylation, the operation is carried out, preferably at elevated temperature, with an excess of acrylonitrile [J. Am. Chem. Soc. , 1951, p. 1641-1644].

B) The reduction of the nitrile function to the amine is carried out by catalytic hydrogenation in a basic medium or by any other method known to a person skilled in the art. For example,
Platinum oxide or Raney nickel [J. Org. Chem. 1988, p. 3
108-3111] can be used as a catalyst. Preferably, the solvent selected is an alcohol (eg, methanol, ethanol, etc.) in the presence of a base, such as sodium hydroxide, potassium hydroxide, and the like.

3) Synthesis of building block Rep-R: The building block Rep-R is obtained by a peptide bond between the acid Rep obtained in the previous step and the amine R.

Peptide conjugation is carried out according to conventional methods known to those skilled in the art (Bodanski M., Principles and Practice of Peptide Synthesis, edited by Springe-Verlag) or by similar methods known in the art. In particular, in a suitable aprotic solvent (such as chloroform, dimethylformamide, methylpyrrolidine, acetonitrile, dichloromethane, etc.) in the presence of a non-nucleophilic base at a temperature of 0 to 100 ° C., p
The reaction can be performed while adjusting H from 9 to 11.

4) Synthesis of the compound CA-Rep-R according to the invention: The compounds according to the invention are obtained by several possible methods: a) According to conventional methods known to those skilled in the art, by coupling in basic medium between terminal amine present on Rep-R obtained and CA-S-CH ₃ in stages. Such a procedure is preferably performed using a chlorinated solvent (eg, dichloromethane, chloroform, etc.)
The presence of a base (eg, triethylamine, sodium hydroxide, potassium hydroxide, N-ethyldiisopropylamine, etc.) in or in other organic solvents that are compatible with the reaction, such as, for example, water, alcohols, dimethylformamide, etc. Below room temperature (
(About 20 ° C.).

The building block CA-S-CH ₃ can be obtained from commercial sources (eg, 2-methylthio-2-imidazoline hydroiodide) or a suitable diamine (ie, (Which is selected as a functional group of a cycloamidine group), which is reacted with carbon disulfide, followed by methylation. For example, the reaction scheme may be illustrated as follows:

[0053]

Embedded image

Preferably, the reaction step is performed in an alcohol (eg, ethanol). The methylation step can be carried out by the action of halomethyl, the halogen atom being for example an iodine atom [J. Am. Chem. Soc. , 1956, p. 1618-1
620 and Bioorg. Med. Chem. Lett. , 1994, p. 35
1-354].

B) By intramolecular cyclization of the cycloamidine group from the amino function present on Rep-R by the action of hydrogen O-methylisourea sulfate or S-methylisothiourea hemisulfate. Preferably, such a procedure is carried out in an aqueous medium at elevated temperature in the presence of a base [J. Med. Chem. 1985, p. 694-698
And J. Med. Chem. , 1996, p. 669-672]. Preferred solvents include water / alcohol mixtures or dimethylformamide. As the base, triethylamine, N-ethyldiisopropylamine, sodium hydroxide, potassium hydroxide and the like can be used. The temperature is preferably from 40 ° C to 60 ° C, more preferably the reaction is carried out at 50 ° C.

C) As described above, CA-COOH and R according to conventional techniques known to those skilled in the art.
By a peptide bond between ep-R.

The building block CA-COOH can be obtained in various ways: • According to methods known to those skilled in the art or by other similar methods [J. Am. Ch
em. Soc. , 1956, p. 1618-1629], building block CA-S
By the action of -CH ₃ amino acid or polyamino acid. Building Block CA-S-CH ₃ is obtained in the same manner as described above, amino or polyamino acids, or like according to known methods or selected, or - to a person skilled in the art as a functional group of the desired compound according to the present invention [J. Org. Chem
. , 1971, p. 46-48], by acting S, S-dimethyltosyliminothiocarbonimidate or one of its derivatives on polyamino acids. Preferably, such a procedure is performed in an ethanolic medium in the presence of a base (eg, sodium hydroxide) at the reflux temperature of the mixture.

Examples of the building blocks CA-COOH obtained by one of the methods described above include the following building blocks:

[0059]

Embedded image

In all of the reactions disclosed above, when the amino substituents present in the various groups may interfere with the reaction being carried out, without affecting the rest of the molecule It is preferred to pre-protect with compatible radicals that can be introduced and removed. By way of example, the protected radical is T.I. W. GREENE, "
Protecting Groups in Organic Synthesis ", J. Am. Wiley-Interscience Pu
or a radical described in McOmie, "Protection groups in organic chemistry", Plenum Press (1973).

Another subject of the present invention is a compound of formula (I) as defined above.
Compositions comprising the compounds. In particular, another object according to the invention is:
It comprises one compound of formula (I) and one nucleic acid as defined above. When the compound according to the invention and the nucleic acid come into contact, a complex is formed by the interaction between the positive charge present at physiological pH and the negative charge of the nucleic acid on the compound according to the invention. This complex is referred to as "nucleolipid complex" in the text below. Preferably, the compound and the nucleic acid according to the invention are present in an amount such that the ratio of the positive charge of the compound to the negative charge of the nucleic acid is between 0.1 and 50, preferably between 0.1 and 20. This ratio can be easily adjusted by one skilled in the art according to the compound used, the nucleic acid and the desired application, especially the type of cells to be transfected.

For the purposes of the present invention, “nucleic acid” is understood to mean both deoxyribonucleic acid and ribonucleic acid. They may be natural or artificial sequences, especially genomic DNA
(GDNA), complementary DNA (cDNA), messenger RNA (mRNA)
, Transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid or synthetic or semi-synthetic sequences, modified or unmodified oligonucleotides. These nucleic acids can be from humans, animals, plants, bacteria or viruses, and the like. They can be obtained by techniques known to those skilled in the art, in particular by screening methods for libraries, chemical synthesis or mixed methods involving chemical or enzymatic modification of the sequences obtained by library screening. They may be chemically modified.

More specifically, the deoxyribonucleic acid may be single-stranded or double-stranded, and may be either a short oligonucleotide or a longer sequence. In particular, the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes and the like. These deoxyribonucleic acids may be functional or non-functional in the target cell, an origin of replication, one or more marker genes, sequences that regulate transcription or replication, genes to be treated, modified or unmodified It may include antisense sequences, regions for binding to other cellular components, and the like.

[0064] Preferably, the nucleic acid comprises an expression cassette consisting of one or more genes to be treated under the control of one or more promoters and transcription terminators active in the target cell.

For the purposes of the present invention, “cassette for the expression of the gene of interest” is understood to mean a DNA fragment which can be inserted into a vector at a specific restriction site. The DNA fragment is R
It contains a nucleic acid sequence encoding NA or the polypeptide of interest, and further contains sequences necessary for expression of the sequence (enhancer, promoter, polyadenylation sequence, etc.). Cassettes and restriction sites are designed to ensure that the expression cassette is inserted into the appropriate reading frame for transcription and translation.

Such a DNA fragment is generally a plasmid or episome carrying one or more genes to be treated. Examples include the plasmids described in patent applications WO 96/26270 and WO / 10343, which are incorporated herein by reference.

For the purposes of the present invention, a gene to be treated is understood to mean in particular a gene coding for a protein product having a therapeutic effect. Thus, the encoded protein product may be in particular a protein or a peptide. The protein product can be exogenous, cognate or endogenous with respect to the target cell, ie, a product that is normally expressed in the target cell when the target cell is not in a pathological state. In this case, expression of the protein makes it possible, for example, to repair the expression of a protein which is inactive or weakly active, due to insufficient expression or modification in the cell, or to overexpress the protein. The gene to be treated may also encode a mutant of a cellular protein with high stability, altered activity, etc. The protein product may also be non-cognate with respect to the target cell. In this case, the expressed protein makes it possible, for example, to supplement or provide the activity deficient in the cell, to fight a pathological condition or to stimulate an immune response.

Among the therapeutic products for the present invention, in particular enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF and the like (FR 92/03)
210), growth factors, neurotransmitters or their precursors or synthases, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3
, NT5, HARP / pleiotrophin, etc.), apolipoprotein (ApoAI,
ApoAIV, ApoE, etc., FR 93/05125), dystrophin or mini-dystrophin (FR 91/11947), CFT associated with cystic fibrosis
R protein, tumor suppressor gene (p53, Rb, Rap1A, DCC, k-rev, etc., FR 93/04745, factors involved in coagulation (VII, VIII, I)
X), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), genes relating to hemoglobin or other protein carriers, metabolic enzymes, catabolic enzymes, and the like.

The nucleic acid to be treated may also be a gene or an antisense sequence that allows expression of the gene or transcription of cellular mRNA to be regulated by expression in the target cell. Such sequences can be transcribed into RNA complementary to cellular mRNA in target cells, for example according to the procedure described in patent EP 140 308, thereby blocking their translation into proteins. be able to. Therapeutic genes also provide ribozymes (
EP 321 201). As mentioned above, the nucleic acids may also include one or more genes encoding antigenic peptides capable of raising an immune response in a human or animal. In this particular embodiment, the invention allows the production of vaccines or the implementation of immunotherapy applied to humans or animals, especially against microorganisms, viruses or cancer. They are, in particular, Epstein-Barr virus, HIV virus, hepatitis B virus (EP 185 55).
No. 73), pseudorabies virus, fusion cell forming virus, other viruses, or tumor-specific antigenic peptides (EP 259 212).

Preferably, the nucleic acid also comprises a sequence that allows the expression of the gene to be treated and / or the gene encoding the antigenic peptide in the desired cell or organ.
They may, of course, be sequences that play a role in the expression of the genes considered, when these sequences can function in infected cells. They may also be sequences of different origin (playing a role in the expression of other proteins, or even synthetic). In particular, they can be promoter sequences for eukaryotic or viral genes. For example, they can be promoter sequences derived from the genome of the cell desired to be infected. Similarly, a promoter sequence derived from the genome of the virus may be used. In this connection, for example, the E1A, MLP, CMV and RSV genes are mentioned. Furthermore, these expression sequences may be modified by the addition of activation or regulatory sequences and the like. Promoters may also be inducible or repressible.

In addition, the nucleic acid can also include a signal sequence that directs a therapeutic product synthesized in the secretory pathway of the target cell, especially upstream of the gene to be treated. This signal sequence may be the natural signal sequence of the therapeutic product, but may be another functional signal sequence or an artificial signal sequence. The nucleic acid may also include a signal sequence that directs the synthesized therapeutic product to a particular fraction of the cell.

The composition according to the invention can further be combined with a complex formed between a compound according to the invention and a nucleic acid, and one or more of which can improve its transfection input Adjuvant. In another embodiment, the present invention therefore relates to nucleic acids, compounds of formula (I) as defined above, and compounds (I)
) / Compositions comprising one or more adjuvants that can be combined with the nucleolipid complex of nucleic acids and improve its transfection input. The presence of such adjuvants (eg, lipids, peptides or proteins) advantageously allows for increased transfection input of the compound.

In this regard, the compositions of the present invention may include one or more neutral lipids as an adjuvant. Such a composition is particularly advantageous, especially when the charge ratio R is low. Applicants have indeed shown that the addition of neutral lipids can improve the formation of nucleolipid particles and promote penetration of the particles into cells by destabilizing cell membranes.

More preferably, the neutral lipids used within the framework of the present invention are lipids comprising two fatty chains. Particular preference is given to using natural or synthetic lipids which are zwitterionic or have no ionic charge under physiological conditions. They are, in particular, dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidylethanolamine, and their N-methylated 1-3 times It can be selected from derivatives, phosphatidylglycerol, diacylglycerol, glycosyldiacylglycerol, cerebroside (particularly galactocerebroside, etc.), sphingolipid (particularly sphingomyelin, etc.) or asialoganglioside (particularly asialo GM1, GM2, etc.).

[0075] These various lipids can be synthesized or synthesized using conventional techniques well known to those skilled in the art.
For example, from brain) or from eggs. In particular, extraction of natural lipids can be performed with organic solvents (see also Lehninger, "Biochemistry").

Recently, the applicant has shown that it is also particularly advantageous to use compounds which directly or indirectly participate in the condensation of nucleic acids as adjuvants (WO
96/25508). The presence of such a substance in the composition according to the invention can reduce the amount of the compound of formula (I) without impairing its effect on the transfection activity, from a toxicological point of view. Produces beneficial results. Substances involved in the condensation of nucleic acids are intended to define substances that directly or indirectly densify nucleic acids. More precisely, the substance may act directly at the level of the nucleic acid to be transfected, or at the level of additional substances directly involved in the condensation of the nucleic acid. Preferably, it acts directly at the nucleic acid level. For example, the pre-compacting agent can be a polycation, such as polylysine. According to a preferred embodiment,
This substance involved in the condensation of nucleic acids is wholly or partly derived from protamine, histone or nucleolin and / or one of its derivatives. Such substances can also be wholly or partly composed of peptide units (KTPKK
AKKP) and / or (ATPAKKAA), and the number of units is 2 to 10
Range. In the structure of the compounds according to the invention, these units may or may not be repeated continuously. Thus, they can be separated by a bond of biochemical nature, for example by one or more amino acids, or by a bond of chemical nature.

Preferably, the composition of the present invention comprises from 0.1 to 1.0 equivalent of nucleic acid in mol / mol.
It contains from 01 to 20 equivalents, more preferably 0.5 to 5 equivalents of adjuvant.

In a particularly advantageous embodiment, the composition according to the invention further comprises a targeting element capable of directing the transfer of the nucleic acid. The targeting element can be an extracellular targeting element that can direct DNA transfer to a particular cell type or a particular desired tissue (tumor cells, hepatocytes, hematopoietic cells, etc.). It may also be an intracellular target element capable of directing the transfer of a nucleic acid to a particular preferred cell fraction (mitochondria, nucleus, etc.). The targeting element may also bind to a compound according to the invention or a nucleic acid as defined above.

Among the targeting elements that can be used within the framework of the present invention, mention may be made in particular of sugars, peptides, proteins, oligonucleotides, lipids, nerve mediators, hormones, vitamins or derivatives thereof. Preference is given to proteins such as sugars, peptides or antibodies or antibody fragments, ligands for cell receptors or fragments thereof, receptors or receptor fragments. In particular, it may be a ligand of a growth factor receptor, a cytokine receptor, a lectin receptor of a cell, or an RGD sequence-containing ligand having an affinity for a receptor for an adhesion protein such as an integrin. Also included are receptors for transferrin, HDL and LDL, or folate transporters. The targeting element may also be a receptor for asialoglycoprotein or sialide Lewis X.
Lectins, such as receptors for sialides, or alternatively, Fab fragments of antibodies, or sugars that allow targeting of single chain antibodies (ScFv).

The combination of a nucleolipid complex of the invention and a targeting element may be attached by any means known to those skilled in the art, for example, by attaching a hydrophobic moiety or a nucleic acid-interacting moiety of a compound of general formula (I) according to the invention. Or alternatively, a compound of the general formula (I
)) Or by binding to a group that interacts with the compound or nucleic acid. The interaction may be of an ionic or covalent nature, according to a preferred mode.

According to another variant, the compositions according to the invention also comprise compounds of general formula (I)
A sufficient amount of at least one nonionic surfactant can be incorporated to stabilize the particle size of the nucleic acid nucleolipid complex. Introducing a non-ionic surfactant prevents the formation of aggregates, thereby making the composition particularly suitable for administration in vivo. Compositions according to the invention incorporating such surfactants have advantages from a safety standpoint. They also have the added advantage that they reduce the overall charge of the nucleolipid complex composition and reduce the risk of interference with other proteins.

The surfactant advantageously consists of at least one hydrophobic segment and at least one hydrophilic segment. Preferably, the hydrophobic segments are selected from fatty chains, polyoxyalkylenes, alkylidene polyesters, polyethylene glycols with benzyl polyether heads and cholesterol, and the hydrophilic segments are advantageously polyoxyalkylenes, polyvinyl alcohol, polyvinylpyrrolidone or saccharides. Is selected from Such nonionic surfactants are described in patent application WO 98/34648.

The subject of the present invention also relates to the manufacture of a medicament for treating a disease by transferring a nucleic acid (more generally a polyanion) into progenitor cells or established lineages, as described above. The use of a compound of formula (I). They are, in particular, differentiated or pluripotent forms (progenitors), fibroblasts, muscle cells, nerve cells (neurons, astrocytes, glial cells), hepatocytes, hematopoietic cell lines (lymphocytes) , CD34, dendritic cells, etc.), epithelial cells and the like.

Such use is particularly advantageous since the compounds of the general formula (I) according to the invention have a lower cytotoxicity compared to the prior art cationic lipids. Applicants have shown in particular that no apparent cytotoxicity is detected at very high charge ratios, which usually results in animal death after transfection. The compounds of the invention are particularly useful for nucleic acid in vivo.
It can be used for in vitro, ex vivo or in vivo transfection. For use in vivo, for example, in therapy or for studying the regulation of genes or the creation of animal models of pathological conditions, the compositions according to the invention may be administered topically, dermally, orally, rectally, It can be formulated for administration by vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal routes. Preferably, the compositions of the present invention are injectable preparations, especially pharmaceutically acceptable excipients for direct injection into the desired organ or for administration by topical route (on the skin and / or mucosa). Agent. They can in particular be isotonic sterile solutions, or dry compositions, in particular lyophilized compositions, in which case sterile water or saline can be added to make up the injection solution. The dose of nucleic acid used for injection as well as the number of doses may be adapted according to various parameters, in particular according to the mode of administration used, the pathological condition involved, the genes expressed or the desired duration of treatment. More specifically, with regard to the mode of administration, for example, direct injection into the tissue at the level of the tumor, or direct injection into the circulatory system, or treatment of cultured cells followed by their reimplantation in vivo by injection or transplantation. It can be either. Related tissues within the framework of the present invention include, for example, muscle, skin, brain, lung, liver, spleen,
Bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testis, ovary, rectum, nervous system, eyes, glands, connective tissue, and the like. Conveniently, the transfected tissues are muscle and lung.

The present invention further provides: (1) contacting a nucleic acid with a compound of general formula (I) as defined above to form a nucleolipid complex; and (2) the nucleolipid formed in (1) Contacting a cell with a complex. A method for transferring nucleic acid into a cell, comprising incubating a cell with the complex (in vitro).
Or for ex vivo use) or by injecting the complex into an organism (for in vivo use), the cells can be contacted with the nucleolipid complex. Incubation is preferably carried out in the presence of 1000μg from 10 ⁶ cells per nucleic 0.01. For in vivo administration, a nucleic acid dose of, for example, 0.01 to 10 mg can be used.

If the composition according to the invention furthermore comprises one or more adjuvants as defined above, the adjuvant (s) can be prepared in advance by means of a compound of general formula (I) according to the invention Or mixed with nucleic acids.

The present invention therefore provides a nucleic acid encoding a protein capable of correcting the disease or a nucleic acid which can be transcribed into a nucleic acid capable of correcting the disease, as defined above under the conditions defined above. A particularly advantageous method for treating diseases by administering in combination with such a compound of general formula (I) is provided. More specifically,
The method is applicable to diseases resulting from protein or nuclear product deficiency, wherein the administered nucleic acid encodes the protein product, is transcribed into the nuclear product, or constitutes the nucleic acid. I do.

The present invention extends to any use of a compound of formula (I) according to the present invention for transfection of cells in vivo, ex vivo or in vitro.

In addition to the above, the present invention also includes other features and advantages that will become apparent from the following examples and figures, which examples and figures do not limit the scope of the invention. It should be regarded as illustrative of the invention. In particular, Applicants provide, without implying any limitation, various operational protocols and reaction intermediates that can be used to prepare compounds of general formula (I). Of course, the general formula (
I) It is within the ability of those skilled in the art to derive teachings from these protocols and / or intermediate products to develop similar methods for deriving other compounds.

Drawings FIG. 1: The synthetic vectors described in patent application WO 97/18185, referred to in the present invention as lipid A, lipid B, lipid C and lipid D, which are incorporated by reference into the present application. Construction.

FIG. 2: Schematic representation of the plasmid pXL2772.

FIG. 3: Phase diagram for compound (1) / DNA nucleolipid complex. The binding of compound (1) to DNA was determined by the decrease in fluorescence (%, naked DNA) of ethidium bromide (EtBr) (symbol, solid line) as indicated on the y-axis located on the right.
Is 100%). The size (nm) of the particles of the complex is indicated on the y-axis located on the left. The x-axis represents the transfer agent / DNA charge ratio. The size of the nucleolipid complex without colipid is indicated by a solid line with the symbol ■. The size of the nucleolipid complex containing 25% cholesterol is represented by the □ symbol as a dashed line. The size of the nucleolipid complex containing 40% DOPE is indicated by a dashed line with a dashed line. In this method 3
It is not possible to measure the size of particles exceeding μm.

FIG. 4: No colipid (dark shaded middle bar), with 25% cholesterol (medium shaded left shaded bar), 4 compared to naked DNA.
Activity for gene transfer in vitro to HeLa cells of a nucleolipid complex containing compound (1) according to the invention, containing 0 mol% of DOPE (lightly shaded right bar). Only nucleolipid conjugates whose DNA was completely saturated with the compounds according to the invention and whose size was between 100 nm and 300 nm were used.

FIG. 5: i of Nucleolipid complex formed from compound (3) on HeLa cells
Gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The x-axis shows the charge ratio between compound (3) and DNA expressed in mol / μg. Expression was measured for the preparations containing no colipide (micelles), containing DOPE, and containing cholesterol, respectively.

FIG. 6: i of Nucleolipid complex formed from compound (5) on HeLa cells
Gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The x-axis shows the charge ratio between compound (5) and DNA expressed in mol / μg. Expression was measured for the preparations containing no colipide (micelles), containing DOPE, and containing cholesterol, respectively.

FIG. 7: i of Nucleolipid complex formed from compound (6) on HeLa cells
Gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The charge ratio between the compound (6) and DNA expressed in mol / μg is shown on the x-axis. Expression was measured for the preparations containing no colipide (micelles), containing DOPE, and containing cholesterol, respectively.

FIG. 8: No colipid (dark shaded bars) compared to naked DNA.
Compound (1) according to the present invention comprising 25% cholesterol (medium shaded bars), 40 mol% DOPE (light shaded bars) or a compound of the formula H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COArgN
In vivo gene transfer activity of a complex containing [(CH ₂ ) ₁₇ CH ₃ ] ₂ (hereinafter referred to as “lipid A”) after direct injection into muscle. Only complexes where the DNA was completely saturated with lipids and between 100 and 300 nm in size were used.

FIG. 9: Through two routes of administration: intravenous (iv) and intramuscular (im)
The importance of the present invention is illustrated by comparing the gene transfer activity of two different lipids, Compound (1) according to the present invention and lipid A, with naked DNA. Only complexes where the DNA was completely saturated with lipids and between 100 and 300 nm in size were used.

FIG. 10: i.p. of nucleolipids containing compounds (5) or (6) according to the invention, with a charge ratio of 0.25 / 1 without colipid compared to naked DNA. m. In vivo gene transfer activity 48 hours after injection. Expression is expressed in pg of luciferase per ml. From left to right, the bars represent:
(b) naked DNA; (c) compound (5) and (d) compound (6)
.

Experimental Materials and Methods A. Experimental Materials—Starting amino acids or polyamino acids (or derivatives thereof) are commercially available. For example, N- (3-aminopropyl) glycine, N- (2-cyanoethyl) glycine or 2,4-diaminobutyric acid corresponds to this, or can be synthesized by a conventional method known to those skilled in the art.

The cyclic isothioureas are also commercially available substances, for example 2-methylthio-2-imidazoline hydroiodide, or can be synthesized by conventional methods known to the person skilled in the art.

The amine substituted with one or more lipids may be obtained commercially or
Alternatively, they are synthesized from the corresponding amines and aldehydes by alkylative reduction.

-Triethylamine, trifluoroacetic acid, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BO
P), dimethylaminopyridine (DMAP), benzyl chloroformate, di-
Materials such as t-butyl dicarbonate are commercially available materials.
Sodium chloride and sodium carbonate solutions are saturated. Potassium sulfate solution is 0.
5M concentration.

B＼ Method 1) Physical Measurements Proton NMR spectra were recorded on Bruker 400 and 600 MHZ spectrometers.

The mass spectrum was examined by API-MS / III.

2) Methods of purification and analysis a) Direct phase chromatography conditions-Thin layer chromatography (TLC) was performed on 0.2 mm thick Merck silica gel plates. Chromatography is described in US Pat. V. Under (254 nm), for ninhydrin, fluorescamine was heated by spraying (small spray) with ethanol solution of ninhydrin (40 mg / 100 cm ³ of ethanol) to reveal amine or amide by heating to 150 ° C. For bromocresol green, by spraying the solution (40 mg / 100 cm ³ of acetone) to reveal the primary amine, (solution 0.1% in 2-propanol) For vanillin, spray an ethanolic solution of vanillin (3%) with 3% sulfuric acid (small spray) and then heat to 120 ° C. or cover the plate with iodine powder for iodine. Expand by things.

Column chromatography was performed on Merck 60 silica gel with a particle size of 0.063-0.200 mm.

B) Preparative HPLC (High Performance Liquid Chromatography) Purification Conditions V. This is a set for gradient type liquid phase chromatography that can be detected. This preparative chain consists of the following components: Pump A: GILSON 305 with 50 SC head Pump B: GILSON 303 with 50 SC head Injection loop: 5 ml Pressure module: GILSON 806 Mixer: GILSON 811C with 23 ml head Type UV detector: GILSON 119 type fraction collector equipped with a preparative cell: No. GIL with 21 racks and 10ml glass tubes
SON202 type integrator: Shimadzu _{C-R 6} A Type Column: length 25cm marketed by VYDAC, stainless steel C4 column having an inner diameter 2.2 cm (10 mm), the solution injected sample to be purified 214 TP 1022 type The column is packed by the loop and the eluate is collected in a single tube fraction for 30 seconds. The detector is set at 220 nm and 254 nm wavelengths.

The mobile phase is defined as follows: Solvent A Solvent B Demineralized water 2500 cm³ Acetonitrile for HPLC 2500cm³  Trifluoroacetic acid 2cm³ Trifluoroacetic acid 2.5cm³  Slope:

[0110]

[Table 1]

C) Analytical chromatography technique-Hitachi D 2500 integrator-calculator, automatic sampler AS-2000A
HPL on a Merck-Hitachi device equipped with an intelligent pump L-6200A and a wavelength-adjustable screw-UV detector L-4000 set at 220 nm
C (High Performance Liquid Chromatography) analysis was performed.

Columns for analytical separations are commercially available from APPLIED BIOSSYSTEM.
With a 3cm long, 0.46cm inside diameter stainless steel Brownie column
is there. The stationary phase consisted of Aquapore Butyl 7 microns. Mobile phase
Water (with trifluoroacetic acid) and acetonitrile (with trifluoroacetic acid)
It is. The injection is 0.1 cm³About 1mg / cm in loop valve³Solution 20μ
l. Flow rate for analysis is 1cm³/ Cm to 4cm³Adjust in minutes
. The separation conditions are summarized below: Solvent A Solvent B Demineralized water 2500 cm³ Acetonitrile for HPLC 2500cm³  Trifluoroacetic acid 2cm³ Trifluoroacetic acid 2.5cm³  Slope:

[0113]

[Table 2]

Example A Synthesis of Compounds According to the Invention Example 1: Condensation formula, NH, hereinafter referred to as "lipid B"₂(CH₂)₃N
H (CH₂)₄NH (CH₂)₃NHCH₂COGlyN [(CH₂)₁₇CH ₃ ]₂Cationic lipids (the preparation of which is described in patent application WO 98/18185)
The compound (1) (N-dioctadecyl) derived from
Rucarbamoylmethyl-2- {3- [4- (2-iminotetrahydropyrimidine
-1-yl) butylamino] propylamino {acetamide).

0.784 mmol of lipid B is dissolved in 25 cm ³ of methanol in a round bottom flask equipped with a magnetic bar, and 10.21 mmol of triethylamine is added. Then O- Mechiriso urea and an aqueous solution of sulfuric acid (1.173mmol) (9cm ³⁾ was slowly poured into a mixture (5 minutes). The mixture is kept in an oil bath at 50 ° C. for 20 hours.

Next, the mixture is concentrated and dried on a rotary evaporator. Dry extract in aqueous solution (4 cm ³ )
And trifluoroacetic acid (1 cm ³ ). This solution was divided into two portions,
Inject into PLC.

The fractions of interest (quantified by analytical HPLC) were collected together, frozen and
And freeze-dried. Thus, 194 mg (0.163 mmol) of the chloride product
Get) Yield: 20.8% For HPLC analysis: R_t= 15.99 minutes.¹ 1 H NMR spectrum (400 MHz, (CD₃)₂SO d₆, In ppm
δ): 0.88 (t, J = 6.5 Hz, 6H: CH of fat chain)₃); 1.24 (m
t, 60H: center CH of fatty chain₂); 1.35 to 1.70 (mt, 4H: each fat)
One CH in the fatty chain₂); 1.57 (mt, 4H: butyl (CH₂)₂1.8
8 and 1.96 (2 mts, each 2H: propyl center CH₂And the center CH of the ring ₂ ); 2.85 to 3.35 (2 mts, total 16H: 8NCH)₂); 3.81
(Wide s, NCH₂CON); 4.03 (d, J = 5 Hz, 2H: C of glycyl
ONCH₂CON); 7.25 and 7.84 (s and wide s, respectively, 1H each)
8.61 (t, J = 5.5 Hz, 1H = NHCO);
70 and 9.02 (2unres.comp., Each 1H: 2 NH). MH⁺= 846.

Example 2: Condensation formula, NH ₂ (CH ₂ ) ₃ N, hereinafter referred to as “lipid C”
Compounds having H (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₃ ] ₂ (the preparation of which is described in patent application WO 97/18185,
Compound (2) (N-ditetradecylcarbamoylmethyl-2- {3- [4- (2-iminotetrahydropyrimidin-1-yl) butylamino] propylamino} acetamide) whose structure is shown in FIG. Synthesis of

Dissolve 1.036 mmol of lipid C in 30 cm ³ of methanol in a round bottom flask equipped with a magnetic bar and add 13.13 mmol of triethylamine. Then O- Mechiriso urea and an aqueous solution of sulfuric acid (1.554mmol) (9cm ³⁾ was slowly poured into a mixture (5 minutes). The mixture is kept at 50 ° C. in an oil bath for about 20 hours. The mixture is then concentrated to dryness on a rotary evaporator. The dry extract is solubilized with an aqueous solution (3 cm ³ ), ethanol (2 cm ³ ) and trifluoroacetic acid (0.5 cm ³ ). This solution is injected on a preparative HPLC.

The target fractions (quantified by analytical HPLC) are collected together, frozen and lyophilized. 218 mg (0.2022 mmol) of the final salified product
Get. Yield: Y = 19.5% HPLC _analytical: R t = 10.76 min. ¹ H NMR spectrum (400 MHz, (CD ₃ ) ₂ SO d ₆ , δ in ppm): 0.88 (t, J = 7 Hz, 6H: CH _{3 of} fatty chain);
40 (mt, 44H: center of the fatty chain (CH ₂ ) ₁₁ ); 1.45 and 1.50 to 1.65 (2 mts, each 2H: one CH ₂ of each fatty chain); 1.59 (mt
, 4H: _CH 2) butyl; 1.91 and 1.97 (2mts, each 2H: central _CH 2) propyl; 2.85 from 3.10 (mt, 10H: 2 N-butyl
CH ₂ -one of _two NCH ₂ -of two propyls and one of two NHC ₂ of the other propyl; 3.23 and 3.30 to 3.50 (2 mts, 5H and 1H, respectively) : other NCH ₂ and fatty chains other propyl _NCH 2); 3.
79 (unres.comp., 2H: NCH ₂ CON); 4.03 (d, J =
5 Hz, 2H: CONCH ₂ CON of glycyl); 7.27 and 8.40 to 9
. 30 (broad s and unres.comp., 2H and 4H: NH ₂ + respectively)
_{CH 3 COO-; NH + CF 3} COO- and = NH); 7.88 and 8.61 (
Each s and wide s, each 1H: NHC = N and CONH, respectively. MH ⁺ = 734.

Example 3 Compound (3) (2- (3- {4 [3- (4,5-dihydro-1H-imidazole-) from lipid C (see Example 2 and FIG. 1 for its structure) 2-
Synthesis of ylamino) propylamino {-N-ditetradecylcarbamoylmethylacetamide).

0.36 mmol of 2-methylmercapto-2-imidazolinium iodide is
0.36c of 1N sodium hydroxide in a round bottom flask equipped with a foamer and a magnetic bar
m³Dissolve in 1N sodium hydroxide 1.44cm in advance³Dissolved in
0.36 mmol of lipid C, 5 cm of water³And ethanol 4cm³The solution
Add to The mixture is kept stirring until the evolution of methyl mercaptan ceases (24
time). Then the mixture is concentrated to dryness on a rotary evaporator. The dried extract was added to an aqueous solution (4 cm ³ ), Ethanol (4cm³) And trifluoroacetic acid (0.5 cm)³) Solubilized
I do. This solution is injected into the preparative HPLC in two portions.

The fractions of interest (quantified by analytical HPLC) were collected together, frozen and
And freeze-dried. Finally 213 mg (0.1727 mmol) of chloride product
obtain. Yield: Y = 48% For HPLC analysis: R_t= 8.90 minutes.¹ 1 H NMR spectrum (400 MHz, CD₃A few drops of COOD d4 were added (
CD₃)₂SO d₆, Δ in ppm): 0.87 (t, J = 7 Hz, 6H: fat)
Fat chain CH₃); 1.15 to 1.40 (mt, 44H: center of fatty chain (CH₂ )₁₁); 1.45 and 1.55 (2 mts, each 2H: one CH of each fat chain) ₂ ); 1.65 (mt, 4H: two central CHs of butyl)₂); 1.80 to 1.
95 (mt, 4H: propyl center CH₂); 2.80 to 3.05 (mt, 1
0H: two NCHs of butyl₂-Two NCHs of one of the two propyls₂−
And the other two propyl HCH₂One); 3.24 (mt, 6H: other
Propyl other NCH₂And NCH of the fatty chain₂); 3.56 (s, 2H: NCH) ₂ CON); 3.62 (s, 4H: NCH₂CH₂CH₂N); 4.02 (d,
J = 5Hz, 2H: Glycyl CONCH₂CON). MH⁺= 777.

Example 4: Compound (4) (2) by method for synthesizing “building block”
Synthesis of-(3- {bis [3- (4,5-dihydro-1H-imidazol-2-ylamino) propyl] amino} propylamino-N-ditetradecylcarbamoylmethylacetamide).

I) Synthesis of Boc-Gly-ditetradecylamine (a) A Gly group (10 mmol) in which an amine is protected by a Boc group and ditetradecylamine (10 mmol) were placed in a 250 ml round bottom flask, and dichloromethane 1 was added.
Add 00cm ^3. Stir the mixture until completely dissolved. Next, 30 mmol of N-ethyldiisopropylamine (DIEA) and 11 mmol of benzotriazol-1-yloxytrisdimethylamine phosphonium (BOP) are added. D
The pH is kept at 10 by IEA and the mixture is stirred for 2 hours. When the reaction is complete (monitored by TLC and / or HPLC), the dichloromethane is evaporated and the solid obtained is taken up in ethyl acetate (300 cm ³ ). The organic phase potassium sulfate (100 cm ³ four times), washed with a solution of sodium carbonate (100 cm ³ four times) and sodium chloride (100 cm ³ four times). The organic phase is then dried over magnesium sulfate, filtered and evaporated under vacuum. Product (a) in 93% yield
Get. _{TLC: R f = 0.9 (CHCL} 3 / MeOH, 9: 1) MH + = 567.

II) [Z-NH (CH₂)₃]₂-N- (CH₂)₃-NH-Boc-CH ₂ Synthesis of —COOH (b) 1) NC— (CH₂)₂-NH-Boc-CH₂Synthesis of —COOH (c) N- (cyanoethyl) glycine amine (0.1 mol / amine, commercially available product)
1N sodium hydroxide (200cm³/ Amine) and dioxane (200cm)³)
Dissolve in The solution is stirred in an ice bath and then O- (t-butoxycarbonyl) 2 or
Di-p-chlorobenzyloxycarbonyl (ClZ, 0.14 mol / amine)
Oxane 200cm³The medium solution is added dropwise. The pH is maintained at a value of 9 or higher. That
The mixture is subsequently stirred at about 20 ° C. overnight. Evaporate dioxane under vacuum, then
The mixture is acidified to pH 3 using potassium sulfate solution. Remove insolubles with ethyl acetate
Extract (100cm³3 times), wash with sodium chloride solution (100 cm³ Twice). Dry the organic phase over magnesium sulfate, filter and evaporate under vacuum
. The formula, NC- (CH₂)₂-NH-Boc-CH₂COOH product (c) is 9
Obtained in 8% yield. TLC: R_f= 0.66 (CHCl₃/ MeOH, 8: 2) MH⁺= 229.

2) Synthesis of NH ₂ — (CH ₂ ) ₃ —NH—Boc—CH ₂ —COOH (d) Formula (c): Product of NC— (CH ₂ ) ₂ —NH—Boc—CH ₂ COOH 50
The mmol is injected into a 1 liter stainless steel autoclave. ethanol(
A solution (80 mol) of 10 cm ³ (95%) and 3.3 g of sodium hydroxide is prepared simultaneously in a beaker. When the sodium hydroxide is dissolved, Raney nickel on carbon 2
cm ³ is introduced. Close the autoclave. The initial hydrogenation pressure is about 52 bar,
Reduce to about 48.5 bar overnight at room temperature (20 ° C.). The suspension strained by filter paper, the filter washed with ethanol (25 cm ³ four times), and concentrated to dryness under vacuum the filtrate. The product (d) is obtained, which is used in the next step without further purification. _{TLC: R f = 0.12 (CHCl} 3 / MeOH, 6: 4) MH + = 233.

3) [NC (CH ₂ ) ₂ ] ₂ -N- (CH ₂ ) ₃ -NH-Boc-CH ₂ -C
Synthesis formula _{OOH (e), NH 2 -} (CH 2) 3 -NH-Boc-CH 2 -COOH product (d)
(0.05 mol) and sodium hydroxide (0.1 mol) are dissolved in 150 cm ³ of water in a round bottom flask. Cool the solution in an ice bath. Acrylonitrile (0.12
mol) is slowly poured with vigorous stirring to keep the mass temperature below 20 ° C. The reaction mixture is left overnight at room temperature (20 ° C.). Thereafter, the mixture is kept at 50 ° C. for 2 hours. The solvent is evaporated off under vacuum and the mixture is then diluted with potassium sulphate solution.
Acidify to H3. Extract the insolubles with ethyl acetate (3 × 200 cm ³ ) and wash with sodium chloride solution (2 × 100 cm ³ ). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The "crude product" is optionally purified on a silica column. The product (e) is obtained with a yield of 50%. _{TLC: R f = 0.75 (CHCl} 3 / MeOH, 6: 4) MH + = 339.

4) Synthesis of [Z—NH (CH ₂ ) ₃ ] ₂ —N— (CH ₂ ) ₃ —NH—Boc—CH ₂ —COOH (b) Formula [NC (CH ₂ ) ₂ ] ₂ —N _{- (CH 2) 3 -NH-} Boc-CH 2 -CO
The product of OH (e) (50 mmol) is injected into a 1 liter stainless steel autoclave. A solution (80 mol) of 10 cm ^{3 of} ethanol (95%) and 3.3 g of sodium hydroxide is simultaneously prepared in a beaker. When the sodium hydroxide has dissolved, the solution is introduced into the autoclave. A stream of nitrogen is passed through the autoclave and 2 cm ³ of Raney nickel on carbon is introduced. Close the autoclave. The initial hydrogenation pressure is about 52 bar and drops to about 48.5 bar overnight at room temperature (20 ° C.). The suspension strained by filter paper, the filter washed with ethanol (25 cm ³ four times), and concentrated to dryness under vacuum the filtrate. A white solid is obtained, which is used without further purification after TLC analysis. _{TLC: R f = 0.14 (CHCl} 3 / MeOH, 6: 4).

The solid obtained above is dissolved in 1N sodium hydroxide (200 cm ³ / amine) and dioxane (200 cm ³ ). The solution is stirred in an ice bath and then a solution of (t-butoxycarbonyl) 2 O or p-chlorobenzyloxycarbonyl (0.14 mol / amine) in 200 cm ³ of dioxane is added dropwise. The pH is maintained at a value of 9 or higher. The mixture is subsequently stirred at about 20 ° C. overnight. Evaporate the dioxane under vacuum and then acidify the mixture to pH 3 using potassium sulfate solution. Extract the insolubles with ethyl acetate (3 times with 100 cm ³ ) and wash with sodium chloride solution (10
0 cm ³ twice). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product is analyzed by TLC and / or HPLC.

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

The product (b) is obtained in a yield of 66% relative to the product (d). _{TLC: R f = 0.42 (CHCl} 3 / MeOH, 6: 4) MH + = 615.

III) [Z-NH (CH ₂ ) ₃ ] ₂ -N- (CH ₂ ) ₃ -NH-Boc-C
Synthesis of H ₂ —COGlyN [(CH ₂ ) ₁₃ —CH ₃ ] ₂ (f) The product (a) (1 mmol) in which the amine is protected with a Boc group is injected into a round bottom flask equipped with a magnetic bar. . 30 cm ³ of trifluoroacetic acid at 4 ° C. are added and the solution is stirred for 1 hour. When the reaction is complete (monitored by TLC and / or HPLC), the trifluoroacetic acid is evaporated under vacuum and the product is
Co-evaporate with m ³ three times and dry. _HPLC: R t = 12.86 min _(H 2 O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

The obtained product (Gly-ditetradecylamine, 10 mmol) and the product (
b) (10 mmol) was placed in a 250 cm ³ round bottom flask and dichloromethane (
100 cm ³ ) is added and the mixture is stirred until completely dissolved. Next, 30 mmol of N-ethyldiisopropylamine (DIEA) and 11 mmol of BOP hexafluorophosphate are added. The pH is kept at 10 by DIEA and the mixture is stirred for 2 hours. When the reaction is complete (monitored by TLC and / or HPLC), the dichloromethane is evaporated and the solid obtained is ethyl acetate (300 cm ³ )
Take inside. The organic phase is washed with a solution of potassium sulphate solution (4 times at 100 cm ³ ), sodium carbonate (4 times at 100 cm ³ ) and sodium chloride solution (4 times at 100 cm ³ ). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product is used without further purification. After purification on a silica column (dichloromethane / methanol, 8: 2), the product (f) is obtained in a yield of 75%. _{TLC: R f = 0.86 (CHCL} 3 / MeOH, 8: 2) HPLC: R t = 17.44 min _(H 2 O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

[0135] _{IV) [NH 2 (CH 2} ) 3] 2-N- (CH 2) Synthesis amine 3 -NH-Boc-CH 2 -COGlyN [(CH 2) 13 -CH 3] 2 (g) is The protected product (f) is placed in a round bottom flask equipped with a magnetic bar and dissolved in 10 cm ³ of methanol per gram of product. Palladium on carbon (10%, 1 g / g product) and ammonium formate (1 g / g product) are added at room temperature. Monitor the hydrogenolysis by HPLC. After 2 hours, the reaction is terminated, the mixture is filtered and the filter is washed three times with 10 cm ³ of methanol per gram of product. Double-distilled water was added and the solution was frozen, or lyophilized, or filtrate is concentrated to dryness, take the solid up in ethyl acetate (300 cm ^3). The organic phase with sodium carbonate solution (100 cm ³ twice) and sodium chloride solution (100 cm ³ with 2
), Then it is dried over magnesium sulfate, filtered and evaporated under vacuum. The product is analyzed by HPLC and used without further purification.
The product (g) is obtained in a yield of 40% based on the product (f). _HPLC: R t = 9.62 _{min, (H 2 O / MeCN:} 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100]. MH +: 795.

V) Synthesis of Compound (4) The product (g) (1 mmol / amine) containing the primary amine to be modified is dissolved in dichloromethane (10 cm ³ ) and then 2-methylthioimidazoline hydroiodide (1.2 mmol) / Amine) and triethylamine (3 mmol / amine). Stir the mixture at room temperature (20 ° C.) until the evolution of methyl sulfide has ceased. When the reaction is over (monitored by HPLC), the dichloromethane is evaporated under vacuum.

The product obtained (1 mmol), in which the amine is protected by a Boc group, is placed in a round-bottomed flask equipped with a magnetic bar. 30 cm ³ of trifluoroacetic acid at 4 ° C. are added and the solution is stirred for 1 hour. When the reaction is completed (TLC and / or HPL
C), the trifluoroacetic acid is evaporated under vacuum and the product is dried by co-evaporation with 30 cm ³ of ethyl ether three times.

The product obtained is purified by preparative HPLC and the fractions are analyzed by HPLC. The compound (4) according to the invention is thus obtained in a yield of 34%. _HPLC: R t = 10.07 _{min, (H 2 O / MeCN:} 3 min [40/60], 3
-20 minutes [0/100], 35 minutes [0/100]. ¹ H NMR spectrum ((CD ₃ ) ₂ SO at 400 MHz, temperature 383 K)
d ₆ , δ in ppm): 0.92 (t, J = 7 Hz, 6H: CH _{3 of the} fatty chain);
1.25 to 1.45 (mt, 44H: center of fatty chain (CH ₂ ) ₁₁ ); 1.5
7 (mt, 4H: propyl center CH ₂ ); 2.50 to 3.40 (mt, 16
H: two NHC _{2 of} propyl and NCH _{2 of} fatty chain); 3.68 (s, 8H:
Two _{NCH 2 CH 2 N); 3.72} ( broad _{s, 2H: NCH 2 CON)} ; 4.
06 (s, 2H: glycyl the CONCH ₂ CON). MH +: 831.

Example 5 Compound (5) by the Method of Synthesis of “Building Block”: (N-ditetradecylcarbamoylmethyl-2- {3- [3- (1,4,5,6-tetrahydropyrimidine-2) Synthesis of -ylamino) propylamino] propylaminodiacetamide.

I) Synthesis of Boc-Gly-ditetradecylamine (a) The procedure is carried out as in the above example. The product (a) is obtained with a yield of 93%. _{TLC: R f = 0.9 (CHCl} 3 / MeOH, 9: 1) MH +: 567.

II) Z—NH (CH₂)₃-N-Boc- (CH₂)₃-N-Boc-CH ₂ Synthesis of —CHOOH (b) 1) NC— (CH₂)₂-NH-Boc-CH₂Synthesis of —COOH (c) The procedure is performed as in Example 4 above. The product (c) was obtained in a yield of 98%.
obtain. TLC: R_f= 0.66 (CHCl₃/ MeOH, 8: 2) MH +: 229.

2) Synthesis of NH ₂ — (CH ₂ ) ₃ —NH—Boc—CH ₂ —COOH (d) A product (d) is obtained in the same manner as in Example 4 above. _{TLC: R f = 0.12 (CHCl} 3 / MeOH, 6: 4) MH +: 233.

3) NC (CH ₂ ) ₂ -N-Boc- (CH ₂ ) ₃ -NH-Boc-CH _2-
Synthesis of COOH (e) The product (d) (0.05 mol) and sodium hydroxide (0.1 mol) are dissolved in 150 cm ³ of water in a round bottom flask. Cool the solution in an ice bath. Acrylonitrile (0.05 mol) was slowly poured with vigorous stirring, and the temperature of
Keep below 0 ° C. The reaction mixture is left overnight at room temperature (20 ° C.).

The solvent is evaporated under vacuum, then the mixture is acidified to pH 3 with potassium sulfate solution. Extract the insolubles with ethyl acetate (3 × 200 cm ³ ) and wash with sodium chloride solution (2 × 100 cm 2). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product obtained is optionally purified on a silica column.

The obtained product (0.1 mol / amine) was added to 1N sodium hydroxide (200 c
m ³ / amine) and dioxane (200 cm ³ ). The solution was stirred in an ice bath, then _{(Boc) 2} O or p- chloro benzyloxycarbonyl (0.14Mo
1 / amine) in 200 cm ³ of dioxane is added dropwise. The pH is maintained at a value of 9 or higher. Then the mixture is stirred overnight at room temperature (20 ° C.). Evaporate the dioxane under vacuum and then acidify the mixture to pH 3 using potassium sulfate solution. Extract the insolubles with ethyl acetate (3 × 100 cm ³ ) and wash with sodium chloride solution (2 × 100 cm ³ ). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product is analyzed by TLC and / or HPLC.

The product (e) is thus obtained in a yield of 93%. _{TLC: R f = 0.75 (CHCl} 3 / MeOH, 8: 2) MH +: 386.

4) Z—NH— (CH₂)₃-N-Boc- (CH₂)₃-N-Boc-CH ₂ Synthesis of —COOH (b) The product (e) (50 mmol) was placed in a 1 liter stainless steel autoclave.
inject. Ethanol (95%) 10cm³And 3.3 g of sodium hydroxide solution
(80 mol) are simultaneously prepared in a beaker. Once the sodium hydroxide is dissolved,
Is introduced into the autoclave. Pass a stream of nitrogen through the autoclave and
Raney nickel 2cm³Is introduced. Close the autoclave. Initial hydrogenation pressure is
At about 52 bar, drop to about 48.5 bar at room temperature (20 ° C.) overnight. Suspension
The solution is filtered with filter paper, and the filter is washed with ethanol (25 cm).³4 times), filtration
The liquid is concentrated to dryness under vacuum. A white solid was obtained, which after TLC analysis was further
Used without purification. TLC: R_f= 0.14 (CHCl₃/ MeOH, 6: 4).

The obtained product (0.1 mol / amine) was added to 1N sodium hydroxide (200 c
m ³ / amine) and dioxane (200 cm ³ ). The solution is stirred in an ice bath and then a solution of p-chlorobenzyloxycarbonyl (0.14 mol / amine) in 200 cm ³ of dioxane is added dropwise. The pH is maintained at a value of 9 or higher. Then the mixture is stirred overnight at room temperature (20 ° C.). Evaporate the dioxane under vacuum and then acidify the mixture to pH 3 using potassium sulfate solution. Extract insolubles with ethyl acetate (3 times with 100 cm ³ ) and wash with sodium chloride solution (100 c
m ³ in 2 times). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product obtained is applied to a silica column (dichloromethane / methanol, 9: 1)
To purify. The product is analyzed by TLC and / or HPLC. Product (b)
In a yield of 32% relative to the product (d). _{TLC: R f = 0.63 (CHCl} 3 / MeOH, 9: 1) MH +: 523.

III) Synthesis of 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) 3,4,5,6-tetrahydro-2-pyrimidinethiol (0.0103mo
1) was charged into a round bottom flask under a stream of nitrogen while stirring, and 5 cm ^{3 of} methanol and 0.65 cm ³ (0.0105 mol) of methyl iodide were added. The mixture is heated under reflux for 1 hour and then cooled to room temperature (20 ° C.). The product is precipitated by adding 5 cm ³ of ethyl ether. The precipitate is filtered and then washed with ethyl ether. The product is then dried overnight at a pressure of 34 mbar.

1.5 g (0.0041 mol) of product (VI) are obtained, ie a yield of 40%. _{TLC: R f = 0.25 (CHCl} 3 / MeOH, 9: 1) MH +: 131.

D) Z-NH (CH ₂ ) ₃ -N-Boc (CH ₂ ) ₃ -N-Boc-CH _{2-
COGlyN [(CH 2) 13 -CH} 3] 2 (g) product synthesized amine is protected with Boc group of (a) (1mmol), it is injected in a round bottom flask equipped with a magnetic bar. 30 cm ³ of trifluoroacetic acid at 4 ° C. are added and the solution is stirred for 1 hour. When the reaction is complete (monitored by TLC and / or HPLC), the trifluoroacetic acid is evaporated in vacuo and the product obtained is dried by co-evaporation with 30 cm ³ of ethyl ether three times. _HPLC: R t = 12.86 min _(H 2 O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

The obtained product (10 mmol) and the product (b) (10 mmol) were mixed with 250c
placed in a round-bottom flask of m ^3. Dichloromethane (100 cm ³ ) is added and the mixture is stirred until completely dissolved. Next, DIEA 30 mmol and BOP 11 mm
ol. The pH is kept at 10 by DIEA and the mixture is stirred for 2 hours. When the reaction is complete (monitored by TLC and / or HPLC), the dichloromethane is evaporated and the solid obtained is taken up in ethyl acetate (300 cm ³ ). The organic phase potassium sulfate solution (100 cm ³ four times), washed with sodium carbonate (100 cm ³ four times) and sodium chloride solution (100 cm ³ four times). The organic phase is dried with magnesium sulfate, filtered and evaporated under vacuum. The product is used without further purification.

After purification on a silica column (dichloromethane / methanol, 8: 2), 85%
The product (g) is obtained in a yield of TLC: R _f = 0.9 (CHCL ₃ / MeOH, 9: 1) HPLC: R _t = 19.79 min (H ₂ O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

V) [NH ₂ (CH ₂ ) ₃ ] 2-N-Boc- (CH ₂ ) ₃ -NH-Boc-
_{CH 2 -COGlyN [(CH 2)} 13 -CH 3] synthesis product of 2 (h) a (g), placed in a round bottom flask equipped with a magnetic bar, dissolved in methanol 10 cm ³ / g product. Palladium on carbon (10%, 1 g / g product) and ammonium formate (1 g / g product) are added at room temperature (20 ° C.). Monitor the hydrogenolysis by HPLC. After 2 hours, the reaction is terminated, the mixture is filtered and the filter is washed three times with 10 cm ³ / g product of methanol. Add double distilled water,
The solution is frozen and lyophilized, or the filtrate is concentrated to dryness and the solid is ethyl acetate (
Take in 300 cm ³ ). The organic phase was washed with sodium carbonate solution (2 times at 100 cm ³ )
And washed with sodium chloride solution (2 × 100 cm ³ ), then it is dried over magnesium sulfate, filtered and evaporated under vacuum. The product is analyzed by HPLC and used without further purification. The product (h) is obtained in a yield of 93% based on the product (g). _{TLC: R f = 0.42 (CHCL} 3 / MeOH, 6: 4) HPLC: R t = 14.66 _{min, (H 2 O / MeCN:} 3 min [40/60], 3
-20 minutes [0/100], 35 minutes [0/100]. MH +: 838.

VI) Synthesis of Compound (5) The product (h) (1 mmol / amine) containing the primary amine to be modified is dissolved in dichloromethane (10 cm ³ ) and then the product (f) (1.2 mmol / amine) )
And triethylamine (1.3 mmol / amine). Stir the mixture at room temperature (20 ° C.) until the evolution of methyl sulfide has ceased. When the reaction is completed (HPLC
), Evaporate the dichloromethane under vacuum.

The product obtained is purified by preparative HPLC and the fractions are analyzed by HPLC. Thus, compound (5) is obtained in a yield of 38%. _HPLC: R t = 8.42 _{min, (H 2 O / MeCN:} 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100]. ¹ H NMR spectrum (400 MHz, (CD ₃ ) ₂ SO d ₆ , δ in ppm): 0.86 (t, J = 7 Hz, 6H: CH _{3 in} fatty chain);
35 (mt, 44H: center of the fatty chain (CH ₂ ) ₁₁ ); 1.44 and 1.53 (
2Mts, each 2H: the one of each fatty chain _CH 2); 1.80 from 2.00 (mt
, 6H: propyl center CH ₂ and 1,4,5,6-tetrahydropyrimidine CH ₂ ); 2.80 to 3.10 (mt, 10H: propyl NCH ₂ and 1,
_4,5,6-NCH 2 tetrahydropyrimidine); 3.14 from 3.45 (m
t, = 4,5,6-tetrahydropyrimidine = NCH ₂ and fatty chain = NC
. Corresponding to _{H 2 6H); 3.81 (unres.comp} , 2H: NCH 2 C
ON); 4.04 (d, J = 5 Hz, 2H: CONCH ₂ CON of glycyl);
7.89 = 8.62-8.75 and 9.01 (4unres.comp, total 8H:. OH exchangeable group and _CF 3 COOH). MH +: 720.

Example 6 Compound (6): Synthetic Method of “Building Block”: N-Dioctadecylcarbamoylmethyl-2- {3- [3- (1,4,5,6-tetrahydropyrimidin-2-ylamino) ) Synthesis of propylamino] propylaminodiacetamide.

I) Synthesis of Boc-Gly-ditetradecylamine (a) The procedure is carried out as in the above example. The product (a) is obtained with a yield of 93%. _{TLC: R f = 0.9 (CHCl} 3 / MeOH, 9: 1) MH +: 567.

II) Z—NH (CH₂)₃-N-Boc- (CH₂)₃-N-Boc-CH ₂ Synthesis of —CHOOH (b) 1) NC— (CH₂)₂-NH-Boc-CH₂Synthesis of —COOH (c) The procedure is performed as in Example 5 above. The product (c) was obtained in a yield of 98%.
obtain. TLC: R_f= 0.66 (CHCl₃/ MeOH, 8: 2) MH +: 229.

2) Synthesis of NH ₂ — (CH ₂ ) ₃ —NH—Boc—CH ₂ —COOH (d) The product (d) is obtained in the same manner as in Example 5 above. _{TLC: R f = 0.12 (CHCl} 3 / MeOH, 6: 4) MH +: 233.

3) NC (CH ₂ ) ₂ -N-Boc- (CH ₂ ) ₃ -NH-Boc-CH _2-
Synthesis of COOH (e) The procedure is carried out as in Example 5 above. The compound (e) is thus obtained in a 93% yield. _{TLC: R f = 0.75 (CHCl} 3 / MeOH, 8: 2) MH +: 386.

4) Z-NH- (CH₂)₃-N-Boc- (CH₂)₃-N-Boc-CH ₂ Synthesis of —COOH (b) The procedure is carried out as in Example 5 above. A white solid was obtained and analyzed by TLC
Later, it is used without further purification. TLC: R_f= 0.14 (CHCl₃/ MeOH, 6: 4)

The resulting product is used as described above, so that the terminal amine is protected with a benzyloxycarbonyl group. The product (b) is thus obtained in a yield of 32% relative to the product (d). _{TLC: R f = 0.63 (CHCl} 3 / MeOH, 9: 1) MH +: 523.

III) Synthesis of 2-methylsulfanyl-1,4,5,6-tetrahydropyrimidine (f) The procedure is carried out as in Example 5 above. As such, 1.5 g (0.
0041 mol) of product (f), ie a yield of 40%. _{TLC: R f = 0.25 (CHCl} 3 / MeOH, 9: 1) MH +: 131.

IV) Synthesis of Z-NH (CH ₂ ) ₃ -N-Boc (CH ₂ ) ₃ -N-Boc-CH ₂ -COGlyN [(CH ₂ ) ₁₇ -CH ₃ ] ₂ (g) The procedure is carried out as in Example 5. The product (a) in which the Boc group has been cleaved in this way is obtained. _HPLC: R t = 19.44 min _(H 2 O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

The product obtained is used analogously to the product (b) of Example 5 above. After purification on a silica column (dichloromethane / methanol, 8: 2), the product (g)
Obtained in 4% yield. TLC: R _f = 0.9 (CHCL ₃ / MeOH, 9: 1) HPLC: R _t = 23.95 min (H ₂ O / MeCN: 3 min [40/60], 3-
20 minutes [0/100], 35 minutes [0/100].

V) [NH ₂ (CH ₂ ) ₃ ] 2-N-Boc- (CH ₂ ) ₃ -NH-Boc-
_{CH 2 -COGlyN [(CH 2)} 17 -CH 3] out the procedure in the same manner as in Synthesis of Example 5 2 (h). The product (h) is obtained with a yield of 73% based on the product (g). _{TLC: R f = 0.28 (CHCL} 3 / MeOH, 6: 4) HPLC: R t = 20.59 _{min, (H 2 O / MeCN:} 3 min [40/60], 3
-20 minutes [0/100], 35 minutes [0/100]. MH +: 838.

VI) Synthesis of Compound (6) The procedure is carried out as in Example 5 above. Thus, compound (6) is obtained with a yield of 68%. _HPLC: R t = 15.83 _{min, (H 2 O / MeCN:} 3 min [40/60], 3
-20 minutes [0/100], 35 minutes [0/100]. ¹ H NMR spectrum (500 MHz, (CD ₃ ) ₂ SO d ₆ , δ in ppm): 0.88 (t, J = 7 Hz, 6H: CH _{3 of} fatty chain);
35 (mt, 44H: center of the fatty chain (CH ₂ ) ₁₅ ); 1.46 and 1.54 (
2Mts, each 2H: the one of each fatty chain _CH 2); 1.80 from 2.00 (mt
, 6H: propyl center CH ₂ and 1,4,5,6-tetrahydropyrimidine CH ₂ ); 2.85 to 3.05 (mt, 10H: propyl NCH ₂ and 1,
_4,5,6-NCH 2 tetrahydropyrimidine); 3.15 from 3.45 (m
t, = 4,5,6-tetrahydropyrimidine = NCH ₂ and fatty chain = NC
. Corresponding to _{H 2 6H); 3.81 (unres.comp} , 2H: NCH 2 C
ON); 4:04 (d, J = 5 Hz, 2H: CONCH ₂ CON of glycyl);
7.88 = 8.61-8.74 and 8.99 (4unres.comp, total 8H:. OH exchangeable group and _CF 3 COOH). MH +: 832.

B—Use of the Transfer Agent According to the Invention

Example 7: Preparation of nucleolipid conjugate This example illustrates the preparation of a nucleolipid conjugate according to the present invention.

The compound used in this example is a chloroform solution of the compound (1). D
An amount of 10 nmol of compound (1) (ie 11.8 μg) per μg NA was used. In some cases, neutral colipide, cholesterol or DOPE is premixed with the compound. The chloroform is evaporated using a weak argon flow to form a thin lipid film, which is then rehydrated in a mixture of 5% dextrose and 10 mM sodium chloride at 4 ° C. overnight. The sample is then treated with ultrasound for 5 minutes,
Heat at 65 ° C. for 30 minutes and finally treat again with ultrasound for 5 minutes. A lipid suspension is thus obtained, which is stored at 4 ° C. until use.

The DNA used was 0.5 mg / ml or 1.0 mg / ml of plasmid pXL2772 (FIG. 2) in a mixture of 5% dextrose and 20 mM sodium chloride.
Solution. Plasmid pXL2772 has the following properties: an endotoxin level of less than 50 EU / mg, a helical DNA level of more than 60%, an RNA of less than 5%, ie mRNA, tRNA and ribosomal RNA (
Content as determined by HPLC), chromosomal DNA levels of less than 1%, protein content of less than 1%, osmolarity of less than 15 mosmol / kg.

The nucleolipid complex according to the invention is prepared by rapidly mixing an equal volume of the DNA solution and the lipid suspension as described above. The amount of compound that binds to DNA ranges from 0.5 nmol / μg DNA to 12 nmol / μg DNA.

Example 8 Behavior of Complexes Formed at Various Charge Ratios This example illustrates the behavior of nucleolipid complexes according to the present invention at various charge ratios. In addition, the influence of the addition of neutral colipid is also exemplified.

First, dynamic light scattering (Dynam) was performed using a Coulter N4plus instrument.
The size of the complex was analyzed by measuring the hydrodynamic diameter by ic Laser Light Scattering. The sample is diluted 20-fold in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple diffusion. The effects of cycloamidine groups, lipid composition and charge ratio on the size of the nucleolipid complex according to the present invention were studied.

When the charge ratio between compound (1) and DNA according to the invention is high, three possible phases can be distinguished. These three phases determine the therapeutic potential of compound (1). FIG. 3 illustrates these three phases for compound (1). The same behavior is observed for other compounds according to the invention.

At low charge ratios, the DNA is not saturated with compound (1). There is still naked DNA left and the complex as a whole is negatively charged. Particle size is small (1
Between 00 and 300 nm). This stage is referred to as "Phase A".

The fact that the DNA is not completely saturated with compound (1) means that the DNA is not completely protected by compound (1). Therefore DNA is an enzyme (
DNAse). In addition, the complex is generally negatively charged, making it difficult to cross the cell membrane. For these reasons, the nucleolipid complex in phase A has much lower transfection efficiency.

At intermediate charge ratios, the DNA is completely saturated with compound (1) and the complex is generally neutral or slightly positive. This phase is unstable because of the low ionic repulsion and possible "cross-linking" phenomena. The particle size is well above the limit of detection by dynamic light scattering (much larger than 3 μm). This unstable phase is called “B phase”. The size of such a complex is not suitable for use by injection. However, this does not necessarily mean that the conjugate is inert in phase B, just that it is a formulation that is not suitable for injection for pharmaceutical purposes.

At relatively high charge ratios, the DNA is oversaturated with compound (1) and the complex is overall positively charged. This phase is stable because of the strong repulsion between positive charges.
This phase is called "C phase". Unlike phase A, the nucleolipid complex is a form in which the DNA is well protected against the enzyme, and its overall positive charge facilitates the passage through anionic cell membranes. Therefore, the "phase C" complexes are particularly suitable for use for transfer of nucleic acids into cells.

In addition to the cycloamidine group of the compounds according to the invention, the use of neutral colipids
As illustrated in FIG. 3, it strongly affects the stability of the complex. The added colipid is DOPE (cationic lipid / DOPE = 3/2) or cholesterol (cationic lipid / cholesterol = 3/1). In general, the addition of neutral colipids increases the instability of the complex, resulting in an increase in the amount of compound required to obtain phase C. This is shown very clearly by comparing the charge ratios at which the phase C is obtained in the presence and absence of colipid in FIG.

The value of the charge ratio defining the three phases A, B and C depends on the compound used. Thus, these numbers can vary very widely from compound to compound.

Finally, the affinity of the compound for DNA as a function of the charge ratio was examined. For this purpose, the decrease in fluorescence after addition of 3 μg of ethidium bromide (EtBr) was measured. In fact, the replacement of ethidium bromide in DNA by a compound
Is an indicator of binding to

The formulation used is diluted 20-fold to a final concentration of 25 μg DNA / ml. The relative fluorescence measured on naked DNA is defined as 100%. The level of binding to compound (1) is represented by a decrease in the relative fluorescence of the sample. FIG. 3 shows that the fluorescence decreases when the charge ratio is high, which is due to the higher amount of compound (1).
Can be used for binding to DNA (the lower the fluorescence, the greater the amount of compound bound to DNA until saturation is reached).

Thus, it was shown that the affinity of compound (1) according to the present invention for DNA was determined by the cycloamidine group, not by the addition of colipide.

Example 9: Transfection of compound (1) in vitro This example demonstrates that the compound (1
) Illustrates the ability to transfer DNA into cells in vitro.

A 24-well microplate is inoculated with 60,000 HeLa cells (ATCC) / well and transduced 24 hours later. The complex containing 1 μg of DNA is diluted in 0.5 ml of serum-free DMEM medium (Gibco / BRL) and then added to each well. The cells are cultured at 37 ° C. for 4 hours. The medium containing the complex is removed and replaced with a mixture of DMEM and 10% fetal calf serum. Next, the cells are again
Incubate for hours. Finally, the cells are lysed and a luciferase test kit (Prome
ga) and test using a Dynex MLX luminometer.

The results shown in FIG. 4 highlight the difference in the performance of naked DNA compared to the fully saturated compound (1) / DNA complex of the invention: naked DNA in vitro
No luciferase activity was detectable after transfection with o (the sensitivity of the device was below 1 pg / well), whereas the transfection activity of the complex according to the invention was in the range from 200 pg / well to 8000 pg / well. is there.

This example therefore clearly shows that the use of compound (1) according to the invention for the transfection of cells in vitro is beneficial.

Example 10: Transfection in vitro with compounds (3), (5) and (6) This example demonstrates the comparison of the compound (3
), (5) and (6) illustrate the ability to transfer DNA into cells in vitro.

Transfection is performed on HeLa cells according to the protocol of Example 9 above. The results are shown in FIGS. These three compounds are used in vivo
It has been found to have good transfection levels at tro.

Example 11 Transfection of Compound (1) In Vivo This example describes the use of unformulated DNA and patent application WO 97/18185.
Mentioned the condensation formula: NH₂(CH₂)₃NH (CH₂)₄NH (CH₂) ₃ NHCH₂COArgN [(CH₂)₁₇CH₃]₂Fig. 1
The compound (1) according to the present invention is compared with the lipid A shown in FIG.
The ability to transfer DNA into is illustrated.

In vivo gene transfer was performed on Bulb / C mice by intramuscular and intravenous administration. The formulations compared were a formulation of naked DNA, a formulation containing lipid A, or a formulation containing compound (1) according to the present invention.

For intramuscular injection, 30 μl of a formulation containing 15 μg of DNA was administered to the tibialis anterior muscle of each mouse. Tissues are harvested 7 days after injection, frozen and stored at -80 ° C until the luciferase activity test is performed. The measurement of luciferase activity was performed in Example 8.
Perform as in. In the case of injection by the intravenous route, each mouse has D
200 μl of a formulation containing 50 μl of NA was administered. In this case, the tissue is collected 24 hours after the injection, frozen, and stored as above.

The results of the gene transfer in vivo are shown in FIGS. 8 and 9. The ratio of compound (1) to DNA is 10 nmol / μg DNA. The ratio of lipid A to DNA is 4n
mol / μg DNA.

FIG. 8 illustrates the in vivo in vivo activity of compound (1) according to the invention compared to naked DNA and lipid A. The level of luciferase activity is comparable between naked DNA and compound (1), the latter furthermore being found to have greatly improved activity compared to lipid A. The mechanism of transfer involved is naked DNA
And the use of compound (1) according to the invention. In fact, the complexes according to the invention used do not contain free DNA (phase C) and, moreover, their i.
The results in n vitro far exceed the results for naked DNA.

FIG. 9 compares the activity of compound (1), naked DNA and lipid A according to the invention by the intravenous and intramuscular routes.

It can be seen that the transfection efficiency by the intravenous route is almost equivalent for lipid A and compound (1). On the other hand, in the intramuscular route, the transfection efficiency of the compound (1) according to the present invention far exceeds that of lipid A.

As compared to naked DNA, compound (1) shows transfection by the intravenous route in addition to transfection by the intramuscular route which is at least equivalent.

Thus, the in vivo nucleic acid transfer efficiency for compound (1) according to the present invention is expected to exceed the efficiency of lipid A, a known cationic lipid, and unformulated DNA as a whole.

Finally, the complex according to the invention has the advantage of protecting the DNA from nuclease degradation as compared to naked DNA transfection,
Therefore, it seems to contribute to a significant improvement in the stability of the formulation. The compounds of the invention can also be used to protect DNA from damage during lyophilization, again improving the stability of the formulation.

Example 12 Transfection of Compounds (5) and (6) In Vivo This example illustrates the ability of compounds (5) and (6) to transfer nucleic acids efficiently in vivo. .

[0203] The same protocol is used as in the above example. FIG. 10 does not contain colipids,
Compounds (5) and (6) formulated at a charge ratio of 0.25: 1 with DNA were administered i.p. m.
48 hours post-injection reveals transfection levels above or equal to naked DNA in vivo.

The table below shows the results obtained for compounds (5) and (6) in various formulations.

[0205]

[Table 3]

[Brief description of the drawings]

FIG. 1 shows the structure of a synthetic vector, referred to in the present invention as Lipid A, Lipid B and Lipid C and described in Patent Application WO 97/18185, incorporated herein by reference.

FIG. 2 shows schematically the plasmid pXL2772.

FIG. 3 is a phase diagram for the compound (1) / DNA nucleolipid complex. The binding of compound (1) to DNA was determined by the decrease in fluorescence (%, naked DNA) of ethidium bromide (EtBr) (symbol, solid line) as indicated on the y-axis located on the right.
Is 100%). The size (nm) of the particles of the complex is indicated on the y-axis located on the left. The x-axis represents the transfer agent / DNA charge ratio. The size of the nucleolipid complex without colipid is indicated by a solid line with the symbol ■. The size of the nucleolipid complex containing 25% cholesterol is represented by the □ symbol as a dashed line. The size of the nucleolipid complex containing 40% DOPE is indicated by a dashed line with a dashed line.

FIG. 4. No colipid (dark shaded middle bar) compared to naked DNA.
Contains 25% cholesterol (middle shaded left bar), 40 mo
A compound according to the invention containing 1% DOPE (light shaded right bar) (
FIG. 2 shows the activity of the nucleolipid complex containing 1) for in vitro gene transfer into HeLa cells.

FIG. 5 shows in vivo of nucleolipid complex formed from compound (3) on HeLa cells.
5 shows the gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The x-axis shows the charge ratio between compound (3) and DNA expressed in mol / μg.

FIG. 6 shows in vivo of nucleolipid complex formed from compound (5) on HeLa cells.
5 shows the gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The x-axis shows the charge ratio between compound (5) and DNA expressed in mol / μg.

FIG. 7 shows in vivo of nucleolipid complex formed from compound (6) on HeLa cells
5 shows the gene transfer activity in vitro. Luciferase expression in pg per transfected well is shown on the y-axis. The charge ratio between the compound (6) and DNA expressed in mol / μg is shown on the x-axis.

FIG. 8: No colipid (dark shaded bars), 25% compared to naked DNA
40mol% DOP with cholesterol (medium shaded bars)
Compound (1) or a compound of the formula H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COArgN containing E (lightly shaded bar) according to the invention
FIG. ₂ shows in vivo gene transfer activity of a complex comprising [(CH ₂ ) ₁₇ CH ₃ ] ₂ (“lipid A”) after direct injection into muscle.

FIG. 9. Gene transfer activity of two different lipids, compound (1) and lipid A according to the invention and lipid A, via two routes of administration: intravenous (iv) and intramuscular (im). Is shown.

FIG. 10 shows the i.p. of nucleolipids comprising compounds (5) or (6) according to the invention at a charge ratio of 0.25 / 1 without colipid compared to naked DNA. m. 48 from injection
5 shows the in vivo gene transfer activity after time. Expression is expressed in pg of luciferase per ml. From left to right, the bars represent: (a
A) negative control; (b) naked DNA; (c) compound (5) and (d) compound (6).

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 28, 2000 (2000.1.28)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0090

[Correction method] Change

[Correction contents]

Drawings FIG. 1: Structure of a synthetic vector, referred to in the present invention as Lipid A, Lipid B and Lipid C, and described in patent application WO 97/18185, which is incorporated herein by reference.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AT, BA, BB, BG, BR, CA, CN, CU , CZ, EE, GD, GE, HU, ID, IL, IN, IS, JP, KP, KR, LC, LK, LR, LT, LV, MG, MK, MN, MX, NO , NZ, PL, RO, RU, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU (72) Inventor Hoffland, Hans United States of America, Calif. Rain 126 (72) Inventor Cielman, Daniel France, France 75012 Paris, Lille Erard, 10F term (reference) 4B024 AA01 AA11 DA02 GA11 HA17 4C076 AA95 EE41 4C084 AA13 BA23 MA55 MA66 NA13 NA20 4C086 AA01 AA02 DA08 EA16 ZC80

Claims (28)

[Claims] 1. Compounds of the formula (I) in D, L or DL form and salts thereof: CA-Rep-R (I) wherein CA is a cycloamidine group of the formula (II) and its mesomeric form Represents
S:Wherein m and n are, independently of one another, both ends such that m + n is 1 or more
An integer from 0 to 3 including₁Represents a group of the general formula (III):In the formula, p and q are each independently an integer from 0 to 10 including both ends, and Y is
A carbonyl, amino, methylamino or methylene group, wherein Y is a different group [(
CH₂)_p-Y] can have different meanings,^*) Is hydrogen field
Is a moiety that represents a bond or binds to a Rep group;₁Is a general formula including Z
May be bonded to any atom of formula (II), and one R₁ It is understood that the group is present, where X is NR₂Or CHR₂R represents a group₂Is a hydrogen atom or as defined above
Una R₁A bond to a group,Represents one of the following:^*First case: group of general formula (IV):Wherein W 'represents CHR "or NR", and R "and R" are, independently of one another, hydrogen
Atom, methyl or R as defined above₁Represents a bond to a group, or^*Second case: group of general formula (V):Wherein W 'represents CHR "or NR", and R' and R "independently represent hydrogen
Atom, methyl or R as defined above₁Rep is absent or represents a spacer of general formula (VI), representing a bond to the group:Wherein the nitrogen atom is an X, V, W or Z atom or optionally R₁Group substituent Y
And t is an integer from 0 to 8 inclusive, r is an integer from 0 to 10 inclusive, and r is a different group -NR₄− (C
H)_rIt is possible to have a different meaning within-R₃Is a different group NR₄-(CH)_rR₃Can have different meanings in the hydrogen
Represents an atom, a methyl group or a group of the general formula (VII):Wherein u is an integer from 1 to 10 inclusive, and s is a different group-(CH₂ )_s-NR₅Can have different meanings within and is an integer from 2 to 8 inclusive
, R₅Is a hydrogen atom, a CA group as defined above, provided that the CA groups are independently of each other
It is understood that they can be different or are groups of the general formula (VII)
It is understood that the groups of formula (VII) are independent of one another and may have different meanings.
・ R₄Is R₃Or as defined above, or a CA group as defined above
With the proviso that the CA groups are independent of each other and may differ.
R is attached to the carbonyl function of the Rep group of general formula (VI), or
If no Rep group is present, R is directly bonded to the CA group and one of the following
Forget:^*NR₆R₇A group having the formula₆And R₇Are, independently of one another, a hydrogen atom or 1
Optionally fluorinated linear or branched, saturated or unsaturated, containing from 22 to 22 carbon atoms
A sum aliphatic radical, wherein at least one of the two substituents R₆Or R ₇ Is not hydrogen, the other contains 10 to 22 carbon atoms,^*Or a steroid derivative,^*Alternatively, a group of the general formula (VIII):Wherein x is an integer from 1 to 8 including both ends, and y is from 1 to 10 including both ends.
And Q is R₆And R₇Is as defined above for C (O) NR₆R ₇ Or Q represents R₈Represents a group of formula (IX): C (O) R₈Of
Represents:Wherein z is an integer from 2 to 8 inclusive,₉Is 8 to 22 carbons
Optionally fluorinated saturated or unsaturated aliphatic radicals or atoms containing atoms
And two substituents R₆Are, independently of one another, as defined above
Or R₈Is R₉Is as defined above for -OR₉Table
I forgot. 2. The compound according to claim 1, wherein the R ₁ group is linked on the one hand to either Z or V, and on the other hand to the Rep group via Y. 3. The method according to claim 1, wherein the cycloamidine head CA of the formula (II) is 5, 6, 7 or 8.
Compound according to claim 1, characterized in that it comprises at least one member. _{4. A process according} to claim 1, wherein R ₃ represents a hydrogen atom or methyl, R ₄ is as defined in claim 1, or R ₃ and R ₄ present in formula (VI) represent a hydrogen atom. Or the compound of claim 1, wherein R ₄ is a hydrogen atom and R ₃ is a group of formula (VII), wherein R ₅ represents a CA group. 5. The compound according to claim 1, wherein in formula (V), p and q are independently selected from 2, 3 or 4. 6. An optionally fluorinated linear or branched, saturated or unsaturated aliphatic chain, wherein the R ₆ and R ₇ groups are the same or different and each contain from 10 to 22 carbon atoms. The compound according to claim 1, wherein _{7. The} groups R ₆ and R ₇ are the same or different and each has 12,1
2. The compound according to claim 1, characterized in that it represents an optionally fluorinated straight or branched, saturated or unsaturated aliphatic chain containing 4, 16, 17, 18 or 19 carbon atoms. 8. R is a steroid derivative, wherein said steroid derivative is cholesterol, cholestanol, 3-α-5-cyclo-5-α-cholestan-6.
-Β-ol, cholic acid, cholesteryl formate, cotestanyl formate, 3α, 5 formate
-Cyclo-5α-cholestan-6β-yl, cholesterylamine, 6- (1,5
-Dimethylhexyl) -3a, 5a-dimethylhexadecahydrocyclopenta [
a] Cyclopropa [2,3] cyclopenta [1,2-f] -naphthalene-10-
2. The method according to claim 1, wherein the at least one of ylamine and cholestanylamine is selected.
The compound according to the above. 9. The following formula: The compound according to claim 1. 10. Synthesis of a building block bearing a cycloamidine functional group,
10. A process for preparing compounds according to claims 1 to 9, characterized in that these building blocks are subsequently grafted onto lipids comprising spacers. 11. A process for preparing compounds according to claims 1 to 8, characterized in that analogous lipopolyamines are synthesized, followed by cyclization of the cycloamidine group. 12. A composition comprising at least one compound of the general formula (I). 13. The composition according to claim 12, comprising one compound of the general formula (I) and one nucleic acid. 14. The composition according to claim 12, further comprising one or more adjuvants. 15. The composition according to claim 14, wherein the adjuvant is one or more neutral lipids comprising two fatty chains. 16. The neutral lipid may be, for example, dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidylethanolamine, and 1 to 3 times N Selected from methylated derivatives thereof, phosphatidylglycerol, diacylglycerol, glycosyldiacylglycerol, cerebroside (especially galactocerebroside, etc.), sphingolipids (especially sphingomyelin, etc.) or asialogangliosides (especially asialo GM1 and GM2, etc.) The composition according to claim 15, characterized in that it is a natural or synthetic lipid which is zwitterionic under physiological conditions or has no ionic charge. 17. The composition according to claim 14, wherein the adjuvant is a compound directly or indirectly involved in the condensation of nucleic acids. 18. The adjuvant may be derived in whole or in part from protamine, histone or nucleolin and / or one of its derivatives, or may be derived in whole or in part from a peptide unit (KTPKKAK).
18. Composition according to claim 17, characterized in that it consists of (KP) and / or (ATPAKKAA), the number of units is in the range from 2 to 10, and can be repeated continuously or not. object. 19. The composition according to claim 12, which comprises one or more nonionic surfactants in an amount sufficient to stabilize the particle size of the nucleolipid complex. object. 20. The composition according to claim 12, comprising a pharmaceutically acceptable excipient for injection. 21. Composition according to claims 12 to 19, characterized in that it comprises a pharmaceutically acceptable excipient for application to the skin and / or mucous membranes. 22. The composition according to claim 13, wherein the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid. 23. The nucleic acid of claim 23, wherein the nucleic acid comprises an expression cassette consisting of one or more genes to be treated under the control of one or more promoters and transcription terminators active in the target cell. 23. The composition according to 22. 24. Use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for treating a disease. 25. Use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for treating a disease by transferring nucleic acids into cells by an intramuscular route. 26. (1) contacting the nucleic acid with a compound of general formula (I) as defined above to form a nucleolipid complex, and (2) placing the nucleolipid complex formed in (1) in a cell. A method for transferring nucleic acids into cells, comprising the step of: 27. The method for transferring a nucleic acid according to claim 26, wherein the nucleic acid and / or the compound is previously mixed with one or more adjuvants. 28. A method comprising administering a nucleic acid encoding a protein capable of correcting a disease or being transcribed to a nucleic acid capable of correcting the disease in combination with a compound of the general formula (I) to thereby reduce the disease. How to treat.