Pro-ligands, conjugates comprising said pro-ligands and their uses for therapeutic applications

Abstract

The present invention relates to new compounds called pro-ligands or inactivated ligands of formula (I) as defined in claim 1. It also relates to conjugates comprising said pro-ligands and their uses for therapeutic applications.

Description

Title of the invention: Pro-ligands, conjugates comprising said proligands and their uses for therapeutic applications Technical field

[0001] The present invention relates to the field of therapeutic chemistry. It relates in particular to new compounds called “pro-ligands” or “inactivated ligands”, the conjugates comprising said pro-ligands and their uses for therapeutic applications.

[0002] The compound called pro-ligand within the meaning of the invention designates a ligand which has been rendered temporarily inactive, for example by the addition, within said ligand, of one or more labile groups. In the present application the pro-ligand may also be called inactivated ligand. The role of a ligand being to be recognized by its membrane receptor, a pro-ligand thus designates a ligand which is no longer recognized by its membrane receptor since the pro-ligand is rendered temporarily inactive. The pro-ligands of the invention are mannose 6-phosphate (M6P) analogs or mannose analogs (Man) rendered temporarily inactive. Mannose 6-phosphate (M6P) analogs refer to compounds that are different from natural M6P but exhibit high recognition by the cation-independent mannose 6-phosphate receptor (CM6P-CI). Similarly, mannose analogues refer to compounds that are different from natural mannose but exhibit high recognition by mannose receptors. The M6P analogs are called “AMFA” (“Synthetic Analogues of Mannose 6-Phosphate Functionalized in Anomer Position”) and the mannose analogs are called “Man”. The pro-ligands obtained after inactivation of the AMFA analogs or the Man analogs are called in the present application “ProAMFA” or “ProMan”. ProAMFA pro-ligands are thus no longer recognized by RM6P-CI and ProMan pro-ligands are no longer recognized by mannose receptors.

[0003] The conjugate of the invention comprises the pro-ligand on the one hand and a product of interest on the other hand. The conjugate of the invention, of structure “Pro-ligand - Product of interest”, is capable of selectively binding a target, via the product of interest capable of recognizing said target, which makes it possible to obtain a complex having the structure “Pro-ligand - Product of interest - Target”. Prior art

[0004] Documents EP 2 448 600 B1 and EP 3 350 192 B1 describe conjugates comprising M6P analogues which are linked, via an arm spacer, to a product of interest, such as a glycoprotein. M6P analogs have several advantages for the functionalization of glycoproteins, in particular for the functionalization of lysosomal enzymes, in particular because they are small, are not very immunogenic and can be easily linked with the lysosomal enzyme. These M6P analogs are also modular, on the one hand in terms of the length and structure of the spacer arm and on the other hand in terms of the terminal reactive group of the spacer arm depending on the type of binding desired with the product of interest. In these documents, M6P analogs are more particularly used to internalize, via RM6P-CI, lysosomal enzymes into the lysosomes of the cell, so that said enzymes can play their physiological role.M6P analogues are also modular at the phosphate group in position 6, which can be replaced by a bioisostere group, i.e. a group that fulfills the same biological functions as the phosphate group. The phosphate group can, for example, be replaced by a phosphonate, carboxylate or malonate group.

[0005] In documents EP 2 448 600 B1 and EP 3 350 192 B1, however, it is not envisaged to temporarily inactive the M6P analogues, which means that the conjugates, of structure "M6P analogues - Product of interest", are, once introduced into an organism, immediately recognized by the cation-independent mannose 6-phosphate (RM6P-CI) receptors and therefore bound by them. RM6P-CI is a ubiquitous receptor which is present both in intracellular vesicles and on the membrane of all cells; it is therefore not a receptor specific to a particular tissue or cell type. This means more specifically that the conjugates, once in the organism, will not necessarily have the time necessary to reach and bind a specific target capable of being recognized by the product of interest since the M6P analogue can be quickly recognized by the RM6P-CI present in the immediate environment.The product of interest will thus undergo a reduction in its free movement to reach and fix the biological target that it is able to recognize. In other words, this means that the conjugates described in these documents could reduce the detection of the predetermined target.

[0006] The inventors have now developed new conjugates that are specific for previously determined targets. To do this, they had the original idea of ​​developing pro-ligands that are analogues of M6P or analogues of mannose and that are temporarily inactivated, and then preparing conjugates with the structure “Pro-ligand - Product of interest”. Thus, when the conjugates of the invention are introduced into an organism, they will not be directly recognized by the ligand receptor (RM6P-CI or one of the mannose receptors), which will allow them to circulate freely in order to more effectively reach a target specifically recognized by the product of interest. The product of interest binds to the target, which results in the formation of a “Pro-ligand - Product of interest - Target” complex. This complex is then activated so that the Pro-ligand becomes a ligand. The activated complex thus obtained, with the structure “Ligand - Product of interest - Target”, will then be recognized by the ligand receptor and bound by the latter. Summary

[0007] According to a first aspect, the present invention relates to a pro-ligand of general formula (I) as described in the detailed description below.

[0008] According to a second aspect, the present invention relates to a conjugate of general formula (II) as described in the detailed description below, of structure “Pro-ligand - Product of interest”.

[0009] According to a third aspect, the invention relates to the process for preparing the proligands of formula (I) and the conjugates of formula (II).

[0010] According to another aspect, the invention relates to a complex of structure “Pro-ligand - Product of interest - Biological target”.

[0011] According to yet another aspect, the present invention relates to a conjugate of formula (II) for specific targeting of a previously determined target. As an example of such a target, mention may be made of a non-membrane or membrane target, chosen from the group comprising proteins, in particular receptors, autoantibodies, glycoproteins, protein aggregates, protein or non-protein antigens, protein activators or inhibitors, cells, viruses, bacteria, bone tissue, collagen or fiber aggregates. The conjugate (II) and the target are linked to each other by a non-covalent or covalent type bond.

[0012] According to yet another aspect, the invention relates to a conjugate of formula (II) for use as a medicament in the treatment of pathologies chosen from the group comprising neurodegenerative diseases (such as, for example, Alzheimer's disease), cancers, autoimmune diseases, rare and genetic diseases (such as, for example, lysosomal diseases), inflammatory diseases and infectious diseases. Brief description of the drawings

[0013] Other characteristics, details and advantages will appear on reading the detailed description below, and on analyzing the attached drawings, in which: Fig.l

[0014] [Fig.l] illustrates the sequence of events occurring when the conjugate of the invention, of structure “Pro-ligand - Product of interest”, is introduced into an organism for the purpose of binding a non-membrane target. Fig. 2

[0015] [Fig.2] illustrates the sequence of events occurring when a conjugate of the invention, of structure “Pro-ligand - Product of interest”, is introduced into an organism with the aim of binding a membrane target. Fig. 3

[0016] [Fig.3] is an illustration of four possible embodiments for the synthesis of conjugates of formula (II) obtained by the reaction between a pro-ligand of general formula (I) with a product of interest Y. Fig. 4

[0017] [Fig.4] illustrates the detailed synthesis scheme of the pro-ligand of general formula (I) called ProAMFAl. Fig. 5

[0018] [Fig.5] illustrates the results obtained by SDS-PAGE electrophoresis of the enzyme Commercial Myozyme® alone or linked to the AMFA1 compound (obtaining the Myo-AMFAl conjugate) or to the ProAMFAl pro-ligand (obtaining the Myo-ProAMFAl conjugate). The enzyme alone or linked to the AMFA1 or ProAMFAl compounds is detected by Coomassie blue staining. Fig. 6

[0019] [Fig.6] illustrates the results obtained by SDS-PAGE electrophoresis of the In- antibody flixhnab alone or bound to the AMFA1 compound (obtaining the Infli-AMFAl conjugate) or to the Pro-AMFAl pro-ligand (obtaining the Infli-ProAMFAl conjugate). The antibody alone or bound to the AMFA1 or ProAMFAl compounds is detected by Coomassie blue staining. Fig. 7

[0020] [Fig.7] illustrates the cytotoxicity on human MCF-7 breast cancer cells of the pro-ligand ProAMFAl in comparison with compound M6P and compound AMFA1. The x-axis represents the molar concentration of the compounds M6P (white histogram), AMFA1 (hatched histogram) and ProAMFAl (black histogram). The y-axis represents the cell survival of the compounds. The cell survival observed in the presence of M6P was taken as 100%. Fig 8

[0021] [Fig.8] illustrates the cytotoxicity on human fibroblasts FS01035 of ProAMFAl compared with M6P and AMFA1. The description of the axes and histograms in [Fig.7] applies to [Fig.8]. Fig. 9

[0022] [Fig.9] illustrates the interactions between RM6P-CI and the pro-ligand ProAMFAl in comparison with compounds M6P and AMFA1. The x-axis represents the Molar concentration of compounds M6P (dotted curve), AMFA1 (dashed curve) and ProAMFAl (solid curve). The y-axis represents the percentage binding of these compounds to RM6P-CI. Fig. 10

[0023] [Fig. 10] is a series of NMR spectra at different times (t = 0, t = 1 h, t = 1 h, t = 24h) showing the kinetics of the degradation of two pro-ligands ProAMFAl and ProAMFAlox into ligands AMFA1 and AMFAlox, in the presence of an alkaline phosphatase isolated from human placenta. The difference between ProAMFAl and ProAMFAlox lies in the Li radical which represents -O-NH2 in ProAMFAl versus -ON=C(CH3)2 in ProAMFAlox. The peaks on the left of the spectra are characteristic of the radical X equal to phosphonate (i.e. X = CH2-P(O)(OH)2) presented by each of ProAMFAl and ProAMFAlox. The peaks on the right of the spectra are characteristic of the PI radical equal to phosphate (i.e. X = P(O)(OH)2) presented by each of ProAMFAl and ProAMFAlox. Fig. 11

[0024] [Fig. 11] illustrates the cytotoxicity, on human MCF-7 breast cancer cells, of the Myozyme® enzyme (white histogram) and the Myo-AMFAl (hatched histogram) and Myo-ProAMFAl (black histogram) conjugates. The x-axis represents the molar concentration of the compounds and the y-axis the cell survival of the compounds. The cell survival observed in the presence of Myozyme® was taken as 100%. Fig. 12

[0025] [Fig. 12] illustrates the cytotoxicity, on human fibroblasts FS01035, of the enzyme Myozyme® (white histogram) and the conjugates Myo-AMFAl (hatched histogram) and Myo-ProAMFAl (black histogram). The x-axis represents the molar concentration of the compounds and the y-axis the cell survival of the compounds. The cell survival observed in the presence of Myozyme® was taken as 100%. Fig. 13

[0026] [Fig. 13] illustrates the interactions between RM6P-CI and the compound M6P or between the RM6P-CI and rinfliximab alone or bound to AMFA1 or ProAMFAl. The x-axis represents the molar concentration of the compounds M6P (cross curve), Infliximab (dotted curve), Infli-AMFAl (dashed curve) and Infli-ProAMFAl (solid curve). The y-axis represents the percentage binding of these compounds to RM6P-CI. Fig. 14

[0027] [Fig. 14] illustrates the detection by Western blots of the enzyme Myozyme®, of Myo-AMFAl, Myo-ProAMFAl and Myo-ProAMFAl conjugates incubated in the presence of alkaline phosphatase isolated from human placenta, at different times (t = 1h, t = 24h and t = 96h). Detailed description

[0028] Pro-ligands of formula (I)

[0029] The subject of the present invention is a pro-ligand characterized in that it has the following general formula (I):

[0030] [Chem.l] FS —L—L, (I) in which: X represents -OH; -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; =CH-CO2Z; =CH-P(O)(OZ)2; -CHF-CO2Z; -CHF-P(O)(OZ)2; -CF2-CO2Z; -CF2-P(O)(OZ)2; =CF-CO2Z; =CF-P(O)(OZ)2 ; -CH(CO2Z)(P(O)(OZ)2); with Z independently representing H; Na; K or NH4; PI, P2 and P3 representing independently of each other H; P(O)(OZ)2; -S(O)2 (OZ); with Z as defined previously and with the condition that at least one of PI, P2 and P3 is other than a hydrogen atom; or P2 and P3 together forming an acetonide group (also called isopro-pylidene)

[0031] [Chem.2] - H ÿC A represents a divalent radical chosen from -O-; -S-; -NH-; -CH2-; L represents oo -(CH2)2-(O-CH2-CH2)n- with n representing an integer ranging from 0 to 6; oo -H ; -NH2 ; -(CH2)ni-CH=CH2 or -(CH2)ni-C=CH with ni representing an integer ranging from 0 to 4, which means that in each of these cases Li is absent; oo a saturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 1 to 30 carbon atoms; an unsaturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 2 to 30 carbon atoms; oo a saturated or unsaturated divalent hydrocarbon radical as defined above of which one or more -CH2-, -CH=CH- and / or -C=C- groups of the saturated or unsaturated hydrocarbon radical is (are) replaced independently of one another by a -O- group; -S- ; -CO-NH- ; -NH-CO-O- ; -NH- ; -NRr with Ri representing a C1-C5 alkyl; -ON=CH- ; -CO-NH-N=CH- ; a cyclic or heterocyclic system, saturated or unsaturated, substituted or not; Li represents 00 -O-NH2 ; 00 a cyclic or heterocyclic system chosen from

[0032] [Chem.3]

[0033] with Ri as defined above, and is preferably ethyl;

[0034] [Chem.4]

[0035] [Chem.5]

[0036] [Chem.6]

[0037] [Chem.7]

[0038] [Chem.8] oo -ON=C(CH3)2; oo -(CH2)nl-CH=CH2; -(CH2)nl-C=CH; -(CH2)nl-N3; -(CH2)nl-SH; -(CH2)nl-NH2; -(CH2)nl-N=C=O; -(CH2)nl-N=C=S; -(CH2)nl-NHR1; -(CH2)nl-ArNH2; -(CH^-A! -NHR, ; -(CH2)nl-NHCO-CH2Hal; -(CH2)nl-COZ1; -(CH2)nl-A1COZ1; -(CH2)nl -ON=CH2; -(CH2)nl-CO-NH-N=CH2; with nor as defined above; Ri representing a C1-C5 alkyl; Ai representing -O-; -NH-; Hal representing Cl; Br or I; Zi representing -OH; -ORi; -NHRi; -NH-NH2; -NH-NHRi with Ri and R2 as defined above; oo a halogen chosen from F, Cl, Br or I.

[0039] As examples of a saturated, linear or branched divalent hydrocarbon radical having from 1 to 30 carbon atoms, we may notably cite: -CH2-; -CH2-CH2-; -CH2-(CH2)m-; -(CH2)m-CH(Ci-C7)-(CH2)m-; -(CH2)m-CH(Ci-C7)-(CH2)m-CH(Ci-C7)-(CH2)m-; -(CH2)mC(Ci-C7)2-(CH2)m; -(CH2)mC(Ci-C7)2-(CH2)m-CH(Ci-C7)-(CH2)m-; -(CH2)mC(Ci-C7)2-(CH2)mC(Ci-C7)2-(CH2)m-; with m representing independently of each other an integer ranging from 0 to 30 with the condition that the length of the main hydrocarbon chain does not exceed 30 carbon atoms, and CrC7 representing an alkyl having from 1 to 7 carbon atoms. Examples of CrC7 alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl. An unsaturated, linear or branched, divalent hydrocarbon radical having from 2 to 30 carbon atoms, denotes a hydrocarbon radical comprising one or more carbon-carbon double bonds and / or one or more carbon-carbon triple bonds. Examples of an unsaturated divalent hydrocarbon radical include: -CH=CH-; -(CH2)m-CH=CH-(CH2)m-; -(CH2)m-CH=CH-(CH2)m-CH=CH-(CH2)m-; -(CH2)m-CH=CH-CH(CrC7)-(CH2)m-; -(CH2)m-CH(C1-C7)-(CH2)m-CH=CH-CH(C1-C7)-(CH2)m-; -(CH2)m-CH(C1-C7)-CH=CH-(CH2)m-; -(CH2)m-CH(C1-C7)-CH=CH-(CH2)m-CH=CH-(CH2)m-; -(CH2)m-CH=CH-C(CrC7)2-(CH2)m-; -(CH2)m-CH=CH-C(C1-C7)2-(CH2)mC(C1-C7)2-(CH2)m-CH=CH; -(CH2)m-CH=CH-C(C1-C7)2-(CH2)m-CH(C1-C7)-(CH2)m-CH=CH; -C=C- ; -(CH2)mC=C-(CH2)m-; -(CH2)mC=C-(CH2)mC=C-(CH2)m-; -(CH2)m-CH=CH-CH(CrC7)-(CH2)mC=C-(CH2)m-CH(Ci-C7)-; -(CH2)m-CH=CH-C(C1-C7)2-(CH2)mC=C-(CH2)mC(C1-C7)2-(CH2)m-; -(CH2)mC=C-CH(C1-C7)-(CH2)mC=C-(CH2)m-CH(C1-C7)-(CH2)m; -(CH2)mC=CC(C1-C7)2-(CH2)m-CH=CH-(CH2)m-CH(C1-C7)-; -(CH2)m-CH=CH-C(C1-C7)2-(CH2)m-CH(C1-C7)-(CH2)mC=C-CH=CH-(CH2)m-; with m as defined previously. The term "cyclic or heterocyclic system" refers to the divalent radical originating from a cyclic or heterocyclic compound, saturated or unsaturated. As an example of a saturated or unsaturated cyclic or heterocyclic compound, mention may in particular be made of azetidine, oxetane, thietane, pyrrole, pyranose, furanose, furan, pyrroline, tetrahydrofuran, thiophene, tetrahydrothiophene, pyrazole, imidazole, oxazole, isoxazole, pyrazoline, imidazoline, pyrazolidine, imidazolidine, dioxolane, thiazole, isothiazole, thiazolidine, isoxazolidine, triazole, oxadiazole, thiadiazole, thiosuccinimide, tetrazole, pyridine, naphthyridine, phthalimide, pyran, dihydropyran, piperidine, pyridazine, pyridinium, pyrimidine, purine, pyrazine, pteridine, oxazine, dioxin, piperazine, maleimide, morpholine, dioxane, thiazine, thiomorpholine, oxathiane, dithiane, triazine, trioxane, thiadiazine, dithiazine, trithiane, 3-cyclobutene-l,2-dione, cyclobutane, cyclobutene, cyclopentane,cyclopentene, cyclohexane, cyclohexene, cycloheptane, cycloheptene, benzene, toluene, naphthalene, indene, indane, indolizine, indole, benzofuran, indoline, benzothiophene, indazole, benzimidazole, benzthiazole, tetralin, tetrazine, quinoline, chromene, , chroman, cinnoline, quinazoline, quinoxaline, phthalazine.

[0040] According to an advantageous embodiment of the invention, the substituent L of the proligand of formula (I) more particularly represents: 00 a saturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 1 to 10 carbon atoms; an unsaturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 2 to 10 carbon atoms; a saturated or unsaturated divalent hydrocarbon radical as defined above, of which one or more -CH2-, -CH=CH- and / or -C=C- groups of the saturated or unsaturated hydrocarbon radical is (are) replaced independently of one another by: 000 a group -O-; -NH-; -S-; -CO-NH-; -NH-CO-O-; and / or 000 a cyclic or heterocyclic system chosen from those listed in Table 1 below [Table 1]

[0041] According to a particularly advantageous embodiment of the invention, the proligand as defined above is more particularly characterized in that: X represents -OH; -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2 with Z as defined above, and preferably Z equal to H; PI, P2 and P3 representing independently of each other H or P(O)(OZ)2 with Z as defined above, and preferably Z equal to H, with the condition that at least one of PI, P2 and P3 is other than a hydrogen atom; or P2 and P3 together forming an acetonide group of formula [Chem2] such that defined above; A represents oxygen -O-; L represents -(CH2)2-(O-CH2-CH2)n- with n representing an integer ranging from 0 to 6, and preferably n equal to 0; Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above.

[0042] When X represents -CH2-CO2Z, -CH2-P(O)(OZ)2, -CH(CO2Z)2 or -CH(P(O)(OZ)2) 2 then the pro-ligands (I) of the invention are the ProAMFA pro-ligands, namely the analogues of M6P (mannose 6-phosphate). When X represents -OH then the pro-ligands (I) of the invention are the ProMan pro-ligands, namely the mannose analogues.

[0043] Table 2 below exemplifies ProAMFAs of formula (I) in which: X represents a phosphonate group of formula -CH2-P(O)(OH)2; PI, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of PI, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents oxygen; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Table 2]

[0044] Table 3 below exemplifies ProAMFAs of formula (I) in which: X represents a carboxylate group of formula -CH2-COOH; PI, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of PI, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents oxygen; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Table 3]

[0045] Table 4 below exemplifies ProAMFAs of formula (I) in which: X represents a malonate group of formula -CH(CO2H)2; PI, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of PI, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents oxygen; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Table 4] V->. ' ' ?' b <• JA ..-'y J; ' Z""* >X.' •• X»** k'- •x '•>. : .•X to*' : ' >■ Hi / \ Q' 1 x'_ .•>. X.$SSL; ><„: / .-•'■■■ :"4 'W4.. : U-J'' '' Cv .J ...-'"■yy ■: ..Q..s ■ S$s. ' ■''x

[0046] Table 5 below exemplifies ProAMFAs of formula (I) in which: X represents a bisphosphonate group of formula -CH(P(O)(OH)2)2; PI, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of PI, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents oxygen; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Table 5]

[0047] Table 6 below exemplifies ProMan of formula (I) in which: X represents an -OH group; PI, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of PI, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2] as defined above; A represents oxygen; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Tableauô] V—- . * : S : y :- y y--- W; »■< y... - ' X. ,X U..X 1 » ry. y* v ' .y J 1 O -"vAx-y XU y. ' :yy y., v 'X-OS '•yy Vÿ : .XS ; yy .. y-” ; y? -fy to y* yy?-- y''.:, ,yy.- ! iys - ! i X * s P- i / 1 p % !> sLs ,.y .. y . ■ -: ÿî ................................XX: K «:< yy

[0048] The conjugates of formula (II)

[0049] According to another aspect, the present invention relates to a conjugate characterized in that it has the following general formula (II):

[0050] [Chem.9] (II) in which n2 is an integer ranging from 1 to 1000, preferably from 1 to 20, and more preferably from 1 to 10, PI, P2, P3, X, A and L are as defined above, The i represents the substituent Lx as defined above when said Li is found involved in a covalent bond with Y b Yi represents a product of interest Y, said product of interest Y being chosen from the group comprising proteins, in particular antibodies and ly- enzymes sosomal, nanoparticles, protein activators or inhibitors, cytotoxic compounds and markers for medical imaging, Yi forming n2 covalent bond(s) with L'b

[0051] For the purposes of the invention, the term "conjugate" means a compound comprising two parts linked together by a covalent bond. The first part of the conjugate thus represents the pro-ligand while the second part of the conjugate represents the product of interest. The product of interest Y designates the “free” product of interest when it does not form a bond with the pro-ligand of formula (I). The product of interest Yi denotes the product of interest Y when the latter is covalently linked to the pro-ligand. In formula (II) of the conjugate of the invention, the compound represented in the parenthesis corresponds to the pro-ligand of formula (I) as defined above when the latter is involved in a covalent bond with a product of interest Yb. The integer n2 indicates the number of pro-ligand(s) bound to the product of interest Y,. According to the invention, n2 pro-ligand(s) can be covalently bound to a product of interest Yb. These n2 pro-ligands are bound to the product of interest via the Li or LLi radical of the pro-ligand of formula (I).

[0052] Preparation of conjugates of formula fil) The conjugate of formula (II) as defined above is prepared by reacting: - a product of interest Y as defined above, with - n2 pro-ligand(s) of general formula (I) as defined above. The free product of interest Y can be represented by Yi'-L', with L' representing a functional group or a reactive function carried by the product of interest Y. In other words, Yf represents the product of interest Y without its functional group L'. It is the functional group L' of Y which will react with a functional group or a reactive function carried by the pro-ligand (I) (in this case Li or LLJ in order to form a covalent bond between the product of interest Y and the pro-ligand (I) and thus obtain the conjugate of formula (II) of the invention. In the present application, the terms “functional group”, “reactive function” or “reactive chemical group” may be used interchangeably. Each of these terms designates a group carried by the product of interest Y (or carried by the pro-ligand (I)) capable of entering into a reaction with a group carried by the pro-ligand (I) (or carried by the product of interest Y).

[0053] According to one embodiment of the invention, in the conjugate of formula (II) as defined above, n2 is an integer equal to 1. [Fig. 3] is an illustration of four possible embodiments for the synthesis of conjugates of formula (II) obtained by the reaction between a pro-ligand of general formula (I) with a product of interest Y. In the general formulas (I) and (II) represented in points 1 / to 4 / : - X, PI, P2 and P3 are as defined above, - n2 is equal to 1, - A represents oxygen, - L represents -(CH2)2-(O-CH2-CH2)n- with n being an integer ranging from 0 to 2. The variable lies in the Li radical of the pro-ligand (I) and in the functional group carried by the product of interest Y. [Fig.3] point 1 / illustrates the reaction between the functional group Li of the pro-ligand (I) in which Li represents -ONH2 with an aldehyde function (-CHO) of a product of interest Y which is an antibody or a lysosomal enzyme, said aldehyde function having previously been generated during an oxidative process on an oligosaccharide chain / part of the glycosidic part of the antibody or lysosomal enzyme. The product Y is represented by Yi'-CHO, Yf representing the product Y without its functional group L' which is equal to -CHO. [Fig.3] point 2 / illustrates the reaction between the functional group Li of the pro-ligand (I) in which Li represents [Chem3] with an amine function (-NH2) carried by a product of interest Y such as an antibody or a lysosomal enzyme. The product of interest Y is represented by Yf-NH2, Yf representing Y without its functional group L' equal to -NH2. The amine function comes for example from a lysine amino acid residue. [Fig.3] point 3 / illustrates the reaction between the functional group Li of the pro-ligand (I) in which Li represents [Chem4] with a thiol function (-SH) carried by a product of interest Y such as an antibody or a lysosomal enzyme. The product of interest Y is represented by Yf-SH, Yf representing Y without its functional group L' equal to -SH. The thiol function comes for example from a cysteine ​​amino acid residue. [Fig.3] point 4 / illustrates the reaction between the functional group Li of the pro-ligand (I) in which Li represents [Chem5] with a thiol function (-SH) of a product of interest Y (such as an antibody or a lysosomal enzyme). The product of interest Y is represented by Yf-SH, Yf representing Y without its functional group L' equal to -SH.

[0054] Use of the conjugates (II) of the invention.

[0055] The conjugates (II) of the invention are used to bind, covalently or non-covalently, a membrane or non-membrane target of an organism. Thus, the invention also relates to the use of the conjugates of formula (II) to bind, covalently or non-covalently, a membrane or non-membrane target of an organism. Examples of a membrane or non-membrane target include proteins, autoantibodies, glycoproteins, protein aggregates, protein antigens, non-protein antigens, activators or inhibitors of proteins, cells, viruses, bacteria, bone tissue, collagen and fiber aggregates.

[0056] Figures 1 and 2 illustrate the interest of the pro-ligands (I) and the conjugates (II) of the invention. [Fig.l] illustrates the sequence of events occurring when the conjugate (II) of the invention, of structure “Pro-ligand - Product of interest”, is introduced into an organism for the purpose of binding a non-membrane target (the non-membrane target is represented by a star in [Fig.l]). The conjugate (II), once introduced into the organism, binds, via the product of interest to a non-membrane target in order to form a complex of structure "Pro-ligand - Product of interest - Non-membrane target". The bond between the product of interest and the non-membrane target can be covalent or non-covalent in nature. The non-membrane target serves as an anchoring point for the product of interest. The non-membrane target makes it possible more particularly to concentrate the product of interest at a particular and desired location in the organism. Then the pro-ligand is activated, by an in situ mechanism in the organism, into a ligand which leads to the activated complex with the structure “Ligand - Product of interest - Non-membrane target”. The mechanism of pro-ligand activation occurring in the body can be an enzymatic process or a chemical process. An enzymatic process means hydrolysis by the action of an enzyme naturally present in the body, such as a phosphatase, an esterase, a protease, a nuclease, a sulfatase. According to an advantageous embodiment, the activation of the pro-ligand into ligand is carried out using alkaline phosphatases present in different tissues of the body. A chemical process is understood to mean either hydrolysis under acidic, basic, or neutral conditions, or a redox reaction. The organism naturally has acidic conditions in its environment that can hydrolyze, for example, the acetonide of the proligand to form the ligand, particularly in acidic compartments generated by solid tumors or bone tissue. The activation of the pro-ligand into a ligand thus allows the transformation of the complex with the structure “Pro-ligand - Product of interest - Non-membrane target” into an activated complex with the structure “Ligand - Product of interest - Non-membrane target”. The transformation of the pro-ligand into ligand (by the in situ mechanism described above) allows the ligand to be recognized and fixed again at the level of its extracellular membrane receptor. Once the activated complex is attached to the cell membrane receptor via the ligand, two cases are possible. In case 1, only the conjugate is internalized (therefore without the non-membrane target) because the main objective is to internalize the product of interest into the cell. In case 2, the entire complex is internalized into the cell because the main objective is to eliminate the non-membrane target by internalizing it into the cell. [Fig.2] illustrates the sequence of events occurring when a conjugate of the invention, of structure “Pro-ligand - Product of interest”, is introduced into an organism for the purpose of binding a membrane target (the membrane target is represented by a star in [Fig.2]). The conjugate (II), once introduced into the organism, binds, via the product of interest to a membrane target in order to form a complex of structure "Pro-ligand - Product of interest - Membrane target". The bond between the product of interest and the membrane target can be covalent or non-covalent in nature. The membrane target serves as an anchoring point on the surface of a particular cell: it allows more particularly to concentrate the product of interest at a particular and desired location in the organism. Then the pro-ligand is activated, by an in situ mechanism in the organism, into a ligand which leads to the activated complex of structure "Ligand - Product of interest - Membrane target". The in situ mechanism occurring is that described previously with regard to [Fig. 1]. The ligand, which has become active again, is recognized and fixed at the level of the cell's extracellular membrane receptor. Once the activated complex is attached to the cell's membrane receptor via the ligand, two scenarios are possible. In case 1, only the conjugate is internalized (i.e. without the membrane target) because the main objective is to internalize the product of interest into the cell. In case 2, the entire complex is internalized into the cell because the main objective is to internalize the product of interest and the target into the cell.

[0057] In the present invention, the extracellular membrane receptor of the cell is: - the M6P receptor (namely RM6P-CI), when the pro-ligand (I) of the invention is a ProAMFA, namely an analogue of mannose 6-phosphate (M6P) or, - one of the mannose receptors, when the pro-ligand (I) of the invention is a ProMan, namely an analogue of mannose (Man).

[0058] As indicated above, the bond between the conjugate (II) of the invention and the target (non-membrane or membrane) may be of a covalent or non-covalent nature. The compound obtained after the bonding of the conjugate with the target will be called "complex" in the present application. The present invention also relates to a complex consisting of: - a conjugate of formula (II) as defined above, - a non-membrane or membrane target, said target being chosen from the group comprising proteins, in particular receptors, autoantibodies, glycoproteins, protein aggregates, protein or non-protein antigens, protein activators or inhibitors, cells, viruses, bacteria, bone tissue, collagen and fiber aggregates, the conjugate (II) and the target being linked to each other by a non-covalent or covalent type bond.

[0059] The activated ligand obtained at the end of the in situ mechanism described above corresponds to a compound of general formula (I) in which the radicals PI, P2 and P3 would each represent a hydrogen atom. The activated ligand AMFA is able to be recognized and bound by RM6P-CI. The activated ligand Man is able to be recognized and bound by one of the mannose receptors.

[0060] The activated conjugate obtained at the end of the in situ mechanism occurring in the organism can be represented by the following general formula (II bis):

[0061] [Chem. 10] ,.F'3 (II bis) in which: X, A, L, L'i, n2 and Yi are as defined above, P'1, P'2 and P'3 each represent a hydrogen.

[0062] Activation of the conjugate (II) within the complex generates an activated complex consisting of: - an activated conjugate of formula (II bis) - a target as defined above, the activated conjugate of general formula (II bis) and the target being linked to each other by a non-covalent or covalent type bond. The activated complex thus obtained is used to bind a lectin-type membrane receptor selected from the group comprising mannose receptors and the cation-independent mannose 6-phosphate receptor (RM6P-CI). According to an advantageous embodiment of the invention, when the substituent X of the activated conjugate of formula (II bis) represents OH, then the activated complex binds to the mannose receptors. According to another advantageous embodiment of the invention, when the substituent X of the activated conjugate of formula (II bis) represents -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; =CH-CO2Z; =CH-P(O)(OZ)2; -CHF-CO2Z; -CHF-P(O)(OZ)2; -CF2-CO2Z ; -CF2-P(O)(OZ)2 ; =CF-CO2Z ; =CF-P(O)(OZ)2 ; -CH(CO2Z)(P(O)(OZ)2), with Z as defined above, then the activated complex binds to the cation-independent mannose 6-phosphate receptor (RM6P-CI).

[0063] According to another embodiment of the invention, when, in the activated conjugate of formula (II bis), X represents OH, then the activated complex has an affinity, measured by the 50% inhibiting concentration (IC50), with respect to mannose receptors ranging from 10 4 to 107 M.

[0064] According to another embodiment of the invention, when, in the activated conjugate of formula (II bis), X represents: -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; =CH-CO2Z; =CH-P(O)(OZ)2; -CHF-CO2Z; -CHF-P(O)(OZ)2; -CF2-CO2Z; -CF2-P(O)(OZ)2; =CF-CO2Z; =CF-P(O)(OZ)2 ; -CH(CO2Z)(P(O)(OZ)2; then the activated complex has an affinity, measured by the 50% inhibiting concentration (IC5o), towards the mannose 6-phosphate cation-independent receptor (RM6P-CI) ranging from 105 to 109 M.

[0065] The present invention also relates to the use of the conjugate of formula (II) as defined above, for specific targeting of a given cell. Depending on the target to which the conjugate of formula (II) binds, then the conjugate or complex can, after activation, be internalized into a specific cell, via recognition of the ligand by the membrane receptor of said cell, this specific cell being linked to the nature of the target. For example, if the biological target is a membrane receptor or antigen, then the activated complex will be internalized into the cell and the biological target will be degraded or not in the lysosomes. If the target is hydroxyapatite present in bone tissue, then the activated conjugate or complex can be internalized into a bone tissue cell. If the biological target consists of protein clumps such as amyloid plaque, then the conjugate or activated complex can be internalized into the surrounding cells.

[0066] According to another aspect of the invention, there is provided a pharmaceutical composition characterized in that it comprises a conjugate of formula (II) as defined above.

[0067] According to yet another aspect, the invention relates to a conjugate of formula (II) as defined above or a pharmaceutical composition as defined above, for medical use, of the therapeutic or diagnostic type.

[0068] The invention more particularly relates to a conjugate of formula (II) as defined above or a pharmaceutical composition as defined above, for use as a medicament aimed at the intracellular degradation of a non-membrane target.

[0069] The invention more particularly relates to a conjugate of formula (II) as defined above or a pharmaceutical composition as defined above, for use as a medicament in the treatment of pathologies chosen from the group comprising neurodegenerative diseases, cancers, autoimmune diseases, rare diseases, inflammatory diseases and infectious diseases.

[0070] The subject of the invention is also a conjugate of formula (II) as defined above or a pharmaceutical composition as defined above, for use as a gene therapy medicament.

[0071] The invention also relates to a conjugate of formula (II) for use as defined above, characterized in that the conjugate is in a form suitable for administration by the parenteral, intravenous or subcutaneous route. Examples

[0072] The following examples refer in particular to the figures and describe the synthesis of pro-ligands of formula (I), of conjugates (II) of the invention, as well as the study of their biological effects, and in particular of the activation of the pro-ligand into ligand. The pro-ligand ProAMFAl (I) of the invention is compared to the compound AMFA1 and to the compound mannose 6-phosphate (M6P).

[0073] Example 1: Synthesis of the pro-ligand ProAMFAl of formula (I)

[0074] The pro-ligand ProAMFAl has the following developed formula:

[0075] [Chem. 11] H\?" A' '''' ÏW4MFA1 The pro-ligand ProAMFAl corresponds to the general formula (I) in which: X represents CH2-P(O)(OZ)2 with Z= H (therefore the phosphonate group CH2-P(O)(OH) 2); PI represents P(O)(OZ)2 with Z= H (therefore the phosphate group P(O)(OH)2); P2 = P3 = H ; A represents oxygen; L represents -(CH2)2-(O-CH2-CH2)n- with n = 0 (therefore the group (CH2)2) and Li represents ONH2. The synthesis scheme of ProAMFAl is illustrated in Figure 4 and is detailed below. below.

[0076] “2. 3: 4. 6-di-O-isopropylidene-qD-mannopvranoside of 2-bromoethyl” (2) To 14.300 g (1.0 eq; 49.81 mmol) of 1 (“2-bromoethyl a,D-mannopyranoside”) are added 50 mL of anhydrous acetone. 31 mL (5.0 eq; 249.05 mmol) of 2,2-dimethoxypropane are then added, followed by 0.430 g (0.05 eq; 2.49 mmol) of paratoluenesulfonic acid. After one hour of reaction, compound 2 is obtained; it is not isolated and is used as is in the following reaction. R / : 0.83 (AcOEt / cyclohexane (8:2)) ESI+m / z:367 [M+H]+ SMHR: 367.0765 Da; Conf (%): 100.00%

[0077] “2-Bromoethyl 2.3-O-Isopropylidene-aD-mannopyranoside” (3) Following the previous reaction, 10 mL of mQ water are added. After 5h30, the medium is neutralized with NaHCO3 (sodium bicarbonate), the organic phase is dried and evaporated. The crude product is purified on a silica gel column, using the eluent AcOEt / cyclohexane (1:9). Compound 3 is obtained with a yield of 66% over 2 steps. R / : 0.43 (AcOEt / cyclohexane (8:2)) ESI+m / z:327 [M+H]+ SMHR: 327.0439 Da; Conf (%): 100.00%

[0078] “2.3-O-Isopropvlidene-aD-manno-hexodialdo-L5-pvranoside of 2-bromoethyl” ( 41 2.2 g (1.0 eq; 6.72 mmol) of compound 3 and 0.105 g (0.1 eq; 0.67 mmol) of TEMPO ((2,2,6,6-Tetramethylpiperidin-l-yl)oxy) are weighed and conditioned under an inert atmosphere then 100 mL of anhydrous dichloromethane are added. 1.496 g (1.0 eq; 6.72 mmol) of TCCA (trichloroisocyanuric acid) are added at 0°C. Once the reaction is complete, 10 mL of anhydrous methanol are added and the medium is filtered through Celite. After evaporation, a white foam is obtained. Product 4 is not isolated and is used as such during the next reaction. R / : 0.54 (AcOEt / cyclohexane (8:2))

[0079] “(6E)-2.3-O-Isopropylidene-6.7-dideoxy-7-diethoxyphosphinyl-qD-manno-hept-6 2-bromoethyl-enopyranoside » (5) 0.269 g (1.0 eq; 6.72 mmol) of 60% NaH (sodium hydride) are packaged under an inert atmosphere. 100 mL of anhydrous THF (tetrahydrofuran) are added to form a suspension. 10 minutes later, 1.67 mL (1.0 eq; 6.72 mmol) of tetraethyl methylenediphosphonate (MDPTE) is added to the solution, with stirring. After 30 minutes, the anion solution is cooled to 0°C and then cannulated onto the crude compound 4 previously obtained. At the end of the reaction, the medium is diluted in 300 mL of ethyl acetate and washed with brine and then with distilled water. The organic phase is dried and then filtered before being evaporated to dryness. The crude product is purified on a silica gel column using the eluent AcOEt / cyclohexane (8:2). The fractions are combined and allow compound 5 to be obtained with a yield of 51% over 2 steps. R / : 0.18 (AcOEt / cyclohexane (8:2)) SMHR: 459.0786 Da; Conf (%): 100.00%

[0080] “2.3-O-Isopropylidene-6-deoxy-6-diethoxyphosphinylmethylene-aD-mannopyran 2-bromoethyl ester" (6) 1.250 g (1.0 eq; 2.72 mmol) of compound 5 and 0.125 g (10% wt) of Pd / C are packaged under an inert atmosphere. 27 mL of anhydrous methanol are added to dissolve compound 5 and then 2.17 mL (5.0 eq; 13.61 mmol) of triethylsilane are added dropwise. After reaction, the medium is filtered through Celite and the solvent is evaporated. 1.200 g of compound 6 are finally obtained, i.e. a yield of 96%. Rf: 0.23 (AcOEt) SMHR: 461.0924 Da; Conf (%): 100.00%

[0081] “2.3-O-Isopropylidene-6-deoxy-6-diethoxyphosphinylmethylene-aD-mannopyran 2-phthalimidooxyethyl oside » 17) 0.469 g (1.1 eq; 2.87 mmol) of A-hydroxyphthalimide are weighed and conditioned under an inert atmosphere, then 5 mL of anhydrous THF are added. 0.470 mL (1.2 eq; 3.14 mmol) of DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) are added and the solution is brought to 45°C. 30 minutes later, 1.2 g (1.0 eq; 2.61 mmol) of intermediate 6 dissolved in 8 mL of THF are added dropwise onto the anion. After 22 hours of reaction, the reaction medium is diluted in dichloromethane and then washed with brine. The organic phase is dried over magnesium sulfate (MgSO4) and then evaporated. The crude product obtained is purified on a silica gel column with ethyl acetate (AcOEt). 0.915 g of compound 7 are obtained for a yield of 64%. R / : (CH2Cl2 / ethyl ether 95 / 5 (v / v)) SMHR: 544.1946 Da; Conf (%): 100.00%

[0082] “2.3-O-Isopropylidene-4-O-dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylm 2-(phthalimidooxy)ethyl ethylene-aD-mannopyranoside » (8) 0.915 g (1.0 eq; 1.69 mmol) of compound 7 and 1.296 g (4.0; 6.74 mmol) of 5-(Benzylthio)-1 H-tetrazole are weighed, conditioned under an inert atmosphere and dissolved in 35 mL of dichloromethane. The solution is cooled before adding dropwise 0.724 mL (2.0 eq; 3.37 mmol) of dimethyl A,A-diisopropylphosphoramidite. At the end of the reaction, 0.670 mL (13.0 eq; 21.8 mmol) of 30% H2O2 (hydrogen peroxide) is directly added to the solution at room temperature. After 10 min, the The medium is diluted in dichloromethane and then washed twice with NaHCO3. The organic phase is dried over MgSO4 and then concentrated. Purification on a silica gel column is carried out with AcOEt. After evaporation of the fractions, 0.953 g of compound 8 is obtained. The yield is 87%. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) SMHR: 652.1922 Da; Conf (%): 100.00%

[0083] “4-O-dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylmethylene-qD-mannop 2-(phthalimidooxy)ethyl yranoside » (9) 0.953 g (1.0 eq; 1.46 mmol) of compound 8 are weighed and then 7.3 mL of THF are added. 1.625 g of Dowex H+ resin are then introduced into the reaction medium. After 18 hours, the reaction is complete and the resin is removed by filtration. The filtrate after evaporation allows compound 9 to be obtained with a yield of 34%. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) SMHR: 612.1610 Da; Conf (%): 100.00%

[0084] “4-O-dihydroxyphosphinyl-6-deoxy-6-dihydroxyphosphinylmethylene-aD-manno 2-aminooxyethyl pyranoside » fProAMFAl) 0.300 g (1.0 eq; 0.49 mmol) of compound 9 are conditioned in the presence of 0.882 g (12.0 eq; 5.89 mmol) of sodium iodide (NaI) and then 1.1 mL of anhydrous acetonitrile, 0.55 mL (9.0 eq; 3.93 mmol) of triethylamine and 0.745 mL (12.0 eq; 5.89 mmol) of trimethylsilyl chloride (TMSC1) are added. The solution is stirred at room temperature under stirring and under an inert atmosphere for 24 h then the solvents are evaporated. The residue is taken up in methanol and the excess salts are removed by filtration. The filtrate is treated with a cation exchange resin (DOWEX® 50WX2, in the H+ form) then the resin is removed by filtration and a solution of 0.337 mL (15.0 eq; 7.37 mmol) of hydrazine monohydrate diluted in 2.5 mL of methanol is added to the filtrate. After reaction and evaporation to dryness of the reaction medium, the latter is purified on a 100 C[8] silica gel column (eluent: H2O). 68 mg of ProAMFAl were collected.The yield over 2 stages is 35%. Rf: 0.69 (2-Propanol / NH4OH / H2O 30 / 40 / 30 (v / v)) SMHR: 398.0607 Da; Conf (%): 100.00% [q]D20: + 32.5° (4 mg per 1 mL of H2O)

[0085] Preparation of the AMFA1 compound The compound AMFA1 is an analogue of mannose 6-phosphate (M6P). It is synthesized according to a protocol described in EP 2 448 600 B1 and has the following structural formula:

[0086] [Chem. 12] The difference between ProAMFAl and AMFA1 lies in the definition of the PI radical which represents the phosphate group P(O)(OH)2 in ProAMFAl versus the hydrogen atom in AMFA1.

[0087] Mannose 6-phosphate compound (M6P) The compound mannose 6-phosphate (M6P) is purchased commercially and has the following structural formula:

[0088] [Chem. 13] 'OH

[0089] Example 2: Preparation of the conjugates of the invention of formula (II)

[0090] Two products of interest Y, namely a lysosomal enzyme and an antibody, were respectively functionalized with the compounds Pro-AMFAl or AMFA1.

[0091] The lysosomal enzyme tested is commercially available under the name Myozyme®. It is the enzyme alpha alglucosidase (or acid alpha glucosidase). It can be represented in the following by "Myo". It contains in its structure M6P residues allowing the targeting of RM6P-CI. The lysosomal enzyme Myo is used for the treatment of Pompe disease by enzyme replacement therapy. The antibody tested is infliximab, a chimeric monoclonal antibody of the IgG1 type that does not have M6P residues in its structure. It can be represented in the following as "Infli".

[0092] The conjugates thus formed can indifferently be represented in the present application by “AMFAl-Myo” or “Myo-AMFAl”, “ProAMFAl-Myo” or “Myo-ProAMFAl”, “AMFAl-Infli” or “Infli-AMFAl”, “ProAMFAl-Infli” or “Infli-ProAMFAl”. The conjugates of the invention are those which comprise the ProAMFAl part. Advantageously according to the invention the ProAMFAl part allows the conjugate, once introduced into an organism, not to be immediately recognized by the receptor RM6P-CI, which thus allows the specific targeting of a predetermined biological target (membrane or non-membrane).

[0093] Coupling and quantification of the AMFA1 and ProAMFAl compounds with a product of interest Y (Mvo or Infliximab) The coupling of AMFA1 or ProAMFAl with a product of interest Y (Myo or Infliximab) is achieved thanks to the ethyloxyamine function present on the spacer arm of AMFA1 or ProAMFAl, which allows the formation of a covalent bond of the oxime type with the aldehyde functions previously generated by controlled oxidation on the oligosaccharide chains of the product of interest Y (Myo or Infliximab). The coupling of a product of interest Y comprising an aldehyde function (previously generated by oxidation) with ProAMFAl of formula (I) (or with AMFA1) comprising a spacer arm with an ethyloxyamine function (L= -(CH2)2- and Li= -ONH2) is notably illustrated in [Fig.3] point 1 / . Coupling of AMFA1 or ProAMFAl with Myozyme^ First, the Myozyme® enzyme is oxidized at the level of its oligosaccharide chains. To do this, the Myozyme® is filtered through Sepharose G25 before being oxidized with 1 mM sodium metaperiodate for 30 min at 4°C. The addition of glycerol (20 μL per mL) for 10 min at 4°C stops the oxidation. The enzyme solution is then filtered through Sepharose G25 and the ProAMFAl or AMFA1 compound is added with a 100-fold molar excess. After 2 h at 37°C, the Myozyme® is coupled and the excess of uncoupled ProAMFA 1 or AMFA1 present in the solution is removed by dialysis on buffer containing, for 1 L: 2% mannitol, 3 mM Na2HPO4, 22.1 mM NaH2PO4 and 0.005% polysorbate 80. The Myo-AMFAl and Myo-ProAMFAl conjugates thus obtained are stored in this buffer at 4°C. Quantification MALDLTOF mass spectrometry analysis estimated the number of ProAMFAl or AMFAl coupled to the Myozyme® enzyme to be approximately 1. Coupling of AMFA1 or ProAMFAl with infliximab First, the infliximab antibody is oxidized at the level of its oligosaccharide chains. To do this, infliximab (5 mg) previously filtered on Sepharose G25 is oxidized by 1 mM sodium metaperiodate for 30 min at 4°C. The addition of glycerol (20 μL per mL) for 10 min at 4°C stops the oxidation. The antibody solution is then filtered through G25 sepharose and the ProAMFAl or AMFA1 compound is added with a 200-fold molar excess. After 2 h at 37°C, infliximab is coupled and the excess uncoupled ProAMFAl or AMFA1 present in the solution is removed by dialysis on buffer containing, for 1 L: 2% mannitol, 3 mM Na2HPO4, 22.1 mM NaH2PO4 and 0.005% polysorbate 80. The Infli-AMFAl and Infli-ProAMFAl conjugates thus obtained are stored in this buffer at 4°C. Quantification MALDLTOF mass spectrometry analysis estimated the number of ProAMFAl or AMFAl coupled to the infliximab antibody to be approximately 5.

[0094] Characterization of the conjugates obtained by Coomassie blue staining The integrity of the product of interest (Myozyme® or infliximab) after coupling with AMFA1 or ProAMFAl is verified by electrophoresis on a 12% polyacrylamide-SDS gel followed by Coomassie blue staining. After migration, the gel is stained for 1 h with a Coomassie blue solution for labeling the product of interest. It is indeed important to ensure that the coupling between the product of interest and the pro-ligand has not caused degradation. Myozyme®, Myo-AMFAl and Myo-ProAMFAl samples were loaded onto polyacrylamide gel under denaturing conditions and followed by Coomassie blue staining. The results obtained are illustrated in [Fig.5]. It can be concluded that the coupling did not cause any degradation since the same characteristic bands of the 110 kDa enzyme are observed before and after coupling. Similarly, samples of Infliximab, Infli-AMFAl and Infli-ProAMFAl were loaded onto polyacrylamide gel under denaturing conditions and followed by Coomassie blue staining. The results obtained are illustrated in [Fig.6]. It can be concluded that the coupling did not cause any degradation since the same characteristic bands of the 50 kDa and 25 kDa antibody are observed before and after coupling.

[0095] Example 3: Biological evaluation of pro-ligands (I)

[0096] Evaluation of the cytotoxicity of ProAMFAl The cytotoxicity of ProAMFAl of formula (I) was evaluated in comparison with that of M6P and AMFAl on human MCF-7 breast cancer cells ([Fig.7]) and on healthy human fibroblasts ([Fig.8]) according to the protocol described below. Experimental protocol: Human breast cancer cells (MCF-7) and human fibroblasts (FS01035) were cultured in DMEM / F12 and DMEM medium supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin, respectively. The cells were maintained at 37°C in the presence of 5% CO2. After trypsin treatment, the cells were seeded in 96-well plates at 20% confluence for MCF-7 cells and 40% for fibroblasts. The next day, adherent cells were treated. Treatments with the compounds M6P, AMFA1 and ProAMFAl were carried out. at concentrations between 10 4 and 107 M (Figures 7 and 8), while treatments with coupled or native enzymes are done at 100, 500 and 1000 ng / mL (Figures 11 and 12). Each condition is analyzed in triplicate. After 72 h of incubation, cell viability is assessed by an assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

[0097] The results concerning the study of cytotoxicity are illustrated in [Fig.7] for breast cancer cells and in [Fig.8] for fibroblasts. It is evident from these figures that the ProAMFAl compound does not exhibit significant toxicity during a 72-h treatment up to a concentration of 10 4 M on the selected cell models. The AMFA1 compound is also non-toxic compared to the natural M6P compound.

[0098] Affinity of ProAMFAl for RM6P-CI ProAMFAl was evaluated for its possible affinity for the RM6P-CI receptor in comparison with AMFAl and M6P. As already mentioned, affinity refers to the intensity of interaction of the ligand, e.g. AMFAl, with its receptor, namely RM6P-CI. To do this, a test based on interaction competition with RM6P-CI is performed according to the protocol described below. Experimental protocol: Affinity assays are performed using biotinylated RM6P-CI (RM6P-CIb) that recognizes pentamannose 6-phosphate (PMP) pre-adsorbed onto MaxiSorp™ 96-well plates. Before being dispensed into the wells, RM6P-CIb is pre-incubated for 2 h in the presence of M6P, AMFA1 or ProAMFAl at different concentrations. The pre-incubated solutions are then dispensed into the wells containing PMP. After 2 h at room temperature, the amount of RM6P-CIb bound to PMP is determined using a peroxidase-coupled streptavidin solution in the presence of OPD (o-phenylenediamine) substrate and by optical density measurement at 450 nm. The results obtained are illustrated in [Fig.9] and in Table 7 below. Table 7 indicates the necessary concentrations of M6P, AMFA1 and ProAMFAl to have a 50% interaction with RM6P-CI (IC50).

[0099] [Tables7] Compounds IC50 (M) M6P 2.4 x 105 AMF Al 4.5 x 105 ProAMFAl > 1.0 x 103

[0100] It is clear from [Fig.9] and Table 7 that ProAMFAl has no affinity for the RM6P-CI. This very interesting characteristic allows us to validate its potential for pro-ligand. The lack of affinity of ProAMFAl towards RM6P-CI makes it possible to avoid cellular internalization by these receptors of the active ingredients with which ProAMFAl will be associated (such as a product of interest and possibly a biological target).

[0101] Activation of ProAMFAl in AMFA1 To verify its therapeutic potential as a pro-ligand, the ability of ProAMFAl to be devoid of its PI = phosphate group (P(O)(OH)2) was studied. A kinetic dephosphorylation study was performed by incubating ProAMFAl in the presence of an alkaline phosphatase isolated from human placenta and followed by 31P NMR analysis according to the protocol described below. ProAMFAl alkaline phosphatase enzymatic digestion test An aliquot of 2.3 mg of ProAMFAl is dissolved in 200 pL of D2O and 300 pL of 50 mM Tris buffer pH 7.4. A first analysis of the sample placed in the NMR tube is carried out at 37°C using a Bruker AVANCE 400 MHz spectrometer with the following parameters: 162 MHz, ns=16, Di=2s, pulse width: 15°, 310 K. An aliquot of 2.3 mg of alkaline phosphatase isolated from human placenta is dissolved in 100 pL of 50 mM Tris pH 7.4 and then added to the NMR tube. The indicated parameters are used for a kinetic study of the enzymatic digestion. The kinetics of conversion of ProAMFAl to AMFA1 could be established after integration of the different signals observed in NMR. The results obtained are illustrated in [Fig. 10]. As shown in the spectra series in this figure, the decrease in the phosphate signal of ProAMFAl in favor of the appearance of mineral phosphate (formed during the hydrolysis reaction by phosphatase) is observed in the presence of phosphatase. At the same time, the 31P signal of the phosphonate of ProAMFAl disappears in favor of the 31P signal of the phosphonate of AMFAl. The formation of AMFAl was confirmed by the addition of a known amount of AMFAl resulting in the increase of the specific signal of AMFAl. In the test, a sample of ProAMFAl with a purity of 87% was used. The by-product present is ProAMFAl-oxime (ProAMFAlox), which comes from the reaction between ProAMFAl and acetone. ProAMFAlox has the following structural formula:

[0102] [Chem. 14] It is also evident from the spectra series in Figure 10 that alkaline phosphatase similarly hydrolyzes the phosphate of ProAMFAlox leading to the formation of the ligand AMFAl-oxime (AMFAlox).

[0103] Example 4: Biological evaluation of conjugates (II)

[0104] Evaluation of the cytotoxicity of the Mvo-ProAMFAl conjugate The cytotoxicity of the Myo-ProAMFAl conjugate of the invention and the Myo-AMFA1 conjugate was evaluated in comparison with the lysosomal enzyme Myo alone, on human MCF-7 breast cancer cells ([Fig. 11]) and on healthy human fibroblasts ([Fig. 12]) according to the same protocol as that described in Example 3.

[0105] The results illustrated in Figures 11 and 12 demonstrate that the Myo-ProAMFAl and Myo-AMFAl conjugates do not exhibit significant cytotoxicity in the chosen cell models compared to Myozyme® or compared to untreated control cells (100% ± 10%).

[0106] Affinity of the Infli-ProAMFAl conjugate for RM6P-CI The Infli-ProAMFAl conjugate of the invention and the Infli-AMFAl conjugate were evaluated for their affinity for the RM6P-CI receptor according to the same protocol as described in Example 3. The results obtained are illustrated in [Fig. 13] and in Table 8 below. Table 8 indicates the necessary concentrations of M6P and Infli-AMFAl to have a 50% interaction with RM6P-CI (IC50). The concentrations of Infliximab and Infli-ProAMFAl could not be determined in the concentration range tested and are greater than 105 M.

[0107] [Tables8] Compounds IC50 (M) M6P 7.0 x 106 Infliximab not measurable (>105) Infli-AMFAl 1.0 x 106 Infli-ProAMFAl not measurable (>105)

[0108] The affinity test for RM6P-CI of the Infli-ProAMFAl conjugate shows that said Infli-ProAMFAl conjugate has a very low affinity for RM6P-CI ([Fig. 13] and Table 8). This affinity is comparable to that of the infliximab antibody alone, whereas the Infli-AMFAl conjugate has a high affinity for RM6P-CI.

[0109] Activation of Myo-ProAMFAl conjugate to Myo-AMFAl conjugate In order to verify its therapeutic potential and its ability to be activated, the transformation of the Myo-ProAMFAl conjugate, presenting a PI = phosphate group, into Myo-AMFAl conjugate, presenting a PI = H group, was studied. A kinetic study of dephosphorylation was performed by incubating Myo- ProAMFAl in the presence of an alkaline phosphatase isolated from human placenta according to the protocol described below. Protocol for enzymatic digestion by alkaline phosphatase of Myo-AMFA1 and Myo-ProAMFAl conjugates The Myo-ProAMFAl conjugate is incubated with alkaline phosphatase at 37°C. After 1 h, 24 h and 96 h the reaction is stopped in ice and the samples are prepared for analysis by Western blotting using a SDS-PAGE polyacrylamide gel and are compared to the enzyme alone (Myozyme®) and the Myo-AMFAl conjugate. After transfer to a membrane, the fraction of Myo-ProAMFAl that has become Myo-AMFAl is characterized using a specific antibody against AMFAl followed by a secondary antibody anti-rabbit IgG conjugated to peroxidase. The membranes are then revealed by a chemiluminescent substrate. To check the quantities of enzymes present on the membrane, the enzyme is recognized by a rabbit antibody specific to said enzyme (Anti-Myo), then by a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase and followed by chemiluminescent revelation. The results obtained are illustrated in [Fig. 14]. Over time, treatment of the Myo-ProAMFAl conjugate with phosphatase induced an increase in signal intensity at 110 kDa corresponding to Myo-AMFAl recognized by the anti-AMFAl antibody. This result demonstrates the ability of the Myo-ProAMFAl conjugate to be dephosphorylated to Myo-AMFAl in the presence of alkaline phosphatase.

[0110] The present disclosure is not limited to the examples described above, only by way of example, but it encompasses all the variants that a person skilled in the art may envisage within the framework of the protection sought. List of cited documents Patent documents

[0111] For all useful purposes, the following patent documents are cited: - patcitl: EP 2 448 600 B1; -patcitl: EP3 350 192 B1.

Claims

Claims

1. Pro-ligand characterized in that it has the following general formula (I) [Cheml] (I) x—^ o J GA y Pi / m 1 A—LL, in which: X represents -OH; -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; =CH-CO2Z; =CH-P(O)(OZ)2; -CHF-CO2Z; -CHF-P(O)(OZ)2; -CF2 -CO2Z; -CF2-P(O)(OZ)2; =CF-CO2Z; =CF-P(O)(OZ)2; -CH(CO2Z)(P(O)(OZ)2 ); with Z representing independently of each other H; Na; K or NH4; PI, P2 and P3 representing independently of each other H; P(O)(OZ)2 ; -S(O)2(OZ); with Z as defined above and with the condition that at least one of P1, P2 and P3 is other than a hydrogen atom; or P2 and P3 together forming an acetonide group [Chem.2] A represents a divalent radical selected from -0-; -S-; -NH-; -CH2-; L represents oo -(CH2)2-(O-CH2-CH2)n- with n representing an integer from 0 to 6; oo -H; -NH2; -(CH2)nrCH=CH2 or -(CH2)nrC=CH with ni representing an integer from 0 to 4, which means that in each of these cases Li is absent; oo a saturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 1 to 30 carbon atoms; an unsaturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 2 to 30 carbon atoms; oo a saturated or unsaturated divalent hydrocarbon radical as defined above in which one or more -CH2-, -CH=CH- and / or -C=C- groups of the saturated or unsaturated hydrocarbon radical is (are) replaced inde-. hanging from each other by an -O- group; -S- ; -CO-NH- ; -NH-CO-O- ; -NH- ; -NRr with Ri representing a C1-C5 alkyl; -ON=CH- ; -CO-NH-N=CH- ; a cyclic or heterocyclic system, saturated or unsaturated, substituted or not; Li represents 00 -O-NH2 ; 00 a cyclic or heterocyclic system chosen from [Chem. 3] i U .on- with Ri as defined above, and is preferably ethyl; [Chem. 4] [Chem. 5] [Chem. 6] [Chem. 7] [Chem. 8] O

2. oo -ON=C(CH3)2; oo -(CH2)nrCH=CH2; -(CH2)nrC=CH; -(CH2)nrN3; -(CH2)nl-SH; -(CH2)nl-NH2; -(CH2)nl-N=C=O; -(CH2)nl-N=C=S; -(CH2)nl-NHR1; -(CH2)nl-ArNH2; -(CH2)ni-ArNHRi ; -(CH2)nl-NHCO-CH2Hal; -(CH2)nl-COZ1; -(CH2)nrAiCOZi; -(CH2)nl -ON=CH2; -(CH2)nl-CO-NH-N=CH2 ; with nor as defined above; Ri representing a C1-C5 alkyl; Ai representing -O-; -NH-; Hal representing Cl; Br or I; Zi representing -OH; -ORj; -NHRi; -NH-NH2; -NH-NHRi with Ri as defined above; oo a halogen chosen from F, Cl, Br or I. Pro-ligand according to claim 1, characterized in that L represents: oo a saturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 1 to 10 carbon atoms; an unsaturated, linear or branched, substituted or unsubstituted divalent hydrocarbon radical having from 2 to 10 carbon atoms; a saturated or unsaturated divalent hydrocarbon radical as defined above, of which one or more -CH2-, -CH=CH- and / or -C=C- groups of the saturated or unsaturated hydrocarbon radical is (are) replaced independently of one another by: 000 a group -O-; -NH-; -S-; -CO-NH-; -NH-CO-O-; and / or 000 a cyclic or heterocyclic system chosen from those listed in Table 1 below [Table 1] V ..... ^ 1 1_ / x_ uAj > X s "K" \\ <. C-.-.-.v / r""' y—? X >■ V_4 "T >1 H 1 )■.....? / $ > ■?.

3.

4. Pro-ligand according to claim 1 characterized in that: X represents -OH; -CH2-CO2Z; -CH2-P(O)(OZ)2; -CH(CO2Z)2; -CH(P(O)(OZ)2)2 with Z as defined in claim 1, and preferably Z equal to H; PI, P2 and P3 independently representing H or P(O)(OZ)2 with Z as defined in claim 1, and preferably Z equal to H, with the condition that at least one of PI, P2 and P3 is other than a hydrogen atom; or P2 and P3 together forming an acetonide group of formula [Chem2] as defined in claim 1; A represents oxygen -O-; L represents -(CH2)2-(O-CH2-CH2)n- with n representing an integer ranging from 0 to 6, and preferably n equal to 0; Li represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined in claim 1. Conjugate characterized in that it has the following general formula (II): [Chem.9] / .. P® X / X.....\ O" \ (II) in which n2 is an integer ranging from 1 to 1000, preferably from 1 to 20, and more preferably from 1 to 10, PI, P2, P3, X, A and L are as defined in any one of claims 1 to 3, L'i represents the substituent Li as defined in any one of claims 1 to 3 when said Li is involved in a covalent bond with Yb Yi represents a product of interest Y, said product of interest Y being chosen from the group comprising proteins, in particular antibodies and lysosomal enzymes, nanoparticles, protein activators or inhibitors, cytotoxic compounds and markers for medical imaging, Yi forming n2 covalent bond(s) with L'b

5. Complex consisting of: - a conjugate of formula (II) as defined in claim 4, - a non-membrane or membrane target, said target being chosen from the group comprising proteins, in particular receptors, autoantibodies, glycoproteins, protein aggregates, protein or non-protein antigens, activators or inhibitors of proteins of cells, viruses, bacteria, bone tissue, collagen and fiber aggregates, the conjugate (II) and the target being linked to each other by a non-covalent or covalent type bond.

6. A conjugate as defined in claim 4, for use as a medicament for the intracellular degradation of a non-membrane or membrane target, said target being as defined in claim 5.

7. Conjugate as defined in claim 4, for use as a medicament in the treatment of pathologies chosen from the group comprising neurodegenerative diseases, cancers, autoimmune diseases, rare diseases, inflammatory diseases and infectious diseases.

8. A conjugate as defined in claim 4, for use as a gene therapy medicament.

9. A conjugate for use according to any one of claims 6 to 8, characterized in that the conjugate is in a form suitable for parenteral, intravenous or subcutaneous administration.

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