JP2003506335A - Use of quinic acid, shikimic acid and their derivatives for preparing mannose receptor ligands - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of quinic acid and shikimic acid and their derivatives for preparing ligands for mannose receptors, and in particular for preparing medicaments, and more particularly targets expressing the mannose receptor or its related receptors. Prepare vaccines and medicaments intended to ensure delivery of active ingredients to cells, transfection of DNA and RNA sequences and their use in gene therapy, and preparation of laboratory reagents, especially diagnostic reagents And the use of these ligands.

Description

Detailed Description of the Invention

The present invention relates to the use of quinic acid and shikimic acid and their derivatives for preparing ligands for the mannose receptor, the ligands thus prepared, and the therapeutic and diagnostic uses of these ligands.

Dendritic cells, which are specific cell lines of white blood cells originating from the bone marrow and present in virtually all tissues of the organism, play an important role in the regulation of the immune response. These cells, in fact, present antigen to native and mature T lymphocytes in vivo and appear to be the only one that can elicit a helper T-type immune response through activation of these T lymphocytes. Yes (J. BLANCHEREAU and RM ST
EINMANN, Nature, 1998, 392 , 245-252).

Activation of T lymphocytes by dendritic cells involves prerecognition and internalization of the antigen by the latter. Here, it was shown that the internalization of antigens by dendritic cells is carried out by macropinocytosis as well as through an endocytosis mechanism called mannose receptor, which has a specific receptor for carbohydrates as a mediator. (A. AVRAMEAS et al., Eur. J. Immun
ol., 1996, 26 , 394-400).

Mannose receptors belong to the family of C-lectins, which represent a group of calcium-dependent glycoproteins that form non-covalent bonds with carbohydrates. As its name suggests, the mannose receptor binds preferentially to D-mannose, while it also, like D-mannose, contains two adjacent carbon atoms carried by two adjacent carbon atoms in the ring forming them. Other carbohydrates containing hydroxyl functional groups (
For example, it appears to bind to L-fucose, D-glucose and N-acetyl-D-glucosamine. The bond between the mannose receptor and these carbohydrates appears to be established by the coordination of calcium ions between the amino acid residues at the recognition site of this receptor and the two adjacent hydroxyl functional groups of the carbohydrate. (WI WEIS et al., Nature, 1992, 360 , 127-1
34).

Furthermore, as with all lectins, recognition of carbohydrates by the mannose receptor is specific, but very low, on the order of 10 ⁻⁶ to 10 ⁻³ M. Simultaneous binding of several carbohydrate molecules to several mannose receptor recognition sites (simu
ltaneous) binding makes it possible to compensate for this low affinity. This phenomenon is known as the "cluster effect".

Internalization of mannosylated antigens by dendritic cells is significantly higher than that of non-mannosed antigens, and in the presence of dendritic cells on the one hand and mannose proteins or peptides on the other Cultures of cloned populations of T lymphocytes with specificity limited to specific class II peptide-HLA complexes performed are more favorable than those obtained when these same proteins and peptides are not mannoseylated. It has also been shown to produce 200-10,000-fold higher stimulation of these lymphocytes (MCAA TAN et al., Eur. J. Imm.
unol., 1997, 27 , 2426-2435).

As a result, coupling of this antigen with chemical structures that carry several molecules of carbohydrates that are specifically recognized by this receptor results in dendritic cell-mediated It has been shown that it is possible to induce, stimulate or regulate the immune response by helping to internalize (MCAA TAN et al., Ibid.).

This is very particularly important in vaccinology and, obviously, for the development of vaccines based on synthetic peptides. In fact, the latter are chemically defined with precise composition, at the same time purified, and finally easy to distribute due to their stability, without the difficulty of biological production. While having the advantage of being, they generally have the major drawback of being only slightly immunogenic.

This leads to the fact that we have several glycosyl residues and especially D
-L-lysine tree type core (L-lysine tree) linked to the rest of the mannose
That is why we proposed to modify peptide antigens by grafting them onto dendrimer structures containing (type core) (C. GRANDJEAN et al., 11 ^th Meand
ing on Peptides and Proteins (11 ^eme Reunion Peptides and Proteines), 17-
22 January 1999, AUSSOIS, FRANCE).

However, from the point of view of industrial scale development of medicines and especially vaccines, the use of natural carbohydrates (eg D-mannose or D-glucose) makes it difficult for these compounds to attach to other chemical structures. It is clear that both technical and financial are prone to numerous problems due to the fact that they are and are unstable in many reaction media (especially acidic media) and many biological media. .

Thus, the problem is with respect to the mannose receptor, a compound having affinity and specificity that is recognized by and can bind selectively to this receptor, while avoiding the disadvantages inherent in natural carbohydrates. How to get it.

Here, again, within the scope of our work on the mannose receptor, we have made the following surprising discoveries: quinic acid and shikimic acid and their derivatives [these are these The carbocyclic nature indicates compounds that are chemically more stable than natural carbohydrates and therefore easier to use in the synthetic process], but may mimic D-mannose with respect to its receptor (ie, specifically for the latter). Binding and, as a consequence, favoring the natural carbohydrates in the preparation of ligands designed to aid in the internalization of substances by cells expressing this receptor or related receptors of the family of C-lectins. It can be replaced.

The object of the present invention therefore corresponds to the general formula (Ia) as a ligand for, or for the preparation of, a mannose receptor or any receptor of the C-lectin family related to a mannose receptor. The use of at least one compound and / or at least one compound corresponding to general formula (Ib):

[0014]

[Chemical 4]

Here, R ₁ , R ₂ , R ₃ and R ₄ independently of each other represent a hydrogen atom or a protecting group, and R ₅ is —OH group, —SH group, —NH—NH. ₂ groups, halogen atom, or 1
1 selected from 18 carbon atoms and optionally oxygen, sulfur and nitrogen
A saturated or unsaturated, straight-chain, branched or cyclic carbon chain containing from 1 to 12 atoms, said carbon chain being optionally substituted by 1 to 16 halogen atoms, and optionally at least It carries one nucleophilic group or at least one electrophilic group.

Within the meaning of the present invention, a “protecting” group is a Protective Groups in Organic Syn
thesis (TW GREENE and PGM WUTS, Second Edition 1991, J. WILEY
and Sons) as defined in the study, hydroxyl functional groups or diol-protecting groups, in which two groups selected from R ₁ , R ₂ , R ₃ and R ₄ are covalently bonded to each other. Can form a ring together). By way of example and not limitation, trimethylsilyl, triethylsilyl, triisopropylsilyl, tertiobutyldimethylsil.
yle), tertiary butyldiphenylsilyl (tertiobutyldiphenylsilyle), trimethylsilylethyl ether (trimethylsilylethylether), tert-butoxymethyl, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyle (2-methoxyethoxymethyle), methylthiomethyl (methylthiomethyle), acetyl, Or 2 ', 3'-dimethoxybutane-2', 3'-diyl (2 ', 3'-dime
thoxybutane-2 ', 3'-diyle) group.

Further, a “nucleophilic” group is understood to mean a group having a free electron pair (ie, an electron donating group), while an “electrophilic” group interacts with a free electron pair of a nucleophilic group. It is understood to mean a group having a possible low energy empty molecular orbital. Formula (Ia)
And the presence of at least one nucleophilic or electrophilic group in the compound of (Ib) advantageously corresponds to the corresponding nucleophilic or electrophilic group carried by the structure, as described in detail below. Reacting with reactive functional groups allows them to be incorporated into other structures (eg carrier structures).

Within the scope of the present invention, the nucleophilic group is preferably selected from among the following groups: amine, thiol, β-aminothiol, alcohol, hydrazide and hydrazide derivatives, hydrazine and hydrazine derivatives, hydroxyl. Amine and hydroxylamine derivatives. On the other hand, the electrophilic group is preferably selected from the following groups: α-oxo-aldehyde, —COR ^a (for example, R ^a ═H, halogen, imidazolyl, aromatic heterocyclic compound), CO ₂ R ^a (for example, R ^a = H
, Aryl, succinimidyl, sulfosuccinimidyl, aromatic heterocyclic compound) and its derivatives (symmetric anhydride, mixed anhydride, active ester), -OC
OR ^a (for example, R ^a = halogen, imidazolyl, aromatic heterocyclic compound), −
OCO ₂ R ^a (eg, R ^a = aryl, heteroaryl, succinimidyl, sulfosuccinimidyl), —OCSR ^a (eg, R ^a = halogen, imidazolyl, heterocyclic aromatic compound), —OCSOR ^a (eg, , R ^a = aryl, succinimidyl, sulfosuccinimidyl, heterocyclic aromatic compound), —NR ^a C
OR ^b (eg, R ^a = methyl and R ^b = halogen, imidazolyl, aromatic heterocyclic compound), —NR ^a COOR ^b (eg, R ^a = H and R ^b = aryl, succinimidyl, sulfosuccinimidyl) ), -NR ^a CSR ^b (for example, R ^a =
H and R ^b = halogen, imidazolyl, aromatic heterocyclic compound), —NR ^a C
SOR ^b (eg R ^a = H and R ^b = aryl, succinimidyl, sulfosuccinimidyl), —NCO, —NCS, maleimide and alkyl halides.

By way of example and not limitation, in the general formulas (Ia) and (Ib): suitable halogen atoms are bromine, chlorine and fluorine; and suitable carbon chains are alkyl (methyl) , Ethyl, propyl, isopropyl,
Tertiary butyl, pentyl, etc. groups, alkenyl (vinyl, allyl, butenyl, pentenyl, etc.) groups, alkynyl (ethynyl, propynyl, butynyl, pentynyl, etc.) groups, cycloalkyl (cyclopentyl, cyclohexyl, etc.) groups, hetero Cyclic compounds (such as piperazine), aryl (phenyl, cresyl, etc.) groups, heteroaryl (pyridine, pyrimidine, pyrazine, etc.) groups, polyethylene glycol groups, or polyamines.

The compounds corresponding to general formulas (Ia) and (Ib) form D-mannose mimics. They can be used by themselves as ligands for the D-mannose receptor, or they can be linked to other compounds leading to the production of ligands where they play a part of a mimetic of D-mannose. Thus, in ligands (eg, ready-to-use), the general formulas (Ia) and (I
The compound of b) has at least two adjacent free hydroxyl groups, so that
While it is preferred to be able to mimic D-mannose and to allow these ligands to be recognized by the mannose receptor and bind to the latter, however, these compounds are It is not necessary to have a local hydroxyl functionality.

Accordingly, in accordance with the present invention, for preparing a ligand, comprising two hydroxyl groups protected by a protecting group at two adjacent carbon atoms in the ring,
At least one compound of formula (Ia) and / or at least one compound of formula (Ib)
It is entirely possible to use the compounds, while the other groups carried by the ring atoms may optionally also be protected themselves. This is often the case, inter alia, when the group carried by the carbon atom in the ring of the general formula (Ia) or (Ib) interacts during coupling of the compound with intramolecular cyclization or Even desired to prevent leading to dimerization. Of course,
The group protecting the two hydroxyl functional groups carried by the two adjacent carbon atoms in the ring can be removed prior to use of the ligand, the removal of other protecting groups
It is optional.

According to one preferred arrangement of the invention, the compounds of general formula (Ia) and / or (Ib) are in trans relationship with the —OR ₂ and —OR ₃ groups so that they are carried by the ring of D-mannose. Are selected in such a manner as to mimic the two hydroxyls that are involved, which appear to be involved in the binding between this compound and its receptor.

Preferably, the compounds of formula (Ia) and (Ib) are those wherein R ₅ is a hydroxyl group (
In this case, the ring is such that it possesses a carboxyl group in the 1-position).

The presence of a carboxyl group in the ring of the compounds of formulas (Ia) and (Ib) does in fact lead to these compounds, for example via amide, ester, thioester, hydrazide or hydroxamate type bonds, It has the advantage of providing a large number of coupling possibilities. Moreover, such coupling can be readily carried out by simply activating the carboxyl group according to the activation techniques conventionally used for peptide synthesis and especially for solid support peptide synthesis. These techniques are described, for example, in The Practice of Peptides Synthesis (M. a.
nd A. BODANSKY, 1984, SPRINGLER-VERLAG, Berlin).

According to one particularly preferred arrangement of the invention, the compound of formula (Ib) is
R ₁ , R ₂ and R ₃ are hydrogen atoms and R ₅ is a hydroxyl group (—OH). Further, the -OR ₁ and -OR ₂ groups are in a cis relationship and-
When the OR ₂ and —OR ₃ groups are in the trans relationship, the configurations are 3R, 4S and 5R, and the resulting compound is (−)-shikimic acid (ie 3,4,5-trihydroxy-1- Cyclohexene-1-carboxylic acid).

According to another preferred arrangement of the invention, the compound of formula (Ia) is R ₁ ,
R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is a hydroxyl group (—OH). Furthermore, there -OR ₂ and -OR ₃ groups in trans relationship, whereas, if the -OR ₁ and -OR ₄ groups are in cis relationship to each other with respect to -OR ₂ group,
The configurations are 1s _n , 3R, 4s _n and 5R, and the resulting compound is (-)-
It consists of quinic acid (ie 1,3,4,5-tetrahydroxy-cyclohexane-carboxylic acid).

(−)-Shikimic acid and (−)-quinic acid are, for example, ALDRICH or AC
Commercially available from ROS.

According to another preferred arrangement of the invention, the compound of formula (Ia) has the formula R ₁ ,
R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is a —NH— (CH ₂ ) ₂ —SH group. We then have a derivative of quinic acid.

Generally speaking, the substitution of the hydroxyl carried at the 2, 3 and 4 positions of the ring (and optionally the 1 position) and the carbonyl functional group (positioned at the 1 position of the ring (
The formulas (Ia) and (Ib), which represent some of the quinic and shikimic acids and their derivatives, obtained by substitution of the R ₅ group) are not limiting, and that of the quinic and shikimic acids It is clearly understood that any other derivative can be used without departing from the scope of the invention.

The present invention also relates to compounds which can be used as ligands for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor or as intermediate compounds for preparing such ligands,
The compound is characterized in that it corresponds to the following general formula (III): (B ¹ M) _m (XN) _n A (PY) _y (III) where: B ¹ is one or more of the following: Of the general formula (IIa) or (IIb):

[0031]

[Chemical 5]

[Wherein W is a bond or a saturated or unsaturated, straight-chain, branched chain containing 1 to 18 carbon atoms and optionally 1 to 12 atoms selected from oxygen, sulfur and nitrogen. A branched or cyclic carbon chain, wherein the carbon chain may be 1 to
Substituted with 16 halogen atoms], -X is the remainder of the X'oligopeptide containing 1 to 6 amino acids, -A is the remainder of the at least bifunctional compound A '. Yes, -Y is the remainder of the compound Y'of interest, -m is an integer from 1 to 32, -n is an integer from 0 to 32, and -y is an integer from 1 to 4. And -M, N and P are each between B ¹ and A (when n = 0) or B ¹ and X (when n is different from 0), X and A linking group between A (when n is different from 0) and between A and Y, and independently of each other, including a functional group selected from the following functional groups: oxime, hydrazone, Amides, esters,
Thioesters, hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines.

Examples of suitable halogen atoms and carbon chains in formula (III), and in all following, are as described above in connection with compounds of general formula (Ia) and (Ib).

Accordingly, the compounds corresponding to general formula (III) include: * as essential elements: one or more balances B ¹ corresponding to one of the general formulas (IIa) or (IIb) [this balance Or the rest of these are (vis-a-vis) D for the mannose receptor
One or more compounds intended to act as part of a mimetic of mannose and corresponding to general formula (Ia) and / or (Ib) as defined above and compound A
From the bond with '(optionally via X'), and deprotection of the hydroxyl group if the compound of general formula (Ia) and / or (Ib) contains an originally protected hydroxyl group. Obtained];-one or more residues Y of compound Y'corresponding to the compound of interest; -remainder A of compound A'having at least difunctionality [because its role is the balance (s) B ¹ and the balance (s) Y], and * as an ancillary element, the balance (s) X of compound X ′ [this balance or these balances X are When they are present, they act as spacers between B ¹ and A, so that some of them are present in compounds according to general formula (III) (ie when m is greater than 1) ,different Controlling the distance between parts B ^1. Although the remainder X of the oligopeptide nature has been described, it is outside the scope of the invention to use compounds of different nature as compound X ¹ as long as they can play the role of spacer between B ¹ and A. Absent. By way of example and not limitation, mention may be made of mercaptopropionic acid, thioamine, bromoamine and polyethylene glycol type spacers such as 4,7,10-trioxa-1,13-tridecanediamine.

Within the meaning of the present invention, a “compound of interest” is a cell (such as a dendritic cell, a cell of the monocyte-macrophage lineage, etc.) via the mannose receptor or a receptor of the family of C-lectins related to the latter. ) Is understood to mean a compound which is sought to aid in the internalization by

For this purpose, the compounds of interest are 1 to 5 per compound of general formula (III).
In the proportion of 4 molecules of the compound of interest, it can be A-attached to the rest by the linking group P; each is carried by the compound of interest in the former case and by compound A ′ in the latter case. It results from a reaction between two functional groups. We can specifically bind to the mannose receptor or any receptor related thereto through the presence of the balance (s) B ¹ , and through its binding with the compound of interest. , A lig that can aid in the internalization of the latter by cells expressing this type of receptor.

According to one advantageous arrangement of the invention, the compound A ′ is formed by a linkage comprising several residues of at least trifunctional compounds covalently bound to one another and of these residues At the level of free functional groups (ie, functional groups not involved in covalent bonding), to the compound (s) of general formula (Ia) and / or (Ib) or to the compounds where X is present. It is suitable for providing a large number of anchoring sites for X ′ and compound Y ′.

According to one preferred mode of this arrangement, the compound A ′ comprises the remainder of the trifunctional compound 2-32, and preferably 4-16.

According to the invention, the compound A ′ can be in either a linear or branched form and in particular of the arborescent (so-called “dendrimeric”) type. Examples of branched structures that are particularly suitable for preparing ligands for the mannose receptor according to the invention are provided in Figures 1A and 1B, which show very schematically compound A'in the form of two different dendrimers. .

Preferably, compound A ′ comprises a linkage of the same or different amino acids selected from the group formed by lysine, hydroxylysine, serine, threonine, cysteine, ornithine, aspartic acid and glutamic acid. This amino acid can be in both the L and D forms, and compound A ′ contains the remainder in both the L and D forms.

Preference is given to lysine-based compounds A ′, since the use of lysine provides a ligand for the mannose receptor according to the invention, so long as the lysine-based macromolecule is shown to lack intrinsic immunogenicity. This is because it is particularly suitable for preparation (TAM, Proc. Natl. Acad. Sci. USA, 1988, 85 , 540).

However, as long as they provide a satisfactory biocompatibility, as compounds A ′,
It is possible to use chains of trifunctional compounds other than the amino acids mentioned above. By way of example and not limitation, 3,3'-iminobis (propylamine), 2,2'-
Iminobis (ethylamine), gallic acid, tris (2-carboxyethyl) nitromethane (M. BRANDTEICH et al., Synlandt, 1998, 1396), tris (hydroxymethyl) aminomethane (PR ASHTON et al., J. Org. Chem., 1998, 63 , 3429) and (dihydroxy) propylamine.

The supplemental compound A ′ may further comprise one or more balances corresponding to one or more different compounds of the at least trifunctional compound forming its basic element. This or the rest of them is in principle not designed to provide any binding between the various elements making up the ligand. The presence of these is, in fact, intended to confer on the ligand additional features such as one or more additional coupling capacities (eg for the purpose of subsequently producing dimers of this ligand). Or
Either linked to the need to use additional compounds (eg spacers) during their preparation. It goes without saying that these compounds differ from the at least trifunctional compounds forming the basic element of compound A ′, but preferably have the same properties as the latter. Thus, for example, they are preferably amino acids when compound A ′ comprises amino acids.

According to the invention, n, which indicates the number of the balance X present in the compounds of general formula (III), can be: • equal to 0, in which case the balance (one or more) ) B ¹ is directly bound to the balance A), or: 1 to 32 (where n is equal to 1 and the balance (s) B ¹ are linked via a single balance X) To the balance A, while n is different from 1, each residue X may serve as an element of coupling between one or more of the balances B ¹ and the balance A).

With respect to the linking groups M, N and P, these are, independently of one another, selected from linking groups containing functional groups selected from the following functional groups: oximes, hydrazones, amides, esters, thioesters, hydrazides. , Hydroxamate, ether, thioether, amine, carbonate, carbamate, thiocarbonate, thiocarbamate, urea, thiourea and thiazolidine. By way of example and not limitation: an oxime bond may be formed by reacting an O-alkylamine group with a carbonyl group, a hydrazone bond may be formed by reacting a hydrazine group with a carbonyl group, -Amide (respectively ester) bond is amine (respectively alcohol)
A group may be formed by reacting a group with an activated acid, anhydride or acid halide group; a thioester group may be formed by reacting a thiol group with an activated acid, anhydride or acid halide group; a hydrazide The bond may be formed by reacting a hydrazine group with an activated acid, anhydride or acid halide group, an ether bond may be formed by reacting an alcohol group with an alkyl halide group, and a thioether bond may be A thiol can be formed by reacting with an alkyl halide group, an amine bond can be formed by a reaction between an amino group and an alkyl halide, a carbonate bond can react a chloroformate group with an alcohol group. A carbamate bond can be formed by a chloroformate group A thiocarbonate bond may be formed by reacting a chlorothioformate group with an alcohol group, and a thiocarbamate bond may be formed by reacting a chlorothioformate group with an amino group. A urea bond can be formed by reacting an isocyanate group with an amino group, a thiourea bond can be formed by reacting an isothiocyanate group with an amino group, and a thiazolidine bond can be formed by: , A β-aminothiol group can be formed by reacting a carbonyl group, and a hydroxamate bond can be formed by reacting an activated acid group and a hydroxylamine group.

Accordingly, in the general formula (III), B ¹ , A and X are naturally functional groups suitable for leading to the production of the linking groups M, N and P defined above by reacting with each other. The compounds possessed or the remainder of the compounds modified in such a way as to possess such functional groups are indicated.

In this regard, it should be noted that, in accordance with the present invention, compounds corresponding to general formula (III) may be prepared by any of the following: -convergent synthesis [ie, In some cases, the general formula (
III), commercially available compounds modified for the purpose of preparing compounds corresponding to
Or combination of pre-synthesized compounds].

Thus, for example, a compound according to general formula (III) may be prepared by first coupling the compound (s) corresponding to general formula (Ia) and / or (Ib) with compound X ′, and then Can be obtained by coupling the compound resulting from this first coupling with compound A ′ and, finally, the compound resulting from this second coupling with compound Y ′, the latter coupling When compound (Ia) and / or (Ib) bears such a group,
Optionally followed by deprotection of the hydroxyl group carried by the balance (s) B ¹ .

Alternatively, the compound of general formula (III) may be represented by general formula (Ia) and / or (Ib
) By coupling a compound obtained from the coupling of one or more compounds corresponding to) with compound X ′ with a compound which is obtained as a part thereof from the coupling of compound A ′ and compound Y ′. This final coupling can be obtained, again optionally following the deprotection described above.

Of course, any other order of combination may also be envisaged, provided that the compound corresponding to general formula (III) may be obtained.

Using a recurrent synthesis strategy, especially when the compounds Y ′, A ′ and X ′ are formed by amino acids. Thus, compounds Y ′, A ′ and X ′ can be prepared, for example, by the method first described by MERRIFIELD (The Chemistry of Solid Phase Peptide Synthesis, STEWART and
According to solid support peptide synthesis techniques derived from YOUNG Ed., 1984), which can be formed by successive additions of the amino acids that compose them and be formed and combined with each other, or alternatively partially convergent and partially Use a synthetic strategy that repeats dynamically.

According to one preferred means of the present invention, the compound has the general formula (III) [wherein
m is an integer of 4-16, n is an integer of 2-8, X is the rest of the oligopeptide containing amino acid residues of 2-4, while A is a lysine residue of 2-8. Are included and show the rest of the chains in the form of dendrimers].

According to another preferred arrangement of the invention, the compounds according to general formula (III) represent the balance of one or more compounds in which B ¹ is selected from (−)-shikimic acid and (−)-quinic acid. Is like.

According to the intended use of the ligand, the compound of interest Y'is an antigen, and in particular a vaccine antigen (eg a synthetic peptide corresponding to one or more epitopes of a protein of a pathogenic agent), a protein (proteic). ) Or polysaccharides (polysaccharidic)
) Subunit antigens, purified bacterial or viral fractions, antibodies (eg immunoglobulin or anti-lymphocyte sera), nucleic acids or nucleic acids encoding the protein of interest or one or more antigenic determinants of this protein ( RNA or DN
A sequence), a medicinal active ingredient such as an immunostimulatory agent, an immunosuppressive agent, a cytostatic agent or an antibiotic, and as such, a protein, peptide, glycoside or polyglycoside, lipid, lipoprotein. It can be both a protein, a lipopolyglycoside, a substance with the properties of polynucleotides and a chemically produced compound.

The compound of interest may also be a suitable detectable marker to allow the presence of the ligand to be revealed in the medium, where a radioisotope, fluorescent dye or enzyme such as Things are arranged.

Of course, it is also possible that the compound Y ′ of interest is constructed by the association of several substances with different properties (eg an antigen or antibody linked to a detectable marker). .

The present invention also relates to compounds that can be used as intermediate compounds for preparing ligands for mannose receptors or any receptor of the C-lectin family related to mannose receptors, which compounds are of the general formula: V) corresponding to: (B ² M) _m (XN) _n A (V) where: B ² is one or more of the following general formulas (IVa) or (IVb):

[0058]

[Chemical 6]

[Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are the same as those represented by the general formulas (Ia) and (Ib)]
Is as defined above for, and W is as defined above for compounds of general formula (IIa) and (IIb)], X is from 1 to 6 The remainder of the X'oligopeptide containing an amino acid is shown, -A shows the remainder of at least bifunctional compound A ', -m is an integer of 1 to 32, and -n is an integer of 0 to 32. And -M and N are respectively between B ² and A (when n = 0) or B ² and X (when n is different from 0), and X and A, respectively. Between (when n is different from 0) and independently of each other include a functional group selected from the following functional groups: oxime, hydrazone, amide, ester, thioester, hydrazide. , Hydroxamate, ether, thioether, amine, Carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines.

Therefore, in the general formula (V), the balance (one or more) B ² is represented by the general formula (Ia
) And / or (Ib) corresponding to one or more compounds (optionally X ′
Via a compound A ′ (via).

In the compound of general formula (V), M, N, m, n and compounds A ′ and X
'Is as described for general formula (III).

Furthermore, the compounds of general formula (V), like the compounds corresponding to general formula (III), are prepared by convergent synthesis, iterative synthesis, or also partially convergent, partially iterative synthesis. Can be done.

According to one preferred arrangement of the invention, the compound of general formula (V) is
B ² is such that it represents the balance of one or more compounds selected from (−)-shikimic acid and (−)-quinic acid, optionally in protected form.

The present invention further relates to compounds which may be used as intermediate compounds for preparing ligands for mannose receptors or any receptor of the C-lectin family related to mannose receptors, which compounds are of the general formula: (VI)
Characterized in that it corresponds _{^{to: (B 1 M) m A}} (VI) wherein: - B ¹ and M have the same meaning as in the general formula (III), - m is 50 Is an integer of 500, and-A indicates the remainder of the polymer A'which can form a non-covalently bound complex with the compound of interest (eg, nucleic acid).

Within the meaning of the invention, the term “polymer” is understood to mean a large number of molecules of varying molecular weight, which result from the joining of several monomers.

The polymer is selected from compounds that are capable of contacting the compound of interest to form a non-covalently bound complex with the compound, for example via ionic, dipolar or electrostatic interactions. Selected and then the resulting complex can be used as a ligand for the mannose receptor or any receptor of the C-lectin family related thereto.

Preferably the A ′ polymer is a cationic polymer. By way of example, and not limitation, suitable cationic polymers are lysine polymers (eg SIGM).
From Company A, reference numbers P0296, P6403, P4408, P7886, P08
99, P1149 or also P0124), and polyethyleneimine (eg, commercially available from FERMENTAS under the reference EXGEN 500 as a cell transfection reagent).

The present invention also relates to a compound which can be used as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, said compound corresponding to general formula (VI) as defined above. It is characterized in that it takes the form of a non-covalent complex between the compound and the compound of interest (eg nucleic acid).

The ligands according to the invention are of considerable interest. While they can be recognized by and specifically bind to the mannose receptor in a highly satisfactory manner, they are practically industrial, especially using a number of techniques of solid support peptide synthesis. It has the advantage of being easy to prepare, at a cost compatible with use.

These ligands can be used in a number of applications, such as: -Preparation of medicaments, and more particularly delivery of active ingredients to target cells expressing the mannose receptor or related receptors. Preparation of vaccines and medicaments designed to ensure (vectorization).

Its multiple uses in transfection of DNA and RNA sequences and gene therapy [in the latter case, a ligand in the form of a non-covalent complex between the compound of general formula (VI) and the polynucleotide sequence. Is used], and-preparation of laboratory reagents, and in particular diagnostic reagents.

The present invention further provides at least one compound corresponding to general formula (Ia) and / or for preparing a medicament capable of binding to the mannose receptor or any receptor of the C-lectin family related to the mannose receptor. Or relates to the use of at least one compound corresponding to general formula (Ib).

The present invention further relates to compounds which can be used as ligands for mannose receptors as defined above or for any receptor of the C-lectin family related to mannose receptors as defined above.

Such medicaments are primarily easy to use for applications in all therapeutic applications,
Here, it is sought to elicit, regulate or suppress the immune response, and more particularly what is under the control of T cells. By way of example and not limitation, the following may be mentioned: -prevention and treatment of infectious diseases, and especially vaccinology, where the compound of interest is a vaccine antigen or vaccine protein or such protein. A nucleic acid or nucleic acid fragment encoding one or more antigenic determinants), and non-specific anti-infectious immunotherapy; -autoimmune diseases (eg systemic lupus erythematosus, rheumatoid arthritis or Wegener) Disease; treatment of congenital or acquired immunodeficiency; prevention and treatment of hypersensitivity conditions; treatment of cancer pathology; and prevention of organ transplant rejection.

This medicament can also be used to vectorize the active ingredient of a non-immunomodulatory agent to target cells expressing the C-lectin family of receptors.
Thus, for example, a compound that can be used as a ligand according to the invention can be introduced into cells of the monocyte-macrophage system infected by bacteria or mycobacteria,
Antibiotics (eg fluoroquinones or colistin)
stine)) in the treatment of antibiotics, or in the cellular enzyme deficiency (c
treatment of diseases related to ellular intentional deficit (eg Gaucher disease) (
In this case, they may find utility in delivering defective enzymes to Kupfer cells).

The invention further relates to compounds for use in vitro, which can be used as ligands for the mannose receptor as defined above or for any receptor of the C-lectin family related to the mannose receptor.

The present invention further relates to a diagnostic reagent, characterized in that it comprises a compound which can be used as a ligand for a mannose receptor or any receptor of the family of C-lectins related to the mannose receptor defined above.

Apart from the above arrangements, the invention also provides examples of the preparation of compounds which can be used as intermediate compounds for preparing ligands for the mannose receptor according to the invention, and the biological activity of these ligands. Examples for demonstrating properties, as well as other arrangements, will be apparent from the further description below in connection with the accompanying drawings.

However, it should be clearly understood that these examples are provided solely for the purpose of illustrating the invention, and without constituting a limitation thereof.

Hereinafter, the following formula is used: Ac ₂ O: acetic anhydride Boc: t-butyloxycarbonyl ^t Bu: tert-butyl DCC: dicyclohexylcarbodiimide DCM: dichloromethane DIEA: diisopropylethylamine DMF: dimethylformamide eq. : Equivalent EDTA: Ethylenediaminotetraacetic acid EIS-MS: Electrospray mass spectrometry Fmoc: 9-fluorenylmethoxycarbonyl HOBt: N-hydroxybenzotriazole HBTU: [(1H-benzotriazol-1-yl) (dimethylamino)- Methylene] -N-ethylmethanaminium hexafluorophosphate N-oxide MBHA: 4-methylbenzhydrylamine Mtt: 4-methyltrityl NMP: N-methylpyrrolidone PBS: phosphate buffer solution Pmc: 2, 2, 2, 5 , 7,8-Pentamethylchroman-6-sulfonyl NMR: Nuclear magnetic resonance RP-HPLC: Reversed phase high performance liquid chromatography TFA: Trifluoroacetic acid TNBS: 2,4,6-Trinitrobenzenesulfonic acid TOF-PDMS: Flight Time type plasma Desorption mass spectrometry (time of flight plasma
desorption mass spectrometry) Trt: trityl.

In addition, in the framework of the examples, the following is followed: The optical rotation is measured using a PERKIN-ELMER 21 polarimeter and the value [α] _D is 10 ⁻¹ degrees. Expressed as cm ² / g; semi-preparative analytical reverse phase high performance liquid chromatographic measurements using a SHIMADZU LC-9A or LC-4A system, ultrapore C-8 (300). A. 5 μm, 4.6 × 250 mm) BEC
KMANN column or hyperprep C-18 (300A,
8 μm, 15 × 500 mm) HYPERSIL column, 1 or 3 ml / min flow rate, detection at 215 or 230 nm, and using the following 5 elution systems: -System A: 0. 05% TFA-System B: 0.05% TFA in acetonitrile / water mixture (80:20).
-System C: 0.05% TFA in acetonitrile / water mixture (60:40).
-System D: phosphate buffer (50 mM, pH 6.95) -System E: phosphate buffer (50 mM, pH 6.95) and acetonitrile mixture (50:50) -System F: water / isopropanol mixture ( 60:40) in 0.05% TF
A; -TOF-PDMS spectrum was analyzed by APPLIED BIOSSYSTEMS Bi
o-Ion 20 plasma desorption mass spectrometry was recorded; EIS-MS spectra were recorded using a Micromass Quattro II electrospray ionization source mass spectrometer,
On the other hand: NMR ¹ H and ¹³ C spectra, BRUKER DRX 300 or DR
Recorded using an X 600 mass spectrometer; chemical shifts in ppm
And tetramethylsilane and sodium deuterated trimethylsilylpropionate sodium salt were used as internal standards.

Example 1 : Preparation of compounds which can be used as intermediate components for the preparation of ligands according to the invention Six compounds in the form of dendrimers, corresponding to the general formula (V) below: an integer equal to 4,8 or 16, · B ² is (-) - shikimic or (-) - a quinic acid balance, · n is (when m is equal to 4, 8, and 16) 2 Is an integer equal to 4 or 8; X is the remainder of the following (S1) sequence tripeptide: Lys-Cys (tert-butylthio) -Gly-OH (S1). , 8 and 16) 2, 4 or 8 L-
2 containing lysine residues, as well as one for binding an equal number of tripeptides
4 or 8 chloroacetyl groups and, on the other hand, the molecule of interest (molecule d'i
having a free amino group that enables binding to nteret), the balance of the dendrimer, · M is an amide group (-CO-NH-), whereas, N is the thioether group (-CH ₂ -S- CH ₂ -CO-
) Was prepared.

These compounds were prepared using a convergent synthetic strategy, ie by doing the following: -First, the A'dendrimer is prepared-Then the sequence (S1) tripeptide is prepared, They are (-)-shikimic acid or (-
) -Coupling with two molecules of quinic acid, and-finally, deprotecting the thiol functional groups of these peptides with A'dendrimers and (-)-shikimic acid or tripeptides with (-)-quinic acid residues Then, a thioether bond is formed between the thiol functional group and the chloroacetyl group of the A ′ dendrimer to bond them.

The chemical structures of these compounds, designated as follows: Compounds 21, 2 in the case of compounds containing 4, 8 and 16 (-)-shikimic acid residues, respectively.
3 and 25, and for compounds containing 4, 8 and 6 (-)-quinic acid residues, respectively, are shown in compounds 22, 24 and 26, FIGS. 2A, 2B and 2C.

1.1-Preparation of A'dendrimers Each A'dendrimer was prepared by solid support synthesis from 15 g of MBHA resin (the SENN CHEMICALS company) initially loaded at 0.4 mmole / g using the following: Prepared on a scale of 0.5 mmole: Boc / benzyl strategy to protect the bound amino acids, HBTU / HOBt / DIEA mixture in DMF as activator for the amino acid coupling procedure, and coupling And the ninhydrin test (KAISER) to control deprotection operations.
Et al., Anal. Biochem., 1970, 34 , 595) and TNBS test (HANCOCK and BATTERSBY, Anal. Biochem., 1976, 71 , 261).

For the free amino functionality of the resin up to a value of 0.1 mmoles / g, in the presence of a HBTU / HOBt / DIEA mixture (0.25: 0.25: 0.75 equivalents per reacting amino functionality) in order to lower the loading. , In DMF, 0.25 equivalents of Boc-β- (per reacting amino functional group)
Ala-OH was first bound to this resin (reaction time: 1 hour). The resin's amino groups that remained free were then protected by acetylation by reacting the resin with an Ac ₂ O / DIEA / DCM mixture (5:10:85) for 10 minutes.

In fact, the second coupling corresponding to the first coupling of L-lysine is (
Using 4 equivalents of Boc-L-Lys (2-chlorobenzyloxycarbonyl) -OH, ie, L-lysine whose side chain is protected by a 2-chlorobenzyloxycarbonyl group, per reacting amino functional group. It was carried out in DMF in the presence of a HBTU / HOBt / DIEA mixture (4: 4: 12 equivalents per reacting amino functional group) (reaction time: 45 minutes).

The third coupling was carried out by using 4 equivalents of Boc-L-Lys (Boc) -OH in a mixture of HBTU / HOBt / DIEA (4: 4 per reacting amino functional group: (12 equivalents) in DMF (reaction time: 45 minutes).

Following this third coupling, one third of the resin was deprotected and then acylated with 8 equivalents of pre-made chloroacetic anhydride (by activation with DCC). It contains two L-lysine residues, while a free amino group (which is exactly β
-Which is the epsilon-amino group of the first L-lysine bound to alanine), while 2
A dendrimer having four chloroacetyl groups (hereinafter referred to as compound 13) was obtained.

The remaining two-thirds of the resin underwent a fourth coupling to obtain two more L-lysine bonds. This coupling is 4 equivalents of Boc-L-Lys (Boc) -OH and HB
It was carried out in DMF in the presence of a TU / HOBt / DIEA mixture (4: 4: 12 equivalents per reacted amino functional group) (reaction time: 45 minutes).

Following the fourth coupling, half of the remaining resin (ie 1/3 of the initial amount of resin) was then deprotected and acylated with 8 equivalents of pre-made chloroacetic anhydride. To give a dendrimer containing 4 L-lysine residues and having a free amino group and 4 chloroacetyl groups (hereinafter referred to as compound 14).

The other half of the remaining resin was subjected to a fifth coupling with 4 equivalents of Boc-L-Lys (Boc) -OH as described above. The operation of deprotecting the resin and acylating it with chloroacetic anhydride was repeated once more to obtain a dendrimer having 8 L-lysine residues, a free amino group and 8 chloroacetyl groups (hereinafter referred to as compound 15). Obtained.

In practice, during each coupling operation, HBTU (1 equivalent to the amino acid to be bound) was added to the bound amino acid (for β-alanine-in the DMF) after dissolution in DMF. 0.25 equivalent to NH ₂ functional group and 4 equivalents for coupling lysine), HOBt (1 equivalent to amino acid) and DIEA (3 equivalent to amino acid)
Was added to the solution containing. The whole was mixed for 1 minute at ambient temperature, then the resin was added and presoaked with DMF. The suspension was mechanically stirred for the duration of the reaction.

Further, during each coupling operation, the resin was continuously filtered to remove DMF (3
x2 min) and DCM (3x2 min) three times, suitable T to allow cleavage of the Boc protecting group
Treat with FA / DCM mixture (50:50, 1x2 min, 1x20 min), wash again with DCM (2x2 min), neutralize with DIEA / DCM mixture (5:95; 3x1 min) and finally DCM (2x1 min). Min) washed.

Cleavage and deprotection of the dendrimer was carried out by reacting the resin at 0 ° C. for 60 minutes with 11 ml of HF / anisole mixture (10: 1) per gram of resin, followed by cold tert-butylmethyl. Sequentially precipitate in ether, centrifuge, dissolve in water, lyophilize and use semi-preparative RP-HPLC [gradient (A / B): 100: 0 to 50:50 at 120 minutes]. It is achieved by purification.

In this way, the following was obtained: 160 mg of dentrimer 13 (yield: 52%); TOF-PDMS spectrum: m / z 498 (M + H).
) ⁺ ; -215 mg of dendrimer 14 (yield: 42%); TOF-PDMS spectrum: m / z 928 (M + N)
a) ⁺ ; and 182 mg of dendrimer 15 (yield: 20%); TOF-PDMS spectrum: m / z 1726 (M +
H) ⁺ .

1.2- Preparation of tripeptides with the balance of (-)-shikimic acid and (-)-quinic acid The tripeptides with the balance of (-)-shikimic acid and (-)-quinic acid are on a solid support. Wherein the (S1) sequence peptide is synthesized in the first step, and one molecule of this tripeptide is a bond between the amino group of the N-terminal L-lysine of said tripeptide and the carboxyl group of said acid. Through formation, in the second step (−)-shikimic acid 2 molecules or (−)-
It is bound to two molecules derived from quinic acid.

In this way, the following was obtained: On the other hand, N ^α , N ^ε -di-[(3R, 4S, 5R) -3,4,5-, referred to below as compound 1 Trihydroxy-1-cyclohexenecarbox-1-yl] -L-lysyl [S- (tert-butylthio)]-
L-cysteyl-glycine, on the other hand, referred to below as compound 3, N ^α , N ^ε -di-[(1s _n , 3R, 4s _n , 5R) -1,3,4,5-
Tetrahydroxy-1-cyclohexanecarbox-1-yl] -L-lysyl [S- (tert-butylthio)]-L-cysteyl-glycine.

A) Preparation of Compound 1 Compound 1 was prepared using Fmoc-glycine p-benzyloxybenzyl resin (available under the trade name Fmoc-Gly-O-Wang from NOVABIOCHEM) loaded at 0.8 mmole / g. And at the following 2 mmole scale: • Fmoc strategy to protect the amino functional group of the amino acid to be bound • HBTU / in NMP as activator for amino acid coupling manipulations DIEA mixture, piperidine / NMP mixture to remove Fmoc protecting group, and ninhydrin and TNBS test to control coupling and deprotection reactions.

The Fmoc group protecting the amino function of the glycine residue of the resin was replaced with piperidine.
After removal by treatment with a 20/80 / NMP mixture for 20 minutes, the coupling of the S- (tert-butylthio) -L-cysteine residue (after reacting amino groups) with 2 equivalents of Fmoc-L -Cys (tert-Butylthio) -OH was used to prepare HBTU / DIEA mixture (2:
(3 equivalents) (reaction time: 40 minutes). While the tert-butylthio group is not sensitive to both reaction conditions used during coupling and cleavage operations,
At the same time, it is easily removed through the action of trialkylphosphine, so L-
The tert-butylthio group was chosen as a protecting group for the thiol functional group of cysteine residues.

This first coupling is followed by a second coupling intended to immobilize the tripeptide L-lysine, which results in 2 equivalents of Fmoc-L-Lys (Fmoc) -OH.
Was carried out in NMP in the presence of 5 equivalents of HBTU / DIEA mixture (2: 3 equivalents) (reaction time: 40 minutes).

Then, two molecules of (−)-shikimic acid were added in NMP for 30 seconds with 2 equivalents of this acid (per reactive amino functional group) in a HBTU / DIEA mixture (1: 1 equivalent). Activate,
The resin was pre-soaked in NMP and bound by adding the acid (reaction time: 4
0 minutes).

At the end of this coupling operation, the solvent was filtered and the resin was continuously washed with NMP (3
x 2 min) and DCM (3 x 2 min), then dried.

Compound 1 was cleaved by treating the resin with 25 ml of TFA / H ₂ O / Me ₂ S mixture (95: 2.5: 2.5) at ambient temperature for 2 hours. The solution was concentrated under reduced pressure, the residue thus obtained was dissolved in water and then freeze-dried to produce 1.64 g of a pale yellow powder. Semi-preparative RP-HPLC [gradient (A / B
): 110 min in 100: 0 to 50:50] and lyophilized to give 15 mg, ie 14
% Pure compound 1 could be obtained.

[0105]   The properties of compound 1 are as follows:

[0106]

[Equation 1]

The coupling constants between the protons 3-H / 4-H and 4-H / 5-H of the two (−)-shikimic acid residues present in compound 1 determined by NMR ¹ H spectroscopy are: 4.6 and 9.2 Hz. These constants are compatible with the quasi-equatorial orientation of the hydroxyl functional groups carried by the carbon atoms located at positions 4 and 5 of the acid, confirming the structural similarity between (−)-shikimic acid and D-mannose.

B) Preparation of Compound 3 Compound 3 was Fmoc-glycine o-methoxy-p- (benzyloxy) benzyl resin (trade name Fmoc-Gly-Sasrin from BACHEM) loaded at 0.8 mmole / g. Prepared on a 2 mmole scale, using: Fmoc / tert-butyl strategy to protect the amino acids to be bound, as an activator for amino acid coupling procedures. HBTU / DIEA mixture in NMP, piperidine / NMP mixture to remove the Fmoc protecting group, and ninhydrin and TNBS tests to control coupling and deprotection procedures.

The coupling operation of S- (tert-butylthio) -L-cysteine and L-lysine was carried out under the same conditions used to prepare compound 1.

On the other hand, the coupling of two (−)-quinic acid molecules is performed using a different operating protocol than that used to couple the (−)-shikimic acid. It was In fact, the activation of (−)-quinic acid immediately leads to the formation of the bicyclic γ-lactone by esterification between the carboxyl group at the 1st carbon atom and the hydroxyl group at the 3rd carbon atom of the acid. It has been found to be a consequence and thus it is necessary to protect the hydroxyl functionality of the (−)-quinic acid before carrying out the coupling.

Thus, we chose to peracetylate (−)-quinic acid with acetic anhydride, so that it is exactly the tetraacetyl derivative of (−)-quinic acid, which is referred to below as compound 9 ( 1s _n , 3R, 4s _n , 5R) -1,3,4,5-tetraacetoxycyclohexane-1-carboxylic acid, and then treating the latter with its acid fluoride by treating compound 9 with cyanuric fluoride. (Referred to below as Compound 18).

To do so, 1 drop of perchloric acid was stirred at ambient temperature while stirring 4 g of (−)-quinic acid (21 mmole) in 30 ml of acetic acid / Ac ₂ O mixture (2: 1). Was added to the suspension containing. After stirring for 12 hours, the resulting mixture was diluted with chloroform and extracted with a solution saturated with sodium bicarbonate and then with water. The organic layer was dried with sodium sulfate, filtered, and concentrated under reduced pressure. Pentane was added and the residue was precipitated to give 6.45 g, ie 86% yield of compound 9.

500 mg (in 6 ml of methylene chloride and 112 μl (1.39 mmole) of pyridine
1.39 mole) of compound 9 in a solution containing 937 μl (11,10 mmole) of cyanuric fluoride, added dropwise with stirring, and the resulting mixture was refluxed for 2 hours.
Acid 9 was converted to its fluoride. A white precipitate formed, the mixture was filtered and then extracted with water. After the organic layer was dried over sodium sulfate, the solvent of the organic phase was removed to give an oil corresponding to compound 18, which was dried under vacuum for several hours.

Compounds 9 and 18 have the following spectral properties: Compound 9 :

[0115]

[Equation 2]

Compound 18 :

[0117]

[Equation 3]

Two molecules of compound 18 firstly acylate the resin with 2 equivalents of compound 18 (per reacting amino functional group) in the presence of 3 equivalents of DIEA in NMP (reaction time: 60 Minutes)
, Then different reagents after washing the resin with NMP (3x2 min) and DCM (3x2 min)
It is attached by a second acylation with 1 equivalent.

At the end of this coupling operation, the solvent was filtered and the resin was continuously washed with NMP (3x
2 minutes) and DCM (3 x 2 minutes), then dried.

Cleavage of compound 3 was accomplished by treating the resin with 20 ml of CH ₂ Cl ₂ / TFA / ⁱ Pr ₃ SiH (95: 2: 3) mixture (4x
5 minutes). The solution was concentrated under reduced pressure and the residue thus obtained was dissolved in water and lyophilized to produce 1.40 g of crude compound, which was
It was dissolved in ml of methanol. The solution thus obtained is brought to 1 M until the pH is about 9.
Of methanolic sodium methylate was added, and then the mixture was reacted for 3 hours, at which time the reaction course was controlled by RP-HPLC. Then the mixture was concentrated under reduced pressure. A sample of 100 mg of the residue was subjected to semi-preparative RP-HPLC [gradient (A / B): 10
Purified by 100: 0 to 90:10 in minutes, then 90:10 to 50:50 in 70 minutes, and after lyophilization, 41 mg, ie 59% yield of pure compound 3 was obtained.

[0121]   Compound 3 has the following properties:

[0122]

[Equation 4]

Coupling constants between the protons 3-H / 4-H and 4-H / 5-H of the two (−)-quinic acid residues present in compound 3 measured using NMR ¹ H spectra. Are 9.6 and 2 respectively
It is .9 Hz. These constants fit the cyclohexane << chassis >> conformation in which the amide group is in the equatorial position, hydroxyls 3 and 4 are in the di-equatorial trans relationship, and hydroxyls 4 and 5 are in the equatorial-axial cis relationship. .

1.3 -(−)-Shikimi and Tripeptides Bearing (−)-Quinic Acid Residue and A ′ Dendrimers
Coupling compounds 13, 14 and 15 and compounds 1 and 3 were combined using a method including two steps, ie, the following method: -Disulfide bond (S-SBu ^t ) of each compound 1 and 3 Is the first step aimed at reducing methane, which is a degassed n-propanol / water mixture (50:50)
These compounds were treated with tri-n-butylphosphine in
And 3 by removing the tert-butyl thiol protecting groups each and deprotecting their thiol functional groups in this way, thus reducing compounds 1 and 3 thus degassed with a degassed DMF / water mixture ( 90:10) Medium,
This is the second step aimed at reacting with compounds 13, 14 and 15 in the presence of potassium carbonate, so that the thiol functional group of compounds 1 and 3 and compounds 13, 14 and 15
The formation of thioether bonds between the chloroacetyl functional groups of can be obtained.

Thus, a solution containing 1.5 equivalents of compound 1 or compound 3 in 1 ml of degassed n-propanol / water mixture (50:50) (per chloroacetyl group which reacts in the second step). To this was added 1 equivalent of tri-n-butylphosphine. Each reaction mixture was left under nitrogen atmosphere with stirring for 24 hours at ambient temperature, then the solvent and the released tert-butyl mercaptan were evaporated under reduced pressure and the residue was dried under vacuum for 15 minutes.

After dissolving each of these residues in a degassed DMF / water mixture (90:10),
Added to 1 eq (1-5 μmole) of compound 13, 14 or 15 and 20 eq of potassium carbonate. Each reaction mixture was left stirring at ambient temperature for 72-96 hours, the reaction was controlled by RP-HPLC, then diluted with water and lyophilized. The residue was then purified by semi-preparative RP-HPLC.

In this way the following was obtained: • Gradient (A / C): 3.39 mg of compound 21 (yield: 61% using 100: 0 to 70:30 in 70 minutes).
), • Gradient (A / C): 100: 0 to 75:25 in 25 minutes, followed by an isocratic gradient of 5
.98 mg of compound 22 (yield: 66%), • Gradient (A / C): 100: 0 to 80:20 in 35 minutes, followed by isocratic gradient to 2
.25 mg of compound 23 (yield: 42%), • Gradient (A / C): 100: 0 to 75:25 in 25 minutes, followed by isocratic gradient, 5
.75 mg of compound 24 (yield: 56%), • Gradient (A / C): 100: 0 to 75:25 in 40 min, followed by 3 with isocratic gradient.
.15 mg of compound 25 (yield: 43%), • Gradient (A / C): 100: 0 to 80:20 in 35 minutes, followed by isocratic gradient to 7
0.03 mg of compound 26 (yield: 61%), all in the form of a white powder.

Compounds 21-26 have the following spectral properties: Compound 21 :

[0129]

[Equation 5]

Compound 22 :

[0131]

[Equation 6]

Compound 23 :

[0133]

[Equation 7]

Compound 24 :

[0135]

[Equation 8]

Compound 25 :

[0137]

[Equation 9]

Compound 26 :

[0139]

[Equation 10]

Example 2 Preparation of Ligands According to the Invention Referred to below as ligands 21F, 22F, 23F and 24F, corresponding to general formula (III) 4
One ligand, where: m is an integer equal to 4 or 8 B ¹ is (−)-shikimic acid or (−)-quinic acid residue, n is (m is 4 and 8 Is an integer equal to 2 or 4 (when equal), X is the remainder of the (S1) sequence tripeptide as defined in Example 1. • A contains 2 or 4 L-lysine residues (when m is equal to 4 and 8 respectively), while 2 or 4 chloro for coupling with an equal number of tripeptides Is the rest of the A'dendrimer with an acetyl group and, on the other hand, a free amino group suitable to allow its attachment to Y. Y is the rest of the fluorescein molecule. M is the amide group ( a -CO-NH-), N while a thioether group _{(-CH 2 -S-CH 2 -CO-} ), P is a thiourea group (-NH-CS-NH-), a compound Compounds 1 and 3 which were previously protected with FITC after deprotecting their thiol functional groups under the same conditions as described in §1.4 of Example 1 above.
Prepared by coupling with 14.

Thus, as described in §1.4 of Example 1, degassed n-propanol /
1.5 in 1 ml of water mixture (50:50) (per chloroacetyl group which is subsequently reacted)
To a solution containing an equivalent amount of Compound 1 or Compound 3, 1 equivalent of tri-n-butylphosphine was added. Each reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours.
Allowed to stand for a period of time, then the solvent was evaporated under reduced pressure and the residue dried under vacuum for 15 minutes.

In addition, 1 equivalent (1 to 2.5 μmole) of compound 13 or 14 was added to 1 ml of degassed DMF.
1.1 eq FITC and 4 eq diisopropylethylamine in the dark at ambient temperature, 4
Each reaction mixture was added to a solution containing one of the previously obtained residues in 300 μl of degassed water, after which the reaction was allowed to react for a period of time. The wall of the vessel containing the reaction mixture was washed with 200 μl of degassed DMF and the pH of the different reaction mixtures was brought to a value of 8-8.5 by adding potassium carbonate.

The reaction mixtures were then left in the dark for 48 hours and the reaction was controlled using RP-HPLC. The reaction mixtures were then diluted with water containing 10% acetic acid, lyophilized and the residue was purified using semi-preparative RP-HPLC.

In this way the following was obtained: Gradient (A / C): 100: 0 to 75:25 in 15 minutes, then 75:25 to 65:35 in 30 minutes, 1
.82 mg of ligand 21F (yield: 46%), • Gradient (A / C): 100: 0 to 75:25 in 15 minutes, then 75:25 to 65:35 in 30 minutes, 1
.42 mg of ligand 22F (yield: 27%), • Gradient (A / C): 100: 0 to 85:15 in 15 minutes, then 85:15 to 70:30 in 30 minutes, 4
.56 mg of ligand 23F (yield: 62%), • Gradient (A / C): 100: 0 to 85:15 in 15 minutes, then 85:15 to 70:30 in 30 minutes, 3
0.57 mg of ligand 24F (yield: 50%), all in the form of a white powder.

[0145]   The chemical structures of these ligands are shown in Figures 3 and 4.

[0146]   Their EIS-MS spectra are as follows:

[0147]

[Equation 11]

Example 3 Preparation of Ligands According to the Invention Two ligands corresponding to general formula (III), wherein: m is an integer equal to 3, B ¹ is (−)-shikimic acid or ( -)-Quinic acid balance, n is an integer equal to 0 (hence X is absent), A is 3 L-lysine residues and (-)-shikimic acid or (-) -The remainder of the A'straight chain with three free amine groups for coupling with three molecules of quinic acid, while for coupling to Y by an amide bond, Y is in this case hen's Ovalbumin 323-339 peptide (hereinafter referred to as Ova 323-339) represents a compound of interest formed by three Arg-Gly-Arg residues bound to a class II epitope, M and P are amides A group (-CO-NH-) was prepared.

[0149]   These ligands were prepared as follows: First, a peptide having the following (S2) sequence is synthesized on a solid support:

[0150]

[Equation 12]

Where: -the 17 amino acid residues located at the C-terminus correspond to the Ova 323-339 peptide sequence, -the three Arg- located upstream of the sequence of said Ova 323-339 peptide sequence. The group formed by the Gly-Arg residue is a group that is sensitive to endopeptidases, the group formed by the three Lys-Lys-Lys residues corresponds to the binding of L-lysine,
On the other hand, the acetylated S- (tert-butylthio) -L-cysteine residue located at the -N-terminus is intended to allow subsequent coupling of ligands, e.g. to obtain dimers. -Then, the L-lysine residue of this peptide and (-)-shikimic acid and (-)-quinic acid (in the form of their tetra-O-acetylated derivatives) are combined with ε of said L-lysine residue; -Binding by forming an amide bond between the amino group and the carboxyl group of the acid.

These ligands are referred to below as ligand 25 and ligand 28, respectively.

The (S2) sequence peptide was synthesized from 337 mg of MBHA Rink amide resin (SENN CHEMICALS company) loaded at 0.74 mmole / g on a 0.25 mmole scale using: Fmoc / tert-butyl strategy for protecting amino acids, -2,2,2,5,7,8-pentamethylchroman-6-sulfonyl group, -L as a side chain protecting group for L-arginine -As a group that protects the side chains of serine and L-glutamic acid, a tert-butyl group,
-L-asparagine, L-glutamine and L-histidine as a side chain protecting group, trityl group, -L-lysine side chain as a protecting group, 4-methyltrityl group, -for coupling amino acids As an activator of HBTU / HOBt / DIEA in DMF
Mixtures, piperidine / DMF mixtures to remove Fmoc protecting groups, and ninhydrin and TNBS tests to control coupling and deprotection procedures.

The first 20 amino acids are automatically linked by the APPLIED BIOSYSTEMS ABI431 synthesizer, while L-lysine and S- (tert-butylthio) -L-cysteine are linked manually.

In all cases, these coupling operations were carried out in the presence of a mixture of HBTU / HOBt / DIEA in DMF (4: 4: 12 equivalents per reacting amino functional group). 4 minutes) with 4 equivalents of amino acid, for 1 minute before adding them to the resin,
This was done by activating an amino acid. Actually, HBTU (4 eq) is DMF
Dissolved in, and then bound amino acids, HOBt (4 eq) and DIEA (8 eq)
Added to the solution contained in DMF. The whole was mixed for 1 minute and then added to the DMF presoaked resin. The resulting reaction mixture was mechanically stirred for the duration of the reaction, ie 40 minutes for automatic coupling and 45 minutes for manual coupling.

Following all manipulations of the coupling of L-lysine with S- (tert-butylthio) -L-cysteine, Ac ₂ O / DIEA was prepared with the intention of acetylating the unbound amino group. / DC
The resin was treated with M mixture (5:10:85) for 10 minutes.

Further, during each coupling operation, the resin was continuously filtered to remove DMF (3
x 2 min) and DCM (3 x 2 min), treated with piperidine / DMF mixture (20:80) resulting in the cleavage of the Fmoc group and another wash with DMF (2 x 2 min) and DCM (3 x 1 min).

Following coupling of S- (tert-butylthio) -L-cysteine and its acetylation, the resin was treated with a TFA / DCM mixture (1:99) in a continuous flow until the yellow color disappeared. The 4-methyltrityl group protecting the L-lysine residue was hydrolyzed by washing.

The operation of coupling the (S2) peptide sequence with (−)-shikimic acid and (−)-quinic acid was carried out by treating the fraction of the resin thus washed in the presence of 12 equivalents of DIEA in DMF. Reacted with 4 equivalents of (-)-shikimic acid or 4 equivalents of tetra-O-acetyl-(-)-quinic acid previously activated with 4 equivalents of HBTU and 4 equivalents of HOBt at ambient temperature for 45 minutes, The reaction was performed by monitoring with ninhydrin and TNBS test.

Cleavage of the resulting products and their deprotection were carried out at ambient temperature for 90 minutes with 10 ml of TFA / H ₂ O / ⁱ Pr ₃ SiH mixture (95: 2.5: 2.5) / g resin. By treating the resins with, after which they were continuously precipitated in cold diethyl ether, centrifuged, dissolved in water, lyophilized, semi-preparative RP-HPLC [gradient (A / B): 120 minutes to 100: 0 to 50:50].

In this way the following was obtained: • Gradient (A / B): 48 mg of ligand 25 (yield: 29 using 100: 50 from 100: 0 to 50:50).
%); And • gradient (A / B): using 100: 0 to 60:40 at 100 minutes, 25 mg of compound 26 (yield: 15%
).

This compound 26 was deacetylated to give the ligand 28. To do so, 300 μl of a 1 M sodium methanolate solution in methanol was added to a solution of 23 mg of compound 28 in 10 ml of methanol distilled over magnesium. The reaction mixture was stirred under a nitrogen atmosphere at ambient temperature for 45 minutes and the reaction was stopped by adding a few drops of acetic acid. The crude reaction was concentrated under reduced pressure and gradient (A / B) in 30 minutes: 100: 0.
Purified by RP-HPLC using -80: 20, then 80: 20-70: 30 in 25 minutes, followed by an isocratic gradient for 10 minutes to produce 15 mg (yield: 75%) of ligand 28.

[0163]   The EIS-MS spectra of ligands 25 and 28 are as follows:

[0164]

[Equation 13]

Example 4 Preparation of Ligands According to the Invention Two ligands corresponding to general formula (III) according to the invention (FIG. 5), where: m is an integer equal to 2, n is 1. Is an integer equal to y y is an integer equal to 1 B ¹ is the balance of (−)-quinic acid (ligand 33F) or (−)-shikimic acid (ligand 34F), A is one The remainder of the dipeptide containing L-lysine residue and one β-alanine (N ^α- (chloroacetyl) -L-lysyl-β-alanine-amide residue, the chemical structure of which is shown in Figure 5) X is a formula (S1) tripeptide as defined in Example 1, Y is the rest of the fluorescein molecule, M, N and P are each an amide (-CO- NH-), thioether (-CH ₂ -S-CH ₂ -CO-
) And a thiourea (-NH-CS-NH-) group were prepared.

A) Preparation of N ^α- (chloroacetyl) -L-lysyl-β-alanine-amide dipeptide This dipeptide was prepared on a 0.7 mmole scale using the Fmoc / tert-butyl strategy.
It is manually synthesized with the help of ink amide-AM-PS resin (0.70 mmol / g, Senn Chemicals). Following coupling, the following TNBS test is performed: HBTU dissolved in NMP.
(4 eq) is added to a mixture of amino acid (4 eq), HOBt (4 eq) and DIEA (8 eq) in NMP. After stirring for 1 minute, the reaction mixture is added to NMP presoaked peptidyl resin (1 equivalent) containing DIEA (4 equivalents) and stirred for 40 minutes. Peptidyl resin (i.e. peptide sequence on the solid support) was filtered, washed with NMP (3x2 min) and CH ₂ Cl ₂ (3x2 min). The Fmoc protecting group is removed by treatment with 20% piperidine in NMP (1 × 2 min, 1 × 20 min), followed by washing with NMP (2 × 2 min) and CH ₂ Cl ₂ (2 × 1 min). After the second coupling step, the peptidyl resin is deprotected and acylated with an excess (4 equivalents) of chloroacetic anhydride prepared by DIC activation. Finally, the dipeptide was separated from the support and the TFA-TIS-H ₂ O mixture (90: 5: 5) for 1.5 hours at ambient temperature.
Deprotect by the action of (11 ml per gram of peptidyl resin), precipitate in a cold diethyl ether-heptane mixture (1: 1), centrifuge, dissolve in water and freeze-dry. After purification by RP-HPLC (with the following gradient: 100: 0 to 90:10 (A / B) in 20 minutes), followed by lyophilization, the dipeptide (184 mg, 65%) was obtained in the form of a white powder. Get at. The analysis is as follows:

[0167]

[Equation 14]

B) Preparation of Ligands Corresponding to General Formula (III) According to the Present Invention, On the Other hand, the Remaining (−)-Shikimic Acid (Compound 1) and (−)-Quinic Acid (Compound 3) Coupling of the tripeptide with 1 described in 1, and on the other hand the fluorescein molecule with the dipeptide prepared in a) is carried out according to the protocol described in Example 2. The ligands 33F and 34F are thus obtained.

RP-HPLC (gradient: 100: 0 to 85:15 (A / C) in 15 minutes, then 85:15 to 75:25 (30 minutes in
A / C), followed by purification using an isocratic gradient), followed by lyophilization,
4 mg (80% yield) of ligand 33F was obtained in the form of a white powder. Ligand 33F
The analysis of is as follows:

[0170]

[Equation 15]

RP-HPLC (gradient: 100: 0 to 85:15 (A / C) in 15 minutes, then 85:15 to 70:30 (in 50 minutes).
2.42 mg (80% yield) of ligand 34 after purification using A / C)) followed by lyophilization.
F was obtained in the form of a white powder. The analysis of ligand 34F is as follows:

[0172]

[Equation 16]

Example 5 : Preparation of ligands according to the invention Four ligands corresponding to general formula (III) according to the invention, wherein: m is an integer equal to 8 n is an integer equal to 4 , Y is an integer equal to 1, B ¹ is the rest of (−)-shikimic acid or (−)-quinic acid, Y is the rest of the fluorescein molecule, A is 4 L -The remainder of the A'dendrimer containing a lysine residue and having four chloroacetyl groups for coupling to four tripeptides and one suitable free amino group to allow coupling to Y , X is the rest of the (S2) or (S3) tripeptide sequence as defined below, M, N and P are amide groups (-CO-NH-), thioethers (-CH, respectively) ₂ -S-CH ₂ -C
O-) and thiourea (-NH-CS-NH-) were prepared according to the following protocol.

A ) Preparation of Compound Q (FIG. 6), a derivative of quinic acid in which the hydroxyls located in the 3 and 4 positions are protected in the form of diacetals Compound Q, ie (1s _n , 3R, 4s _n , 5R , 2'S, 3'S) -3,4-O- (2 ', 3'-dimethoxybutane-
2 ', 3'-diyl) -1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid is 12
980 mg (3.06 mmol) of 3,4-O- dissolved in ml of MeOH-aq / NaOH 0.1 M (1: 1) solution.
(2 ', 3'-dimethoxybutane-2', 3'-diyl) -1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid methyl ester (J. Org. Chem., 1996, 61 , 3897-3899 to J
.L. MONTCHAMP et al.). The reaction mixture is stirred at ambient temperature for 2 hours and concentrated under reduced pressure. The residue was separated with EtOAc and aqueous citric acid (0,1 M). The aqueous phase is extracted twice with EtOAc. The organic phases are combined, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. After crystallization with Et ₂ O-heptane, 780 mg (
80% yield of compound Q is obtained.

[0175]

[Equation 17]

B) Preparation of Tripeptides of Sequences (S2) and (S3) These tripeptides have the following sequence: Lys-Cys (tert-butylthio) -Gly-OH (S2) Lys-Cys (tert- (Butylthio) -Arg-NH ₂ (S3) Negatively charged peptide in physiological medium (S1) described in Example 1
In contrast, the tripeptides (S2) and (S3) are neutrally and positively charged in the physiological medium, respectively.

Example 1 for peptide (S1) synthesis except that they are synthesized on a Rink amide-AM-PS resin (0.70 mmol / g, Senn Chemicals) on a 0.2 mmole scale.
Obtained by the same protocol described in. The Fmoc-L-Arg-OH protecting group is 2,2
It is a 5,5,7,8-pentamethylchroman-6-sulfonyl group.

C) Coupling of shikimic acid and quinic acid derivatives (compound Q) with peptides (S2) and (S3) 0.2 mmole of the respective peptidyl resin (ie solid support peptide sequence),
In other words, 0.2 mmole sequence (S2) on the one hand and 0.2 mmole sequence (S3) on the other hand in the presence of BOP (177 mg, 0.4 mmol, 2 eq) and DIEA (140 μl, 0.8 mmol, 4 eq) in DMF. , Contact with 123 mg (0.4 mmol, 2 eq) of compound Q for 1 h at ambient temperature.
Then wash the peptidyl resin with DMF (2x2 min) and CH ₂ Cl ₂ (3x1 min). The coupling operation is performed twice.

In addition, 0.2 mmole of each peptidyl resin was added to HBTU-HOBt-DIEA [(
166 mg, 0.44 mmol, 2.2 eq), (67 mg, 0.44 mmol, 2.2 eq), (146 mg, 0
.84 mmol, 4.2 eq)] at ambient temperature for 40 min, 61 mg (0,4
Contact with 4 mmol, 2.2 equivalents) of shikimic acid. Then, the peptidyl resin was added to DMF.
It washed with (2x2 min) and CH ₂ Cl ₂ (3x1 min). The coupling is done twice.

The peptidyl resin is then washed with Et ₂ O. 1 hour at ambient temperature, TFA / H ₂ O / iPr ₃ SiH
The peptide is separated from the solid support by treatment with a mixture (95: 2.5: 2.5) (10 ml / g of peptidyl resin); precipitated in cold diethyl ether-heptane mixture, centrifuged and dissolved in water. And then freeze-dried. Peptide (S2)
q, (S2) s, (S3) q and (S3) s are obtained, which have two residues of quinic acid (peptides (S2) q and (S S3) q) or 2 of shikimic acid
Corresponds to the tripeptides (S2) and (S3) with one residue (peptides (S2) s and (S3) s).

Peptide (S2) q This is N ^α , N ^ε -di-[(1s _n , 3R, 4s _n , 5R) -1,3,4,5-tetrahydroxycyclo-hexane-1-carbox -1-yl] -L-lysyl [S- (tert-butylthio)]-L-cysteinyl
-Glycine-amide. Purification using RP-HPLC [Gradient: 100: 0 to 72:28 (A / B) for 20 minutes, then 72:28 to 62:38 (A / B) for 30 minutes] followed by lyophilization followed by 35.6. mg (2
4% yield) of this product is obtained in the form of a white powder.

[0182]

[Equation 18]

Peptide (S2) s This is N ^α , N ^ε -di-[(3R, 4S, 5R) -3,4,5-trihydroxy-1-cyclohexene-
Carbox-1-yl] -L-lysyl [S- (tert-butylthio)]-L-cysteinyl-glycine
-It's Amido. RP-HPLC [Gradient: 100: 0 to 90:10 (A / B) in 10 minutes, and 90:10 in 15 minutes.
After purification with ~ 85: 15 (A / B)] followed by lyophilization, 56 mg (40% yield) of this product are obtained in the form of a white powder.

[0184]

[Formula 19]

Peptide (S3) q This is N ^α , N ^ε -di-[(1s _n , 3R, 4s _n , 5R) -1,3,4,5-tetrahydroxycyclo-hexane-1-carbox -1-yl] -L-lysyl [S- (tert-butylthio)]-L-cysteinyl
-L-arginine-amide. Purification using RP-HPLC [gradient: 100: 0 to 85:15 (A / B) for 15 minutes, then 85:15 to 75:25 (A / B) for 30 minutes], followed by lyophilization, then 45 mg
This product (24% yield) is obtained in the form of a white powder.

[0186]

[Equation 20]

Peptide (S3) s This is N ^α , N ^ε -di-[(3R, 4S, 5R) -3,4,5-trihydroxy-1-cyclohexene-
Carbox-1-yl] -L-lysyl- [S- (tert-butylthio)]-L-cysteinyl-L-arginine-amide. RP-HPLC [Gradient: 100: 0 to 90:10 (A / B) for 10 minutes, then 1
After 2 minutes 90: 10-85: 15 (A / B), then purification using isocratic gradient] followed by lyophilization, 48 mg (26% yield) of this product was obtained as a white powder. Obtained in form.

[0188]

[Equation 21]

D) Preparation of ligand 41F (FIG. 6) Six equivalents of peptide (S2) q are placed in solution in a mixture of degassed nPrOH—H ₂ O 50:50 (1 ml). To this is introduced 6 equivalents of nBu ₃ P. The mixture is stirred at ambient temperature for 24 hours under a nitrogen atmosphere. The solvent was evaporated under reduced pressure and the residue obtained was re-evaporated.
Vacuum dry for 15 minutes. 500 μl degassed mixture DMF-aq NaOAc 0.1 M (90:10)
, From which the dendrimer of L-lysine 14 0.9 μmol (1 equivalent), the preparation of which is described in Example 1. The reaction medium was stirred for 24 hours at ambient temperature and the reaction progress was followed using RP-HPLC. Fluorescein isothiocyanate (4 eq) was added and the medium was stirred under a nitrogen atmosphere overnight with protection from light. The culture was diluted with water, lyophilized and RP-HPLC [Gradient: 15 minutes 100: 0 to 85:15 (A / B).
Purification using 85:15 to 80:20 (A / B) for 15 minutes, followed by an isocratic gradient to give 2.38 mg (72% yield) of ligand 41F in the form of a white powder. The analysis is as follows: EIS-MS, measured 3763.0, theoretical 3765.1; m / z 1883.0 (
M + 2H) ²⁺ , 1255.5 (M + 3H) ³⁺ .

[0190]   e)Preparation of ligands 42F (Figure 6), 43F and 44F (Figure 7)   6 equivalents of peptide (S2) s (or peptide (S3) s, or again peptide (S3) q),
Degassed nPrOH-H₂Place in a solution in a mixture of O 50:50 (1 ml). 6 equivalents of nBu ₃ Introduce P. The mixture is stirred at ambient temperature for 24 hours under a nitrogen atmosphere. The solvent
Was evaporated under reduced pressure and the residue obtained was vacuum dried again for 15 minutes, then
Dissolve in 50 μl water. 1 equivalent (0,5-1) in 300 μl DMF containing 4 equivalents of DIEA.
μmol) L-lysine dendrimer 14, the preparation of which is described in Example 1,
Is added to 1 equivalent of fluorescein isothiocyanate. The mixture is nitrogen atmosphere
Stir for 2 hours with protection from light under. Once the reaction is complete (RP-HPLC
(By monitoring with) the mixture containing the peptide compound.
Add to the dium. The pH of the mixture was adjusted to K₂CO₃By adding 8-8.
Adjust to 5. The mixture was stirred at room temperature for 24 hours under nitrogen atmosphere with protection from light.
Solution, diluted with water, lyophilized, purified using RP-HPLC, ligand 42F, 43F or 4
Get 4F.

RP-HPLC [Gradient: 100: 0 to 82:18 (A / B) for 18 minutes, then 82:18 to 72:28 (30 minutes.
A / B)], followed by lyophilization, followed by 1,3 mg (36% yield) of ligand
42F is obtained in the form of white powder. The analysis is as follows: EIS-MS,
Found 3620.0, theoretical 3621.0; m / z 1207.7 (M + 3H) ³⁺ , 1001.8 (M + 3H-C ₂₅ H ₃₈ N ₅ O _{1 1} ) ³⁺ , 905.9 (M + 4H) ⁴⁺ .

RP-HPLC [Gradient: 18 minutes 100: 0 to 82:18 (A / B), then 30 minutes 82:18 to 72:28 (
A / B)], followed by lyophilization, followed by 1,3 mg (36% yield) of ligand
43F is obtained in the form of white powder. The analysis is as follows: EIS-MS,
Measured 4160.0, Theoretical 4161.7; m / z 1387.6 (M + 3H) ³⁺ , 1041.0 (M + 4H) ⁴⁺ , 833.0
(M + 5H) ⁵⁺ .

RP-HPLC [Gradient: 18 minutes 100: 0 to 82:18 (A / B), then 21 minutes 82:18 to 75:25 (
A / B), followed by purification using an isotactic gradient] followed by lyophilization, 1.
3 mg (36% yield) of ligand 44F is obtained in the form of a white powder. The analysis is as follows: EIS-MS, measured 4016, theoretical 4017.6; m / z 1339.9 (M + 3H) ^{3 +} , 1005.2 (M + 4H) ⁴⁺ , 804.4 (M + 5H) ⁵⁺ .

Example 6 : Preparation of ligands according to the invention Two ligands corresponding to general formula (III) according to the invention, where: m is an integer equal to 4 or 8 n is an integer equal to 0 is (that is, no X), is · y is an integer equal to 1, · B ¹ is (-) - a quinic acid balance, · Y is a remainder of the antigen, · a is (FIG. 10 Is the remainder of the A'dendrimer containing 8 (in the case of the ligand 51 shown in Figure 4) or 4 (in the case of the ligand 52 shown in Figure 11) L and M, and P is Thioether (-CH ₂ -S-CH ₂ -CO-) and hydrazone (-CO-C, respectively)
H = N—NH—CH ₂ —CO—) group was prepared according to the following protocol.

These ligands were derived from quinic acid derivatives, namely quinic acid (compound G) functionalized with thiol functional groups, into lysine dendrimers (dendrimers H or I) and peptides according to the protocol described below. It is obtained by a ligation reaction to an antigen (antigen J or K).

A) Preparation of the quinic acid derivative (Compound G, FIG. 8).

Compound G is 2- {N, N-di-[(1s _n , 3R, 4s _n , 5R) -1,3,4,5-tetrahydroxycyclohexane-1-carboxamido-1-yl]} It is ethyl disulfide. It contained 52 mg (0,3 mmol) of lactone quinic acid and 34 mg (1 equivalent) of cysteamine hydrochloride in degassed water (pH 8-9) containing solid K ₂ CO ₃ at 50 ° C. overnight. Obtained by mixing. The mixture was then stirred at ambient temperature for 24 hours in the presence of air, RP-HPLC [gradient: 15 minutes 100: 0 to 80:20 (A / C), followed by an isotactic gradient.
] And then lyophilized to give compound G (70
mg, 71%) is obtained. The analysis is as follows:

[0198]

[Equation 22]

B) Preparation of Dendrimer H (FIG. 8) and I (FIG. 9).

[0200]   Functionalization of amino-PEGA resin with spacers   At ambient temperature, (+)-2,3-O-isopropylidene-D-tartaric acid (733 μl) in dimethyl ether.
To the Stell solution, 7.2 μl (4 eq) water and 59.6 μl (4 eq) 1,8-diazabishi
Black [5,4,0] -undec-7-ene is added continuously. The reaction mixture was stirred for 1 hour,
Then, 0.1 mmol of amino-PEGA resin (0.4 mmol / g) dipped in a minimal amount of DMF.
), Where the resin is previously CH₂Cl₂Medium 5% DIEA (2 x 1 minute) and DMF (3
It has been neutralized with (x1 min). Then add BOP (177 mg, 4 eq) to DMF (1 ml)
And the resulting mixture is stirred at ambient temperature for 40 minutes. The resin was added to DMF (4 x 2 minutes
) And CH₂Cl₂Wash with (2 x 2 minutes). Soak it in DMF and stir at ambient temperature for 1 hour with 1,3-dia.
Acylate with minopropane (0.65 ml). Finally, add the resin to DMF (3 x 1 min) and CH ₂ Cl₂Wash with (2 x 2 minutes).

[0201]   -L-lysine-based dendrimer synthesis.

These dendrimers were amino-PEGA resins (0.2 mmol / g, Sen, functionalized as shown above using the Fmoc / tert-butyl strategy, on a 0.1 mmole scale.
n Chemicals). In the amino acid, the coupling step is performed in excess with respect to the groupements amines reactionnels, that is, (
10 or 4 equivalents are used (for the first two coupling operations) and activation is carried out with the HBTU-HOBt-DIEA mixture in NMP. The coupling operation is followed by a TNBS test, eg as described in Example 4.

First, the Fmoc-β-Ala-OH is bound to the resin. The lysine residue is then introduced: the first lysine introduced is protected by the Boc group, the other lysines are their F
It is added in the form of a moc-L-Lys (Fmoc) -OH derivative. After the 4th and 5th coupling steps, the peptide was deprotected in the solid phase and in DMF with an excess (4 equivalents) of chloroacetic anhydride prepared using activation with DCC. Acylate. The peptidyl resin is washed with DMF (3 × 1 min), CH ₂ Cl ₂ (3 × ₂ min) and Et ₂ O (2 × 1 min), then dried. The Boc group of the ε-NH ₂ of the first lysine residue and of the isopropylidene group of the spacer fixed on the support are treated with a TFA-H ₂ O-anisole mixture (95: 2.5: 2.5) (10 ml) for removal. The peptidyl resin was washed with CH ₂ Cl ₂ (5 × 1 min) and Et ₂ O (2 × 1 min), then dried and aqueous Ac
Soak in OH. It was dissolved in aqueous AcOH (33%) / H ₂ O mixture (1: 1) (2 ml).
Add NaIO ₄ (128.3 mg, 6 eq). The mixture is stirred for 5 minutes, then 200 μl
Add ethylene glycol from there. The resin is washed with water (2 x 6 ml) and the collected filtrate is purified using RP-HPLC.

After purification using RP-HPLC [Gradient: 100: 0 to 50:50 (A / B) 120 min for 120 minutes] and lyophilization, 28 mg (31% yield) of 28 mg (31% yield) in the form of a white powder. Dendrimer H is obtained. The analysis is as follows: m / z (positive EIS-MS) 1038.4 (M + H ₂ O) ⁺ , 1020.3 (M + H) ⁺
.

RP-HPLC [Gradient: 100: 0 to 90:10 (A / B) for 10 minutes, then 90:10 to 70:30 (40 minutes).
A / B) followed by purification using an isocratic gradient] and freeze-drying yields 45 mg (23.5% yield) of dendrimer I in the form of a white powder. The analysis is as follows: m / z (positive EIS-MS) 1855.5 (M + H ₂ O) ⁺ , 1838.3 (M + H) ⁺ , 919.5 (
M + 2H) ²⁺ .

C) Preparation of peptide antigens J and K (FIG. 8) The epitope (peptide sequence represented by HA ^307-319 and TT ^{830-846 in} FIG. 8)
Is Rink amide-Nleu-AM-PS resin (0.38 mmol / g, Senn
Chemicals) using an Applied Biosystems A431 automated peptide synthesizer. The protecting group for the side chain of Fmoc-L-Lys-OH is the Boc group; Fmoc-L-Ser-O
The protecting group for H, Fmoc-L-Glu-OH, Fmoc-L-Thr-OH, Fmoc-L-Tyr-OH and Fmoc-L-Asp-OH is tert-butyl; Fmoc-L-Asn -OH, Fmoc-L-Gln-OH and Fmoc-L-His-OH
The protecting group for is a trityl group; the protecting group for Fmoc-L-Arg-OH is 2,2,4,6,7
-Pentamethyldihydrobenzofuran-5-sulfonyl group.

Preparation of Antigen J (H ₂ N-Gly-HA ^307-319 ) Once peptide synthesis was completed, the peptidyl resin (0,125 mmol) was deprotected and activity using DIC in DMF (20 min). Was manually coupled twice with an excess of 4 equivalents of bromoacetic anhydride prepared by crystallization, followed by DMF (3 x 1 min) and CH ₂ Cl ₂ (
Wash 3 x 2 minutes). 66 mg (4 equivalents) of tert-butylcarbazate was added to DIEA (174 μl 8
Equivalent weight) in DMF-soaked peptidyl resin. The mixture was stirred overnight,
Then wash with DMF (3 × 1 min), CH ₂ Cl ₂ (3 × 2 min) and Et ₂ O (2 × 1 min). Finally, the peptide was removed from the support, deprotected with TFA-anisole-H ₂ O mixture (95: 2.5: 2.5) (10 ml) at ambient temperature for 3 hours, precipitated in cold diethyl ether and centrifuged. , Dissolve in water and freeze-dry. After purification using semi-preparative RP-HPLC [gradient: 90 minutes 100: 0 to 50:50 (A / F)] and freeze-drying, 101 mg (40% yield) of antigen J is white. Obtained in powder form. Analysis of antigen J using positive EIS-MS is as follows: m / z 1575 (M + H) ⁺ .

The preparation procedure of antigen K (H ₂ N-Gly-TT ^830-846 ) is as described above for the preparation of antigen J, with the following exceptions: separated from the support, 1.5 _{hours, TFA-iPr 3 SiH-H} 2 O mixture (95: 2.
5: 2.5) (10 ml), the gradient for purification of the antigen using -RP-HPLC is as follows: 100: 0 ~ 25 min.
75:25 (A / F) followed by an isocratic gradient (A / F).

After purification using RP-HPLC and freeze-drying, antigen K is obtained in the form of a white powder; yield of 15%. Its analysis using positive EIS-MS is as follows: theoretical 2052.5
, Measured value 2052.0; m / z: 1026.7 [M + 2H] ²⁺ , 684.8 [M + 3H] ³⁺ , 514.0 [M + 4H] ⁴⁺ .

D) Preparation of Ligand 51 and 52 Ligand 51 (FIG. 10) Compound G (1,5 eq / chloroacetyl group for substitution), the preparation of which is described in paragraph a) above. -H ₂ O mixture (1: 1) is dissolved in (500 [mu] l) in. Add tri-n-butylphosphine (1 eq). After standing overnight at ambient temperature under nitrogen atmosphere, the mixture was concentrated under reduced pressure for 20 minutes, taken up in 500 μl water and 600 μl DM.
Add to 0.8 μmol (1 eq) Dendrimer I dissolved in F. The pH is adjusted to 8-8.5 by adding solid potassium carbonate. The mixture is stirred for 24-48 hours at ambient temperature under a nitrogen atmosphere. Then add 350 μl citrate / phosphate buffer and peptide antigen J. Adjust pH to 5.2 by adding aqueous 1N HCl
The mixture is stirred for 12 hours at ambient temperature, diluted with water, frozen well and lyophilized.
RP-HPLC [Gradient: 10 minutes 100: 0 to 90:10 (A / B), then 50 minutes 90:10 to 75:25 (A /
After purification with B)] and lyophilization, 1.52 mg of ligand 51 (35% yield) is obtained in the form of a white powder. The analysis using positive EIS-MS is as follows: theoretical value 5114.1, measured value 5112; m / z: 1704.9 [M + 3H] ³⁺ , 1278.9 [M + 4H] ⁴⁺ , 103.
0.9 [M + 5H + K] ⁵⁺ , 859.3 [M + 5H + K] ⁶⁺ .

Ligand 52 (FIG. 11) Compound G (1,5 eq / chloroacetyl group for substitution) in nPrOH—H ₂ O mixture (1: 1)
) (500 μl). Add tri-n-butylphosphine (1 eq). After standing overnight at ambient temperature under a nitrogen atmosphere, the mixture was concentrated under reduced pressure for 20 minutes. In parallel, 0.4 μmol of dendrimer H is contacted with 1 equivalent of antigen K in a ^t BuOH—H ₂ O mixture (180-20 μl) under nitrogen atmosphere at ambient temperature. Once the reaction was complete (followed by RP-HPLC), add the reaction mixture to compound G prepared above,
The pH is adjusted to 8-8.5 by adding solid potassium carbonate. The ligand 52 is then purified as described for ligand 51 above. RP-HPLC [
Gradient: 100: 0 to 75:25 (A / B) for 20 minutes, then 75:25 to 60:40 (A / B) for 30 minutes, and 0.71 mg of ligand 52 (42 % Yield) is obtained in the form of a white powder. Its analysis using positive EIS-MS is as follows: theoretical value
3913.6, measured value 3912; m / z: 1305.0 [M + 3H] ³⁺ , 979.1 [M + 4H] ⁴⁺ , 783.6 [M + 5H] ⁵⁺ .

Example 7 : Preparation of compounds which can be used as intermediate compounds for preparing ligands according to the invention Two compounds corresponding to general formula (VI) according to the invention, wherein: In the case of the compound called << 5% QuinPEI >> in about 58 and (below <<
For compounds called 10% QuinPEI >>) is an integer equal to about 116, · B ¹ is (-) - quinic acid balance, · M is the amide function (-CO-NH-) , A is the rest of the polyethyleneimine (PEI) linear polymer, according to the following protocol: 1.5-lactone quinic acid (carboxyl group held at the carbon atom located at the 1-position and (-)-quina It was prepared by condensing PEI onto (γ-lactone) resulting from esterification between the hydroxyl groups at the 5-position of the acid.

For example, in Chem. Ber., 1921, 54 , 775-785 by HOL FISCHER, J. Antibiot
ics, 1982, 25 , 1507-1512 and M. PHILIPPE et al. and Tetrahedron Lett., 1995.
, 36 , 8299-8302 as described by PQ HUANG et al., In which D (−)-quinic acid (Aldrich) was heated to bicyclic 1.5-γ by heating in toluene in the presence of p-toluenesulfonic acid. -Convert to a lactone (also called "quinide"). The lactone (10,1 mg, 58,1 μmol) reacted with 50 mg of PEI (25 kDa, Aldrich) to give PEI
Assuming an average molecular weight of 43 Da by weight for the repeating unit of
This corresponds to 1162 μmol of amino group. The reaction is carried out in 2 ml of water for 24 hours at 50 ° C. The reaction medium is then diluted and freeze dried. Then we have 5%
To obtain PEI substituted with quinic acid (hereinafter referred to as “5% QuinPEI”). With twice as much lactone (20.2 mg, 116.2 μmol), we obtain PEI (hereinafter “10% QuinPEI”) displaced by quinic acid in a proportion of 10%.

Completion of the reaction was determined by NMR ¹ H and ¹³ C analysis of the reaction medium, the quinide
) Is indicated by the absence of the corresponding signal (quinide on PEI).
Condensation actually results in the opening of the lactone ring). The results were carried out using NMR ¹ H on a sample of the product obtained, purified by ultrafiltration on an Amicon® membrane (30000 Da, Millipore), bound quinic acid on PEI. Confirmed by quantification of the amount. The quinic acid content of PEI is the C of the backbone of the polymer.
Confirmed by comparing the surface of the H ₂ protons and the peaks of the quinic acid residue protons H-4, H-5 and H-6.

PEI is a branched polymer with secondary and primary amines. By examining the NMR ¹ H spectra it is possible to distinguish between quinic acids linked by secondary or tertiary bonds (the latter being indicated by the mark '). Thus, 5% QuinPEI and 10%
Quin PEI contains 30 and 26% quinic acid residues, respectively, branched by a tertiary amide bond, the rest being secondary amide bonds.

In the following data (NMR ¹ H and ¹³ C analysis performed in D ₂ OH ₂ O 10:90), the internal standard is 3- (trimethylsilyl) [2,2,3,3-d ₄ ]. It is the sodium salt of propionic acid.

[0217]

[Equation 23]

Example 8 : Biological activity of ligands according to the invention The biological activity of the ligands according to the invention is that they are internalized by dendritic cells obtained from human peripheral blood via the mannose receptor. It was confirmed by a series of in vitro experiments for the purpose of testing capacity.

The experiments reported here were carried out with the following compounds: Methyl quinate (ie the methyl ester of quinic acid) described by E. DELFOURNE et al. In J. Chem. Res., 1991, 56-57. -Methyl-α-mannopyranoside sold by LANCASTER-Ligands bound to fluorescein 23F and 24F, whose preparation is described in Example 2 above, -hereafter referred to as compound 30F, 8 (- ) -Shikimic acid or (-)-quinic acid in place of the rest of the galactoconfiguration 8 polyhydroxylated chains in that the ligand 23
A compound distinct from F and 24F, this compound is intended to serve as a negative control because of the low level of affinity of the mannose receptor for D-galactose and compounds related to the latter. Compounds in the form of dendrimers, hereinafter referred to as Compound 31F, composed of 8 L-lysine residues and the balance of 8 D-mannose. This compound is because the mannose receptor preferentially binds to D-mannose. , Intended to serve as a positive control.

[0220] 1) - Protocol a) Compound 30F and 31F Preparation of Compound 30F was prepared in accordance with the following procedure. Initially as described in §1.2 of Example 1 above for the preparation of Compound 1
Synthesizing a peptide of sequence (S1) under conditions similar to those present, and then allowing the resin to react with a TFA / H ₂ O / Me ₂ S mixture (95: 2.5: 2.5) 25 for 2 hours at ambient temperature.
Cleavage of this tripeptide by treatment with ml, followed by D-galactolactone
160mg (0.90mmole) of tripeptide 140mg (0.22mmole) and D in 5ml of methanol
Reaction of refluxing IEA 0.79mmole for 24 hours in solution, then adding 80 mg (0,45mmole) of D-galactolactone to this reaction mixture, and continuing refluxing for another 24 hours to obtain 1 molecule of D-galacto of the tripeptide. Coupling with two molecules of nolactone; and finally, Nα, Nε-di [(2R, 3R, 4S, 5R) -2,3,4,5-tetrahydroxy-1-obtained in this way Hexane-carbox-1-yl] -L-lysyl [S- (tert-butylthio)]-L-cysteyl-glycine was previously reacted with FITC and compound 14 and Example 2 above.
By coupling under the same conditions as described in.

Compound 31F was prepared by treating compound 15 with 2-thioethyl-α-, D-mannopyranoside and then reacting the resulting compound with FITC.

[0222] b) dendritic cells producing dendritic cells, the protocol described by AVRAMEAS et (Eur.J.Immunol., 1996, 2 6 , in accordance derived protocols from 394-400) of human peripheral blood Obtained by the differentiation of monocytes that were themselves isolated from mononuclear cells.

To do so, a sample of human peripheral blood (Etab from Nord-Pas de Calais, FRANCE
mononuclear cells present in lissement de Transfusion Sanguine (supplied by the Blood transfusion center) were separated from other components of blood by centrifugation on a Ficoll / Paque density gradient (supplied by PHARMACIA), then The cells were washed 3 times by centrifugation in RPMI1640 medium (GIBCO) containing 3 mM EDTA.

The mononuclear cells thus separated were treated with 5.10 ⁻⁵ M β-mercaptoethanol (MERCK), L-glutamine (MERCK) 2 mM, and pyruvin at 37 ° C. in a 5% CO ₂ atmosphere. Sodium acidate (GIBCO) 1 mM, heat inactivated 10% fetal bovine serum (GIBCO), penicillin 100
RPMI supplemented with UI / ml and streptomycin 100 μg / ml (both SPECIA)
Suspended in culture medium containing 1640 medium.

The resulting suspension was then distributed into Petri dishes 10 cm in diameter at 8 to 12.10 ⁷ cells per disc. Mononuclear purification was obtained by attaching these cells to dishes for 3 to 4 hours in a humidified (5%) CO ₂ incubator at 37 ° C.

After removing the non-adherent cells, the adherent cells were treated in the above-mentioned medium except that 800 U / ml of granulocyte- and macrophage-based recombinant human growth factor (PEPROTECH) and 1000 U of human recombinant interleukin-4 were added. Recultured in medium containing / ml at a ratio of 10 ⁶ cells per ml of medium. They are then placed in the culture medium of a 24-well cell culture dish,
It was cultured for 6 to 8 days under the same temperature and atmospheric conditions as before, during which the differentiation of monocytes into dendritic cells (the latter slightly attached) occurred.

C) Flow cytometric analysis of collected non-adherent cells In order to confirm that the non-adherent cells collected in step b) really have the surface antigenic properties of dendritic cells, these cells Of a mouse monoclonal antibody and / or dendritic cells, which were washed twice with PBS buffer containing 0.03 mg / ml of bovine serum albumin and then directed against the human mannose receptor and complexed with phycoerythrin (BECTON DICKINSON). Mouse monoclonal antibody directed against human antigen CD1a, one of the major surface antigens, or human antigen CD14, one of the surface antigens specific to monocytes and macrophages
Incubated for 30 minutes on ice with mouse monoclonal antibody, directed against FITC (BECTON D
ICKINSON).

Following these culture procedures, cells were washed with PBS buffer and resuspended in this same buffer. The associated fluorescence was then analyzed by cytofluorometry using an EPICS XL-MCL cytometer (COULTER CORPORATION).

D) Inhibition test of internalization of compound 31F using methylquinate To show that methylquinate mimics mannose in internalization at mannose receptor of dendritic cells, methylquinate or methyl-α-D. -Competition between mannopyranoside (serving as an inhibition control) and compound 31F is studied according to the following protocol. : Dendritic cells are harvested, washed with PBS buffer, pre-incubated at 37 ° C, then with increasing concentrations of methylquinate and compound 31F (final concentration 5 μM) at 37 ° C.
Incubate for 20 minutes. The cells are then washed with PBS, fixed in 1% paraformaldehyde and then analyzed using flow cytofluorometry as described in c) above.

E) Internalization of the ligands according to the invention in dendritic cells Tests aimed at assessing the internalization of the ligands 23F and 24F were carried out according to the following protocol. : The non-adherent cells collected in step b) above are washed twice with PBS buffer containing 1 mM calcium chloride, subsequently these cells are pre-incubated for 10 minutes at 37 ° C., then One to one and
Cultured in different solutions, each containing a concentration between 10 μM. The cells are then cold PBS
It was washed 3 times with buffer and fixed with 2% paraformaldehyde for 20 minutes.

Fluorescence associated with these cells was then assessed by FAC scan analysis using the cytometer.

Furthermore, to confirm that the internalization of ligands 23F and 24F by dendritic cells is indeed linked to the recognition of these ligands by the mannose receptor, and not to any other mechanism, These tests were repeated by pre-culturing the cells in the presence of mannan solution extracted from Saccharomyces cerevisiae (SIGMA). It is known that mannan is indeed selectively internalized via the mannose receptor. For this purpose, dendritic cells are harvested, washed with PBS buffer and incubated at 37 ° C. Mannan 10 ^-4 and 10 extracted from Saccharomyces cerevisiae
It is pre-incubated for 20 minutes at 37 ° C. in the presence of a solution with a concentration of between mg / ml. The ligands 23F and 24F (final concentration 5 μΜ) are then added (incubation: 37 ° C for 20 minutes). Cells are washed with PBS and analyzed using flow cytofluorometry as described in c) above.

F) Effect of the number of mannose mimetics carried by the ligands according to the invention on the internalization of these ligands Optimal construction / structure, ie mimicking mannose which is specifically internalized by the mannose receptor. Dendritic cells contain 2, 4 or 8 residues of mannose, quinic acid or shikimic acid for 20 minutes at 37 ° C to determine the ligand with the smallest number of compounds (to facilitate chemical synthesis of the ligand) Ligand (2-, 4- or 8
-Valent ligand) 10 μM. After fixation in paraformaldehyde, cells are analyzed by flow cytofluorometry as described in c) above.

For these experiments, the use of the following ligands is utilized: Ligands with shikimic acid residues: 34F (divalent ligand), 21F (tetravalent ligand) and 23F (8 valent). Ligands having quinic acid residues: 33F (divalent ligand), 22F (tetravalent ligand) and 24F (octavalent ligand).

2) -Results a) Inhibition test of internalization of compound 31F with methylquinate FIG. 12 shows the ability of methylquinate to inhibit internalization of compound 31F. The percentage inhibition calculated using the following formula is shown on the Y-axis. % Inhibition = (MnX31F-MnXmqui) / MnX31F where: <MnX> is the average fluorescence intensity, <MnX31F> is the signal due to internalization of compound 31F only, and <MnXmqui> is on the X-axis. Shown is the signal from the internalization of compound 31F in the presence of different concentrations of methylquinate (expressed in mM).

The curves marked by the- ◆ -marks the inhibition of the internalization of compound 31F by methylquinate, the curves marked by the- ○ -marks the methyl-, which is a compound that acts as an inhibitory control. 3 shows inhibition of internalization of compound 31F by α-D-mannopyranoside.

In the presence of methylquinate, we note a 75% effective inhibition of specific internalization by the mannose receptor, comparable to that of the reference ligand, methyl-α-D-mannopyranoside.

B) Internalization of ligands according to the invention by dendritic cells FIG. 13 shows ligand 23F (− ● −), ligand 24F (− ◆ −), compound 30F (−Δ).
The average fluorescence intensity value of dendritic cells after culturing in the presence of a solution containing 1 to 10 μM each of −) and Compound 31F (− □ −) is shown in the form of a curve. Average fluorescence intensity value
(UA) is shown on the Y-axis and solution concentration on the X-axis.

FIG. 13 shows that culturing dendritic cells carried out in the presence of a solution of ligand 23F or ligand 24F adjusted to 1 μM for a duration of 20 minutes results in internalization of these ligands by these cells. Is sufficient for. It also shows that internalization of compound 30F (which has polyhydroxylated chains) hardly penetrates into dendritic cells, so whatever the amount of compound used to culture these cells, It is specific, whereas compound 31F (with D-mannose balance) is shown to be very widely distributed in the dendritic cells.

Furthermore, FIG. 14 shows that continuous pre-culture was performed in the presence of mannan solution at a concentration between 10 ⁻⁴ and 10 mg / ml, followed by ligand 23F (− ● −), ligand 24F (− ◆ −). ), Compound 30F (-Δ-) or compound 31F (-□-) in a solution containing 5 µM, the average fluorescence intensity of dendritic cells is shown in the form of a curve. The mean fluorescence value (UA) is shown on the Y-axis and the concentration of mannan solution on the X-axis.

FIG. 14 shows that dendritic cell internalization of ligands 23F and 24F, like that of compound 31F, dramatically decreases in proportion to increasing mannan concentration. This characterizes the existence of a strong competition between these ligands and mannans for the mannose receptor, confirming that dendritic cell internalization of the ligands according to the invention is really occurring via the mannose receptor. is doing.

C) Effect of the number of mannose mimetics carried by the ligands according to the invention on the internalization of these ligands . FIG. 15 shows, on the Y-axis, 2-, 4- or octavalent ligands by the mannose receptor. The percentage of specific internalization (X-axis) is shown. 4-
Alternatively, the octavalent ligand is preferentially internalized to the mannose receptor with respect to the divalent ligand. Thus, the optimal structure sought could be considered to include these four mannose mimetics, which are easier to synthesize than those of the eight mannose mimics. Compared to natural ligands (ligands with mannose residues), their internalization is optimal for octavalent structures, and ligands with quinic acid or shikimic acid residues are optimal for tetravalent structures It can be seen that it will be internalized.

Example 9 : Specificity of internalization of ligand according to the invention in dendritic cells mediated by mannose receptor 1) Analysis using confocal microscopy Internalization and distribution of ligand according to the invention in dendritic cells Are analyzed using confocal microscopy with mannose receptors and dual or triple labeling of cell nuclei.

Dendritic cells that differentiate from mononuclear cells in the presence of IL-4 and the growth factor GM-CSF are used at J + 5. They are incubated with 10 μg of anti-mannose human receptor (PHARMIGEN) and 5 μM of one of the following constructs at 4 ° C. for 45 minutes. 61F (Fig. 16): 4L- with 4 residues of D-mannose and a fluorescent (FITC) molecule
Dendrimer composed of lysine residues. The structure 61F is 2-thioethyl-α,
Prepared as for compound 31F except that D-mannopyranoside was reacted with compound 14 (see Example 1, paragraph 1.1) (paragraph 1.a. of Example 8).
See). 21F, 22F and 64F: (derived from galactonolactone molecule) a structure having a polyhydroxyl chain of galacto configuration, the rest of shikimic acid, and quinic acid. Structure
64F was prepared as for compound 30F (see Example 8, paragraph 1.a), but using compound 13 for coupling (Example 1, paragraph 1.1).
See). The preparation of ligands 21F and 22F is described in Example 2 above.

Construct 61F serves as a positive control, while construct 64F serves as a negative control. Structures 21F and 22F represent ligands according to the present invention.

Cells are maintained at 37 ° C for 5 minutes. Following incubation, they are fixed for 45 minutes at 4 ° C. with a solution having the following composition. : PBS (0.1 mM MgCl ₂ , 0.1 mM Ca ₂ ), 4%
Paraformaldehyde, sucrose. The fixation reaction is quenched with ammonium chloride solution (50 mM) for 10 minutes at ambient temperature. Cells were washed 3 times with PBS, then poly-L
-Attached on a slide of lysine. Fluorescence signal is shown at 37 ° C with anti-FITC rabbit IgG coupled with Alexa Fluor 488 (dilution: 1/200 ^th ) (MOLECULAR PROBES).
Amplified by incubation for 30 minutes and at the same time secondary goat antibody, Alexa Fluor 568 (dilution: 1/5
00 ^th ) (MOLECULAR PROBES) incubated with anti-mouse IgG coupled to allow detection of anti-mannose receptor. Culture with secondary antibody is PBS, BSA medium (1mg / ml)
, Saponin (0.05%).

Cells are washed with PBS containing 1 mg / ml BSA and 0.05% saponin, then PBS / BSA and finally with PBS, and placed between slides and coverslips in VECTASHIELD mounting medium. Slides are krypton / argon laser (excitation 488, 468
And 647) on a LEICA TCS NT instrument with a confocal microscope. The acquisition of two wavelengths (ie, the acquisition of the fluorescence emitted by Alexa 568 corresponding to the mannose receptor and the acquisition of the fluorescence emitted by Alexa 488 corresponding to the structure being tested) is performed simultaneously by superimposing the two images. That is, two markers made on the Z axis of the cells for the same cell slice can be observed simultaneously.

Since the fluorescently labeled ligands 61F, 21F and 22F occupy the same position as the mannose receptor, the fact that the two markers occupy the same position means that the ligand of the present invention really mediated mannose receptors. It will be noted that internalization can be stated in detail.

2) Analysis with Cos Cells Transfected with Mannose Receptor Cos-1 cells do not express the mannose receptor in its native conformation, so with plasmid CD8 containing the coding sequence for the mannose receptor (MR). Be transfected. Cells are harvested 48 hours post-transfection. : 10 to 15% of them express the transgene. These cells are hereafter referred to as "CosMR" cells.

Internalization studies are performed from these cells with the following compounds. 22F: A dendrimer composed of four residues of L-lysine, which retains four residues of quinic acid, like a fluorescent molecule (FITC). This is a ligand according to the invention. 64F: construct carrying polyhydroxylated chains in the galacto conformation (negative control) The internalization test was carried out by incubating the cells with the compound 22F or the compound 22F before the FAC scan analysis using the cytometer described in Example 8. Incubate for 20 minutes at 37 ° C. in the presence of 10 μmoles of 64F, then wash the cells with cold PBS and fix them with 1% paraformaldehyde. FIG. 17 shows the percentage of positive cells detected (on the X-axis) for the following four tests shown on the Y-axis. -Test a): Incubation of CosMR cells with compound 64F. -Test b): Coated in the presence of mannan solution (5 mg / ml) as described in Example 8 with mannan which competes with compound 22F for the mannose receptor.
Incubation of sMR cells with compound 22F. -Test c) Culture of CosMR cells with compound 22F-Test d) Culture of Cos-1 cells (untransfected) with compound 22F-According to Figure 17, untransfected Cos-1 cells (test d) of course does not result in internalization of compound 22F. The signal detected can be absorbed by noise in the background of the device. The same applies to CosMR cells cultured with compound 64F, which is non-specific for the mannose receptor (test a). Test b)
Reflects inhibition of compound 22F internalization by mannan. For construct 22F, it is indeed specifically internalized by cells transfected with the MR receptor (test c).

[Brief description of drawings]

[Figure 1]   1A and 1B provide two examples of possible branched conformations of compound A '.

2A, 2B and 2C show the chemical structures of six compounds that can be used as intermediate compounds to prepare ligands according to the present invention.

2A, 2B and 2C show the chemical structures of six compounds that can be used as intermediate compounds to prepare ligands according to the present invention.

2A, 2B and 2C show the chemical structures of six compounds that can be used as intermediate compounds to prepare ligands according to the present invention.

3 and 4 show the chemical structures of four ligands according to the invention prepared from the compounds shown in FIGS. 2A and 2B.

FIGS. 3 and 4 show the chemical structures of four ligands according to the present invention prepared from the compounds shown in FIGS. 2A and 2B.

FIG. 5 shows the chemical structures of two ligands corresponding to formula (III) according to the invention, referenced at 33F and 34F.

6 and 7 show the chemical structures of four ligands corresponding to formula (III) according to the invention, referred to as 41F, 42F, 43F and 44F.

FIGS. 6 and 7 show the chemical structures of four ligands corresponding to formula (III) according to the invention, referred to as 41F, 42F, 43F and 44F.

8 and 9 are derivatives of quinic acid (compound G), which can be used to form ligands corresponding to formula (III) according to the invention, referenced 51 and 52.
One dendrimer (Compounds H and I) and two peptide antigens (Compound J
And K) shows the chemical structure.

FIGS. 8 and 9 are derivatives of quinic acid (compound G), 2 which can be used to form ligands corresponding to formula (III) according to the invention, referenced 51 and 52.
One dendrimer (Compounds H and I) and two peptide antigens (Compound J
And K) shows the chemical structure.

10 and 11 show the chemical structures of two ligands corresponding to formula (III) according to the invention, referred to as 51 and 52.

11 and 12 show the chemical structures of two ligands corresponding to formula (III) according to the invention, referred to as 51 and 52.

FIG. 12 shows the ability of methylquinate to inhibit carrier internalization of compound 31F, a D-mannose residue, as described in Example 8 above.

FIG. 13: Dendritic cells treated with two ligands according to the invention (− ● − and − ◆ −) and two control compounds (−Δ− and − □ −) at a concentration of 1-10 μM. The average value of the fluorescence intensity of is shown in a curve format.

FIG. 14 shows that in the presence of a mannan solution having a concentration of 10 ^{−4 to} 10 mg / ml,
After preincubation of the cells, two ligands according to the invention (-●-and-◆-) and two control compounds (-Δ- and-□) at a concentration of 5 μM.
The mean value of the fluorescence intensity of dendritic cells treated with −) is shown in a curve format.

FIG. 15 shows the percentage of specific internalization of divalent, tetravalent or octavalent ligands by the mannose receptor, as described in Example 8 above.

FIG. 16 is a dendrimer containing four L-lysine residues carrying four residues of D-mannose as well as fluorescein (F) as described in Example 9 above.
6C shows the chemical structure of the ligand referred to as 61F, which is composed of molecules of ITC).

FIG. 17: Cellular Cos with various compounds as described in Example 9 above.
The percentage of positive cells detected at an incubation time of -1 is shown.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] July 30, 2001 (2001.30)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 17

[Correction method] Change

[Contents of correction]

[Chemical 3] [Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined in any one of claims 1 to 8, and W is defined in claim 9]. X represents the remainder of the X'oligopeptide containing 1 to 6 amino acids, A is at least bifunctional, and lysine, hydroxylysine, serine, threonine, cysteine. , Ornithine, aspartic acid and glutamic acid, the remainder of the compound A ′ comprising a chain of amino acids, the same or different, selected from the group formed by: -m is an integer from 1 to 32; Is an integer from 0 to 32, and -M and N are respectively between B ² and A (when n = 0) or between B ² and X (n is different from 0). , And between X and A (where n is different from 0) And a functional group selected from the following functional groups independently of each other: oxime, hydrazone, amide, ester, thioester,
Hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 18

[Correction method] Change

[Contents of correction]

18. The general formula (17) according to claim 17, wherein M, N, m, n and compounds A ′ and X ′ are as defined in any one of claims 9-12 and 14. Compounds of V).

[Procedure amendment]

[Submission date] April 9, 2002 (2002.4.9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

[Chemical 1] _{Wherein: - R 1, R 2,} R 3 and R ₄ independently _of one another, a hydrogen atom or a protecting group, - R ₅ is, -OH group, -SH group, -NH-NH ₂ group, Halogen atom, or 1 to
1 selected from 18 carbon atoms and optionally oxygen, sulfur and nitrogen
A saturated or unsaturated, straight-chain, branched or cyclic carbon chain containing from 1 to 12 atoms, said carbon chain being optionally substituted by 1 to 16 halogen atoms, and optionally at least It carries one nucleophilic group or at least one electrophilic group.

[Chemical 2] [Wherein W is a bond or a saturated or unsaturated, straight chain, branched chain or branched chain containing 1 to 18 carbon atoms and optionally 1 to 12 atoms selected from oxygen, sulfur and nitrogen. A cyclic carbon chain, the carbon atom chain being optionally substituted by 1 to 16 halogen atoms], -X is the remainder of the X'oligopeptide, comprising 1 to 6 amino acids. , -A is the balance of at least the bifunctional compound A ', -Y is the balance of the compound Y'of interest, -m is an integer from 1 to 32, and -n is , Is an integer of 0 to 32, -y is an integer of 1 to 4, and -M, N and P are each between B ¹ and A (when n = 0) or B, respectively. ¹ and (if different from n is 0) between the X, different fields will be between (n is 0 and X and a ), And a linking group between A and Y, and independently of one another, comprising a functional group selected from the following functional groups: oximes, hydrazones, amides, esters,
Thioesters, hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines.

[Chemical 3] [Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined in any one of claims 1 to 8, and W is defined in claim 9]. X represents the remainder of the X'oligopeptide containing 1 to 6 amino acids, A is at least bifunctional, and lysine, hydroxylysine, serine, threonine, cysteine. , Ornithine, aspartic acid and glutamic acid, the remainder of the compound A ′ comprising a chain of amino acids, the same or different, selected from the group formed by: -m is an integer from 1 to 32; Is an integer from 0 to 32, and -M and N are respectively between B ² and A (when n = 0) or between B ² and X (n is different from 0). , And between X and A (where n is different from 0) And a functional group selected from the following functional groups independently of each other: oxime, hydrazone, amide, ester, thioester,
Hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 37/00 A61P 43/00 111 43/00 111 C07K 14/00 ZNA C07K 14/00 ZNA G01N 33/566 G01N 33/566 33/53 S // G01N 33/53 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, 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, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, M), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Anstitue Pasteur Dolly France F-59019 Lille Cedex BP 245 Lü du Professurer. Calumet 1 (72) Inventor Grandjean Cyril France F-75013 Paris Rue Berneau 9 (72) Inventor Melny Oleg France F-59370 Mont Envalor Ruge. Peri 9 (72) Inventor Glass-Masse Helen France F-59710 Merigny Le de la Rosiele 321 (72) Inventor Angarosi Gerhild France F-59800 Lille Rue de la Porte des Presses 11 (72) Inventor Lomen Corrine France F-59120 Roth Ryu de Gesec 8 (72) Inventor Ouriolt Cloud France F-59310 Nomine Luis Guisline 60 F Term (Reference) 4C084 AA02 BA31 CA59 DC50 NA10 NA13 NA14 ZA962 ZB012 ZB152 ZB322 A0C31 CC0 4C0 AA10 AA20 AA30 BA10 BA50 EA20 EA50 FA34

Claims (25)

[Claims] 1. At least one compound corresponding to the general formula (Ia) as a ligand for a mannose receptor or any receptor of the C-lectin family related to a mannose receptor, or for the preparation of such a ligand, and Use of at least one compound corresponding to general formula (Ib): _{Wherein: - R 1, R 2,} R 3 and R ₄ independently _of one another, a hydrogen atom or a protecting group, - R ₅ is, -OH group, -SH group, -NH-NH ₂ group, Halogen atom, or 1 to
1 selected from 18 carbon atoms and optionally oxygen, sulfur and nitrogen
A saturated or unsaturated, straight-chain, branched or cyclic carbon chain containing from 1 to 12 atoms, said carbon chain being optionally substituted by 1 to 16 halogen atoms, and optionally at least It carries one nucleophilic group or at least one electrophilic group. 2. A compound of the general formula (Ia) and / or (Ib) is --OR. ₂ And -OR₃Use according to claim 1, wherein the groups are such that they are in trans relation.
for. 3. Use according to claim 1 or 2, wherein the compounds of general formula (Ia) and / or (Ib) are such that R ₅ is a hydroxyl group. 4. The compound according to any one of the preceding claims, wherein the compound of (Ib) is such that R ₁ , R ₂ and R ₃ are hydrogen atoms and R ₅ is a hydroxyl group. use. 5. The use according to claim 4, wherein the compound of formula (Ib) is (−)-shikimic acid. 6. A compound of formula (Ia), wherein R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is a hydroxyl group. The use according to item 1. 7. Use according to claim 6, wherein the compound of formula (Ia) is (−)-quinic acid. 8. A compound of formula (Ia) wherein R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is a —NH— (CH ₂ ) ₂ —SH group. The use according to claim 1 or 2. 9. A compound which can be used as a ligand for a mannose receptor or any receptor of the C-lectin family related to a mannose receptor or as an intermediate compound for preparing such a ligand, which comprises Characterized in that it corresponds to formula (III): (B ¹ M) _m (XN) _n A (PY) _y (III) where: B ¹ is one or more of the following general formula (IIa) or (IIb): [Wherein W is a bond or a saturated or unsaturated, straight chain, branched chain or branched chain containing 1 to 18 carbon atoms and optionally 1 to 12 atoms selected from oxygen, sulfur and nitrogen. A cyclic carbon chain, the carbon atom chain being optionally substituted by 1 to 16 halogen atoms], -X is the remainder of the X'oligopeptide, comprising 1 to 6 amino acids. , -A is the balance of at least the bifunctional compound A ', -Y is the balance of the compound Y'of interest, -m is an integer from 1 to 32, and -n is , Is an integer of 0 to 32, -y is an integer of 1 to 4, and -M, N and P are each between B ¹ and A (when n = 0) or B, respectively. ¹ and (if different from n is 0) between the X, different fields will be between (n is 0 and X and a ), And a linking group between A and Y, and independently of one another, comprising a functional group selected from the following functional groups: oximes, hydrazones, amides, esters,
Thioesters, hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines. 10. Compound A ′ is formed by a chain containing several balances of at least trifunctional compounds covalently linked to one another and, with respect to these balances of free functional groups, a compound of the general formula ( Compounds (1a) and / or (Ib) (1
10. A compound according to claim 9, which is suitable for providing multiple anchoring sites for compound X'or compound X'when X is present and for compound Y '. 11. A compound according to claim 10, wherein compound A'comprises the balance of 2-32, and preferably 4-16 of the trifunctional compound. 12. The compound according to any one of claims 9 to 11, wherein the compound A ′ is either in the linear form or in the branched chain form (and especially in the dendritic type). 13. Compound A ′ comprises the same or different chain of amino acids selected from the group formed by lysine, hydroxylysine, serine, threonine, cysteine, ornithine, aspartic acid and glutamic acid. The compound according to any one of 9 to 12. 14. The compound of the general formula (III), wherein m is an integer of 4 to 16,
n is an integer of 2-8, X is the rest of the oligopeptide containing amino acids of 2-4 residues, and A is the rest of the chain containing lysine residues of 2-8 and in the form of a dendrimer. 14. The compound of any one of claims 9-13, which is: 15. A compound of general formula (III), wherein B ¹ is the balance of one or more compounds selected from (−)-shikimic acid and (−) quinic acid. 15. The compound according to any one of to 14. 16. N'and antigens, antibodies, nucleic acids and fragments of nucleic acids encoding the protein of interest or one or more antigenic determinants of this protein, purified bacterial and viral fractions, medicinal actives and detection. 15. A compound according to any one of claims 9-14 selected from among possible markers. 17. A compound which can be used as an intermediate compound for preparing a ligand for a mannose receptor or any receptor of the C-lectin family related to a mannose receptor, which compound corresponds to the following general formula (V): (B ² M) _m (XN) _n A (V) where: B ² is one or more of the following general formulas (IVa) or (IVb): [Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined in any one of claims 1 to 8, and W is defined in claim 9]. X represents the rest of the X'oligopeptide containing 1 to 6 amino acids, -A represents the rest of the at least bifunctional compound A ', and -m represents 1 an integer from to 32, - n is an integer from 0 to 32, and - M and n are each, respectively, (when n = 0) B ² and between the a or B ² and the X Between (when n is different from 0) and between X and A (when n is different from 0), and independently of each other, selected from the following functional groups: Containing functional groups: oximes, hydrazones, amides, esters, thioesters,
Hydrazides, hydroxamates, ethers, thioethers, amines, carbonates, carbamates, thiocarbonates, thiocarbamates, ureas, thioureas and thiazolidines. 18. The general formula (17) according to claim 17, wherein M, N, m, n and compounds A ′ and X ′ are as defined in any one of claims 9-14.
Compounds of V). 19. The method according to claim 17, wherein B ² is the balance of one or more compounds selected from (−)-shikimic acid and (−)-quinic acid, optionally in protected form. 18. The compound according to 18. 20. A compound which can be used as an intermediate compound for preparing a ligand for a mannose receptor or any receptor of the C-lectin family related to a mannose receptor, which compound corresponds to the following general formula (VI): Characterized by: (B ¹ M) _mA (VI) where: B ¹ and M are as defined in claim 9 or claim 15, and m is an integer from 50 to 500. And -A represents the remainder of the polymer A'which can form a non-covalent complex with the compound of interest (eg, nucleic acid). 21. The compound of claim 20, wherein polymer A'is a lysine polymer or a cationic polymer such as polyethyleneimine. 22. A compound that can be used as a ligand for a mannose receptor or any receptor of the C-lectin family related to a mannose receptor, having the general formula (VI) as defined in claim 20 or claim 21. Between the compound corresponding to and the compound of interest (eg nucleic acid) in the form of a non-covalent complex. 23. Use as a ligand for the mannose receptor according to any one of claims 9-16 and 22 or any receptor of the C-lectin family related to mannose receptors for use as a medicament. Compound. 24. A method according to claims 9-16 and 22 for use in vitro.
A compound which can be used as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor according to claim 1. 25. A compound which can be used as a ligand for the mannose receptor according to any one of claims 9 to 16 and 22 or any receptor of the C-lectin family related to the mannose receptor. , Diagnostic reagents.