Pro-ligands in the form of analogues of mannose 6-phosphate or mannose, conjugates comprising said pro-ligands, and uses thereof for therapeutic applications

Abstract

The invention relates to novel compounds, referred to as pro-ligands or inactivated ligands, of formula (I). The invention also relates to conjugates comprising such pro-ligands, said conjugates corresponding to formula (II). The invention further relates to said conjugates (II) for use as a drug.

Description

Description Title: Pro-ligands, conjugates comprising said pro-ligands 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 analogues of mannose 6-phosphate (M6P) rendered temporarily inactive. Mannose 6-phosphate (M6P) analogs refer to compounds that differ from natural M6P but exhibit high recognition by the cation-independent mannose 6-phosphate receptor (CM6P-CI). The M6P analogs are called “AMFA” (“Synthetic Analogues of Mannose 6-Phosphate Functionalized in Anomer Position”). The pro-ligands obtained after inactivation of the AMFA analogs are called in the present application “ProAMFA”. The ProAMFA pro-ligands are thus no longer recognized by the RM6P-CI.

[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] EP 2 448 600 B1 and EP 3 350 192 B1 describe conjugates comprising M6P analogues which are linked, via a spacer arm, to a product of interest, such as a glycoprotein. M6P analogues have several advantages for the functionalization of glycoproteins, in particular for the functionalization of lysosomal enzymes, in particular because they are small in size, are poorly immunogenic and can be easily linked with the lysosomal enzyme. These M6P analogues 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 bond desired with the product of interest. In these documents, the M6P analogues 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. The M6P analogues are also modular at the level of the phosphate group in position 6 which can be replaced by a bioisostere group, namely a group which fulfills the same biological functions as the phosphate group. The phosphate group in position 6 can for example be replaced by a phosphonate, carboxylate, malonate group.Several publications indicate that derivatives substituted in position 6 of M6P possess significant affinities for RM6P-CI (Vidil C. et al., Eur.J. Org. Chem., 1999, 447; Jeanjean A. et al., Bioorg. Med. Chem. Lett., 2008, 18, 6240; El Cheikh K. et al., Angew. Chem. Int. Ed., 2016, 55, 14774).

[0005] In 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 mannose 6-phosphate cation-independent (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 (Ghosh P. et al., Nat. Rev. Mol. Cell. Biol., 2003, 4, 202; Gauthier C. et al. J. Control. Release 2024, 365, 759).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 rapidly recognized by the RM6P-CI present in the immediate environment. The product of interest will thus undergo a reduction in its free circulation to reach and bind 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 Kleeb et al. (Journal of Medicinal Chemistry, 2016, 59, 3163) paper describes the addition of a phosphate group at position 2, 3, or 4 of the mannose ring to improve the solubility and bioavailability of mannose derivatives. However, this paper never considers using the resulting mannose phosphate derivatives to prevent premature recognition of a ligand by its receptor.

[0007] Thus, to the knowledge of the Inventors, a Proligand has never been described to date which does not have an affinity for mannose 6-phosphate receptors or even the in situ activation of said Pro-ligand by a phosphatase.

[0008] 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 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, namely RM6P-CI, 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, namely RM6P-CI, and fixed by the latter.

[0009] The destination and activity of “Ligand - Product of interest” conjugates or “Ligand - Product of interest - Target” complexes have been the subject of several publications in the prior art. Thus, in the case of coupling of Ligand to a lysosomal enzyme, it has been demonstrated that Ligand allows for efficient targeting of this enzyme in lysosomes thanks to internalization by RM6P-CI (Basile I. et al. J. Control. Release, 2018, 269, 15; Godefroy A. et al. J. Cell. Mol. Med., 2019, 23, 6499). In the case of Ligand coupling to an antibody targeting a therapeutic target, entry of the antibody and its target via RM6P-CI has been demonstrated for several antibodies directed against: - soluble extracellular targets (non-membrane targets) such as tumor necrosis factor alpha (TNFa) or vascular endothelial growth factor (VEGF) (Daurat M. et al., Front. Immunol. 2024, 15, 1273280) or, - membrane targets such as human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor 1 (EGFR) (Gauthier et al., Biomed. Pharmacother. 2024, 175, 116707). Furthermore, when the product of interest is an antibody, it has been demonstrated in a mouse model that the internalized “Ligand-Product of Interest” conjugates were recycled into the circulation (Gauthier C. et al., J. Control. Release 2023, 358, 465). In the case of Ligand coupling to nanoparticles for therapeutic purposes, the entry of nanoparticles via RM6P-CI has been demonstrated in the endolysosomal system (Vaillant O. et al., Angewandte Chemie, 2015, 54, 5952; Bouffard et al., Int J Mol Soi., 2019, 20, 2809; Daurat et al., Biomater. Soi., 2020, 8, 3678). On the other hand, it has also been shown by the Inventors that a family of RM6P-CI Ligands was capable of binding to hydroxyapatite which is present in bone tissue (French patent application FR2302097), which made them promising compounds for the treatment of bone pathologies.

[0010] The development of “Pro-ligand - Product of interest” type conjugates and their capacity to bind in situ to a target (membrane or non-membrane type) and then to transform in situ into activated “Ligand - Product of interest - Target” type complexes constitutes the originality and interest of the present invention in view of the promising activity of certain of the “Ligand - Product of interest” and / or “Ligand - Product of interest - Target” compounds described in the prior art cited above. Summary

[0011] According to a first aspect, the present invention relates to a pro-ligand of general formula (I) as described in the detailed description below.

[0012] 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”.

[0013] According to a third aspect, the invention relates to the process for preparing the pro-ligands of formula (I) and the conjugates of formula (II).

[0014] According to another aspect, the present invention relates to a conjugate of formula (II) for use as a medicament.

[0015] According to another aspect, the invention describes a complex of structure “Pro-ligand - Product of interest - Biological target”. Brief description of the drawings

[0016] Other features, details and advantages will become apparent upon reading the detailed description below, and upon analyzing the attached drawings, in which: Fig. 1

[0017] [Fig. 1] 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

[0018] [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. Fig. 3

[0019] [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

[0020] [Fig. 4] illustrates the detailed synthesis scheme of the pro-ligand of general formula (I) called ProAMFAI. Fig. 5

[0021] [Fig. 5] illustrates the results obtained by SDS-PAGE electrophoresis of the commercial Myozyme® enzyme alone or bound to the AMFA1 compound (obtaining the Myo-AMFA1 conjugate) or to the ProAMFAI pro-ligand (obtaining the Myo-ProAMFA1 conjugate). The enzyme alone or bound to the AMFA1 or ProAMFAI compounds is detected by Coomassie blue staining. Fig. 6

[0022] [Fig. 6] illustrates the results obtained by SDS-PAGE electrophoresis of the Infliximab antibody alone or bound to the AMFA1 compound (obtaining the Infli-AMFA1 conjugate) or to the Pro-AMFA1 pro-ligand (obtaining the Infli-ProAMFAI conjugate). The antibody alone or bound to the AMFA1 or ProAMFAI compounds is detected by Coomassie blue staining. Fig. 7

[0023] [Fig. 7] illustrates the cytotoxicity on human MCF-7 breast cancer cells of the pro-ligand ProAMFAI in comparison with the compound M6P and the compound AMFA1. The x-axis represents the molar concentration of the compounds M6P (white histogram), AMFA1 (hatched histogram) and ProAMFAI (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

[0024] [Fig. 8] illustrates the cytotoxicity on human FS01035 fibroblasts of ProAMFAI in comparison with M6P and AMFA1. The description of the axes and histograms in Figure 7 applies to Figure 8. Fig. 9

[0025] [Fig. 9] illustrates the interactions between RM6P-CI and the pro-ligand ProAMFAI in comparison with the compounds M6P and AMFA1. The x-axis represents the molar concentration of the compounds M6P (dotted curve), AMFA1 (dashed curve) and ProAMFAI (solid curve). The y-axis represents the percentage of binding of these compounds to RM6P-CI. Fig. 10

[0026] [Fig. 10] is a series of NMR spectra at different times (t = 0, t = 1 h, t = 11 h, t = 24 h) showing the kinetics of the degradation of two pro-ligands ProAMFAI and ProAMFAI ox into ligands AMFA1 and AMFAIox, in the presence of an alkaline phosphatase isolated from human placenta. The difference between ProAMFAI and ProAMFAI ox lies in the Li radical which represents -O- NH2 in ProAMFAI versus -ON=C(CH3)2 in ProAMFAI ox. 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 ProAMFAI and ProAMFAI ox. The peaks on the right of the spectra are characteristic of the P1 radical equal to phosphate (i.e. X = P(O)(OH)2) presented by each of ProAMFAI and ProAMFAI ox. Fig. 11

[0027] [Fig. 11] illustrates the cytotoxicity of the Myozyme® enzyme (white histogram) and the Myo-AMFA1 (hatched histogram) and Myo-ProAMFA1 (black histogram) conjugates on human MCF-7 breast cancer cells. The x-axis represents the molar concentration of the compounds and the y-axis represents the cell survival of the compounds. The cell survival observed in the presence of Myozyme® was taken as 100%. Fig. 12

[0028] [Fig. 12] illustrates the cytotoxicity, on human fibroblasts FS01035, of the enzyme Myozyme® (white histogram) and the conjugates Myo-AMFA1 (hatched histogram) and Myo-ProAMFA1 (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

[0029] [Fig. 13] illustrates the interactions between RM6P-CI and the compound M6P or between RM6P-CI and Ifliximab alone or bound to AMFA1 or ProAMFAI. The x-axis represents the molar concentration of the compounds M6P (cross curve), Infliximab (cross curve) and dotted line), lnfli-AMFA1 (dashed curve) and Infli-ProAMFAI (solid curve). The y-axis represents the percentage binding of these compounds to RM6P-CI. Fig. 14

[0030] [Fig. 14] illustrates the detection by Western blots of the Myozyme® enzyme, the Myo-AMFA1, Myo-ProAMFA1 and Myo-ProAMFA1 conjugates incubated in the presence of alkaline phosphatase isolated from human placenta, at different times (t = 1 h, t = 24h and t = 96h). Fig. 15

[0031] [Fig. 15] illustrates the detection by Western blots of the lnfli-AMFA1, Infli-ProAMFAI and Infli-ProAMFAI conjugates incubated or not in the presence of acid or alkaline phosphatases at different times. Fig. 16

[0032] [Fig. 16] illustrates the interactions between RM6P-CI and the Infli-ProAMFAI conjugate preincubated or not in the presence of alkaline phosphatases. Fig. 17

[0033] [Fig. 17] illustrates the detection by Western blots of the Infli-ProAMFAI conjugate incubated in the presence of human serum at different times and its quantification by densitometry of the AMFA1 signal. Fig. 18

[0034] [Fig. 18] illustrates the interactions between RM6P-CI and the M6P compound (dashed curve) or between RM6P-CI and the unincubated Infli-ProAMFAI conjugate (solid curve) or after 24 h incubation with human serum (dotted curve). The x-axis represents the molar concentration of the compounds. The y-axis represents the percentage inhibition of RM6P-CI binding by the compounds. Fig. 19

[0035] [Fig. 19] illustrates the results obtained by SDS-PAGE electrophoresis of the Cetuximab antibody alone or bound to the AMFA1 compound (obtaining the Cetux-AMFA1 conjugate) or to the ProAMFAI pro-ligand (obtaining the Cetux-ProAMFA1 conjugate). The antibody alone or bound to the AMFA1 or ProAMFAI compounds is detected by Coomassie blue staining. Fig. 20

[0036] [Fig. 20] illustrates the detection by Western blots of Cetux-ProAMFA1 conjugates after incubation or not in the presence of acid or alkaline phosphatases at different times in comparison with the Cetux-AMFA1 conjugate. Fig. 21

[0037] [Fig. 21] illustrates the internalization at 5 h in Hela cells of the Infli-ProAMFAI conjugate previously incubated or not in the presence of human serum for 48 h. Internalization is analyzed by confocal microscopy and by fluorescence quantification. Fig. 22

[0038] [Fig. 22] illustrates the internalization at 18 h in Hela cells of the Cetux-ProAMFAI conjugate previously incubated or not in the presence of human serum for 24 h. Cellular fluorescence is measured by flow cytometry. Fig. 23 [Fig. 23] illustrates the binding of the Infli-ProAMFAI conjugate and the Infliximab antibody to their antigen, TNFa, measured by ELISA. Fig. 24

[0039] [Fig. 24] illustrates the synthesis scheme of the pro-ligand of general formula (I) called ProAMFA1 acetonide-oxime. Fig. 25

[0040] [Fig. 25] illustrates the transformation of ProAMFA1 acetonide-oxime into AMFAI ox. Fig. 26

[0041] [Fig. 26] is a series of NMR spectra at different times (t = 0, t = 30 min, t = 1 h30, t = 2h30) showing the kinetics of the degradation of the pro-ligand ProAMFAIacetonide-oxime into the ligand AMFAIox. Detailed description

[0042] Pro-ligands of formula (I)

[0043] The subject of the present invention is a pro-ligand characterized in that it has the following general formula (I): [Cheml] in which: the dotted line represents a bond which is present or not; X represents -CH2-P(O)(OZ)2; -CH2-CO2Z ; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; -CHF-CO2Z ; -CHF-P(O)(OZ)2; -CF2-CO2Z ; -CF2-P(O)(OZ)2; -CH(CO2Z)(P(O)(OZ)2), in which case the bond represented by the dotted line is not present; or X represents =CH-CO2Z; =CH-P(O)(OZ)2; =CF-CO2Z; =CF-P(O)(OZ)2, in this case the bond represented by the dotted line is present; with Z representing independently of each other H; Na; K or NF; P1, P2 and P3 independently represent 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 form an acetonide group [Chem2] H3C CH3. A represents a divalent radical chosen from -O-; -S-; -NH-; -CH2-; L represents 00 -(CH2)2-(O-CH2-CH2) n - with n representing an integer ranging from 0 to 6; 00 -H ; -NH2; -(CH2) n i-CH=CH2 or -(CH2) n iC=CH with representing an integer ranging from 0 to 4, which means that in each of these cases L1 is absent; 00 a saturated, linear or branched divalent hydrocarbon radical having from 1 to 30 carbon atoms; an unsaturated, linear or branched divalent hydrocarbon radical having from 2 to 30 carbon atoms; 00 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] Li represents 00 -O-NH2 ; 00 a cyclic or heterocyclic system selected from [Chem3] nt a C1-C5 alkyl, and is preferably an ethyl; [Chem5] [Chem6] [Chem7] [ 00 -ON=C(CH3)2; 00 -(CH2)ni-CH=CH2; -(CH2) n iCCH; -(CH2)m-N3; -(CH2)m-SH; -(CH2) n i-NH2; -(CH2)ni-N=C=O; -(CH2)ni-N=C=S; -(CH2) n i-NHRi ; -(CH2) n i-Ai-NH2; -(CH2) n i-Ai-NHRi ; -(CH2)ni-NHCO-CH2Hal ; -(CH2) n i-COZi ; -(CH2) n i-AiCOZi ; -(CH2) n iO-NH2; -(CH2)ni-CO-NH-NH2; with ni and Ri as defined above; Ai representing -O-; -NH-; Hal representing Cl; Br or I; Z1 representing -OH; -OR1; -NHR1; -NH-NH2; -NH-NHR1 with Ri as defined above; 00 a halogen selected from F, Cl, Br or I.

[0044] Examples of a saturated, linear or branched divalent hydrocarbon radical having 1 to 30 carbon atoms include: -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) m -C(Ci-C7)2-(CH2) m ; -(CH2) m -C(Ci-C7)2-(CH2) m -CH(Ci-C7)-(CH2) m - ; -(CH2) m -C(Ci-C7)2-(CH2) m -C(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 Ci-C7 representing an alkyl having from 1 to 7 carbon atoms. Examples of Ci-C7 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 means 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(Ci-C7)-(CH2) m - ; -(CH2) m -CH(Ci-C7)-(CH2) m -CH=CH-CH(Ci-C7)-(CH2) m - ; -(CH2) m -CH(Ci-C7)-CH=CH-(CH2) m - ; -(CH2) m -CH(Ci-C7)-CH=CH-(CH2) m -CH=CH-(CH2) m - ; -(CH2) m -CH=CH-C(Ci-C7)2-(CH2) m - ; -(CH2) m-CH=CH-C(Ci-C7)2-(CH2) m -C(Ci-C7)2-(CH2) m -CH=CH ; -(CH2) m -CH=CH-C(Ci-C7)2-(CH2) m -CH(Ci-C7)-(CH2) m -CH=CH ; -C C- ; -(CH2) m -C C-(CH2) m - ; -(CH2) m -C C-(CH2) m -C=C-(CH2) m - ; -(CH2) m -CH=CH-CH(Ci-C7)-(CH2) m -C C-(CH2) m -CH(Ci-C7)- ; -(CH2) m -CH=CH-C(Ci-C7)2-(CH2) m -C C-(CH2) m -C(Ci-C7)2-(CH2) m -; -(CH2) m -C C-CH(Ci-C7)-(CH2) m -C C-(CH2) m -CH(Ci-C7)-(CH2) m ; -(CH2) m -C CC(Ci-C7)2-(CH2) m -CH=CH-(CH2) m -CH(Ci-C7)-; -(CH2) m -CH=CH-C(Ci-C7)2-(CH2) m -CH(Ci-C7)-(CH2) m -C C-CH=CH-(CH2) m -; avec m tel que défini précédemment.

[0045] According to one embodiment of the invention, the pro-ligand (I) as defined above is characterized in that: X represents -CH2-P(O)(OZ)2; CH2-CO2Z ; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; -CHF-CO2Z ; -CHF-P(O)(OZ)2; -CF2-CO2Z ; -CF2-P(O)(OZ)2; -CH(CO2Z)(P(O)(OZ)2); =CH-CO2Z; with Z as defined above; P1, P2 and P3 independently represent H or P(O)(OZ)2; 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 form an acetonide group of formula [Chem2] as defined above; A represents a divalent radical chosen from -O-; -S-; -CH2-; L represents: oo -(CH2)2-(O-CH2-CH2) n - with n as defined above; oo -H ; -NH2; -(CH2) n i-CH=CH2 or -(CH2) n iC CH with as defined above, which means that in each of these cases Li is absent;oo a saturated, linear or branched, divalent hydrocarbon radical having from 1 to 30 atoms of carbon; an unsaturated, linear or branched, divalent hydrocarbon radical having from 2 to 30 carbon atoms; Li represents oo -O-NH2 ; oo -ON=C(CH3)2; oo a cyclic or heterocyclic system selected from [Chem3]; [Chem4]; [Chem5]; oo -(CH2)ni-CH=CH2; -(CH2) n iCCH; -(CH2)m-N3; -(CH2)m-SH; -(CH2) n i-NH2; -(CH2)ni-N=C=O; -(CH2)ni-N=C=S; -(CH2) n i-NHCO-CH2Hal; with m and Hal as defined above; oo a halogen selected from Cl, Br or I.

[0046] According to another embodiment of the invention, the pro-ligand as defined above is more particularly characterized in that: X represents -CH2-P(O)(OZ)2; -CH2-CO2Z; -CH(CO2Z)2; -CH(P(O)(OZ)2)2with Z as defined above and preferably Z equal to H; P1, P2 and P3 independently represent H or P(O)(OZ)2 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 form an acetonide group of formula [Chem2] as defined above; A represents an oxygen atom -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; -ON=C(CH3)2; a substituent of formula [Chem3] or [Chem4] as defined previously.

[0047] According to yet another embodiment of the invention, the pro-ligand as defined above is more particularly characterized in that: X represents -CH2-P(O)(OZ)2 with Z as defined above, and preferably Z equal to H; P1, P2 and P3 are as defined in the paragraph above, namely they represent independently of each other H or P(O)(OZ)2with 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 form an acetonide group of formula [Chem2] as defined previously; A, L and L1 are as defined in the paragraph above, namely A represents an oxygen atom; 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; -ON=C(CH3)2; a substituent of formula [Chem3] or [Chem4] as defined previously.

[0048] According to an advantageous embodiment of the invention, the pro-ligand is more particularly characterized in that: X represents -CH2-P(O)(OZ)2 with Z as defined previously and preferably Z equal to H; P1, P2 and P3 independently represent H or P(O)(OZ)2 with Z as defined above, and with the condition that at least one of P1, P2 and P3 is other than a hydrogen atom; A represents an oxygen atom -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 previously.

[0049] As an example of a pro-ligand of the invention, mention may more particularly be made of the pro-ligand of formula (I) in which: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents P(O)(OZ)2 with Z = H and P2 and P3 = H; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; L1 represents -O-NH2.

[0050] As an example, we can also cite the pro-ligand of formula (l) in which: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents H and P2 and P3 together form an acetonide group of formula [Chem2] as defined above; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; L1 represents -O-NH2 or -ON=C(CH3)2.

[0051] Table 2 below exemplifies ProAMFAs of formula (I) in which: X represents a phosphonate group of formula -CH2-P(O)(OH)2; P1, P2 and P3 representing independently of each other H or P(O)(OH)2 with the condition that at least one of P1, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents an oxygen atom; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); L1 represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Table 2]

[0052] Table 3 below exemplifies ProAMFAs of formula (I) in which: X represents a carboxylate group of formula -CH2-COOH; P1, P2 and P3 independently representing H or P(O)(OH)2 with the condition that at least one of P1, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents an oxygen atom; L represents -(CH2)2- (i.e. the group -(CH2)2-(O-CH2-CH2)n- with n equal to 0); L1 represents -O-NH2; a substituent of formula [Chem3] or [Chem4] as defined above. [Tables]

[0053] Table 4 below exemplifies ProAMFAs of formula (I) in which: X represents a malonate group of formula -CH(CO2H)2; P1, P2 and P3 independently representing H or P(O)(OH)2 with the condition that at least one of P1, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents an oxygen atom; 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]

[0054] Table 5 below exemplifies ProAMFAs of formula (I) in which: X represents a bisphosphonate group of formula -CH(P(O)(OH)2)2; P1, P2 and P3 independently representing H or P(O)(OH)2 with the condition that at least one of P1, P2 and P3 is other than hydrogen; or P2 and P3 together forming an acetonide group of formula [Chem2]; A represents an oxygen atom; 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. [Tables]

[0055] Conjugates of formula (II)

[0056] According to another aspect, the present invention relates to a conjugate characterized in that it has the following general formula (II): [Chem9] in which ri2 is an integer ranging from 1 to 1000, preferably from 1 to 20, and more preferably from 1 to 10, P1, P2, P3, X, A and L are as defined above, The i represents the substituent Li as defined above when said Li is involved in a covalent bond with Yi, 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'i. The above-mentioned antibodies include armed antibodies, antibody-drug conjugates and antibody fragments.

[0057] 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 parentheses 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 Yi. The integer n2 indicates the number of pro-ligand(s) bound to the product of interest Yi. According to the invention, n2 pro-ligand(s) may be covalently bound to a product of interest Yi. These n2 pro-ligands are bound to the product of interest via the Li or LLi radical of the pro-ligand of formula (I).

[0058] According to one embodiment of the invention, the product of interest Yi of the conjugate (II) as defined above is an antibody or a lysosomal enzyme.

[0059] As an example of a conjugate of the invention, we may cite that of formula (II) in which: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents P(O)(OZ)2 with Z = H and P2 and P3 = H; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero, The i represents -ON= ; ri2 is an integer ranging from 1 to 7; Yi represents the product of interest Y which is an antibody or a lysosomal enzyme.

[0060] Still as an example of a conjugate of the invention, we can cite that of formula (II) in which: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents H and P2 and P3 together form an acetonide group of formula [Chem2] as defined above; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; The i represents -ON=; ri2 is an integer ranging from 1 to 7; Yi represents the product of interest Y which is an antibody or a lysosomal enzyme.

[0061] Preparation of conjugates of formula (II) 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, Y / 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 LLi) 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).

[0062] According to one embodiment of the invention, in the conjugate of formula (II) as defined above, n2 is an integer equal to 1. Figure 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, P1, P2 and P3 are as defined above, - n2 is equal to 1, - A represents an oxygen atom, - 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. Figure 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, Y1' representing the product Y without its functional group L' which is equal to -CHO. Figure 3 point 21 illustrates the reaction between the functional group L1 of the pro-ligand (I) in which L1 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 Yi'-NH2, Y1' representing Y without its functional group L' equal to -NH2. The amine function comes for example from a lysine amino acid residue. Figure 3 point 3 / illustrates the reaction between the functional group L1 of the pro-ligand (I) in which L1 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 Yi'-SH, Y1' representing Y without its functional group L' equal to -SH. The thiol function comes for example from a cysteine ​​amino acid residue. Figure 3 point 4 / illustrates the reaction between the functional group L1 of the pro-ligand (I) in which L1 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 Yi'-SH, Yi' representing Y without its functional group L' equal to -SH.

[0063] Use of the conjugates (II) of the invention.

[0064] The present invention also relates to the conjugates of formula (II) for use as a medicament.

[0065] The conjugates (II) of the invention bind in situ, covalently or non-covalently, to a membrane or non-membrane target of an organism. Examples of a membrane target include: - growth factor receptors, such as REGF, RFGF, RVEGF, TrK, NGF, RPDGF, insulin receptor, erythropoietin receptor, ephrin receptor or - membrane molecules involved in immune control, such as CD or ILT factors, PD-1, TIGIT, LAG, PD-L1, TIM3. Examples of an extracellular non-membrane target include circulating proteins such as cytokines (TNFa, CD20, interferons, interleukins), growth factors (VEGF, EGF, FGF), antibodies, protein or non-protein blood aggregates, exosomes, hydroxyapatite of bone tissue.

[0066] Depending on the target to which the conjugate of formula (II) binds, the complex can, after activation, be internalized into a specific cell, via recognition of the ligand by the RM6P-CI of said cell. The specificity of targeting this cell may be linked to the nature of the target. For example, if the biological target is an epidermal growth factor receptor (EGFR), then the activated complex will be internalized into the cell and the biological target will be degraded or not in lysosomes. If the target is hydroxyapatite present in bone tissue, then the activated complex can be internalized into a bone tissue cell. If the biological target is a blood aggregate made up of several components of protein type, nucleic acids, collagen or fibrils, then the activated complex can be internalized into the surrounding cells.

[0067] For example, if the product of interest Y of the conjugate of the invention is an antibody such as infliximab, it will be able to bind to a non-membrane target such as the cytokine TNFa, which could make it possible to treat Crohn's disease, ankylosing spondylitis or rheumatoid arthritis. Similarly, if the product of interest is an antibody such as cetuximab, it will be able to bind to a membrane target such as the epidermal growth factor receptor (EGFR), which could make it possible to treat metastatic colorectal cancer or head and neck cancers.

[0068] Examples of pathologies that can be treated using the conjugates of the invention (II) include those chosen from the group comprising: - cancers, in particular solid cancers including prostate, breast, pancreatic, colon, lung, liver or bone cancers, - autoimmune and inflammatory diseases, including ankylosing spondylitis, multiple sclerosis, type 1 diabetes, Crohn's disease, lupus, autoimmune thyroiditis, rheumatoid arthritis, - neurodegenerative diseases (Alzheimer's, Parkinson's, Charcot) and, - infectious diseases.

[0069] Figures 1 and 2 illustrate the interest of the pro-ligands (I) and the conjugates (II) of the invention. Figure 1 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 Figure 1). 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 or acid 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 fixed at 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 in the cell. In case 2, it is the entire complex which is internalized into the cell because the main objective is to eliminate the non-membrane target by internalizing it into the cell. Figure 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 Figure 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 figure 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 fixed at the cell membrane receptor via the ligand, two scenarios are possible. In case 1, only the conjugate is internalized (therefore 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.

[0070] In the present invention, the extracellular membrane receptor of the cell is RM6P-CI since the pro-ligand (I) of the invention is a ProAMFA, namely an analogue of mannose 6-phosphate (M6P).

[0071] As indicated above, the bond between the conjugate (II) of the invention and the target (non-membrane or membrane) may be covalent or non-covalent in 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 describes a complex consisting of: - a conjugate of formula (II) as defined above, - a non-membrane or membrane target, said target being as defined above, the conjugate (II) and the target being linked to each other by a non-covalent or covalent type bond.

[0072] The activated ligand obtained following the in situ mechanism described above corresponds to a compound of general formula (I) in which the radicals P1, P2 and P3 would each represent a hydrogen atom. The activated AMFA ligand is then able to be recognized and bound by RM6P-CI.

[0073] The activated conjugate obtained as a result of the in situ mechanism occurring in the organism can be represented by the following general formula (II bis): [Cheml O] in which: X, A, L, L'i, ri2 and Yi are as defined above, P'1, P'2 and P'3 each represent a hydrogen.

[0074] Activation of the conjugate (II) within the complex generates an activated complex consisting of: - of an activated conjugate of formula (II bis) - of 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 RM6P-CI.

[0075] According to one embodiment of the invention, the activated conjugate of formula (II bis) has an affinity, measured by the 50% inhibiting concentration (IC50), with respect to RM6P-CI, ranging from 10' 5 at 10' 9 Mr.

[0076] 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.

[0077] The invention also relates to a conjugate of formula (II) for use as defined above, namely for use as a medicament, characterized in that the conjugate is in a form suitable for administration by oral, parenteral, intravenous, muscular or subcutaneous route. Examples

[0078] 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 the activation of the pro-ligand into a ligand. The pro-ligand ProAMFAI (I) of the invention is compared to the compound AMFA1 and the compound mannose 6-phosphate (M6P).

[0079] Example 1: Synthesis of the pro-ligand ProAMFAI of formula (I) and the pro-ligand ProAMFAI acetonide-oxime of formula (I)

[0080] ProAMFAI Summary

[0081] The pro-ligand ProAMFAI has the following structural formula: [Chem11] The pro-ligand ProAMFAI 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); P1 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 ProAMFAI synthesis diagram is illustrated in Figure 4 and is detailed below.

[0082] “2, 3; 4, 6-di-O-isopropylidene-aD-mannopyranoside de 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 such in the following reaction. Rf: 0.83 (AcOEt / cyclohexane (8:2)) ESI + m / z: 367 [M+H] + SMHR: 367.0765 Da; Conf (%): 100.00%

[0083] "2,3-O-lsopropylidene-aD-mannopyranoside de 2-bromoéthyle" (3) Following the previous reaction, 10 mL of mQ water are added. After 5h30, the medium is neutralized with NaHCOs (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% on 2 steps. Rf: 0.43 (AcOEt / cyclohexane (8:2)) ESI + m / z: 327 [M+H] + SMHR: 327.0439 Da; Conf (%): 100.00%

[0084] “2,3-O-lsopropylidene-aD-manno-hexodialdo-1,5-pyranoside de 2-bromoéthyle” (4) 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-1-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. Rf: 0.54 (AcOEt / cyclohexane (8:2))

[0085] “2-Bromoethyl (6E)-2,3-O-lsopropylidene-6,7-dideoxy-7-diethoxyphosphinyl-aD-manno-hept-6-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) are 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 washes with brine and then with distilled water are carried out. The organic phase is dried and then filtered before being evaporated to dryness. The crude 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. Rf: 0.18 (AcOEt / cyclohexane (8:2)) SMHR: 459.0786 Da; Conf (%): 100.00%

[0086] "2,3-O-Isopropylidene-6-deoxy-6-diethoxyphosphinylmethylene-aD-2-bromoethyl mannopyranoside" (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%

[0087] "2,3-O-lsopropylidene-6-deoxy-6-diethoxyphosphinylmethylene-aD-2-(phthalimidooxy)ethyl mannopyranoside" (7) 0.469 g (1.1 eq; 2.87 mmol) of / V-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 (MgSC) 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%. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) SMHR: 544.1946 Da; Conf (%): 100.00%

[0088] "2,3-O-Isopropylidene-4-O-dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylmethylene-aD-mannopyranoside de 2-(phtalimidooxy)éthyle" (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 / , / V-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 medium is diluted in dichloromethane and then washed twice with NaHCOs. The organic phase is dried over MgSC 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%

[0089] “4-O-Dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylmethylene-aD-2-(phthalimidooxy)ethyl mannopyranoside” (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: (CF^Ch / ethyl ether 95 / 5 (v / v)) SMHR: 612.1610 Da; Conf (%): 100.00%

[0090] “2-aminooxyethyl 4-O-dihvdroxyphosphinyl-6-deoxy-6-dihvdroxyphosphinylmethylene-aD-mannopyranoside” (ProAMFAI) 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 (TMSCI) are added. The solution is stirred at room temperature under stirring and under an inert atmosphere for 24 hours 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 form H +) 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 silica gel column (eluent: H2O). 68 mg of ProAMFAI were collected. The yield over 2 steps is 35%. Rf: 0.69 (2-Propanol / NH4OH / H2O 30 / 40 / 30 (v / v)) SMHR: 398.0607 Da; Conf (%): 100.00% [has] D 20 : + 32.5° (4 mg per 1 mL of H2O)

[0091] Preparation of 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: [Chem12] The difference between ProAMFAI and AMFA1 lies in the definition of the radical P1 which represents the phosphate group P(O)(OH)2 in ProAMFAI versus the hydrogen atom in AMFA1.

[0092] Mannose 6-phosphate (M6P) compound The compound mannose 6-phosphate (M6P) is purchased commercially and has the following structural formula: [Chem13] The pro-ligand ProAMFA1acetonide-oxime 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); P1 represents H and P2 and P3 together form an acetonide group of formula [Chem2] as defined above; A represents oxygen; L represents -(CH2)2-(O-CH2-CH2)n- with n = 0 (therefore the group (CH2)2) and, Li represents -ON=C(CH3)2. The synthetic scheme of ProAMFA1acetonide-oxime is illustrated in Figure 24 and is detailed below.

[0095] "ProAMFA1acetonide-oxime" or "2,3-O-isopropylidene-6-deoxy-6-dihydroxyphosphinylmethylene-aD-mannopyranoside oxime of 2-aminooxyethyl" An amount of 0.010 g (1.0 eq; 0.032 mmol) of the compound AMFA1 is solubilized in an anhydrous DMSO / acetone (0.5 mL / 0.2 mL) mixture. After 18 h, the solvents are evaporated. Purification is carried out on a silica gel column (220 mg) (eluent: isopropanol) which allows obtaining the compound called AMFAI ox (9 mg) (see figure 24). ESI + m / z: 358 [M+H] +

[0096] The compound AMFAI ox (9 mg) is taken up in anhydrous dimethylformamide (0.2 mL) and 0.3 mg of para-tuoluenesulfonic acid (APTS) (0.05 eq; 0.002 mmol) as well as 2,2-dimethoxypropane (0.02 mL) are added. After 18 hours of stirring, the medium is neutralized with an ammonia solution and the ProAMFA1 acetonide-oxime (I) (9 mg) is obtained after purification on silica gel (eluent: water) (see figure 24). ESI + m / z: 398 [M+H] +

[0097] Activation of ProAMFA1acetonide-oxime to AMFAIox To verify its potential as a pro-ligand, the ability of ProAMFA1acetonide-oxime to be devoid of the acetonide group of formula [Chem2] in P2 and P3 was studied. A kinetic study of the acid hydrolysis of the acetonide group was carried out by solubilizing ProAMFA1acetonide-oxime in deuterated methanol (MeOD), adjusting the pH to 5.5 and the reaction was followed by NMR analysis. 31P according to the protocol described below. The transformation of ProAMFA1acetonide-oxime into AMFAI ox is described in Figure 25. Hydrolysis test of the acetonide group of ProAMFA1 acetonide-oxime ProAMFA1 acetonide-oxime (9 mg) is dissolved in 500 pL of MeOD. The pH is adjusted to 5.5 by the addition of 4 pL of a 1 N hydrochloric acid solution. Analyses of the sample placed in the NMR tube are carried out at 37°C using a Bruker AVANCE 400 MHz spectrometer with the following parameters: 162 MHz, ns=32, D1 =2s, pulse width: 15°, 310 K. The results obtained during this study of the conversion of ProAMFA1acetonide-oxime to AMFAIox are illustrated in Figure 26. As indicated in the series of spectra in this figure, the signal 31 P of the phosphonate of ProAMFA1 acetonide-oxime disappears in favor of the signal 31P of AMFAIox phosphonate. The formation of AMFAIox was confirmed by the addition of a known amount of AMFAIox resulting in an increase in the AMFAIox-specific signal (t = 2h30 + AMFAIox).

[0098] Example 2: Preparation of the conjugates of the invention of formula (II)

[0099] Three products of interest Y, namely a lysosomal enzyme and two antibodies, were respectively functionalized with the compounds Pro-AMFA1 or AMFA1.

[0100] The lysosomal enzyme tested is commercially available under the name Myozyme®. This is the enzyme alpha alglucosidase (or acid alpha glucosidase). It can be represented in the following by "Myo". It contains M6P residues in its structure allowing the targeting of RM6P-CI. The lysosomal enzyme Myo is used for the treatment of Pompe disease by enzyme replacement therapy. The antibodies tested are infliximab and cetuximab, two chimeric monoclonal antibodies of the lgG1 type that do not have M6P residues in their structure. They can be represented in the following by "Infli" and "Cetux"; As already stated: - Infliximab antibody binds to the soluble extracellular cytokine, TNFa, and is used for the treatment of autoimmune and inflammatory diseases (Crohn's disease, ankylosing spondylitis or rheumatoid arthritis) (Melsheimer R et al., Biologies. 2019, 13,139); - the Cetux antibody binds to extracellular and membrane REGF and is used for the treatment of various cancers (metastatic colorectal cancer or head and neck cancers) (Baselga J. et al., J. Clin.Oncol., 2005, 23, 5560; Galizia G et al., Oncogene. 2007, 26, 3654).

[0101] The conjugates thus formed may indifferently be represented in the present application by “AMFA1-Myo” or “Myo-AMFA1”, “ProAMFA1-Myo” or “Myo-ProAMFA1”, “AMFA1-lnfli” or “lnfli-AMFA1”, “ProAMFA1-lnfli” or “Infli-ProAMFAI”, “AMFA1-Cetux” or “Cetux-AMFA1”, “ProAMFAI-Cetux” or “Cetux-ProAMFA1”. The conjugates of the invention are those which comprise the ProAMFAI part. Advantageously according to the invention the ProAMFAI part allows the conjugate, once introduced into an organism, not to be immediately recognized by the RM6P-Cl receptor, which thus allows the specific targeting of a predetermined biological target (membrane or non-membrane) by the compound of interest Y, namely in the case of the examples Myozyme ® or the antibodies Infliximab and Cetuximab.

[0102] Coupling and quantification of AMFA1 and ProAMFAI compounds with a product of interest Y (Myozyme ®, Infliximab or Cetuximab) The coupling of AMFA1 or ProAMFAI with a product of interest Y (Myozyme ®, Infliximab or Cetuximab) is carried out thanks to the ethyloxyamine function present on the spacer arm of AMFA1 or ProAMFAI, which allows the formation of a covalent bond of oxime type with the aldehyde functions previously generated by controlled oxidation on the oligosaccharide chains of the product of interest Y (Myozyme ®, Infliximab or Cetuximab). The coupling of a product of interest Y comprising an aldehyde function (previously generated by oxidation) with ProAMFAI of formula (I) (or with AMFA1) comprising a spacer arm with an ethyloxyamine function (L= -(CH2)2- and Li= -ONH2) is notably illustrated in figure 3 point 1 / . Coupling of AMFA1 or ProAMFAI 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 ProAMFAI or AMFA1 compound is added with a 100-fold molar excess. After 2 h at 37°C, Myozyme® is coupled and the excess of ProAMFA 1 or uncoupled 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-AMFA1 and Myo-ProAMFA1 conjugates thus obtained are stored in this buffer at 4°C. Quantification MALDI-TOF mass spectrometry analysis estimated the number of ProAMFAI or AMFAI coupled to the Myozyme® enzyme to be approximately 1. Coupling of AMFA1 or ProAMFAI 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 ProAMFAI or AMFA1 compound is added with a 200-fold molar excess. After 2 h at 37°C, infliximab is coupled and the excess uncoupled ProAMFAI 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 lnfli-AMFA1 and Infli-ProAMFAI conjugates thus obtained are stored in this buffer at 4°C. Quantification MALDI-TOF mass spectrometry analysis estimated the number of ProAMFAI or AMFAI coupled to the infliximab antibody to be approximately 5. Coupling of AMFA1 or ProAMFAI with cetuximab The protocol is similar to that described for infliximab.

[0103] Characterization of conjugates obtained by Coomassie blue staining The integrity of the product of interest (Myozyme®, infliximab or cetuximab) after coupling with AMFA1 or ProAMFAI is verified by electrophoresis on a 12% SDS-polyacrylamide 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 proligand has not caused any degradation. Myozyme®, Myo-AMFA1 and Myo-ProAMFA1 samples were loaded onto polyacrylamide gel under denaturing conditions and followed by Coomassie blue staining. The results obtained are illustrated in Figure 5. We can conclude that the coupling did not cause any degradation since the same characteristic bands of the enzyme at 110 kDa are observed before and after coupling. Similarly, samples of Infliximab, Infli-AMFA1, and Infli-ProAMFAI were loaded onto polyacrylamide gels under denaturing conditions and followed by Coomassie blue staining. The results obtained are illustrated in Figure 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.

[0104] Example 3: Biological evaluation of pro-ligands (I)

[0105] Evaluation of the cytotoxicity of ProAMFAI The cytotoxicity of ProAMFAI of formula (I) was evaluated in comparison with that of M6P and AMFAI on human MCF-7 breast cancer cells (Figure 7) and on healthy human fibroblasts (Figure 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 ProAMFAI were carried out at concentrations between 10' 4 and 10' 7M (Figures 7 and 8). Each condition was analyzed in triplicate. After 72 h of incubation, cell viability was assessed by an assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The results of the cytotoxicity study are shown in Figure 7 for breast cancer cells and in Figure 8 for fibroblasts. It is clear from these figures that the ProAMFAI compound does not present significant toxicity during a 72-h treatment up to the concentration of 10' 4 M on selected cell models. The AMFA1 compound is also non-toxic compared to the natural M6P compound.

[0106] Affinity of ProAMFAI for RM6P-CI ProAMFAI was evaluated for its possible affinity for the RM6P-CI receptor in comparison with AMFAI and M6P. As already mentioned, affinity refers to the intensity of interaction of the ligand, e.g. AMFAI, with its receptor, namely RM6P-CI. To do this, an assay based on interaction competition with RM6P-CI is performed according to the protocol described below. Experimental protocol: Affinity assays are performed using biotinylated RM6P-Cl (RM6P-Clb) that recognizes pentamannose 6-phosphate (PMP) pre-adsorbed onto MaxiSorp™ 96-well plates. Before being dispensed into the wells, RM6P-Clb is pre-incubated for 2 h in the presence of M6P, AMFA1 or ProAMFAI 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-Clb 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 Figure 9 and Table 7 below. Table 7 indicates the necessary concentrations of M6P, AMFA1 and ProAMFAI to have a 50% interaction with RM6P-CI (IC50).

[0107] [Painting?]

[0108] It is clear from Figure 9 and Table 7 that ProAMFAI has no affinity for RM6P-CI. The lack of affinity of ProAMFAI for RM6P-CI is unexpected because it was not foreseeable for those skilled in the art that the sole addition of a phosphate on M6P (namely P1 = P(O)(OH)2) would allow its interaction with said RM6P-CI receptor to be completely neutralized. This novel characteristic validates its potential as a pro-ligand. The lack of affinity of ProAMFAI for RM6P-CI makes it possible to avoid cellular internalization by these receptors of the active ingredients with which ProAMFAI will be associated (the product of interest and the biological target).

[0109] Activation of ProAMFAI in AMFA1 To verify its therapeutic potential as a pro-ligand, the ability of ProAMFAI to be devoid of its P1 = phosphate group (P(O)(OH)2) was studied. A kinetic dephosphorylation study was performed by incubating ProAMFAI in the presence of alkaline phosphatases isolated from human placenta and followed by NMR analysis. 31 P according to the protocol described below. ProAMFAI Alkaline Phosphatase Enzyme Digestion Test An aliquot of 2.3 mg of ProAMFAI 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 parameters indicated are used for a kinetic study of enzymatic digestion. The conversion kinetics of ProAMFAI to AMFA1 could be established after integration of the different signals observed in NMR. The results obtained are illustrated in Figure 10. As shown in the series of spectra in this figure, the decrease in the phosphate signal of ProAMFAI 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 signal 31 P of the phosphonate of ProAMFAI disappears in favor of the signal 31P of AMFAI phosphonate. The formation of AMFAI was confirmed by the addition of a known amount of AMFAI resulting in an increase in the AMFAI specific signal. In the test, a sample of ProAMFAI with a purity of 87% was used. The by-product present is ProAMFAI - oxime (ProAMFAI ox), which is produced by the reaction between ProAMFAI and acetone. ProAMFAI ox has the following developed formula: [Chem14] It is also evident from the spectra series in Figure 10 that alkaline phosphatase similarly hydrolyzes the phosphate of ProAMFAI ox leading to the formation of the ligand AMFA1 -oxime (AMFAI ox).

[0110] Example 4: Biological evaluation of conjugates (II), Myo-ProAMFA1 and Infli-ProAMFAI: cytotoxicity and affinity for RM6P-CI

[0111] Cytotoxicity assessment of the Myo-ProAMFA1 conjugate The cytotoxicity of the Myo-ProAMFA1 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 (Figure 11) and on healthy human fibroblasts (Figure 12) according to the same protocol as that described in Example 3. The results shown in Figures 11 and 12 demonstrate that Myo-ProAMFA1 and Myo-AMFA1 conjugates do not exhibit significant cytotoxicity in cellular models chosen compared to Myozyme® or compared to untreated control cells (100% ± 10%).

[0112] Evaluation of the affinity of the lnfli-ProAMFA1 conjugate for RM6P-CI The lnfli-ProAMFA1 conjugate of the invention and the lnfli-AMFA1 conjugate were evaluated for their affinity for the RM6P-CI receptor according to the same protocol as that described in Example 3. The results obtained are illustrated in Figure 13 and in Table 8 below. Table 8 indicates the necessary concentrations of M6P and lnfli-AMFA1 to have a 50% interaction with RM6P-CI (IC50). The concentrations of Infliximab and infli-ProAMFA1 could not be determined in the concentration range tested and are greater than 10' 5 Mr.

[0113] [Tables]

[0114] The RM6P-CI affinity assay of the Infli-ProAMFAI conjugate shows that the Infli-ProAMFAI conjugate has a very low affinity for RM6P-CI (Figure 13 and Table 8). This affinity is comparable to that of the infliximab antibody alone, whereas the Infli-AMFAI conjugate has a high affinity for RM6P-CI. The very low affinity of the Infli-ProAMFAI conjugate is consistent with the above results obtained with ProAMFAI alone.

[0115] Example 5: Evaluation of the activation of the conjugate (II) Myo-ProAMFA1 by alkaline phosphatases

[0116] Activation of Myo-ProAMFA1 conjugate to Myo-AMFA1 conjugate by alkaline phosphatases In order to verify its therapeutic potential and its ability to be activated, the transformation of the Myo-ProAMFA1 conjugate, presenting a P1 = phosphate group, into Myo-AMFA1 conjugate, presenting a P1 = H group, was studied. A kinetic dephosphorylation study was performed by incubating Myo-ProAMFA1 in the presence of alkaline phosphatase isolated from human placenta according to the protocol described below. Protocol for enzymatic digestion of conjugates by alkaline phosphatase Myo-AMFAI and Myo-ProAMFA1 The Myo-ProAMFA1 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-AMFA1 conjugate. After transfer to a membrane, the fraction of Myo-ProAMFA1 that has become Myo-AMFA1 is characterized using a specific antibody against AMFAI 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 Figure 14. Over time, treatment of the Myo-ProAMFA1 conjugate with phosphatase induced an increase in signal intensity at 110 kDa corresponding to Myo-AMFA1 recognized by the anti-AMFA1 antibody. This result demonstrates the ability of the Myo-ProAMFA1 conjugate to be dephosphorylated into Myo-AMFA1 in the presence of alkaline phosphatase.

[0117] Example 6: Evaluation of the activation of Infli-ProAMFAI conjugates (II) by phosphatases and human serum

[0118] Evaluation of the activation of Infli-ProAMFAI conjugate to lnfli-AMFA1 conjugate by acid and alkaline phosphatases by Western Blots The Infli-ProAMFAI conjugate is incubated with 8 IU / ml of human placental alkaline phosphatase or with 3 IU / ml of acid phosphatase from Solanum Tuberosum at 37°C. After 16, 24, 48 or 72 h the reaction is stopped in ice and the samples are analyzed by Western blot as described above for Myo-ProAMFA (Figure 14). The samples are revealed with an anti-AMFA1 antibody (which recognizes AMFAI) and with an anti-human IgG antibody (in order to verify that the quantities of antibodies deposited are comparable). The results obtained are illustrated in Figure 15. Over time, treatment of the Infli-ProAMFAI conjugate with phosphatases induced an increase in signal intensity at 50 kDa corresponding to lnfli-AMFA1 recognized by the anti-AMFA1 antibody. This result demonstrates the ability of the Infli-ProAMFAI conjugate to be dephosphorylated to lnfli-AMFA1 in the presence of acid and alkaline phosphatases.

[0119] Affinity of infli-ProAMFAl for RM6P-CI after activation by alkaline phosphatases RM6P-CI receptor affinity of the Infli-ProAMFAI conjugate before and after digestion by alkaline phosphatases (8 Units / ml) at 37°C for 24 h is analyzed by ELISA by binding the antibodies for 90 min to RM6P-CI (0.5 pg / ml) previously adsorbed on a 96-well microplate. The retained antibodies are quantified by a second anti-human IgG antibody conjugated to peroxidase and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The absorbance is read at 650 nm. The results presented in Figure 16 show that the action of phosphatases makes it possible to obtain an affinity from a concentration of 2.10' 7M for the digested / activated Infli-ProAMFAI conjugate (i.e., the lnfli-AMFA1 conjugate), while the affinity of Infli-ProAMFAI is not detectable. This result demonstrates for the first time that alkaline phosphatase activation of Pro-AMFA1 to AMFA1 on the surface of an antibody can create an affinity for RM6P-CI and that this affinity is even much higher than that of M6P described in Figure 13.

[0120] Evaluation of activation of Infli-ProAMFAI conjugate to lnfli-AMFA1 conjugate in human serum by Western Blots The Infli-ProAMFAI conjugate is incubated in the presence of human serum (40 p.1) at 37°C. After 4, 16 or 24 h the reaction is stopped in ice and the samples are analyzed by Western blot and revealed with an anti-AMFA1 antibody and by an anti-human IgG antibody as described above for Figure 15. The results presented in Figure 17 show that over time, treatment of the Infli-ProAMFAI conjugate with human serum induces an increase in signal intensity at 50 kDa corresponding to the lnfli-AMFA1 conjugate recognized by the anti-AMFA1 antibody. This result demonstrates the ability of the Infli-ProAMFAI conjugate to be dephosphorylated to lnfli-AMFA1 by phosphatases present in human serum.

[0121] Affinity of Infli-ProAMFAI for RM6P-CI after activation in human serum The affinity for the RM6P-CI receptor of the Infli-ProAMFAI conjugate is measured according to the same protocol as that described in Example 3, before and after digestion with human serum (40 μl) at 37°C for 24 h. The results presented in Figure 18 show that the action of human serum phosphatases allows detectable affinity to be obtained from a concentration of 10' 7M for the digested Infli-ProAMFAI conjugate, namely the lnfli-AMFA1 conjugate, while the affinity of Infli-ProAMFAI is not detectable.

[0122] Example 7: Evaluation of the activation of Cetuximab-ProAMFAI conjugates (II) by phosphatases

[0123] Coupling of AMFA1 or ProAMFAI with Cetuximab A MALDI-TOF mass spectrometry analysis made it possible to estimate at approximately 7 the number of ProAMFAI or AMFA1 coupled to the Cetuximab antibody. The integrity of the product of interest (Cetux) after coupling with AMFA1 or ProAMFAI is verified by electrophoresis on a 12% SDS-polyacrylamide gel followed by Coomassie blue staining. The results shown in Figure 19 indicate that the couplings did not cause degradation since the same characteristic bands of the 50 kDa and 25 kDa antibody are observed before and after coupling.

[0124] Evaluation of the activation of Cetux-ProAMFA1 conjugate to Cetux-AMFA1 conjugate by acid and alkaline phosphatases by Western Blots The Cetux-ProAMFA1 conjugate is incubated with 8 IU / ml of human placental alkaline phosphatase or 3 IU / ml of acid phosphatase from Solanum Tuberosum at 37°C. After 24, 48, 72 or 96 h, the reaction is stopped on ice and the samples are analyzed by Western blotting as described above for Myo-ProAMFA (Figure 14). The samples are visualized with an anti-AMFA1 antibody and with an anti-human IgG antibody. The results obtained are illustrated in Figure 20. Over time, treatment of the Cetux-ProAMFA1 conjugate by phosphatases induces an increase in signal intensity at 50 kDa corresponding to the Cetux-AMFA1 conjugate recognized by the anti-AMFA1 antibody. This result demonstrates the ability of the Cetux-ProAMFA1 conjugate to be dephosphorylated to Cetux-AMFA1 in the presence of acid and alkaline phosphatases.

[0125] Example 8: Cellular internalization of the conjugates (II) Infli-ProAMFAI and Cetux-ProAMFA1 after activation by phosphatases

[0126] Cellular internalization of the Infli-ProAMFAI conjugate in Hela cells The Infli-ProAMFAI conjugate, which has been previously coupled to the AlexaFluor647 fluorochrome, is then incubated or not for 48 hours in the presence of human serum for its activation by phosphatases. The two batches of antibodies (0.75 mg / ml) are then incubated for 5 hours with human Hela uterine cancer cells. The fluorescence of the cells is observed by confocal microscopy. The results, shown in Figure 21, indicate a 2.7-fold increase in conjugate internalization through digestion of ProAMFAI by serum phosphatases.

[0127] Cellular internalization of Cetux-ProAMFA1 conjugate in Hela cells The Cetux-ProAMFA1 conjugate, which has been previously coupled to the AlexaFluor488 fluorochrome, is then incubated or not for 24 hours in the presence of human serum for its activation by phosphatases. The two batches of antibodies (0.75 g / ml) are then incubated for 18 hours with Hela cells. The fluorescence of the cells is measured by flow cytometry. The results, shown in Figure 22, indicate a 2-fold increase in conjugate internalization after digestion of ProAMFAI by serum phosphatases.

[0128] Example 9: Affinity of the conjugate (II) Infli -ProAMFAI and the antibody Infliximab for its antigen, TNFa

[0129] Antibody affinities for the antigen are quantified by ELISA. Antibodies are incubated with TNFα antigen previously adsorbed on a microplate for 1 h at 37 °C. Antibodies retained on the plate are quantified using a second anti-human IgG antibody coupled to peroxidase and by the addition of o-phenylenediamine hydrochloride (OPD) substrate. Absorbances were read at 450 nm. The results shown in Figure 23 indicate that coupling ProAMFAI with infliximab does not alter the affinity of the Infliximab antibody for its antigen. Therefore, modifying the antibody by grafting several ProAMFAs does not alter the three-dimensional structure of the antibody, preventing its recognition of the antigen.

[0130] This disclosure is not limited to the examples described above, only as an example, but it encompasses all the variations that a person skilled in the art may envisage within the framework of the protection sought. List of cited documents Patent documents

[0131] For convenience, the following patent documents are cited: - patcitl: EP 2 448 600 B1; - patcit2: EP 3 350 192 B1; - patcit3: application FR2302097 filed on 07 / 03 / 2023. Non-patent literature

[0132] For convenience, the following non-patent elements are cited: - nplcitl: Vidil C. et al., Eur.J. Org. Chem., 1999, 447; - nplcit2: Jeanjean A. et al., Bioorg. Med. Chem. Lett., 2008, 18, 6240; - nplcite3: El Cheikh K. et al., Angew. Chem. Int. Ed., 2016, 55, 14774; - nplcite4: Ghosh P. et al., Nat. Rev. Mol. Cell. Biol., 2003, 4, 202; - nplcit5 :Gauthier C. et al. J. Control. Release 2024, 365, 759 ; - nplcit6 : Kleeb et al., Journal of Medicinal Chemistry, 2016, 59, 3163 ;; - nplcit7 : Basile I. et al., J. Control. Release, 2018, 269, 15 ; - nplcit8 : Godefroy A. et al., J. Cell. Mol. Med., 2019, 23, 6499) ; - nplcit9 : Daurat M. et al., Front. Immunol., 2024, 15, 1273280 ; - npIcitIO : Gauthier et al., Biomed. Pharmacother., 2024, 175, 116707 ; - nplcitl 1 : Gauthier C. et al., J. Control. Release, 2023, 358, 465 ; - nplcitl 2 : Vaillant O. et al., Angewandte Chemie, 2015, 54, 5952 ; - nplcitl 3 : Bouffard et al., Int J Mol Sel., 2019, 20, 2809 ; - nplcitl 4 : Daurat et al., Biomater. Sel., 2020, 8, 3678 ; - nplcitl 5 : Melsheimer R et al., Biologies. 2019, 13,139; - nplcitl 6 : Baselga J. et al., J. Clin. Oncol., 2005, 23, 5560 ; - nplcitl 7: Galizia G et al., Oncogene. 2007, 26, 3654.

Claims

Claims

1. Pro-ligand characterized in that it has the following general formula (I): in which: the dotted line represents a bond which is present or not; X represents -CH2-P(O)(OZ)2; -CH2-CO2Z ;- -CH(CO2Z)2; -CH(P(O)(OZ)2)2; -CHF-CO2Z ; -CHF-P(O)(OZ)2; -CF2-CO2Z ; -CF2-P(O)(OZ)2; -CH(CO2Z)(P(O)(OZ)2), in which case the bond represented by the dotted line is not present; or, X represents =CH-CO2Z; =CH-P(O)(OZ)2; =CF-CO2Z; =CF-P(O)(OZ)2, in this case the bond represented by the dotted line is present; with Z representing independently of each other H; Na; K or NF; P1, P2 and P3 independently represent 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 form an acetonide group [Chem2] H3C CH3. 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; °° -H ; -NH2; -(CH2) n i-CH=CH2 or -(CH2) n iC=CH with 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 divalent hydrocarbon radical having from 1 to 30 carbon atoms; an unsaturated, linear or branched 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: 000 a group -O-; -NH-; -S-; -CO-NH-; -NH-CO-O-; and / or 000a cyclic or heterocyclic system chosen from those listed in Table 1 below [Painting"!] Li represents oo -O-NH2; oo a cyclic or heterocyclic system chosen from [Chem3] a C1-C5 alkyl, and is preferably an ethyl; [Chem5] [Chem6] [Chem7] [Chem8] 00 -ON=C(CH3)2; 00 -(CH2)ni-CH=CH2; -(CH2) n iCCH; -(CH2)m-N3; -(CH2)m-SH; -(CH2) n i-NH2; -(CH2)ni-N=C=O; -(CH2)ni-N=C=S; -(CH2) n i-NHRi ; -(CH2) n i-Ai-NH2; -(CH2) n i-Ai-NHRi ; -(CH2)ni-NHCO-CH2Hal ; -(CH2) n i-COZi ; -(CH2) n i-AiCOZi ; -(CH2) n iO-NH2; -(CH2)ni-CO-NH-NH2; with ni and Ri as defined above; Ai representing -O-; -NH-; Hal representing Cl; Br or I; Z1 representing -OH; -OR1; -NHR1; -NH-NH2; -NH-NHR1 with Ri as defined above; 00 a halogen selected from F, Cl, Br or I.

2. Pro-ligand according to claim 1, characterized in that: X represents -CH2-P(O)(OZ)2; CH2-CO2Z ; -CH(CO2Z)2; -CH(P(O)(OZ)2)2; -CHF-CO2Z ; - CHF-P(O)(OZ)2; -CF2-CO2Z ; -CF2-P(O)(OZ)2; -CH(CO2Z)(P(O)(OZ)2); =CH-CO2Z; with Z as defined in claim 1; P1, P2 and P3 independently represent H or P(O)(OZ)2; 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 form an acetonide group of formula [Chem2] as defined in claim 1; A represents a divalent radical chosen from -O-; -S-; -CH2-; L represents: 00 -(CH2)2-(O-CH2-CH2)n- with n as defined in claim 1; 00 -H; -NH2; -(CH2)ni-CH=CH2or -(CH2)ni-C=CH with as defined in claim 1, which means that in each of these cases L1 is absent; 00 a saturated, linear or branched divalent hydrocarbon radical having from 1 to 30 carbon atoms; an unsaturated, linear or branched divalent hydrocarbon radical having from 2 to 30 carbon atoms; L1 represents 00 -O-NH2 ; 00 -ON=C(CH3)2; 00 a cyclic or heterocyclic system selected from [Chem3]; [Chem4]; [Chem5]; 00 -(CH2)ni-CH=CH2; -(CH2)m-CECH; -(CH2)m-N3; -(CH2)m-SH; -(CH2)m-NH2; -(CH2)ni-N=C=O ; -(CH2)ni-N=C=S ; -(CH2)ni-NHCO-CH2Hal ; with m and Hal as defined in claim 1; 00 a halogen selected from Cl, Br or I.

3. Pro-ligand according to claim 1 or 2 characterized in that: X represents -CH2-P(O)(OZ)2; -CH2-CO2Z; -CH(CO2Z)2; -CH(P(O)(OZ)2)2with Z as defined in claim 1 or 2, and preferably Z equal to H; P1, P2 and P3 independently represent H or P(O)(OZ)2 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 form an acetonide group of formula [Chem2] as defined in claim 1; A represents an oxygen atom -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; L1 represents -O-NH2; -ON=C(CH3)2; a substituent of formula [Chem3] or [Chem4] as defined in claim 1.

4. Pro-ligand according to any one of claims 1 to 3, characterized in that: X represents -CH2-P(O)(OZ)2 with Z as defined in claim 3; P1, P2 and P3 are as defined in claim 3; A, L and L1 are as defined in claim 3.

5. Pro-ligand according to any one of claims 1 to 4, characterized in that: X is as defined in claim 4, A, L and L1 are as defined in claim 4, P1, P2 and P3 independently represent H or P(O)(OZ)2 with Z as defined in claim 4, and with the proviso that at least one of P1, P2 and P3 is other than a hydrogen atom.

6. Pro-ligand according to claim 5, characterized in that: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents P(O)(OZ)2 with Z = H and P2 and P3 = H; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; Li represents -O-NH2.

7. Pro-ligand according to any one of claims 1 to 4, characterized in that: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents H and P2 and P3 together form an acetonide group of formula [Chem2] as defined in claim 1; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; L1 represents -ONH2 or -ON=C(CH3)2.

8. Conjugate characterized in that it has the following general formula (II): in which n2 is an integer ranging from 1 to 1000, preferably from 1 to 20, and more preferably from 1 to 10, P1, P2, P3, X, A and L are as defined in any one of claims 1 to 7, L'i represents the substituent L1 as defined in any one of claims 1 to 7 when said L1 is involved in a covalent bond with Y1, 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'i.

9. Conjugate according to claim 8, characterized in that the product of interest is an antibody or a lysosomal enzyme.

10. Conjugate according to claim 8 or 9, characterized in that: X represents CH2-P(O)(OZ)2 with Z = H; P1 represents P(O)(OZ)2 with Z = H and P2 and P3 = H; or, P1 represents H and P2 and P3 together form an acetonide group of formula [Chem2] as defined in claim 1; A represents an oxygen atom, L represents -(CH2)2-(O-CH2-CH2)n- with n equal to zero; The i represents the radical -ON=.

11. A conjugate as defined in any one of claims 8 to 10, for use as a medicament.

12. A conjugate for use according to claim 11, characterized in that the conjugate is in a form suitable for oral, parenteral, intravenous, muscular or subcutaneous administration.

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