EP1200462A2 - Verwendung von china- und shikiminsäure und deren derivate zur herstellung von liganden des mannoserezeptors - Google Patents

Verwendung von china- und shikiminsäure und deren derivate zur herstellung von liganden des mannoserezeptors

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Publication number
EP1200462A2
EP1200462A2 EP00956573A EP00956573A EP1200462A2 EP 1200462 A2 EP1200462 A2 EP 1200462A2 EP 00956573 A EP00956573 A EP 00956573A EP 00956573 A EP00956573 A EP 00956573A EP 1200462 A2 EP1200462 A2 EP 1200462A2
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EP
European Patent Office
Prior art keywords
compound
receptor
group
ligand
mannose receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00956573A
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English (en)
French (fr)
Inventor
Cyrille Grandjean
Oleg Melnyk
Hélène Gras-Masse
Gerhild Angyalosi
Corinne Rommens
Claude Auriault
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut Pasteur
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut Pasteur
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Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut Pasteur de Lille, Institut Pasteur filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1200462A2 publication Critical patent/EP1200462A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • the present invention relates to the use of quinic, shikimic acids and their derivatives for the preparation of mannose receptor ligands, to the ligands thus prepared, as well as to the therapeutic and diagnostic applications of these ligands.
  • Dendritic cells which represent a particular line of leukocytes originating in the bone marrow and which are present in practically all the tissues of the organism, play a key role in the control of the immune responses. Indeed, these cells appear to be the only cells capable in vivo of presenting antigens to naive and mature T lymphocytes, and of inducing, by activation of these T lymphocytes, an immune response of T helper type (J. BLANCHEREAU and RM STEINMANN , Nature, 1998, 392, 245-252). Activation of T cells by 'dendritic cells involves recognition and pre internalising antigens by them.
  • the mannose receptor belongs to the family of C-lectins which represents a group of calcium-dependent glycoproteins forming non-covalent bonds with carbohydrates. If, as the name suggests, the mannose receptor binds preferentially to D-mannose, it also appears to bind to other carbohydrates such as L-fucose, D-glucose and N-acetyl-D -glucosamine which, like D-mannose, comprise two hydroxyl functions carried by two adjacent carbon atoms of the cycle which constitutes them.
  • R 5 represents a group -OH, a group -SH, a group -NH-NH 2 , a halogen atom, or a linear, branched or cyclic, saturated or unsaturated carbon chain, comprising from 1 to 18 carbon atoms and optionally from 1 to 12 atoms chosen from oxygen, sulfur and nitrogen, said carbon chain being optionally substituted with 1 to 16 halogen atoms and optionally carrying at least one nucleophilic group or at least one electrophilic group, as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, or for the preparation of such a ligand.
  • group is understood to mean
  • trimethylsilyl triethylsilyl, triisopropylsilyl, tertiobutyldimethylsilyl, tertiobutyldiphenylsilyl, trimethylsilylethyl ether, terr-butoxymethyl, methoxy-methyl, benzyloxymethyl, 2-methylethyl, methyl-methyloxy, methyl , 3'-dimethoxybutane-2 ', 3'-diyl.
  • nucleophilic group means a group having a pair of free electrons, that is to say an electron donor group, and by "electrophilic” group a group having a vacant low-energy molecular orbital capable of interacting with the pair of free electrons of a nucleophilic group.
  • electrophilic group a group having a vacant low-energy molecular orbital capable of interacting with the pair of free electrons of a nucleophilic group.
  • the nucleophilic group is preferably chosen from amino groups, thiols, ⁇ -aminothiols, alcohols, hydrazide and hydrazide derivatives, hydrazine and hydrazine derivatives, hydroxylamine and hydroxylamine derivatives
  • halogen atoms are bromine, chlorine and fluorine
  • suitable carbon chains are alkyl groups (methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, ...), alkenyl groups
  • the compounds corresponding to the general formulas (la) and (Ib) constitute mimes of D-mannose. They can be used, as such, as ligands for the D-mannose receptor, or be linked to other compounds to lead to the production of ligands in which they will act as mimics of D-mannose. Also, if it is appropriate that, in ligands such as ready to be used, the compounds of general formulas (la) and (Ib) have at least two vicinal free hydroxyl functions, so as to be able to mimic the D- mannose and allow these ligands to be recognized by the mannose receptor and to bind to the latter, it is not however necessary that these compounds initially have two free vicinal hydroxyl functions.
  • the compounds of general formulas (la) and / or (Ib) are chosen so that the groups -OR 2 and -OR 3 are in trans relationship, so as to mimic the two hydroxyls carried by the D-mannose cycle which appear to be involved in the bond between this compound and its receptor.
  • the compounds of formula (Ia) and / or (Ib) are such that R 5 represents a hydroxyl group (in which case the ring carries, in position 1, a carboxyl group).
  • the compound of formula (Ib) is such that Ri, R 2 and R 3 represent hydrogen atoms and R represents a hydroxyl group (-OH). If, in addition, the groups -ORi and -OR 2 are in the cis relationship, and the groups -OR 2 and -OR 3 are in the trans relationship, the configuration being 3R, 4S and 5R, the resulting compound consists of (-) - shikimic acid (or 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid).
  • the compound of formula (la) is such that Ri, R 2 , R 3 and R represent hydrogen atoms and R 5 represents a hydroxyl group (-OH). If, in addition, the groups -OR 2 and -OR 3 are in a tr ⁇ ns relation, while the groups -ORi and -OR are each in a cis relation with respect to the group -OR 2 , and the configuration is lsn, 3R , 4s solicitand 5R, then the resulting compound consists of (-) - quinic acid (or 1,3,4,5-tetrahydroxy-cyclohexane-carboxylic acid).
  • the (-) - shikimic and (-) - quinic acids are commercially available, for example from the companies ALDRICH or ACROS.
  • the compound of formula (la) is such that Ri, R 2 , R 3 and R represent hydrogen atoms and R 5 represents a group -NH- (CH 2 ) 2 SH. It is then a derivative of quinic acid.
  • Ri, R 2 , R 3 and R represent hydrogen atoms and R 5 represents a group -NH- (CH 2 ) 2 SH. It is then a derivative of quinic acid.
  • a subject of the present invention is also a compound useful as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, or as an intermediate compound for the preparation of such a ligand, which compound being characterized in that it corresponds to the general formula (III) below: (B'M) m (XN) n A (PY) v (ffl) in which: - B 1 corresponds to one or more general formulas (Ha) or (Ilb) below
  • W represents a bond or a linear, branched or cyclic, saturated or unsaturated carbon chain comprising from 1 to 18 carbon atoms and possibly from 1 to 12 atoms chosen from oxygen, sulfur and nitrogen, said carbon chain being optionally substituted by 1 to 16 halogen atoms,
  • X represents the remainder of an oligopeptide X ′ comprising from 1 to 6 amino acids
  • - m is an integer between 1 and 32
  • - n is an integer between 0 and 32
  • - y is an integer between 1 and 4, and
  • residues B 1 each corresponding to one of the general formulas (IIa) or (Ilb), this or these residues being intended to act as mimics of D-mannose with respect to the mannose receptor and resulting from the bond between one or more compounds corresponding to the general formula (la) and / or (Ib) previously defined and the compound A ', optionally via X and deprotection of the hydroxyl groups, in the case where the said compound (s) of general formula (la) and / or (Ib) initially has protected hydroxyl groups;
  • residue (s) X of a compound X ' this or these residue X playing the role, when they are present, of a spacer arm between B 1 and A, so as to control the distance between the different residues B 1 , when several of them are present in the compound of general formula (III) (that is to say when m is greater than 1).
  • residues X of oligopeptide nature have been described, it would not be departing from the scope of the present invention to use, as compound X ′, compounds of different nature, provided that they are capable of playing the role of arms spacer between B 1 and A.
  • the term “compound of interest” is intended to mean a compound whose internalization is sought to promote by cells via the mannose receptor or a receptor of the C-lectin family related to the latter (cells dendritics, cells of monocyte-macrophage lines, ).
  • the compound of interest can be linked to the remainder A, at a rate of 1 to 4 molecules of compound of interest per molecule of compound of general formula (III), by linking groups P each resulting from the reaction between two functional groups carried, for the first, by said compound of interest and for the second, by compound A '.
  • a ligand is obtained which is able, by the presence of the residue (s) B 1 , to bind specifically to the mannose receptor or to any receptor related to the latter and, by its bond with the compound of interest, to promote the internalization of the latter by cells expressing this type of receptor.
  • the compound A ′ is formed of a chain comprising several remains of an at least trifunctional compound, linked to each other by covalent bonds, and suitable for providing, at the level of the functional groups free from these residues (that is to say from functional groups which are not engaged in a covalent bond), several anchoring sites for the compound or compounds of general formula (la) and / or (Ib) or, when X is present, compound X ', and for compound Y'.
  • compound A ′ comprises from 2 to 32 and, preferably, from 4 to 16 residues of a trifunctional compound.
  • the compound A ′ can be presented either in a linear form or in a branched form and, in particular, of tree type (called "dendrimeric").
  • Examples of branched structures which are particularly well suited to the preparation of a mannose receptor ligand according to the invention are illustrated in FIGS. 1A and IB which very schematically represent a compound A 'in the form of two different dendrimers .
  • the compound A ′ comprises a chain of amino acids, identical or different, selected from the group consisting of lysine, hydroxylysine, serine, threonine, cysteine, romithine, aspartic acid and glutamic acid.
  • This amino acid can be both of the L series and of the D series and the compound A ′ can even comprise both residues of the L series and residues of the D series.
  • a compound A ′ based on lysine is preferred, the use of lysine being in fact particularly well suited to the production of a mannose receptor ligand according to the invention, insofar as it has been shown that macromolecules based on lysine are devoid of intrinsic immunogenicity (TAM, Proc. Natl. Acad. Sel. USA, 1988, 85, 540).
  • TAM intrinsic immunogenicity
  • the compound A ′ may also comprise one or more residues corresponding to one or more different compounds from the at least trifunctional compound which constitutes the basic element thereof.
  • This or these remains are in principle not intended to provide any connection between the various elements making up the ligand.
  • their presence is either intended to confer on this ligand additional characteristics such as one or more additional possibilities of coupling (for example, with a view to the subsequent production of a dimer of this ligand), or linked to the need to use, during its preparation, additional compounds, such as spacer arms.
  • these compounds, while being different from the at least trifunctional compound constituting the basic element of compound A ′ will preferably be of the same nature as the latter.
  • they will preferably be amino acids in the case where the compound A ′ comprises an amino acid.
  • n which represents the number of X residues present in the compound of general formula (III), can be:
  • linking groups M, N and P they are chosen, independently of one another, from the linking groups which comprise a function chosen from the oxime, hydrazone, amide, ester, thioester, hydrazide, hydroxamate, ether, thioether, amino, carbonate, carbamate, thiocarbonate, thiocarbamate, urea, thiouree and thiazolidine.
  • a function chosen from the oxime, hydrazone, amide, ester, thioester, hydrazide, hydroxamate, ether, thioether, amino, carbonate, carbamate, thiocarbonate, thiocarbamate, urea, thiouree and thiazolidine.
  • an oxime bond can be formed by reaction of an O-alkylamine group with a carbonyl group
  • a hydrazone bond can be formed by reaction of a hydrazine group with a carbonyl group
  • an amide bond (respectively, ester) can be formed by reaction of an amino group (respectively, alcohol) with an activated acid group or anhydride or acid halide
  • a thioester bond can be formed by reaction of a group thiol with an activated acid group or anhydride or acid halide
  • a hydrazide bond can be formed by reaction of a hydrazine group with an activated acid group, anhydride or acid halide,
  • an ether bond can be formed by reaction of an alcohol group with an alkyl halide group
  • a thioether bond can be formed by reaction of a thiol with an alkyl halide group
  • an amino bond can be formed by reaction between an amino group and an alkyl halide
  • a carbonate bond can be formed by reaction of a chloroformate group with an alcohol group
  • - a carbamate bond can be formed by reaction of a chloroformate group with an amino group
  • a thiocarbonate bond can be formed by reaction of a chlorothioformate group with an alcohol group
  • a thiocarbamate bond can be formed by reaction of a chlorothioformate group with an amino group
  • - a urea bond can be formed by reaction of an isocyanate group with an amino group
  • - a thiourean bond can be formed by reaction of an isothiocyanate group with an amino group
  • a thiazolidine bond can be formed by reaction of a ⁇ -aminothiol group with a carbonyl group
  • a hydroxamate bond can be formed by reaction between an activated acid group and a hydroxylamine group. It will therefore be understood that, in the general formula (III), B 1 , A and
  • X represent the residues of compounds naturally carrying functional groups capable of leading, by reaction to each other, to obtain the linking groups M, N and P as defined above, or of compounds having been modified from so as to carry such functional groups.
  • the compound corresponding to the general formula (III) can be prepared:
  • the compound of general formula (III) can be obtained by first coupling the compound or compounds corresponding to the general formula (la) and / or (Ib) with the compound X ', then by coupling the compound resulting from this first coupling with compound A ', and finally by coupling the compound resulting from this second coupling with compound Y', this latter coupling being optionally followed by deprotection of the hydroxyl groups carried by the residue (s) B 1 , in the case where the compound or compounds of formula (la) and / or (Ib) carry such groups.
  • the compound of general formula (III) can be obtained by coupling a compound resulting from the coupling between one or more compounds corresponding to the general formula (la) and / or (Ib) and the compound X ', with a resulting compound , for its part, of the coupling between the compound A 'and the compound Y', this latter coupling being able, here again, to be followed by deprotection as mentioned above.
  • the compounds Y ', A' and X ' are constituted by amino acids. So the compounds Y ', A' and X 'can be formed and assembled with each other by successive addition of the amino acids which compose them, for example according to the peptide synthesis techniques on solid support derived from the method initially described by MERRTPIELD (The Chemistry of Solid Phase Peptide Synthesis, STEWART and YOUNG Ed., 1984).
  • the compound corresponds to the general formula (III) in which m is an integer between 4 and 16, n is an integer between 2 and 8, X represents the remainder of a oligopeptide comprising from 2 to 4 amino acid residues, while A represents the remainder of a chain comprising from 2 to 8 lysine residues and which is in the form of a dendrimer.
  • the compound of general formula (III) is such that B 1 represents the remainder of one or more compounds chosen from (-) - shikimic acid and (-) - acid quinic.
  • the compound of interest Y ′ may be an antigen and, in particular, a vaccine antigen such as a synthetic peptide corresponding to one or more epitopes of a protein of a pathogenic agent.
  • a protein or polysaccharide subunit antigen a purified bacterial or viral fraction, an antibody such as an immunoglobulin or an antilymphocyte serum, a nucleic acid or a fragment of a nucleic acid (RNA or DNA sequence) coding for a protein of interest or for one or more antigenic determinants of this protein, a medicinal active principle such as an immunostimulant, an immunosupressant, a cytostatic or an antibiotic, and may, as such, be as well a substance of protein, peptide, osidic or polysaccharide, lipid, lipoproteinic, lipopolyosidic, polynucleotide nature, than a compound derived from chemistry.
  • the compound of interest can also be a detectable marker capable of allowing revelation of the presence of the ligand in the medium in which it is found, such as a radioisotope, a fluorochrome or an enzyme.
  • the compound of interest Y ' is consisting of an association of several substances of different nature such as, for example, an antigen or an antibody linked to a detectable marker.
  • the present invention also relates to a compound useful as an intermediate compound for the preparation of a ligand of the mannose receptor or of any receptor of the C-lectin family related to the mannose receptor, which compound is characterized in that '' it responds to the general formula (V) below:
  • X represents the remainder of an oligopeptide X ′ comprising from 1 to 6 amino acids
  • - m is an integer between 1 and 32
  • - n is an integer between 0 and 32
  • the residue (s) B 2 result from the coupling of one or more compounds corresponding to the general formula (la) and or (Ib) with the compound A ', optionally via X'.
  • M, N, m, n, as well as compounds A 'and X', are as described in relation to the general formula (III).
  • the compound of general formula (V) can be, like the compound corresponding to general formula (III), prepared by a convergent synthesis, by a recurrent synthesis, or also by a synthesis for partly convergent and for partly recurring.
  • the compound of general formula (V) is such that B 2 represents a residue of one or more compounds chosen from (-) - shikimic acid and (-) - quinic acid , possibly in protected form.
  • the present invention also relates to a compound useful as an intermediate compound for the preparation of a mannose receptor ligand or of any receptor of the C-lectin family related to the mannose receptor, which compound is characterized in that '' it meets the general formula (VI) below:
  • - m is an integer between 50 and 500
  • - A represents the remainder of a polymer A 'capable of forming a non-covalent complex with a compound of interest, such as a nucleic acid.
  • polymer means a set of molecules of variable molecular weight, resulting from the chain of several monomers.
  • This polymer is chosen from compounds capable of giving rise, on contact with a compound of interest, to the formation of a non-covalent complex with this compound, for example by means of ionic bonds, of dipolar bonds or electrostatic interactions, the complex thus obtained then being capable of being used as a ligand for the mannose receptor or any receptor of the C-lectin family related to the latter.
  • the polymer A ' is a cationic polymer.
  • suitable cationic polymers are lysine polymers such as those sold by the company Sigma under the references P0296, P6403, P4408, P7886, P0899, PI 149 or also P0124, and polyethyleneimines, such as that sold by the company FERMENTAS under the reference EXGEN 500 as a cell transfection reagent.
  • the present invention also relates to a compound useful as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, which compound is characterized in that it is in the form of 'a non-covalent complex between a compound corresponding to the general formula (VI) as defined above and a compound of interest, such as a nucleic acid.
  • the ligands in accordance with the invention are of great interest. Indeed, while being able to be recognized very satisfactorily by the mannose receptor and to bind specifically with it, they have the advantage of being able to be easily prepared and at costs compatible with industrial exploitation, in particular by numerous peptide synthesis techniques on solid support.
  • ligands in the form of non-covalent complexes will be used between a compound of general formula (VI) and a polynucleotide sequence, and
  • Another subject of the present invention is the use of at least one compound corresponding to the general formula (la) and / or at least one compound corresponding to the general formula (Ib) for the preparation of a medicament capable of bind to the mannose receptor or to any receptor in the C-lectin family related to the mannose receptor.
  • the present invention also relates to a compound which is useful as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, as defined above, for use as a medicament.
  • a drug is likely to find in the first place applications in all the therapeutic indications in which one seeks an induction, a modulation or a suppression of the immune responses and, more particularly, of those which are under the control of T cells.
  • This drug is also capable of being used for the vectorization of an active non-immunomodulatory principle towards target cells expressing a receptor of the C-lectin family.
  • the compounds useful as ligands in accordance with the invention can find applications in antibiotic therapy, for the vectorization of antibiotics such as fluoroquinones or colistin towards cells of the lines of monocytes-macrophages infected with bacteria. or by mycobacteria, or in the treatment of diseases related to a cellular enzyme deficiency such as GAUCHER's disease, in which case they will be used to vectorize the deficient enzyme to the KUPFER cells.
  • the present invention also relates to a compound useful as a ligand for the mannose receptor or any receptor of the C-lectin family related to the mannose receptor, as defined above, for use in vitro.
  • the present invention further relates to a diagnostic reagent, characterized in that it comprises a compound which is useful as a ligand for the mannose receptor or for any receptor of the C-lectin family related to the mannose receptor, as previously defined.
  • the present invention also comprises other provisions which will emerge from the additional description which follows, which relates to examples of the preparation of compounds useful as intermediate compounds for the preparation of conforming mannose receptor ligands to the invention, mannose receptor ligands in accordance with the invention and for demonstrating the biological properties of these ligands, as well as to the appended drawings in which:
  • FIG. 1A and IB illustrate two examples of branched conformations likely to be presented by the compound A ';
  • - Figures 2A, 2B and 2C illustrate the chemical structures of six compounds useful as intermediate compounds for the preparation of ligands according to the invention;
  • Figures 3 and 4 illustrate the chemical structures of 4 ligands according to the invention prepared from the compounds shown in Figures 2 A and 2B;
  • FIG. 5 illustrates the chemical structures of two ligands corresponding to formula (III) according to the invention, called 33F and 34F;
  • FIG. 6 and 7 illustrate the chemical structures of four ligands corresponding to formula (III) according to the invention, called 41F, 42F, 43F and 44F;
  • FIGS. 8 and 9 illustrate the chemical structures of a quinic acid derivative (compound G), two dendrimers (compounds H and I) and two peptide antigens (compounds J and K) which can be used to form ligands corresponding to formula (III) according to the invention, called 51 and 52;
  • FIGS. 10 and 11 illustrate the chemical structures of two ligands corresponding to formula (III) according to the invention, called 51 and 52;
  • - Figure 12 illustrates the ability of methyl quinate to inhibit the internalization of compound 31F, carrying residues of D-mannose, as described in Example 8 below;
  • FIG. 13 illustrates, in the form of curves, the average values of the fluorescence intensities presented by dendritic cells treated with 2 ligands in accordance with the invention (- • - and - * -) and with 2 control compounds (- ⁇ - and - ⁇ -) at concentrations between 1 and 10 ⁇ M;
  • FIG. 14 illustrates, in the form of curves, the average values of the fluorescence intensities presented by dendritic cells treated with 2 ligands in accordance with the invention (- • - and - • * -) and with 2 control compounds (- ⁇ - and -D-) at a concentration of 5 ⁇ M after pre-incubation of said cells in the presence of mannan solutions of concentrations between 10 ⁇ and 10 mg / ml;
  • FIG. 15 illustrates the percentage of specific internalization of di-, tetra- or octavalent ligands by the mannose receptor, as described in Example 8 below;
  • - Figure 16 illustrates the chemical structure of a ligand called 61F, consisting of a dendrimer comprising 4 L-lysine residues carrying 4 residues of D-mannose, as well as a fluorescein molecule (FITC), as described in Example 9 below;
  • - Figure 17 illustrates the percentage of positive cells detected during the incubation of Cos-1 cells with different compounds, as described in Example 9 below.
  • DCM dichloromethane
  • DIEA diisopropylethylamine
  • PBS phosphate buffer
  • NMR nuclear magnetic resonance RP- ⁇ PLC: high performance liquid chromatography in reverse phase
  • TNBS 2,4,6-trinitrobenzene sulfonic acid
  • TOF-PDMS time of flight spectrometry and plasma desorption
  • Trt trityle
  • TOF-PDMS spectra were recorded using an APPLIED BIOSYSTEMS Bio-Ion 20 plasma desorption mass spectrometer;
  • m represents an integer equal to 4, 8 or 16,
  • B 2 represents an acid (-) - shikimic or (-) - quinic residue
  • n is an integer equal to 2, 4 or 8 (in the case where m is respectively 4, 8 and 16)
  • A represents the remainder of a dendrimer comprising 2, 4 or 8 (in the case where m is equal respectively to 4, 8 and 16) L-lysine residues and having, on the one hand, 2, 4 or 8 chloroacetyl groups for its bond with an equivalent number of tripeptides and, on the other hand, a free amine group specific to allow its connection with a molecule of interest, • M represents an amide group (-CO-NH-), while N represents a thioether group (-CH 2 -S-CH 2 -CO-), have been prepared.
  • the second coupling - corresponding in fact to the first coupling of an L-lysine - was carried out using 4 equivalents (per amine function to react) of Boc-L-Lys (2-chlorobenzyloxycarbonyl) -OH, that is to say -display an L-lysine whose side chain is protected by a 2-chlorobenzyloxycarbonyl group, in the presence of an HBTU / HOBt DIEA mixture (4: 4: 12 eq. per amine function to react) in DMF (duration reaction time: 45 minutes).
  • the third coupling was carried out using 4 equivalents of Boc-L-Lys (Boc) -OH, in the presence of an HBTU / HOBt / DIEA mixture (4: 4: 12 eq. Per amine function to react) in DMF (reaction time: 45 minutes).
  • a third of the resin was deprotected, then acylated with 8 equivalents of preformed chloroacetic anhydride (via activation by DCC), to obtain a dendrimer (referred to as compound 13 in the following) comprising 2 L-lysine residues and having, on the one hand, a free amine group (which is none other than the ⁇ -amine group of the first L-lysine having been coupled to ⁇ -alanine) and, on the other hand, 2 chloroacetyl groups.
  • compound 14 (referred to as compound 14 below) comprising 4 L-lysine residues and having both a free amine group and 4 chloroacetyl groups.
  • the second half of the remaining resin was subjected to a fifth coupling with 4 equivalents of Boc-L-Lys (Boc) -OH, as previously described.
  • the deprotection and acylation of the resin with chloroacetic anhydride were repeated once again to obtain a dendrimer with 8 L-lysine residues (hereinafter called compound 15) having a free amine group and 8 chloroacetyl groups .
  • the HBTU (1 eq. Relative to the amino acid to be coupled), after dissolution in DMF, was added to a solution containing the amino acid to be coupled (0, 25 eq. Relative to the -NH functions for ⁇ -alanine, and 4 eq.
  • HOBt (1 eq. Relative to , amino acid)
  • DIEA 3 eq. with respect to the amino acid
  • the resin was successively filtered, washed 3 times with DMF (3 x 2 minutes) and DCM (3 2 minutes), treated with a TFA DCM mixture (50:50, 1 x 2 minutes, 1 x 20 minutes) suitable for cleavage of the Boc protective groups, washed again with DCM (2 x 2 minutes), neutralized with a DIEA / DCM mixture (5:95; 3 x 1 minute) and washed one last time with DCM (2 x 1 minute).
  • the cleavage of the dendrimers and their deprotection were obtained by reacting the resin with 11 ml of an HF / anisole mixture (10: 1) per gram of resin, at 0 ° C and for 60 minutes. Following which, they were successively precipitated in cold tert-butyl methyl ether, centrifuged, dissolved in water, lyophilized and purified by a semi-preparative RP-HPLC [gradient (AB): 100: 0 to 50: 50 in 120 minutes].
  • the tripeptides carrying the (-) - shikimic and (-) - quinic residues were prepared on a solid support, by first synthesizing the peptide of sequence (SI) and by secondly coupling a molecule of this tripeptide with 2 molecules of (-) - shikimic acid or 2 molecules derived from (-) - quinic acid, by formation of a bond between the amino groups of the N-terminal L-lysine of said tripeptide and the carboxyl group of said acids.
  • Compound 1 was prepared on a 2 mmol scale using an Fmoc-glycine p-benzyloxybenzyl resin (available under the trade name Fmoc-Gly-O-Wang from the company NOVABIOCHEM) loaded at 0.8 mmoles / g, and using:
  • L-cysteine was produced using 2 equivalents (per amine group to react) of
  • the cleavage of compound 1 was carried out by treating the resin with 25 ml of a TFA / H 2 O / Me 2 S mixture (95: 2.5: 2.5) at room temperature for 2 hours. The solution was concentrated under reduced pressure and the residue thus obtained was dissolved in water, then subjected to lyophilization leading to 1.64 g of a pale yellow powder. 107 mg of this powder were purified by a semi-preparative RP-HPLC [gradient (AB): 100: 0 to 50:50 in 110 minutes], which made it possible to obtain, after lyophilization, 15 mg of compound 1 pure, ie a yield of 14%.
  • Compound 1 has the following characteristics:
  • the coupling constants between the 3-H / 4-H and 4-H / 5-H protons of the two (-) - shikimic acid residues present in compound 1 as determined by N NMR spectrometry are 4.6 and 9.2 Hz. These constants are compatible with a pseudo-equatorial orientation of the hydroxyl functions carried by the carbon atoms located in positions 4 and 5 of the said acids and confirm the analogy of structure existing between (-) - shikimic acid. and D-mannose.
  • the cleavage of compound 3 was obtained by treating the resin with 20 ml of a CH 2 h / TFA PrsSiH (95: 2: 3) mixture (4 ⁇ 5 minutes). The solution was concentrated under reduced pressure and the residue thus obtained was dissolved in water, then subjected to lyophilization leading to the production of 1.40 g of a crude compound which has been dissolved in 25 ml of methanol. To the solution thus obtained, 1 M methanolic sodium methoxide was added dropwise with stirring until a pH of approximately 9 was obtained, then this mixture was allowed to react for 3 hours, by monitoring the course of the reaction by RP-HPLC. The mixture was then concentrated under reduced pressure.
  • a 100 mg sample of the residue was purified by semi-preparative RP-HPLC [gradient (A / B): 100: 0 to 90:10 in 10 minutes, then 90:10 to 50:50 in 70 minutes] to lead to the obtaining, after lyophilization, of 41 mg of pure compound 3, ie a yield of 59%.
  • Compound 3 has the following characteristics:
  • the couplings of compounds 13, 14 and 15 and of compounds 1 and 3 were carried out according to a procedure comprising 2 steps, namely: • a first step aimed at reducing the disulfide bond (S-SBu ') that each of the compounds 1 has and 3, by treating these compounds with tri-n-butylphosphine in a degassed n-propanol / water mixture (50:50), so as to obtain the elimination of the protective tert-butylthiol group carried by each of compounds 1 and 3 and, therefore, deprotection of their thiol function, and • a second step aimed at reacting compounds 1 and 3 thus reduced with compounds 13, 14 and 15, in a DMF / degassed water mixture (90:10) and in presence of potassium carbonate, so as to obtain the formation of a thioether bond between the thiol function of compounds 1 and 3 and one of the chloroacetyl functions of compounds 13, 14 and 15.
  • a first step aimed at reducing the disulfide bond
  • ESI-MS spectrum measured: 6382.0, calculated: 6383.0, m / z 1594.5 (M-4H) 4 " , 1275.5 (M- 5H) 5 ⁇ 1062.8 (M-6H) 6 ⁇ 910.8 (M-7H) 7 ' .
  • Compound 26 ESI-MS spectrum: measured: 6671.0, calculated: 6671.2, m / z 2221.9 (M-3H) 3 " , 1666.7 (M-
  • ligands 21F, 22F, 23F and 24F which will be referred to as ligands 21F, 22F, 23F and 24F in the following - and corresponding to the general formula (III) in which: "m represents an integer equal to 4 or 8,
  • B 1 represents an acid (-) - shikimic or (-) - quinic residue
  • N is an integer equal to 2 or 4 (in the case where m is respectively equal to 4 and
  • X represents the remainder of a tripeptide of sequence (SI) as defined in example 1,
  • A represents the remainder of a dendrimer A 'comprising 2 or 4 (in the case where m is respectively equal to 4 and 8) L-lysine residues and having, on the one hand, 2 or 4 chloroacetyl groups for its bond with a equivalent number of tripeptides and, on the other hand, a free amine group capable of allowing its bond with Y, • Y represents the remainder of a fluorescein molecule,
  • M represents an amide group (-CO-NH-), N represents a thioether group
  • reaction mixtures were then left in the dark for 48 hours, the reactions being controlled by RP-HPLC. Then, each reaction mixture was diluted in water containing 10% acetic acid and lyophilized and the residues were purified by semi-preparative RP-HPLC.
  • ligand 23F measured: 3769.0, calculated: 3769.1, m / z 1254.9 (M-3H) 3 * , 941.2 (M- 4H) 4 " • ligand 24F: measured: 3624.0, calculated: 3624.1, m / z 1207.0 (M- 3H) 3 " , 905.4 (M- 4H) 4 ⁇
  • B 1 represents an acid (-) - shikimic or (-) - quinic residue
  • n is an integer equal to 0 (so that X is absent)
  • A represents the remainder of a linear sequence A 'comprising 3 L-lysine residues and having 3 free amino groups for its coupling with the three molecules of (-) - shikimic or (-) - quinic acid and being, of on the other hand, linked to Y via an amide bond,
  • Y represents a compound of interest formed by three Arg-Gly-Arg residues linked to a class II epitope, in this case the chicken ovalbumin peptide 323-339 (which will be called Ova 323-339 in what follows)
  • the group formed by the 3 Arg-Gly-Arg residues located upstream of the sequence of said Ova 323-339 peptide represents a group sensitive to endopeptidases
  • ligand 25 and ligand 28 These ligands will be called ligand 25 and ligand 28 respectively in the following.
  • the peptide of sequence (S2) was synthesized on a 0.25 mmol scale from 337 mg of an MBHA Rink amide resin loaded at 0.74 mmol / g (Company SENN CHEMICALS), and using:
  • the couplings of the first 20 amino acids were performed automatically using an APPLIED BIOSYSTEMS ABI431 synthesizer, while the couplings of L-lysines and S- (tert-butylthio) -L-cysteine were performed manually. In all cases, these couplings were carried out using
  • the cleavage of the resulting products and their deprotection were carried out by treating the resin with 10 ml of a TFA / H 2 O / 'Pr 3 SiH mixture (95: 2.5: 2.5) per gram of resin, at temperature room and for 90 minutes. Following which, they were successively precipitated in cold diethyl ether, centrifuged, dissolved in water, lyophilized and purified by a semi-preparative RP-HPLC [gradient (A / B): 100: 0 to 50: 50 in 120 minutes].
  • This compound 26 was subjected to a deacylation to obtain the ligand 28.
  • To do this to 23 mg of the compound 28 in solution in 10 ml of methanol distilled over magnesium, were added 300 ⁇ l of a 1M solution of sodium methanolate in methanol. The reaction mixture was stirred at room temperature, under nitrogen, for 45 minutes and the reaction was stopped by the addition of a few drops of acetic acid. The reaction crude was concentrated under reduced pressure and was purified by RP-HPLC using a gradient (AB): 100: 0 to 80: 20 in 30 minutes, then 80: 20 to 70: 30 in 25 minutes, then isocratic for 10 minutes to lead to 15 mg of ligand 28 (Yield: 75%).
  • AB a gradient
  • ESI-MS spectra of ligands 25 and 28 are the following:
  • B 1 represents an acid (-) - quinic (ligand 33F) or (-) - shikimic (ligand 34F) residue
  • A represents the remainder of a dipeptide comprising 1 L-lysine residue and 1 ⁇ -alanine residue (N ⁇ - (chloroacetyl) -L-lysyl- ⁇ -alanine-amide, the chemical structure of which is shown in FIG. 5) ,
  • X represents a tripeptide of formula (SI), as defined in example 1, • Y represents the remainder of a molecule of fluorescein,
  • M, N and P respectively represent an amide group (-CO-NH-), thioether (-CH 2 -S-CH 2 -CO-) and thiourea (-NH-CS-NH-), have been prepared.
  • This dipeptide is synthesized manually using a Rink amide-AM-PS resin (0.70 mmol / g, Senn Chemicals) using an ocmocitert-butyl strategy, on a scale of 0.7 mmol.
  • the coupling is followed by a TNBS test: HBTU dissolved in NMP (4 eq.), Is added to a mixture of the amino acid (4 eq.), HOBt (4 eq.) And DIEA ( 8 eq.) In the NMP. After 1 minute of stirring, the reaction mixture is added to the peptidyl-resin (1 eq.) Previously soaked in NMP comprising DIEA (4 eq.), And stirred for 40 minutes.
  • the peptidyl-resin (that is to say the peptide sequence on a solid support) is filtered, washed with NMP (3 x 2 minutes) and CH 2 C1 2 (3 x 2 minutes).
  • the protective groups Fmoc are removed by treatment with 20% piperidine in NMP (1 x 2 minutes, 1 x 20 minutes), followed by washing with NMP (2 x 2 minutes) and CH 2 C1 2 (2 x 1 minutes).
  • the peptidyl-resin is deprotected and acylated using an excess (4 eq.) Of chloroacetic anhydride, prepared via DIC activation.
  • the dipeptide is separated from the support and deprotected under the action of a TFA-TIS-H 2 O 90: 5: 5 mixture (11 ml per gram of peptidyl-resin), for 1.5 h at room temperature, precipitated in a cold diethyl ether-heptane mixture (1: 1), centrifuged, dissolved in water and lyophilized.
  • the dipeptide (184 mg, 65%) is obtained in the form of a white powder after purification by RP-HPLC (with the following gradient: 100: 0 to 90:10 (A / B) in 20 minutes), followed by 'a freeze-drying.
  • ligand 33F 5.04 mg (80% yield) of ligand 33F were obtained in the form of a white powder, after purification by RP-HPLC (gradient: 100: 0 to 85:15 (A / C) in 15 minutes, then 85:15 to 75:25 (A / C) in 30 minutes, then isocratic gradient), followed by lyophilization.
  • ligand 34F 2.42 mg (80% yield) of ligand 34F were obtained in the form of a white powder, after purification by RP-HPLC (gradient: 100: 0 to 85:15 (A / C) in 15 minutes, then 85:15 to 70:30 (A / C) in 50 minutes), followed by lyophilization.
  • B 1 represents an (-) - shikimic or (-) - quinic residue
  • Y represents the remainder of a fluorescein molecule
  • A represents the remainder of a dendrimer
  • X represents the remainder of a tripeptide of sequence (S2) or (S3), as they will be defined below, • M, N and P respectively represent an amide group (-CO-NH-), thioether (- CH -S-CH 2 -CO-) and thiourea (-NH-CS-NH-), were prepared according to the following protocol. a) Preparation of the compound O. derived from quinic acid in which the hvdroxyls located in positions 3 and 4 are protected in the form of a diacetal ( Figure 6).
  • Compound Q i.e. acid (ls n , 3R, 4s n , 5R, 2'S, 3'S) -3.4-0- (2 ', 3' -dimethoxybutane-2 ', 3' -diyl) - 1 , 3, 4,5 -tetrahydroxycyclohexane- 1 -carboxylic, is obtained from 980 mg (3.06 mmol) of the methyl ester of the acid 3,4-0- (2 ', 3'- dimethoxybutane-2', 3 '-diyl) - 1, 3, 4,5 -tetrahydroxycyclohexane- 1 -carboxylic (described by JL MONTCHAMP et al.
  • the tripeptides (S2) and (S3) are respectively neutral and positively charged in physiological medium, in contrast to the peptide (SI) described in Example 1, which is negatively charged in physiological medium.
  • each peptidyl resin i.e. the peptide sequence on solid support
  • 0.2 mmol of sequence (S2) on the one hand
  • 0.2 mmol of sequence ( S3) on the other hand
  • BOP 177 mg, 0.4 mmol, 2 eq.
  • DIEA 140 ⁇ l , 0.8 mmol, 4 eq.
  • each peptidyl-resin is placed in the presence of 61 mg (0.44 mmol, 2.2 eq.) Of shikimic acid, using activation by HBTU-HOBt-DIEA [(166 mg , 0.44 mmol, 2.2 eq.), (67 mg, 0.44 mmol, 2.2 eq.), (146 ml, 0.84 mmol, 4.2 eq.)] In the NMP, during 40 minutes at room temperature. The peptidyl-resin is then washed with DMF (2 x 2 minutes) and CH 2 CI 2 (3 x 1 minute). The coupling is carried out a second time.
  • the peptidyl-resins are then washed with Et 2 O.
  • the peptides are separated from the solid support by means of a treatment with a TFA-H 2 O-iPr 3 SiH 95: 2.5: 2.5 mixture (10 ml per gram of peptidyl-resin), for 1 h at room temperature, precipitated in a diethyl ether mixture - cold heptane, centrifuged, dissolved in water, then lyophilized.
  • the peptides (S2) q, (S2) s, (S3) q and (S3s) are obtained, which correspond respectively to the tripeptides (S2) and (S3) carrying two residues of quinic acid (peptides (S2) q and ( S3) q) or two shikimic acid residues (peptides (S2) s and (S3) s), as shown in Figures 6 and 7. • Peptide (S2) q
  • 35.6 mg (24% yield) of this product are obtained in the form of a white powder, after purification by RP-HPLC [gradient: 100: 0 to 72:28 (A / B) for 20 minutes, then 72: 28 at 62:38 (A / B) for 30 minutes] followed by lyophilization.
  • 48 mg (26% yield) of this product are obtained in the form of a white powder, after purification by RP-HPLC [gradient: 100: 0 to 90:10 (A / B) for 10 minutes, then 90: 10 at 85:15 (AB) for 12 minutes, then isocratic gradient] followed by lyophilization.
  • the medium The reaction mixture is stirred for 24 h at room temperature, the progress of the reaction being followed by RP-HPLC. Fluorescein isothiocyanate (4 eq.) Is added and the medium is stirred under nitrogen and protected from light overnight.
  • the medium is diluted in water, lyophilized and purified by RP-HPLC [gradient: 100: 0 to 85:15 (A / B) for 15 minutes, then 85:15 to 80:20 (A / B) for 15 minutes, then isocratic gradient] to give 2.38 mg (72% yield) of ligand 41F in the form of a white powder.
  • the mixture is added to the reaction medium containing the peptide compounds.
  • the pH of the mixture is adjusted to 8-8.5 by adding solid K 2 CO 3 .
  • the mixture is stirred under nitrogen and protected from light for 24 h, at room temperature, diluted in water, lyophilized and purified by RP-HPLC to give the ligands 42F, 43F or 44F.
  • ligand 42F are obtained in the form of a white powder, after purification by RP-HPLC HPLC [gradient: 100: 0 to 82:18 (A / B) for 18 minutes, then 82:18 to 72:28 (A / B) for 30 minutes], followed by lyophilization. His analysis is as follows: ESI-MS found 3620.0, calculated 3621.0; m / z 1207.7 (M + 3H) 3+ , 1001.8 (M + 3H-C 25 H 38 N 5 On) 3+ , 905.9 (M + 4H) 4+ .
  • ligand 44F are obtained in the form of a white powder, after purification by RP-HPLC HPLC [gradient: 100: 0 to 82:18 (A / B) for 18 minutes, then 82:18 to 75:25 (A / B) for 21 minutes, then isocratic gradient], followed by lyophilization. His analysis is as follows: ESI-MS found 4016, calculated 4017.6; m / z 1339.9 (M + 3H) 3+ , 1005.2 (M + 4H) 4+ , 804.4 (M + 5H) 5 * .
  • B 1 represents an acid (-) - quinic residue
  • Y represents the remainder of an antigen
  • A represents the remainder of a dendrimer A 'comprising 8 (in the case of ligand 51 represented in FIG. 10) or 4 (in the case of ligand 52 represented in FIG. 11) L-lysine residues,
  • ligands are obtained by ligation of derivatives of quinic acid, namely quinic acid functionalized by a thiol function (compound G), to a lysine dendrimer (dendrimer H or I) and to a peptide antigen (J antigen or K), according to the protocol described below.
  • a) Preparation of the quinic acid derivative (compound G. Figure 8).
  • Compound G represents 2- ⁇ N, N-di - [(l-> n, 3-K, 4.s n , 5.r?) - 1,3,4,5-tetrahydroxycyclohexane-l- disulfide carboxamido-l-yl] ethyl ⁇ .
  • the reaction medium is stirred for 1 h, then added to 0.1 mmol of an amino-PEGA resin (0.4 mmol / g) soaked in a minimum amount of DMF, the resin having, beforehand, been neutralized by 5% DIEA in the CH 2 C1 2 (2 x 1 minute) and DMF (3 x 1 minute).
  • BOP 177 mg, 4 eq. Is then added to DMF (1 ml) and the mixture obtained is stirred at room temperature for 40 minutes.
  • the resin is washed with DMF (4 x 2 minutes) and CH 2 C1 2 (2 x 2 minutes). It is soaked in DMF and acylated with 1,3-diaminopropane (0.65 ml) at room temperature for 1 h.
  • Fmoc- ⁇ -Ala-OH is first coupled to the resin.
  • the lysine residues are then introduced: the first lysine introduced is protected by a Boc group, the other lysines being added in the form of their derivatives Fmoc-
  • Boc groups of ⁇ -NH 2 of the first lysine residue and of the isopropylidene group of the spacer arm fixed on the support are removed by treatment with a TFA-H 2 O-anisole 95: 2.5: 2.5 (10 ml) mixture, for 2 h at room temperature.
  • the peptidyl-resin is washed with CH 2 C1 2 (5 x 1 minute) and with Et 2 O (2 x 1 minute), then dried and soaked in aqueous AcOH. NaIO 4 is added
  • the epitopes (peptide sequences designated HA 307 "319 and ⁇ 830 - 846 in FIG. 8) are synthesized on a scale of 0.25 mmol using a resin
  • the protective group of the side chains of Fmoc-L-Lys-OH is the Boc group; the protective group for Fmoc-L-Ser-OH, Fmoc-L-Glu-OH, Fmoc-L-Thr-OH, Fmoc-L-Tyr-OH and Fmoc-
  • L-Asp-OH are tert-butyl; the protective group for Fmoc-L-Asn-OH, Fmoc-
  • L-Gln-OH and Fmoc-L-His-OH is the trityl group; the protective group for Fmoc-L-Arg-OH is the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group.
  • the peptide is separated from the support and deprotected using the TFA-anisole-H 2 O 95: 2.5: 2.5 mixture (10 ml), for 3 h at room temperature, precipitated in cold diethyl ether, centrifuged, dissolved in water and freeze-dried.
  • the peptide is separated from the support and deprotected using the TFA-iPr 3 SiH-H 2 O 95: 2.5: 2.5 mixture (10 ml), for 1.5 h,
  • Ligand 52 is then isolated as described above for ligand 51. 0.71 mg of ligand 52 (yield of 42%) are obtained, in the form of a white powder, after purification by 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 lyophilization. Its analysis by positive ESI-MS is as follows: calculated 3913.6, found 3912; m / z: 1305.0 [M + 3H] 3+ , 979.1 [M + 4H] 4+ , 783.6 [M + 5H] 5+ .
  • B 1 represents an acid (-) - quinic residue, • M represents an amide function (-CO-NH-),
  • A represents the remainder of a linear polyethyleneimine polymer (PEI), were prepared by condensation of PEI on quinic acid 1,5-lactone ( ⁇ -lactone resulting from the esterification between the carboxyl group carried by the carbon located in position 1 and the hydroxyl group located in position 5 of (-) - quinic acid), according to the following protocol.
  • PEI linear polyethyleneimine polymer
  • D (-) - quinic acid (Aldrich) is transformed into bicyclic 1,5- ⁇ -lactone (also called “quinide”) by heating in toluene, in the presence of j-toluenesulfonic acid, as described in particular by HOL FISCHER in Chem. Ber., 1921, 54, 775-785, by M. PHILIPPE et al in J. Antibiotics, 1982, 25, 1507-1512 and by PQ HUANG et al in Tetrahedron Lett., 1995, 36, 8299-8302.
  • the lactone (10.1 mg, 58.1 ⁇ mol) reacts with 50 mg of PEI (25 kDa, Aldrich), which corresponds to 1162 ⁇ mol of amino groups, assuming a weight average molecular weight of 43 Da for the repetitive unit of the IEP.
  • the reaction is carried out in 2 ml of water, for 24 h and at 50 ° C.
  • the reaction medium is then diluted and lyophilized.
  • PEI is obtained substituted, at a rate of 5%, with quinic acid (hereinafter referred to as "5% QuinPEr”).
  • 5% QuinPEr quinic acid
  • PEI is obtained substituted, at a rate of 10%, with quinic acid (hereinafter referred to as "10% QuinPEr").
  • the completion of the reaction is indicated, by ⁇ and 13 C NMR analysis of the reaction medium, by the absence of the signal corresponding to the quinide (the condensation of the quinide on the PEI in fact results in the opening of the cycle of lactone).
  • the results were confirmed by quantitation of the amount of quinic acid coupled on the PEI conducted by ⁇ NMR spectroscopy on samples of the products obtained, purified by ultrafiltration on Amicon membranes ® (30000 Da, Millipore).
  • the quinic acid content of the PEI is confirmed by comparing the peak surfaces of the CH 2 protons of the polymer backbone and of the H-4, H-5 and H-6 protons of the quinic acid residues.
  • PEI is a branched polymer with secondary and primary amines.
  • the study of the N NMR specfre makes it possible to distinguish the quinic acids linked via a secondary or tertiary amide bond (the latter being indicated by an index ').
  • 5% QuinPEI and 10% Quin PEI respectively contain 30 and 26% of quinic acid residues connected via a tertiary amide bond, the rest being secondary amide bonds.
  • the internal reference is the sodium salt of 3- (trimethylsilyl) acid [2,2,3,3- dYI propionic.
  • the biological activity of the ligands in accordance with the invention has been verified by a series of in vitro experiments aimed at testing their ability to be internalized, via the mannose receptor, by dendritic cells obtained from human peripheral blood.
  • Compound 30F was prepared according to an operating protocol consisting of:
  • Compound 31F was, in turn, prepared by treating a compound 15 with 2-thioethyl- ⁇ , D-mannopyranoside, then reacting the resulting compound with FITC. b) Obtaining dendritic cells.
  • the dendritic cells were obtained by differentiation of monocytes, themselves isolated from mononuclear cells of human peripheral blood, according to a protocol derived from that described by AVRAMEAS et al. (Eur. J. Immunol, 1996, 26, 394-400). To do this, the mononuclear cells present in human peripheral blood samples (supplied by the Establishment of Blood Transfusion of Nord-Pas de Calais, FRANCE) were separated from the other elements of the blood by centrifugation in density gradient Ficoll / Passover (PHARMACIA company), then washed 3 times, by centrifugation, with RPMI 1640 medium (GIBCO company) containing 3 mM EDTA.
  • the mononuclear cells thus isolated were suspended, at 37 ° C. and under an atmosphere at 5% CO 2 , in a culture medium comprising RPMI 1640 medium supplemented with 5.10 * 5 M of ⁇ -mercaptoethanol (Company MERCK), 2 mM of L-glutamine (Company MERCK), 1 mM of sodium pyruvate (Company GIBCO), 10% of fetal calf serum inactivated by heat (GIBCO company), 100 IU / ml of penicillin and per 100 ⁇ g / ml of streptomycin (both from the company SPECIA).
  • a culture medium comprising RPMI 1640 medium supplemented with 5.10 * 5 M of ⁇ -mercaptoethanol (Company MERCK), 2 mM of L-glutamine (Company MERCK), 1 mM of sodium pyruvate (Company GIBCO), 10% of fetal calf serum inactivated by heat (
  • the resulting suspension was distributed over 10 cm diameter petri dishes, at a rate of 8 to 12 ⁇ 10 7 cells per disc.
  • Monocyte enrichment was obtained by allowing these cells to adhere to the dishes, at 37 ° C. and in a humidified CO 2 (5%) incubator, for 3 to 4 hours.
  • the adherent cells were re-cultured in the medium described above, but also comprising 800 U / ml of growth factor from recombinant human granulocytic and macrophagic lines (Company PEPRO TECH) and 1000 U / ml of recombinant human interleukin-4 (Company PEPRO TECH), at a rate of 10 6 cells per ml of medium.
  • step b) above clearly present the surface antigens characteristic of dendritic cells
  • part of these cells were washed 2 times with PBS buffer containing 0.03 mg / ml of bovine serum albumin, then incubated, on ice and for 30 minutes, with a mouse monoclonal antibody directed against the human mannose receptor and conjugated with phycoerythrin (Company BECTON DICKINSON) and: either a monoclonal antibody mouse directed against the human CD antigen, which represents one of the main surface antigens of dendritic cells, namely a mouse monoclonal antibody directed against the human CD 14 antigen, which represents one of the antigens monocyte surface and macrophages, these last two antibodies being labeled with FITC (BECTON DICKINSON Company).
  • methyl-quinate is a mimic of mannose in internalization with the mannose receptor of dendritic cells
  • competition between methyl-quinate, or methyl- ⁇ -D-mannopyranoside (serving as inhibition control ), and compound 31F is studied according to the following protocol: the dendritic cells are recovered, washed with PBS buffer, preincubated at 37 ° C., then incubated for 20 minutes, at 37 ° C., with solutions of increasing concentrations of methyl -quinate and compound 31F (in final concentration of 5 ⁇ M). The cells are then washed with PBS, fixed in 1% paraformaldehyde, then analyzed by flow cytofluorometry, as described in c) above.
  • non-adherent cells collected in step b) above were washed 2 times with PBS buffer containing 1 mM of chloride calcium. Following which, these cells were preincubated, at 37 ° C and for 10 minutes, then incubated, at 37 ° C and for 20 minutes, with different solutions each containing one of the compounds to be tested, at a concentration between 1 and 10 ⁇ M. The cells were then washed 3 times with cold PBS buffer and fixed for 20 minutes in 2% paraformaldehyde. The fluorescence associated with these cells was then assessed by a FACScan analysis using the abovementioned cytometer.
  • these tests were repeated by preincubating the cells in the presence of mannan solutions extracted from Saccharomyces cerevisiae (Company SIGMA), mannan being, in fact, known to be selectively internalized via the mannose receptor.
  • mannan solutions extracted from Saccharomyces cerevisiae Company SIGMA
  • the dendritic cells are recovered, washed with PBS buffer, incubated at 37 ° C., then preincubated for 20 minutes, at 37 ° C., in the presence of mannan solutions extracted from Saccharomyces cerevisiae of concentrations between 10 " * and 10 mg / ml.
  • ligands 23 F and 24F (in a final concentration of 5 ⁇ M) are added (incubation: 20 minutes at 37 ° C.)
  • the cells are washed with PBS, fixed in 1% paraformaldehyde and analyzed. by flow cytofluorometry, as described in c) above f) Influence of the number of mannose mimes carried by the ligands in accordance with the invention on the internalization of these ligands
  • the optimal construction i.e.
  • the dendritic cells are incubated for 20 min nutes at 37 ° C with 10 ⁇ M of ligands comprising 2, 4 or 8 residues of mannose, quinic acid or shikimic acid (di-, tetra- or octavalent ligands). After fixation in paraformaldehyde, the cells are analyzed by flow cytofluorometry, as described in c) above.
  • ligands carrying shikimic acid residues 34F (divalent ligand), 21F (tetravalent ligand) and 23F (octavalent ligand),
  • ligands carrying quinic acid residues 33F (divalent ligand), 22F (tetravalent ligand) and 24F (octavalent ligand).
  • Figure 12 illustrates the ability of methyl quinate to inhibit the internalization of compound 31F. The percentages are shown on the ordinate inhibition, calculated by the formula:
  • MnX represents the mean value of the fluorescence intensity
  • MnX 3 IF represents the signal due to the internalization of the compound 31F alone
  • MnX mqui represents the signal due to the internalization of the compound 31F in the presence of different concentrations (expressed in mM) of methyl quinate, indicated on the abscissa.
  • the curve indicated by the symbols - * - represents the inhibition of the internalization of the compound 31F by methyl quinate, and the curve indicated by the symbols - represents the inhibition of the internalization of the compound 31F by methyl - ⁇ -D-mannopyranoside, compound serving as inhibition control.
  • FIG. 13 illustrates, in the form of curves, the average values of the fluorescence intensities presented by the dendritic cells after an incubation in the presence of solutions comprising respectively from 1 to 10 ⁇ M of ligand 23F (- • -), of ligand 24F ( - • * -), of compound 30F (- ⁇ -) and of compound 31F (-D-).
  • solutions comprising respectively from 1 to 10 ⁇ M of ligand 23F (- • -), of ligand 24F ( - • * -), of compound 30F (- ⁇ -) and of compound 31F (-D-).
  • AU average values of the fluorescence intensities
  • abscissa the concentrations of the solutions.
  • Figure 13 shows that an incubation of dendritic cells, for a duration of 20 minutes and carried out in the presence of a solution dosed with 1 ⁇ M of ligand 23F or of ligand 24F is sufficient to obtain an internalization of these ligands by these cells. . It also shows that this internalization is specific since the compound 30F (carrying polyhydroxylated chains) practically does not penetrate into the dendritic cells, and this whatever the dose of this compound used for the incubation of these cells, whereas the compound 31F (carrier of D-mannose residues) is found very widely in said dendritic cells. Furthermore, FIG.
  • Figure 14 shows that the internalization, by dendritic cells, of ligands 23F and 24F, as well as that of compound 31F decreases drastically as the concentration of mannan increases. This indicates the existence of a strong competition between these ligands and the mannan with respect to the mannose receptor and confirms that the internalization of the ligands in accordance with the invention by dendritic cells is effected by the mannose receptor. c) Influence of the number of mannose mimes carried by the ligands in accordance with the invention on the internalization of these ligands.
  • Figure 15 shows, on the ordinate, the percentage of specific internalization of the di-, tetra- or octavalent ligands (abscissa) by the mannose receptor.
  • the tetra- or octavalent ligands are preferably internalized by the mannose receptor, compared to the divalent ligands.
  • the optimal structures sought are those comprising 4 mimes of mannose, which are more easily synthesized than those comprising 8 mimes of mannose.
  • Dendritic cells differentiated from mononuclear cells in the presence of IL-4 and the growth factor GM-CSF, are used on D + 5. They are incubated for 45 minutes at 4 ° C with 10 ⁇ g of human receptor anti-mannose (PHARMIGEN) and 5 ⁇ M of one of the following constructions:
  • 61F ( Figure 16): dendrimer consisting of 4 L-lysine residues carrying 4 residues of D-mannose, as well as a fluorescein molecule (FITC). Construction 61F was prepared like compound 31F (cf. example 8, paragraph a), but by reacting 2-thioethyl- ⁇ , D-mannopyrannoside with a compound
  • 21F, 22F and 64F constructions carrying respectively residues of shikimic acid, quinic acid and polyhydroxylated chains of galacto configuration (coming from molecules of galactonolactones).
  • Construction 64F was prepared like compound 30F (cf. example 8, paragraph a), but using a compound 13 for coupling (cf. example 1, paragraph 1.1).
  • the preparation of ligands 21F and 22F is described in Example 2 above.
  • Structure 61 F serves as a positive control, while structure 64F serves as a negative control.
  • the structures 21F and 22F represent ligands in accordance with the present invention.
  • the cells are placed at 37 ° C for 5 minutes. After incubation, they are fixed for 45 minutes at 4 ° C. with a solution of the following composition: PBS (0.1 mM MgCl 2 , 0.1 mM Ca 2 ), paraformaldehyde at 4%, sucrose. The fixing reaction is stopped using an ammonium chloride solution (50 mM), for 10 minutes and at room temperature. The cells are washed 3 times with PBS, then deposited on poly-L-lysine slides.
  • the fluorescein signal is amplified by incubation at 37 ° C for 30 minutes with a Rabbit anti-FITC IgG coupled to Alexa Fluor 488 (dilution: 1/200 th ) (MOLECULAR PROBES) at the same time as the incubation with the second goat antibody, anti-mouse IgG coupled to Alexa Fluor 568 (dilution: 1/500 th ) (MOLECULAR PROBES), allowing the detection of ranti-mannose receptor.
  • Incubation with the second antibodies is carried out in PBS, BSA (1 mg / ml), saponin (0.05%) medium.
  • the cells are washed with PBS containing 1 mg / ml of BSA and 0.05% of saponin, then with PBS / BSA and finally with PBS, then mounted between slide and coverslip in VECTASHIELD mounting medium.
  • the observation of the slides is carried out by confocal microscopy on a LEICA TCS NT device having a Krypton / Argon laser (excitation 488, 468 and 647).
  • the acquisition of the two wavelengths (namely the acquisition of the fluorescence emitted by the Alexa 568, corresponding to the mannose receptor, and the acquisition of the fluorescence emitted by the Alexa 488, corresponding to the structures tested) is performed simultaneously and the superposition of the two images makes it possible to view the two markers at the same time, on the same cell section, carried out along the z axis of the cell.
  • Cos-1 cells which do not express the mannose receptor in their native state, are transfected with the plasmid CD8 containing a coding sequence for the mannose receptor (MR). 48 hours after transfection, the cells are collected: 10 to 15% of them express the transgene. In what follows, these cells are called “Cos MR”.
  • 22F dendrimer consisting of 4 L-lysine residues carrying 4 residues of quinic acid, as well as a fluorescein molecule (FITC). It is a ligand in accordance with the present invention.
  • 64F construction carrying polyhydroxylated chains of galacto configuration (negative control).
  • test c) incubation of Cos MR cells with compound 22F
  • test d incubation of Cos-1 cells (not transfected) with compound 22F.
  • the non-transfected Cos-1 cells do not, of course, give rise to internalization of compound 22F: the detected signal is comparable to the background noise of the device.
  • the detected signal is comparable to the background noise of the device.
  • Test b) shows the inhibition of the internalization of compound 22F by the mannan.
  • construct 22F it is well internalized in a specific manner by the cells transfected by the MR receptor (test c).

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EP00956573A 1999-07-30 2000-07-28 Verwendung von china- und shikiminsäure und deren derivate zur herstellung von liganden des mannoserezeptors Withdrawn EP1200462A2 (de)

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FR9909928A FR2797441B1 (fr) 1999-07-30 1999-07-30 Derives des cyclohexane, cyclohexene, cyclohexadiene et benzene pour la preparation de ligands du recepteur du mannose
FR9909928 1999-07-30
PCT/FR2000/002194 WO2001009173A2 (fr) 1999-07-30 2000-07-28 Utilisation des acides quinique, shikimique et de leurs derives pour la preparation de ligands du recepteur du mannose

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Cited By (4)

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WO2008116385A1 (fr) * 2007-03-23 2008-10-02 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Dérivés d'acide caféyl quinique contenant de l'azote, et procédé de préparation, composition pharmaceutique et leur utilisation
US8338372B2 (en) 2007-09-20 2012-12-25 Zhejiang Medicine Co., Ltd. Dehydroxy vancomycin, the preparation, pharmaceutical composition and the use
US8748495B2 (en) 2009-03-09 2014-06-10 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Method of preparing oil suspensions of carotenoid with low viscosity and high fluidity and use thereof
US8937076B2 (en) 2007-02-14 2015-01-20 Zhejiang Medicine Co., Ltd. Xinchang Pharaceutical Factory Crystalline form of entecavir, its preparation and the pharmaceutical composition and uses thereof

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CN103415531B (zh) 2011-02-09 2016-10-26 器官平衡有限责任公司 用于治疗皮肤病症的肽

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US5432260A (en) * 1991-05-03 1995-07-11 Washington University High affinity mannose receptor ligands
JP3662926B2 (ja) * 1993-08-06 2005-06-22 タツプ・ホールデイングス・インコーポレイテツド Ｌｈｒｈのｎ末端改変類似体

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8937076B2 (en) 2007-02-14 2015-01-20 Zhejiang Medicine Co., Ltd. Xinchang Pharaceutical Factory Crystalline form of entecavir, its preparation and the pharmaceutical composition and uses thereof
WO2008116385A1 (fr) * 2007-03-23 2008-10-02 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Dérivés d'acide caféyl quinique contenant de l'azote, et procédé de préparation, composition pharmaceutique et leur utilisation
US8722725B2 (en) 2007-03-23 2014-05-13 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Caffeoylquinic acid derivatives containing nitrogen, and preparation method, pharmaceutical composition and usage thereof
US8338372B2 (en) 2007-09-20 2012-12-25 Zhejiang Medicine Co., Ltd. Dehydroxy vancomycin, the preparation, pharmaceutical composition and the use
US8748495B2 (en) 2009-03-09 2014-06-10 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Method of preparing oil suspensions of carotenoid with low viscosity and high fluidity and use thereof

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