Proligands of the form of mannose 6-phosphate or mannose analogs, conjugates containing the above proligands, and their use for therapeutic application.

Abstract

The present invention relates to a novel compound referred to as a proligand or inactivating ligand of formula (I). The present invention also relates to a conjugate comprising the above-mentioned proligand, and to the above-mentioned conjugate corresponding to formula (II). The present invention also relates to a conjugate (II) for use as a pharmaceutical agent.

Description

[Technical Field]

[0001] The present invention belongs to the field of therapeutic chemistry. More specifically, the present invention relates to novel compounds referred to as "proligands" or "inactivating ligands," conjugates containing the above-mentioned proligands, and their use in therapeutic applications.

[0002] In the context of this invention, a compound referred to as a proligand refers to a ligand that has been temporarily inactivated, for example, by adding one or more unstable groups to the ligand. In this application, a proligand may also be referred to as an inactivated ligand. Since the role of a ligand is to be recognized by its membrane receptor, a proligand, being temporarily inactivated, refers to a ligand that is no longer recognized by its membrane receptor. The proligand of the present invention is an analog of mannose 6-phosphate (M6P) that has been temporarily inactivated. Unlike natural M6P, mannose 6-phosphate (M6P) analogs are compounds that exhibit high levels of recognition by the cation-independent mannose 6-phosphate acceptor (CI-M6PR). M6P analogs are referred to as "AMFA" ("Analogues of Mannose 6-phosphate Functionalized at the Anomeric Position"). The proligand obtained after inactivation of the AMFA analog is referred to as "ProAMFA" in this application. The proligand, ProAMFA, is no longer recognized by CI-M6PR.

[0003] The conjugate of the present invention comprises a proligand on one side and a product of interest on the other. The conjugate of the present invention having the structure "proligand-product of interest" is selectively capable of binding to the target via a target-recognizable product of interest, thus yielding a complex having the structure "proligand-product of interest-target". [Background technology]

[0004] Documents EP 2 448 600 B1 and EP 3 350 192 B1 describe conjugates containing M6P analogs linked to a product of interest, for example, a glycoprotein, via spacer arms. M6P analogs offer several advantages with respect to the functionalization of glycoproteins, and in particular to the functionalization of lysosomal enzymes, mainly because these analogs are small in size, have low immunogenicity, and can readily bind to lysosomal enzymes. These M6P analogs are also modular with respect to the length and structure of the spacer arms, as well as the terminal reactive groups of the spacer arms, depending on the desired type of binding with the product of interest. In these documents, M6P analogues are used more specifically to internalize lysosomal enzymes into cellular lysosomes via CI-M6PR, enabling the enzymes to perform their physiological functions. The M6P analog is also modular with respect to the phosphate group at position 6, which may be substituted with a bioisoster group, i.e., a group that performs the same biological function as the phosphate group. The phosphate group at position 6 may be substituted with, for example, a phosphonic acid group, a carboxylic acid group, or a malonic acid group. Several publications have shown that M6P derivatives substituted at position 6 possess significant affinity for CI-M6PR (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, 1).

[0005] However, in EP 2 448 600 B1 and EP 3 350 192 B1, it is not assumed that the M6P analogues become temporarily inactive. This means that once the "M6P analogue-product of interest" conjugate is introduced into a living organism, it is immediately recognized by the cation-independent mannose 6-phosphate acceptor (CI-M6PR) and therefore fixed by them. CI-M6PR is a ubiquitous receptor present in both intracellular vesicles and membranes of all cells; therefore, it is not a receptor specific to any 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). More specifically, this means that once inside the body, the conjugate does not necessarily have the time required to reach and bind to a specific target recognizable by the product of interest, because the M6P analog can be rapidly recognized by CI-RM6P present in the immediate environment. The product of interest will therefore undergo degradation while circulating freely before reaching and binding to a recognizable biological target. In other words, the conjugates described in these documents may reduce the detection of predetermined targets.

[0006] A paper by Kleeb et al. (Journal of Medicinal Chemistry, 2016, 59, 3163) describes how the addition of phosphate groups at the 2, 3, or 4 positions of the mannose cycle improves the solubility and bioavailability of mannose derivatives. However, this paper does not consider using these phosphated mannose derivatives to prevent immature recognition of ligands by their receptors.

[0007] Therefore, to the best of our knowledge, no in situ activation of the above-mentioned proligand by a phosphatase or a proligand that does not possess affinity for the mannose 6-phosphate receptor has been described to date.

[0008] The inventors have developed novel conjugates specific to predetermined targets. To do this, the inventors conceived the original idea of ​​developing a proligand that is an M6P analog and transiently inactivated, and then preparing a conjugate with a "proligand-product of interest" structure. Thus, when the conjugates of the present invention are introduced into a living organism, they will not be directly recognized by the ligand receptor, i.e., CI-M6PR, thereby allowing them to circulate freely and more effectively reach the target that is specifically recognized by the product of interest. The product of interest binds to its target, resulting in the formation of a complex "proligand-product of interest-target". This complex is then activated, and thus the proligand becomes the ligand. The activated complex of the "ligand-product of interest-target" structure thus obtained will then be recognized and bound by the ligand receptor, i.e., CI-M6PR.

[0009] The purpose and activity of "ligand-product of interest" conjugates or "ligand-product of interest-target" complexes have been the subject of several prior art publications. Therefore, when a ligand is coupled to a lysosomal enzyme, it has been shown that the ligand enables efficient targeting of this enzyme in lysosomes through internalization via CI-M6PR (Basile I. et al., J. Control. Release, 2018, 269, 15; Godefroy A. et al., J. Cell. Mol. Med., 2019, 23, 6499). When a ligand is coupled to an antibody directed against a therapeutic target, the entry of the antibody and its target via CI-M6PR has been demonstrated for several antibodies directed against the following: -Soluble extracellular targets (non-membrane targets), such as tumor necrosis factor alpha (TNFα) 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 shown that the internalized "ligand-product of interest" conjugate is recycled within the cycle in mouse models (Gauthier C. et al., J. Control. Release 2023, 358, 465). When ligands are coupled to nanoparticles intended for therapeutic use, nanoparticle entry via CI-M6PR has been demonstrated in the endolysosome system (Vaillant O. et al., Angewandte Chemie, 2015, 54, 5952; Bouffard et al., Int J Mol Sci., 2019, 20,2809; Daurat et al., Biomater. Sci. 2020, 8, 3678). On the other hand, the inventors have shown that the ligand family of CI-M6PR can bind to hydroxyapatite present in bone tissue (French patent application FR2302097), and therefore these are promising compounds for the treatment of osteopathology.

[0010] The development of "proligand-product of interest" type conjugates, and their ability to conjugate in situ to a target (membrane-bound or non-membrane-bound) and subsequently convert in situ to a "ligand-product of interest-target" type activated complex, constitutes the originality and interest of the present invention with respect to some of the promising activities of "ligand-product of interest" and / or "ligand-product of interest-target" compounds described in the prior art described above. [Overview of the Initiative]

[0011] In accordance with the first aspect, the object of the present invention is a proligand of general formula (I), as described in the following detailed description.

[0012] In a second aspect, the object of the present invention is a conjugate of general formula (II) having the structure “proligand-product of interest”, as described in the following detailed description.

[0013] According to a third aspect, the object of the present invention is a process for producing the proligand of formula (I) and the conjugate of formula (II).

[0014] In another embodiment, an object of the present invention is a conjugate of formula (II) for use as a pharmaceutical agent.

[0015] In another embodiment, the present invention describes a complex having the structure “proligand-product of interest-biological target”. [Brief explanation of the drawing]

[0016] Further features, details, and advantages will become clear from the detailed description below and the analysis of the accompanying diagrams.

[0017] [Figure 1] This illustrates a sequence of events that occur when the present invention's conjugate, a "proligand-product of interest" structure, is introduced into a living organism for the purpose of binding to a non-membrane target.

[0018] [Figure 2] This illustrates a sequence of events that occur when the present invention's conjugate, a "proligand-product of interest" structure, is introduced into a living organism for the purpose of binding to a membrane target.

[0019] [Figure 3] These are four possible embodiments for the synthesis of the conjugate of formula (II) obtained by the reaction between a proligand of general formula (I) and a product of interest Y.

[0020] [Figure 4] A detailed synthesis scheme for the proligand of general formula (I), known as ProAMFA1, is provided as an example.

[0021] [Figure 5] Examples of results obtained by SDS-PAGE electrophoresis of a single commercially available Myozyme® enzyme, Myozyme® conjugated with the AMFA1 compound (to obtain a Myo-AMFA1 conjugate), or Myozyme® conjugated with the proligand ProAMFA1 (to obtain a Myo-ProAMFA1 conjugate) are shown. The enzyme, either alone or conjugated with the AMFA1 or ProAMFA1 compound, is detected by Coomassie blue staining.

[0022] [Figure 6] Examples of results obtained by SDS-PAGE electrophoresis of a single infliximab antibody, an infliximab antibody conjugated to the AMFA1 compound (to obtain an Infli-AMFA1 conjugate), or an infliximab antibody conjugated to the proligand ProAMFA1 (to obtain an Infli-ProAMFA1 conjugate) are provided. The antibody, either alone or conjugated to the AMFA1 or ProAMFA1 compound, is detected by Coomassie blue staining.

[0023] [Figure 7]This paper illustrates the cytotoxicity of the proligand ProAMFA1 against human MCF-7 breast cancer cells compared to the compounds M6P and AMFA1. The x-axis shows the molar concentrations of M6P (white histogram), AMFA1 (shaded histogram), and ProAMFA1 (black histogram). The y-axis shows cell survival for each compound. Cell survival observed in the presence of M6P is set as the 100% value.

[0024] [Figure 8] The cytotoxicity of ProAMFA1 against human fibroblast FS01035 cells compared to M6P and AMFA1 is illustrated. The explanations of the axes and histogram in Figure 7 also apply to Figure 8.

[0025] [Figure 9] The interaction between CI-M6PR and the ProAMFA1 proligand is illustrated compared to that between M6P and AMFA1. The x-axis shows the molar concentrations of compounds M6P (dotted line), AMFA1 (dashed line), and ProAMFA1 (solid line). The y-axis shows the binding rate of these compounds to CI-M6PR.

[0026] [Figure 10] These are a series of NMR spectra at different time points (t=0, t=1h, t=11h, t=24h) showing the degradation kinetics of two proligands, ProAMFA1 and ProAMFA1ox, to their AMFA1 and AMFA1ox ligands in the presence of alkaline phosphatase isolated from human placenta. The difference between ProAMFA1 and ProAMFA1ox lies in the L1 radical, which is -O-NH2 in ProAMFA1 compared to -ON=C(CH3)2 in ProAMFA1ox. The peaks on the left of the spectra are characteristic of the X radical, which is equivalent to the phosphonic acid (i.e., X=CH2-P(O)(OH)2) present in each ProAMFA1 and ProAMFA1ox. The peaks on the right of the spectra are characteristic of the P1 radical, which is equivalent to the phosphate (i.e., X=P(O)(OH)2) present in each ProAMFA1 and ProAMFA1ox.

[0027] [Figure 11] This diagram illustrates the cytotoxic effects of Myozyme® enzyme (white histogram), Myo-AMFA1 conjugate (shaded histogram), and Myo-ProAMFA1 conjugate (black histogram) on human MCF-7 breast cancer cells. The x-axis represents the molar concentration of the compound, and the y-axis represents cell survival with respect to the compound. Cell survival observed in the presence of Myozyme® is set as the 100% value.

[0028] [Figure 12] This diagram illustrates the cytotoxic effects of Myozyme® enzyme (white histogram), Myo-AMFA1 conjugate (shaded histogram), and Myo-ProAMFA1 conjugate (black histogram) on human FS01035 fibroblasts. The x-axis represents the molar concentration of the compound, and the y-axis represents cell survival with respect to the compound. Cell survival observed in the presence of Myozyme® is set as the 100% value.

[0029] [Figure 13] The diagram illustrates the interactions between CI-M6PR and compound M6P, or between CI-M6PR and infliximab alone or linked to either AMFA1 or ProAMFA1. The x-axis shows the molar concentrations of compound M6P (crossed line), infliximab (dotted line), Infli-AMFA1 (dashed line), and Infli-ProAMFA1 (solid line). The y-axis shows the proportion of each compound bound to CI-M6PR.

[0030] [Figure 14] This paper illustrates the detection of Myozyme® enzyme, Myo-AMFA1 conjugate and Myo-ProAMFA1 conjugate by Western blotting at different time points (t=1h, t=24h, and t=96h) in the presence of alkaline phosphatase isolated from human placenta.

[0031] [Figure 15] The detection of Infli-AMFA1 and Infli-ProAMFA1 conjugates, as well as Infli-ProAMFA1 conjugates at different time points, incubated and unincubated, in the presence of alkaline phosphatase or acid phosphatase, is illustrated by Western blotting.

[0032] [Figure 16] This paper illustrates the interaction between CI-M6PR and Infli-ProAMFA1 conjugates that are pre-incubated or not pre-incubated in the presence of alkaline phosphatase.

[0033] [Figure 17] This example illustrates Western blotting detection of Infli-ProAMFA1 conjugates incubated in the presence of human serum at different time points and quantified by measuring the concentration of the AMFA1 signal.

[0034] [Figure 18] The graph illustrates interactions between CI-M6PR and M6P compounds (dashed line), or between CI-M6PR and Infli-ProAMFA1 conjugates that are not incubated with human serum (solid line) or that have been incubated for 24 hours (dotted line). The x-axis represents the molar concentration of the compound. The y-axis represents the percentage of inhibition of CI-M6PR binding by the compound.

[0035] [Figure 19] The results obtained by SDS-PAGE electrophoresis of a single cetuximab antibody, a cetuximab antibody conjugated to an AMFA1 compound (to obtain a Cetux-AMFA1 conjugate), or a cetuximab antibody conjugated to the proligand ProAMFA1 (to obtain a Cetux-ProAMFA1 conjugate) are illustrated. The antibody, either alone or conjugated to an AMFA1 or ProAMFA1 compound, is detected by Coomassie blue staining.

[0036] [Figure 20] The detection of Cetux-ProAMFA1 conjugates by Western blotting, after and without incubation in the presence of alkaline phosphatase or acid phosphatase at different time points, is illustrated.

[0037] [Figure 21] We illustrate the 5-hour internalization of Infli-ProAMFA1 conjugates in HeLa cells, with or without pre-incubation for 48 hours in the presence of human serum. Internalization is analyzed by confocal microscopy and fluorescence quantification.

[0038] [Figure 22] This example illustrates the 18-hour internalization of Cetux-ProAMFA1 conjugates in HeLa cells, with or without prior 24-hour incubation in the presence of human serum. Internalization is measured by flow cytometry.

[0039] [Figure 23] This illustrates the binding of the Infli-ProAMFA1 conjugate and the infliximab antibody to their respective antigens, TNFα, as measured by ELISA.

[0040] [Figure 24] A synthesis scheme for the proligand of general formula (I), known as ProAMFA1 acetonide oxime, is illustrated below.

[0041] [Figure 25] The conversion of ProAMFA1 acetonide oxime to AMFA1ox is illustrated as an example.

[0042] [Figure 26]These are a series of NMR spectra at different time points (t=0, t=30min, t=1h30, t=2h30) showing the decomposition kinetics of the proligand ProAMFA1 acetonide oxime to AMFA1ox. [Modes for carrying out the invention]

[0043] Proligand of formula (I)

[0044] The object of the present invention is a proligand characterized by having the following general formula (I). [ka] During the ceremony: The dotted line indicates a connection that may or may not exist; 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 shown by the dotted line does not exist; or X represents =CH-CO2Z;=CH-P(O)(OZ)2;=CF-CO2Z;=CF-P(O)(OZ)2, in which case the bond shown by the dotted line exists; Z independently represents H;Na;K or NH4; P1, P2, and P3 independently represent H;P(O)(OZ)2;-S(O)2(OZ); Z is as previously defined, and the condition is that at least one of P1, P2, and P3 is different from a hydrogen atom; Alternatively, P2 and P3 together form an acetonide group. [ka] form; A represents a divalent radical among -O-;-S-;-NH-;-CH2-; L is -(CH2)2-(O-CH2-CH2) n-, where n is an integer in the range of 0 to 6; -H;-NH2;-(CH2) n1 -CH=CH2 or -(CH2) n1 -C≡CH, where n1 is an integer in the range of 0 to 4, and in each of these cases, L1 is nonexistent; Saturated linear or branched divalent hydrocarbon radicals having 1 to 30 carbon atoms; unsaturated linear or branched divalent hydrocarbon radicals having 2 to 30 carbon atoms; A saturated or unsaturated divalent hydrocarbon radical as defined above, wherein one or more of the saturated or unsaturated hydrocarbon radicals -CH2-, -CH=CH- and / or -C≡C- radicals are independently of each other. -O-;-NH-;-S-;-CO-NH-;-NH-CO-O-; and / or Table 1 below [Table 1] The saturated or unsaturated divalent hydrocarbon radicals described above are substituted by a ring system or heterocyclic system derived from those listed above. Show; L1 is -O-NH2; [ka] In the formula, R1 represents a C1-C5 alkyl group, preferably ethyl. [ka] [ka] [ka] [ka] [ka] A more selected system of rings or heterocyclic rings; -O-N=C(CH3)2; -(CH2) n1 -CH=CH2;-(CH2) n1 -C≡CH;-(CH2) n1 -N3;-(CH2) n1 -SH;-(CH2) n1 -NH2; -(CH2) n1 -N=C=O;-(CH2) n1 -N=C=S;-(CH2) n1 -NHR1;-(CH2) n1 -A1-NH2;-(CH2) n1 -A1-NHR1; -(CH2) n1 -NHCO-CH2Hal;-(CH2) n1 -COZ1;-(CH2) n1 -A1COZ1;-(CH2) n1 -O-NH2; -(CH2) n1 n1 -CO-NH-NH2; In the formula, n1 and R1 are as defined above; A1 is -O-; -NH-; Hal is Cl; Br or I; Z1 is -OH; -OR1, -NHR1; -NH-NH2; -NH-NHR1, and R1 is as defined above; [ Halogen of F, Cl, Br or I is shown.

[0045] The saturated straight-chain or branched-chain divalent hydrocarbon radicals having 1 to 30 carbon atoms include, in particular: -CH2-; -CH2-CH2-; -CH2-(CH2) m -; -(CH2) m -CH(C1-C7)-(CH2) m -; -(CH2) m -CH(C1-C7)-(CH2) m -CH(C1-C7)-(CH2) m -; -(CH2) m -C(C1-C7)2-(CH2) m ; -(CH2) m It should be noted that there may be some inaccuracies in the original text, especially in the chemical formula and related descriptions. It is recommended to double-check with the original technical materials for a more accurate understanding.-C(C1~C7)2-(CH2) m -CH(C1~C7)-(CH2) m -; -(CH2) m -C(C1~C7)2-(CH2) m -C(C1~C7)2-(CH2) m -; In the formula, m is an integer in the range of 0 to 30, provided that the length of the main chain hydrocarbon chain does not exceed 30 carbon atoms, and C1 to C7 represents an alkyl group having 1 to 7 carbon atoms. This includes C1-C7 alkyl groups, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and heptyl. An unsaturated linear or branched divalent hydrocarbon radical having 2 to 30 carbon atoms refers to a hydrocarbon radical having one or more carbon-carbon double bonds and / or one or more carbon-carbon triple bonds. Examples of divalent unsaturated hydrocarbon radicals include: -CH=CH-;-(CH2) m -CH=CH-(CH2) m -; -(CH2) m -CH=CH-(CH2) m -CH=CH-(CH2) m -; -(CH2) m -CH=CH-CH(C1~C7)-(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(C1~C7)2-(CH2)m -; -(CH2) m -CH=CH-C(C1~C7)2-(CH2) m -C(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) m -C≡C-(CH2) m - ;-(CH2) m -C≡C-(CH2) m -C=C-(CH2) m -; -(CH2) m -CH=CH-CH(C1~C7)-(CH2) m -C≡C-(CH2) m -CH(C1~C7)-; -(CH2) m -CH=CH-C(C1~C7)2-(CH2) m -C≡C-(CH2) m -C(C1~C_{7})_{2}-(CH_{2}) m -;<6000433>-(CH2) m -C≡C-CH(C1~C7)-(CH2) m -C≡C-(CH2) m -CH(C1~C7)-(CH2) m -; -(CH2) m -C≡C-C(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) m -C≡C-CH=CH-(CH2) m - where m is as defined previously [[ID=#79]]is included.

[0046] Note: In the translation of chemical formulas, it is necessary to ensure the accuracy of chemical symbols and subscripts. Here, in the formula in item 45, "C1~C7" is translated as "C1~C_{7}" to better represent the chemical range, and "C(C1~C7)2" is translated as "C(C1~C_{7})_{2}" to accurately represent the chemical structure. Also, in item 79, "が含まれる" is translated as "is included" to make the sentence conform to English expression habits. According to one embodiment of the present invention, a proligand (I) as defined above is: X shows that -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; Z is as defined above; P1, P2, and P3 independently represent H or P(O)(OZ)2; Z is as previously defined, and at least one of P1, P2, and P3 is not a hydrogen atom; or P2 and P3 together form an acetonide group of formula “Chemical Formula 2” as defined above; A exhibits a divalent radical of -O-;-S-;-CH2-; L, -(CH2)2-(O-CH2-CH2) n -, where n is defined above; -H;-NH2;-(CH2) n1 -CH=CH2 or -(CH2) n1 -C≡CH, where n1 is as defined above, and in each of these cases, L1 is nonexistent; Saturated linear or branched divalent hydrocarbon radicals having 1 to 30 carbon atoms; unsaturated linear or branched divalent hydrocarbon radicals having 2 to 30 carbon atoms Show; L1, -O-NH2; -ON=C(CH3)2; A ring system or heterocyclic ring system selected from "Chemical Formula 3", "Chemical Formula 4", and "Chemical Formula 5". -(CH2) n1 -CH=CH2;-(CH2) n1 C≡CH;-(CH2) n1 -N3;-(CH2) n1 -SH;-(CH2) n1 -NH2;-(CH2) n1 -N=C=O;-(CH2) n1 -N=C=S;-(CH2)n1 -NHCO-CH2Hal; wherein n1 and Hal are as defined above a halogen selected from Cl, Br or I is shown is characterized in that

[0047] According to another embodiment of the present invention, the proligand is as defined above and is characterized in the following aspects: X is -CH2-P(O)(OZ)2; -CH2-CO2Z; -CH(CO2Z)2; -CH(P(O)(OZ)2)2, wherein Z is as defined above, preferably Z is equal to H; P1, P2 and P3 are independently H or P(O)(OZ)2, wherein Z is as defined above, provided that at least one of P1, P2 and P3 is different from a hydrogen atom; or P2 and P3 together form an acetonide group of the formula "Formula 2" as defined above; A represents an oxygen atom -O-; L is -(CH2)2-(O-CH2-CH2) n - is shown, wherein n represents an integer in the range of 0 to 6, preferably n is equal to 0; L1 is -O-NH2; -O-N=C(CH3)2; a substituent of the formula "Formula 3" or "Formula 4" as defined above.

[0048] According to another embodiment of the present invention, the proligand as defined above is more specifically: X represents -CH2-P(O)(OZ)2, wherein Z is as defined above, preferably Z is equal to H; P1, P2 and P3 are as defined in the above paragraph, independently representing H or P(O)(OZ)2, wherein Z is as defined above, provided that at least one of P1, P2 and P3 is different from a hydrogen atom; or P2 and P3 together form an acetonide group of the formula "Formula 2" as defined above; A, L, and L1 are as defined in the paragraph above, where A is an oxygen atom; and L is -(CH2)2-(O-CH2-CH2) n - indicates a substituent in the formula where n is an integer in the range of 0 to 6, preferably n is equal to 0; L1 is -O-NH2; -ON=C(CH3)2; and it indicates a substituent of formula "Chemical Formula 3" or "Chemical Formula 4" as previously defined. It is characterized by points.

[0049] According to preferred embodiments of the present invention, a proligand as defined above is more specifically: X represents -CH2-P(O)(OZ)2, where Z is as previously defined, preferably Z is equal to H; The conditions are that P1, P2, and P3 independently represent H or P(O)(OZ)2, where Z is as defined above, and at least one of P1, P2, and P3 is not a hydrogen atom; A represents the oxygen atom -O-; L is -(CH2)2-(O-CH2-CH2) n - indicates that in the formula n is an integer in the range of 0 to 6, preferably n is equal to 0; L1 represents a substituent of formula "Chemical Formula 3" or "Chemical Formula 4" as defined above -O-NH2; It is characterized by points.

[0050] Examples of proligands of the present invention include: X represents -CH2-P(O)(OZ)2, and Z=H; P1 represents P(O)(OZ)2, where Z=H, and P2 and P3=H; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n is equal to 0; L1 shows -O-NH2 It is the proligand of formula (I).

[0051] Another example of the proligand of the present invention is: X represents -CH2-P(O)(OZ)2, and Z=H; P1 is H, and together P2 and P3 form an acetonide group of formula “Chemical Formula 2” as defined earlier; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n is equal to 0; L1 indicates -O-NH2 or -ON=C(CH3)2. It is the proligand of formula (I).

[0052] Table 2 below: X represents the phosphonic acid group of the formula -CH2-P(O)(OH)2; The conditions are that P1, P2, and P3 independently represent H or P(O)(OH)2, and at least one of P1, P2, and P3 is not a hydrogen atom; or that P2 and P3 together form the acetonide group of formula "Chemical Formula 2"; A represents an oxygen atom; L is -(CH2)2- (or n is equal to 0 -(CH2)2-(O-CH2-CH2)) n -) indicates; L1 is -O-NH2; it represents a substituent of formula "Chemical Formula 3" or "Chemical Formula 4" as defined earlier. An example of ProAMFA in formula (I) is shown. [Table 2]

[0053] Table 3 below: X represents the carboxylic acid group of the formula -CH2-COOH; The conditions are that P1, P2, and P3 independently represent H or P(O)(OH)2, and at least one of P1, P2, and P3 is not a hydrogen atom; or P2 and P3 together form the acetonide group of formula "Chemical Formula 2"; A represents an oxygen atom; L is -(CH2)2- (or n is equal to 0 -(CH2)2-(O-CH2-CH2)) n -) indicates; L1 represents -O-NH2; a substituent of the formula "Formula 3" or "Formula 4" as defined above Examples of ProAMFA of formula (I) are shown. [Table 3]

[0054] The following Table 4 shows: X represents a malonic acid group of the formula -CH(CO2H)2; P1, P2 and P3 each independently represent H or P(O)(OH)2, provided that at least one of P1, P2 and P3 is different from a hydrogen atom; or P2 and P3 together form an acetonide group of the formula "Formula 2"; A represents an oxygen atom; L represents -(CH2)2- (or -(CH2)2-(O-CH2-CH2) where n is equal to 0 n -) L1 represents -O-NH2; a substituent of the formula "Formula 3" or "Formula 4" as defined above Examples of ProAMFA of formula (I) are shown. [Table 4]

[0055] The following Table 5 shows: X represents a bisphosphonic acid group of the formula -CH(P(O)(OH)2)2; P1, P2 and P3 each independently represent H or P(O)(OH)2, provided that at least one of P1, P2 and P3 is different from a hydrogen atom; or P2 and P3 together form an acetonide group of the formula "Formula 2"; A represents an oxygen atom; L represents -(CH2)2- (or -(CH2)2-(O-CH2-CH2) where n is equal to 0 n -) L1 represents -O-NH2; a substituent of the formula "Formula 3" or "Formula 4" as defined above Examples of ProAMFA of formula (I) are shown. [Table 5]

[0056] Conjugate of equation (II)

[0057] In another aspect, an object of the present invention is a conjugate characterized in that it has the following general formula (II). [ka] During the ceremony, n2 is an integer in the range of 1 to 1000, preferably 1 to 20, and more preferably 1 to 10. P1, P2, P3, X, A, and L are defined above, L'1 represents the substituent L1 as defined above when L1 is involved in the covalent bond with Y1. Y1 represents the product of interest Y, selected from the group including proteins, particularly antibodies and lysosomal enzymes, nanoparticles, protein activators or inhibitors, cytotoxic compounds, and markers for medical imaging. Y1 forms n2 covalent bonds (or more) with L'1. The antibodies mentioned above include armed antibodies, antibody-drug conjugates, and antibody fragments.

[0058] In the context of this invention, "conjugate" refers to a compound comprising two parts linked together by a covalent bond. Thus, the first part of the conjugate represents a proligand, while the second part of the conjugate represents the product of interest. The product of interest Y refers to the "free" product of interest when it does not form a bond with the proligand of formula (I). The product of interest Y1 refers to the product of interest when covalently linked to a proligand. In formula (II) of the conjugate of the present invention, the compound shown in parentheses corresponds to the proligand of formula (I) as defined above, when the proligand of formula (I) as defined above is involved in covalent bonding with the product of interest Y1. The integer n2 indicates the number of proligands(s) linked to the product of interest Y1. According to the present invention, n2 proligands(s) may be linked to the product of interest Y1. These n2 proligands are linked to the product of interest via the L1 or LL1 radical of the proligand of formula (I).

[0059] According to one embodiment of the present invention, the product Y1 of interest of the conjugate (II) as defined above is an antibody or a lysosomal enzyme.

[0060] Examples of the conjugates of the present invention include: X represents CH2-P(O)(OZ)2, and Z=H; P1 represents P(O)(OZ)2, where Z=H, and P2 and P3=H; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n is equal to 0; L'1 indicates -ON=; n² is an integer in the range of 1 to 7; Y1 represents product Y of interest, which is an antibody or lysosomal enzyme. It is the one in equation (II).

[0061] Another example of the conjugate of the present invention is: X represents CH2-P(O)(OZ)2, and Z=; P1 represents H, and together P2 and P3 form an acetonide group as defined in formula "Chemical Formula 2" above; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n is equal to 0; L'1 indicates -ON=; n² is an integer in the range of 1 to 7; Y1 represents product Y of interest, which is an antibody or lysosomal enzyme. It is the one in equation (II).

[0062] Preparation of the conjugate of formula (II) The conjugate of equation (II) as defined above is: -The product Y of the object of interest as defined above and - n2 proligands (or more) of the general formula (I) as defined above It is prepared by reacting the following. The free product Y of interest may also be represented by Y1'-L', where L' represents a functional group or reactive functional group possessed by the product Y of interest. In other words, Y1' represents the product Y of interest that does not contain the functional group L'. The functional group L' of Y will react with a functional group or reactive functional group (in this case L1 or LL1) possessed by proligand (I) to form a covalent bond between product Y of interest and proligand (I), thus yielding the conjugate of formula (II) of the present invention. In this application, the terms “functional group,” “reactive functional group,” or “reactive chemical group” may be used interchangeably. These terms each refer to a group possessed by product Y of interest (or possessed by proligand (I)) that is capable of reacting with a group possessed by proligand (I) (or possessed by product Y of interest).

[0063] In one embodiment of the present invention, in the conjugate of equation (II) as defined above, n2 is an integer equal to 1. Figure 3 illustrates four possible embodiments of the synthesis of the conjugate of formula (II) obtained by a reaction between the proligand of formula (I) and the product of interest Y. In the general formulas (I) and (II) shown in points 1-4: -X, P1, P2, and P3 are as defined above, -n2 is equal to 1, -A indicates an oxygen atom. -L is -(CH2)2-(O-CH2-CH2) n This indicates a negative value where n is an integer between 0 and 2. The variable region is located in the radical L1 of proligand (I) and in the functional group possessed by the product Y of interest. Figure 3, point 1 illustrates the reaction between the functional group L1 of proligand (I), which represents -ONH2, and the aldehyde functional group (-CHO) of product Y of interest, which is an antibody or lysosomal enzyme, where the aldehyde functional group is generated beforehand during an oxidation process to a portion of the oligosaccharide / glycoside moiety of the antibody or lysosomal enzyme. Product Y is represented by Y1'-CHO, where Y1' represents product Y without the functional group L' equivalent to -CHO. Figure 3, point 2 illustrates a reaction between the functional group L1 of proligand (I), where L1 represents "Chemical Formula 3," and an amine functional group (-NH2) possessed by product Y of interest, such as an antibody or lysosomal enzyme. Product Y of interest is represented by Y1'-NH2, where Y1' represents Y without the functional group L' equivalent to -NH2. The amine functional group is derived, for example, from a lysine amino acid residue. Figure 3, point 3 illustrates a reaction between the functional group L1 of proligand (I), where L1 represents "Chemical Formula 4," and the thiol functional group (-SH) possessed by product Y of interest, such as an antibody or lysosomal enzyme. Product Y of interest is represented by Y1'-SH, where Y1' represents Y without the functional group L' equivalent to -SH. The thiol functional group is derived, for example, from a cysteine ​​amino acid residue. Figure 3, point 4 illustrates a reaction between the functional group L1 of proligand (I), where L1 represents "Chemical Formula 5," and the thiol functional group (-SH) possessed by the product of interest Y (e.g., an antibody or lysosomal enzyme). The product of interest Y is represented by Y1'-SH, where Y1' represents Y without the functional group L' equivalent to -SH.

[0064] Use of the Conjugate (II) of the present invention

[0065] The present invention further relates to a conjugate of formula (II) for use as a pharmaceutical agent.

[0066] The conjugate (II) of the present invention binds in situ to a biological membrane or non-membrane target, either covalently or non-covalently. Examples of membrane targets are: - Growth factor receptors, e.g., EGFR, FGFR, VEGFR, TrK, NGF, PDGFR, insulin receptor, erythropoietin receptor, ephrin receptor or - Membrane molecules involved in immune regulation, such as CD or ILT factors, PD-1, TIGIT, LAG, PD-L1, TIM3 This is likely the case. Examples of extracellular non-membrane targets include cytokines (TNFα, CD20, interferon, interleukin), growth factors (VEGF, EGF, FGF), antibodies, proteins or non-protein hematological aggregates, exosomes, and circulating proteins such as bone tissue hydroxyapatite.

[0067] Depending on the target to which the conjugate of formula (II) binds, the complex may then be internalized into the cell after activation through ligand recognition by CI-M6PR in that particular cell. The specificity of this cell targeting may be related to the nature of the target. For example, if the biological target is the epidermal growth factor receptor EGFR, the activated complex will be internalized into the cell, while the biological target will be degraded or not present in the lysosome. If the target is hydroxyapatite present in bone tissue, the activated complex can be internalized within bone tissue cells. If the biological target consists of blood aggregates made up of several protein-like components, nucleic acids, collagen, or fibrils, the activated complex can be internalized into the surrounding cells.

[0068] For example, if the product Y of interest of the conjugate of the present invention is an antibody, such as infliximab, it can bind to non-membrane targets such as TNFα cytokines and may treat Crohn's disease, ankylosing spondylitis, or rheumatoid arthritis. Similarly, if the product of interest is an antibody, such as cetuximab, it can bind to membrane targets such as the epidermal growth factor receptor EGFR, potentially enabling the treatment of metastatic colorectal cancer or head and neck cancer.

[0069] Examples of pathologies treatable with the conjugate (II) of the present invention include: - Cancer, especially solid cancers including prostate cancer, breast cancer, pancreatic cancer, colon cancer, lung cancer, liver cancer, or bone cancer, - Inflammatory and autoimmune diseases, especially ankylosing spondylitis, multiple sclerosis, type 1 diabetes, Crohn's disease, lupus, autoimmune thyroiditis, rheumatoid arthritis, - Neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Charcot disease), and -Infectious diseases This includes items selected from the group that includes the following:

[0070] Figures 1 and 2 illustrate examples of the proligand (I) and conjugate (II) of the present invention. Figure 1 illustrates the sequence of events that occur when the conjugate (II) of the present invention, which has a “proligand-product of interest” structure, is introduced into a living organism for the purpose of binding to a non-membrane target (the non-membrane target is indicated by an asterisk in Figure 1). Once introduced into the body, conjugate(II) binds to a non-membrane target via the product of interest, forming a complex of the "proligand-product of interest-non-membrane target" structure. The binding between the product of interest and the non-membrane target may be covalent or non-covalent. The non-membrane target acts as an anchoring point for the product of interest. More specifically, the non-membrane target allows the product of interest to be concentrated at specific and desired locations in the organism. Next, the proligand is activated in situ through a biological mechanism by the ligand, which leads to the activation complex of the "ligand-product of interest-non-membrane target" structure. The mechanism of proligand activation in the body may be either enzymatic or chemical. By enzymatic process, the inventors mean hydrolysis by the action of enzymes naturally present in the body, such as phosphatases, esterases, proteases, nucleases, and sulfatases. According to a preferred embodiment, activation of the proligand to its ligand is carried out by alkaline phosphatases or acid phosphatases present in different tissues in the body. By chemical process, the inventors mean either hydrolysis under acidic, basic, or neutral conditions, or a redox reaction. In fact, living organisms naturally present acidic conditions in their environment, which can hydrolyze, for example, the acetonide of a proligand to form a ligand, particularly in acidic compartments generated by solid tumors or bone tissue. Activation of a proligand onto its ligand enables the conversion of a "proligand-product of interest-non-membrane target" complex into an activated "ligand-product of interest-non-membrane target" complex. The conversion of proligands to ligands (via the in situ mechanism described above) allows the ligands to be recognized and bind to extracellular membrane receptors. Once the activation complex binds to the cell's membrane receptor via its ligand, two scenarios are possible. In Case 1, since the primary objective is the internalization of the product of interest into the cell, only the conjugate (i.e., without the non-membrane target) is internalized. In Case 2, since the primary objective is to remove the non-membrane target by being internalized into the cell, the entire complex is internalized into the cell. Figure 2 illustrates a sequence of events that occur when the conjugate of the present invention, having a “proligand-product of interest” structure, is introduced into a living organism for the purpose of binding to a membrane target (the membrane target is indicated by a star in Figure 2). Once introduced into the body, conjugate(II) binds to the membrane target via the product of interest, forming a “proligand-product of interest-membrane target” complex. The binding between the product of interest and the membrane target may be covalent or non-covalent. The membrane target acts as an anchoring point on the surface of a particular cell: more specifically, the membrane target allows the product of interest to be concentrated at a specific and desired location in the organism. Subsequently, the proligand is activated in the body via an in situ mechanism, which leads to the activation complex of the "ligand-product of interest-membrane target" structure. The in situ mechanism that occurs is described above in relation to Figure 1. The reactivated ligand recognizes and binds to the extracellular membrane receptor of the cell. Once the activation complex binds to the cell's membrane receptor via its ligand, two scenarios are possible. In Case 1, since the primary objective is to internalize the product of interest into the cell, only the conjugate (i.e., without the membrane target) is internalized. In Case 2, since the primary objective is to internalize both the product of interest and the target into the cell, the entire complex is internalized into the cell.

[0071] In the present invention, if the proligand (I) of the present invention is an analog of ProAMFA, i.e., mannose-6-phosphate (M6P), then the extracellular membrane receptor of the cell is CI-M6PR.

[0072] As shown above, the binding between the conjugate (II) of the present invention and the target (non-membrane or membrane) may be covalent or non-covalent. The compound obtained after the binding of the conjugate to the target will be referred to in this application as a “complex”. Another objective of the present invention is: - Conjugate of equation (II) as defined above - A non-membrane or membrane target, wherein the target is as defined above. The complex consists of the conjugate (II) and the target, which are linked to each other by non-covalent or covalent bonds.

[0073] The activated ligands obtained as a result of the in situ mechanism described above correspond to compounds of general formula (I), where radicals P1, P2, and P3 each represent hydrogen atoms. Activated AMFA ligands can be recognized and bound by CI-M6PR.

[0074] Activation conjugates resulting from in situ mechanisms occurring in living organisms can be represented by the following general formula (II-bis). [ka] During the ceremony: X, A, L, L'1, n2, and Y1 are as defined above. P'1, P'2, and P'3 each represent hydrogen.

[0075] Activation of conjugate (II) within the complex is: -Activation conjugate of formula (II-bis) -Target as defined above This generates an activation complex consisting of the following components, and the activation conjugate and target of general formula (II-bis) are linked to each other by non-covalent or covalent bonds. The resulting activated complex is used to bind to CI-M6PR.

[0076] According to one embodiment of the present invention, the activation conjugate of formula (II-bis) is 10 for CI-M6PR -5 ~10 -9 50% inhibitory concentration in the M range (IC 50 It has affinity measured by ).

[0077] In another aspect of the present invention, a pharmaceutical composition is proposed, characterized in that it comprises a conjugate of formula (II) as defined above.

[0078] The present invention also relates to a conjugate of formula (II) for use as defined above, i.e., for use as a pharmacopoeia, characterized in that the conjugate is of a type suitable for oral, parenteral, intravenous, intramuscular, or subcutaneous administration. [Examples]

[0079] The following examples, with particular reference to the figures, describe the synthesis of the proligand and conjugate (II) of formula (I) of the present invention, and their biological effects, especially the study of the activation of the proligand to its ligand. The proligand ProAMFA1(I) of the present invention is compared with the compound AMFA1 and the compound mannose 6-phosphate (M6P).

[0080] Example 1: Synthesis of the proligand ProAMFA1 of formula (I) and the proligand ProAMFA1 acetonide oxime of formula (I).

[0081] Synthesis of ProAMA1

[0082] The proligand ProAMFA1 presents the following structural formula: [ka] The proligand ProAMFA1 corresponds to general formula (I), where: X represents CH2-P(O)(OZ)2, where Z=H (i.e., the phosphonic acid group CH2-P(O)(OH)2); P1 represents P(O)(OZ)2, where Z=H (i.e., the phosphate group P(O)(OH)2); P2=P3=H; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n=0 (i.e., (CH2)2). L1 indicates ONH2. The ProAMFA1 synthesis pathway is illustrated in Figure 4 and described in detail below.

[0083] "2-bromoethyl 2,3;4,6-di-O-isopropylidene-α-D-mannopyranoside" (2) 14.300 g (1.0 eq; 49.81 mmol) of 1 ("2-bromoethyl α, D-mannopyranoside") was mixed with 50 mL of anhydrous acetone. Next, 31 mL (5.0 eq; 249.05 mmol) of 2,2-dimethoxypropane was added, followed by 0.430 g (0.05 eq; 2.49 mmol) of p-toluenesulfonic acid. After reacting for 1 hour, compound 2 was obtained, but this was not isolated and was used directly in the following reaction. Rf: 0.83 (AcOEt / Cyclohexane (8:2)) ESI + m / z:367[M+H] + HRMS:367,0765Da;Conf(%):100.00%

[0084] "2-bromoethyl 2,3-O-isopropylidene-α-D-mannopyranoside" (3) After the previous reaction, 10 mL of mQ water was added. After 5.5 hours, the medium was neutralized with NaHCO3 (sodium bicarbonate), the organic phase was dried, and the mixture was evaporated. The unpurified product was purified on a silica gel column using AcOEt / cyclohexane (1:9) eluate. Compound 3 was obtained in 2 steps in 66% yield: Rf: 0.43 (AcOEt / Cyclohexane (8:2)) ESI + m / z:327[M+H] + HRMS:327.0439Da;Conf(%):100.00%.

[0085] "2-bromoethyl 2,3-O-isopropylidene-α-D-manno-hexodialdo-1,5-pyranoside" (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-tetramethylpiperidine-1-yl)oxy) were weighed, conditioned under inert air, and then 100 mL of anhydrous dichloromethane was added. 1.496 g (1.0 eq; 6.72 mmol) of TCCA (trichloroisocyanuric acid) was added at 0°C. After the reaction was complete, 10 mL of anhydrous methanol was added, and the medium was then filtered through celite. After evaporation, a white foam was obtained. Product 4 was not isolated and was used directly in the following reaction. Rf: 0.54 (AcOEt / Cyclohexane (8:2))

[0086] "2-bromoethyl(6E)-2,3-O-isopropylidene-6,7-didesoxy-7-diethoxyphosphfinyl-α-D-mannohept-6-enopyranoside" (5) 0.269 g (1.0 eq; 6.72 mmol) of 60% NaH (sodium hydride) was conditioned under inert air. 100 mL of anhydrous THF (tetrahydrofuran) was added to form a suspension. After 10 minutes, 1.67 mL (1.0 eq; 6.72 mmol) of tetraethyl methylenediphosphonate (TEMDP) was added to the solution with stirring. After 30 minutes, the anionic solution was cooled to 0°C and then cannulated onto the previously obtained unpurified compound 4. At the end of the reaction, the medium was diluted with 300 mL of ethyl acetate and washed with brine and then water. The organic phase was dried, filtered, and then evaporated to dryness. The unpurified product was purified on a silica gel column using AcOEt / cyclohexane (8:2) eluate. The fractions were pooled, and compound 5 was obtained in 2 steps in 51% yield. Rf: 0.18 (AcOEt / Cyclohexane 8:2) HRMS:459.0786Da;Conf(%):100.00%

[0087] "2-bromoethyl 2,3-O-isopropylidene-6-deoxy-6-diethoxyphosphenylmethylene-α-D-mannopyranoside" (6) 1.250 g (1.0 eq; 2.72 mmol) of compound 5 and 0.125 g (10% by weight) of Pd / C were packed under inert air. 27 mL of anhydrous methanol was added to dissolve compound 5, and then 2.17 mL (5.0 eq; 13.61 mmol) of triethylsilane was added dropwise. After the reaction, the medium was filtered over celite and the solvent was evaporated. Finally, 1.200 g of compound 6 was obtained in 96% yield. Rf: 0.23 (AcOEt) HRMS:461.0924Da;Conf (%):100.00%.

[0088] "2-(phthalimidooxy)ethyl 2,3-O-isopropylidene-6-deoxy-diethoxyphosphenylmethylene-α-D-mannopyranoside" (7) 0.469 g (1.1 eq; 2.87 mmol) of N-hydroxyphthalimide was weighed and conditioned under inert air, and then 5 mL of anhydrous THF was added. 0.470 mL (1.2 eq; 3.14 mmol) of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) was added, and the solution was heated to 45°C. After 30 minutes, 1.2 g (1.0 eq; 2.61 mmol) of intermediate 6, dissolved in 8 mL of THF, was added dropwise to the anion. After reacting for 22 hours, the reaction medium was diluted in dichloromethane and then washed with brine. The organic phase was dried over magnesium sulfate (MgSO4) and evaporated. The resulting unpurified product was purified on a silica gel column with ethyl acetate (AcOEt). 0.915 g of compound 7 was obtained in 64% yield. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) HRMS:544.1946Da;Conf(%):100.00%.

[0089] "2-(phthalimidooxy)ethyl 2,3-O-isopropylidene-4-O-dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylmethylene-α-D-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)-1H-tetrazole were weighed, packed under inert air, and dissolved in 35 mL of dichloromethane. After cooling the solution, 0.724 mL (2.0 eq; 3.37 mmol) of dimethyl N,N-diisopropyl phosphoramidite was added dropwise. At the end of the reaction, 0.670 mL (13.0 eq; 21.8 mmol) of 30% H2O2 (hydrogen peroxide) was added directly to the solution at room temperature. After 10 minutes, the medium was diluted in dichloromethane and washed twice with NaHCO3 solution. The organic phase was dried over MgSO4 and concentrated. Purification on a silica gel column was performed using AcOEt. After evaporation of the fraction, 0.953 g of compound 8 was obtained. The yield was 87%. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) HRMS:652.1922Da;Conf(%):100.00%.

[0090] "2-(phthalimidooxy)ethyl 4-O-dimethoxyphosphinyl-6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranoside" (9) 0.953 g (1.0 eq; 1.46 mmol) of compound 8 was weighed, and 7.3 mL of THF was added. Then, 1.625 g of Dowex H was added to the reaction medium. + Resin was added. After 18 hours, the reaction was complete, and the resin was removed by filtration. After evaporation, the filtrate yielded compound 9 in 34% yield. Rf: (CH2Cl2 / ethyl ether 95 / 5 (v / v)) HRMS:612.1610Da;Conf(%):100.00%.

[0091] "2-aminooxyethyl 4-O-dihydroxyphosphinyl-6-deoxy-6-dihydroxyphosphinylmethylene-α-D-mannopyranoside" (ProAMFA1) 0.300 g (1.0 eq; 0.49 mmol) of compound 9 was 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, and 0.55 mL (9.0 eq; 3.93 mmol) of triethylamine and 0.745 mL (12.0 eq; 5.89 mmol) of trimethylsilyl chloride (TMSCl) were added. The solution was stirred at room temperature under inert air for 24 hours, and then the solvent was evaporated. The residue was collected in methanol and excess salt was removed by filtration. The filtrate was then filtered using a cation exchange resin (DOWEX® 50WX2, H + The mixture was treated with (as), then the resin was 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 was added to the filtrate. After evaporation of the reaction and reaction medium to dryness, the latter was heated to 100 C 18 The solution was purified on a silica gel column (eluent: H2O). 68 mg of ProAMFA1 was collected. The yield over the two steps was 35%. Rf: 0.69 (2-propanol / NH4OH / H2O 30 / 40 / 30 (v / v)) HRMS:398.0607Da;Conf(%):100.00%. [α] D 20 : +32.5° (4 mg per 1 mL of H2O)

[0092] Preparation of compound AMFA1 Compound AMFA1 is a mannose 6-phosphate (M6P) analog. This compound was synthesized according to the protocol described in EP 2 448 600 B1 and has the following structural formula: [ka] The difference between ProAMFA1 and AMFA1 lies in the definition of the radical P1: ProAMFA1 represents a phosphate group P(O)(OH)2, while AMFA1 represents a hydrogen atom.

[0093] Mannose 6-phosphate (M6P) compounds We commercially purchased a compound called mannose 6-phosphate (M6P), which has the following structural formula: [ka]

[0094] Synthesis of ProAMFA1 acetonide oxime

[0095] The proligand ProAMFA1 acetonide oxime presents the following evolutionary formula: [ka] The proligand ProAMFA1 acetonide oxime is: X represents CH2-P(O)(OZ)2, and Z=H (i.e., the phosphonic acid group CH2-P(O)(OH)2); P1 represents H, and together P2 and P3 form an acetonide group as defined in formula "Chemical Formula 2" above; A represents an oxygen atom; L is -(CH2)2-(O-CH2-CH2) n - indicates that n=0 (i.e., (CH2)2); L1 indicates -ON=C(CH3)2 This corresponds to general formula (I). The ProAMFA1 acetonide-oxime synthesis scheme is illustrated in Figure 24 and described in detail below.

[0096] "ProAMFA1 acetonide oxime" or "2-aminooxyethyl 2,3-O-isopropylidene-6-deoxy-6-dihydroxyphosphinylmethylene-α-D-mannopyranoside oxime" 0.010 g (1.0 eq; 0.032 mmol) of compound AMFA1 was dissolved in anhydrous DMSO / acetone mixture (0.5 mL / 0.2 mL). After 18 hours, the solvent was evaporated. Purification was performed on a silica gel column (220 mg) (eluent: isopropanol) to obtain compound AMFA1ox (9 mg) (see Figure 24). ESI + m / z:358[M+H] +

[0097] Compound AMFA1ox (9 mg) was dissolved in anhydrous dimethylformamide (0.2 mL). 0.3 mg of p-toluenesulfonic acid (PTSA) (0.05 eq; 0.002 mmol) and 2,2-dimethoxypropane (0.02 mL) were added. After stirring for 18 hours, the medium was neutralized with ammonia solution. After purification on a silica gel column (eluent: water), ProAMFA1 acetonide oxime (9 mg) was obtained (see Figure 24). ESI + m / z:398[M+H] +

[0098] Activation of ProAMFA1 acetonide oxime to AMFA1ox To verify its potential as a proligand, the ability of ProAMFA1 acetonide oxime to release the acetonide group of formula "Chemical Formula 2" in P2 and P3 was studied. The kinetics of the acid hydrolysis of the acetonide group were studied by dissolving ProAMFA1 acetonide oxime in deuterium methanol (MeOD) and adjusting the pH to 5.5, according to the following protocol. 31 The reaction was tracked by 1P NMR analysis. The conversion of ProAMFA1 acetonide oxime to AMFA1ox is shown in Figure 25. Hydrolysis test of the acetonide group of ProAMFA1 acetonide oxime ProAMFA1 acetonide oxime (9 mg) was dissolved in 500 μL of MeOD. The pH was adjusted to 5.5 by adding 4 μL of 1N hydrochloric acid solution. The sample in an NMR test tube was analyzed at 37°C using a Bruker AVANCE 400 MHz spectrophotometer with the following parameters: 162 MHz, ns=32, D1=2s, pulse width; 15°, 310 K. The results of this study on the conversion of ProAMFA1 acetonide oxime to AMFA1ox are illustrated in Figure 26. As shown in the series of spectra in this figure, the phosphonic acid of ProAMFA1 acetonide oxime 31 The P signal is from AMFA1ox phosphonic acid. 31 The P signal is supported and then disappears. AMFA1ox formation was confirmed by adding a known amount of AMFA1ox, which resulted in an increase in the AMFA1ox-specific signal (t=2h30+AMFA1ox).

[0099] Example 2: Preparation of the present invention conjugate of formula (II)

[0100] The three products Y of interest, namely lysosomal enzymes and two antibodies, were functionalized with either the compound ProAMFA1 or AMFA1, respectively.

[0101] The tested lysosomal enzyme is commercially available under the name Myozyme®. It is an enzyme, alpha-alglucosidase (or acid alpha-glucosidase). Hereafter, this enzyme may be referred to as "Myo". It contains an M6P residue in its structure, making it capable of targeting CI-M6PR. The lysosomal enzyme Myo is used in the treatment of Pompe disease through enzyme substitution therapy. The antibodies being tested are infliximab and cetuximab, two chimeric monoclonal IgG1 antibodies lacking the M6P residue in their structure. These may hereafter be referred to as "Infli" and "Cetux". As already shown: - The antibody infliximab binds to the soluble extracellular cytokine TNFα and is used to treat autoimmune and inflammatory diseases (Crohn's disease, ankylosing spondylitis, rheumatoid arthritis (Melsheimer R et al., Biologics. 2019, 13, 139)); - The antibody cetuximab binds to extracellular and membrane EGFR and is used to treat different cancers (metastatic colorectal cancer or head and neck cancer) (Baselga J. et al., J. Clin. Oncol., 2005, 23, 5560; Galizia G. et al., Oncogene. 2007, 26, 3654).

[0102] The conjugate thus formed may be referred to in this application as "AMFA1_Myo" or "Myo_AMFA1", "ProAMFA1_Myo" or "Myo_ProAMFA1", "AMFA1_Infli" or "Infli_AMFA1", "ProAMFA1_Infli" or "Infli_ProAMFA1", "AMFA1-Cetux" or "Cetux-AMFA1", "ProAMFA1-Cetux" or "Cetux-ProAMFA1". The conjugate of the present invention includes the ProAMFA1 portion. Preferably, according to the present invention, the ProAMFA1 portion, once the conjugate is introduced into a living organism, is not immediately recognized by the CI-M6PR receptor, and therefore allows for specific targeting of a predetermined biological target (membrane or non-membrane) by the compound of interest Y, i.e., Myozyme® or infliximab and cetuximab antibodies.

[0103] Coupling and quantification of compounds AMFA1 and ProAMFA1 with product Y of interest (Myozyme®, infliximab, or cetuximab). By using the ethyloxyamine functionality present on the spacer arm of AMFA1 or ProAMFA1 to enable the formation of an oxime-type covalent bond with the aldehyde functionality pre-generated by the controlled oxidation of the oligosaccharide chain of the product Y of interest (Myozyme®, infliximab or cetuximab), the coupling of AMFA1 or ProAMFA1 with the product Y of interest (Myozyme®, infliximab or cetuximab) is achieved. The coupling of product Y of interest containing an aldehyde functionality (pre-generated by oxidation) with ProAMFA1 (or AMFA1) of formula (I) containing a spacer arm with an ethyloxyamine functionality (L = -(CH2)2- and L1 = -ONH2) is illustrated in Figure 3, point 1. Coupling of AMFA1 or ProAMFA1 with Myozyme(registered trademark) First, the Myozyme® enzyme is oxidized at the oligosaccharide chain level. To achieve this, after filtering Myozyme® through Sepharose G25, it is oxidized with 1 mM sodium metaperiodate at 4 °C for 30 minutes. Glycerol (20 μL per mL) is added at 4 °C for 10 minutes to stop the oxidation. Next, the enzyme solution is filtered through Sepharose G25, and compound ProAMFA1 or AMFA1 is added in a 100-fold molar excess. After 2 hours at 37 °C, Myozyme® is coupled, and the excess uncoupled ProAMFA1 or AMFA1 present in the solution is removed by dialysis against a buffer containing 2% mannitol, 3 mM Na2HPO4, 22.1 mM NaH2PO4 and 0.005% polysorbate 80 per liter. The conjugates Myo-AMFA1 and Myo-ProAMFA1 thus obtained are stored at 4 °C in this buffer. Quantification Analysis by MALDI-TOF mass spectrometry made it possible to approximately estimate the number of ProAMFA1 or AMFA1 coupled to the Myozyme® enzyme to be about 1. Coupling of AMFA1 or ProAMFA1 with infliximab First, infliximab antibody is oxidized at the level of oligosaccharide chains. To do this, infliximab (5 mg), which has been pre-filtered on Sepharose G25, is oxidized with 1 mM sodium metaperiodate at 4 °C for 30 minutes. Glycerol (20 μL per mL) is added at 4 °C for 10 minutes to stop the oxidation. Next, the antibody solution is filtered on Sepharose G25, and compound ProAMFA1 or AMFA1 is added in a 200-fold molar excess. After 2 hours at 37 °C, infliximab is coupled, and the excess uncoupled ProAMFA1 or AMFA1 present in the solution is removed by dialysis against a buffer containing 2% mannitol, 3 mM Na2HPO4, 22.1 mM NaH2PO4 and 0.005% polysorbate 80 per liter. The thus obtained Infli-AMFA1 and Infli -ProAMFA1 conjugates are stored in this buffer at 4 °C. Quantification Analysis by MALDI-TOF mass spectrometry made it possible to roughly estimate the number of ProAMFA1 or AMFA1 coupled to the infliximab antibody to be approximately 5. Coupling of AMFA1 or ProAMFA1 with cetuximab The protocol is the same as that described for infliximab.

[0104] Characterization of conjugates obtained by Coomassie blue staining The integrity of the product of interest (Myozyme®, infliximab or cetuximab) after coupling with AMFA1 or ProAMFA1 is verified by Coomassie blue staining after electrophoresis on a 12% SDS-polyacrylamide gel. After electrophoresis, the gel is stained with Coomassie blue solution for 1 hour to label the product of interest. It is important to ensure that the coupling between the product of interest and the proligand does not cause degradation. Samples of Myozyme®, Myo-AMFA1, and Myo-ProAMFA1 were loaded onto polyacrylamide gels under denaturing conditions and then stained with Coomassie blue. The results are shown in Figure 5. Since the same characteristic band of the enzyme is observed at 110 kDa both before and after coupling, it is possible to conclude that the coupling did not cause degradation. Using the same method, samples of infliximab, Infli-AMFA1, and Infli-ProAMFA1 were loaded onto polyacrylamide gels under denaturing conditions, and then stained with Coomassie blue. The results are shown in Figure 6. Since the same characteristic bands of the antibody are observed at 50kDa and 25kDa before and after coupling, it can be concluded that the coupling did not cause degradation.

[0105] Example 3: Biological evaluation of proligand(I)

[0106] Cytotoxicity assessment of ProAMFA1 The cytotoxicity of ProAMFA1 of formula (I) was evaluated in comparison with M6P and AMFA1 in human MCF-7 breast cancer cells (Figure 7) and healthy human fibroblasts (Figure 8) according to the protocol described below. Experimental protocol: Human breast cancer cells (MCF-7) and human fibroblasts (FS01035) are cultured in DMEM / F12 and DMEM supplemented with 10% fetal bovine serum (FCS) and penicillin-streptomycin, respectively. The cells are maintained at 37°C in the presence of 5% CO2. After trypsin treatment, the cells are seeded into 96-well plates at 20% density for MCF-7 cells and 40% density for fibroblasts. The adherent cells are treated the following day. 10 -4 ~10 -7The cells were treated with compounds M6P, AMFA1, and ProAMFA1 at concentrations between M (Figures 7 and 8). Each condition was analyzed in triplicates. After 72 hours of incubation, cell viability was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The results of the cytotoxicity studies are shown in Figure 7 for breast cancer cells and in Figure 8 for fibroblasts. These figures show that the ProAMFA1 compound is effective against selected cell models. -4 We demonstrate that no significant toxicity is observed during a 72-hour treatment up to the concentration of M. The AMFA1 compound is also non-toxic compared to the natural M6P compound.

[0107] ProAMFA1 affinity for CI-M6PR ProAMFA1 was evaluated in terms of its potential affinity for CI-M6PR compared to AMFA1 and M6P. As already mentioned, affinity refers to the strength of the interaction between a ligand, e.g., AMFA1, and its receptor, i.e., CI-M6PR. For this purpose, an assay based on interaction competition with CI-M6PR was performed according to the protocol described below. Experimental protocol Pre-installed 96-well MaxiSorp TM An affinity assay is performed using biotinylated CI-M6PR (b-CI-M6PR) that recognizes pentamannose 6-phosphate (PMP) adsorbed on a plate. Before distribution into wells, b-CI-M6PR is pre-incubated for 2 hours in the presence of different concentrations of M6P, AMFA1, or ProAMFA1. The pre-incubated solution is then distributed into PMP-containing wells. After 2 hours at room temperature, the amount of b-CI-M6PR bound to PMP is determined by measuring the optical density at 450 nm using a solution of streptavidin coupled to peroxidase in the presence of an OPD (o-phenylenediamine) substrate. The results obtained are shown in Figure 9 and Table 7 below. Table 7 shows the concentrations (IC) of M6P, AMFA1, and ProAMFA1 required to achieve 50% interaction with CI-M6PR. 50 ) indicates.

[0108] [Table 7]

[0109] Figure 9 and Table 7 show that ProAMFA1 has no affinity for CI-M6PR. The lack of affinity of ProAMFA1 to CI-M6PR is unexpected, as it was unpredictable to those skilled in the art that simply adding a phosphate group to M6P (i.e., P1=P(O)(OH)2) would completely neutralize its interaction with the receptor CI-M6PR. This unprecedented feature demonstrates its capability as a proligand. The lack of affinity of ProAMFA1 to CI-M6PR prevents the intracellular integration of the active substances (products of interest and biological targets) to which ProAMFA1 associates by these receptors.

[0110] Activation of ProAMFA1 to AMFA1 To validate its therapeutic potential as a proligand, we studied the ability of ProAMFA1 to lose its P1=P(O)(OH)2 phosphate group. According to the following protocol, ProAMFA1 was incubated in the presence of alkaline phosphatase isolated from human placenta, and then... 31 We conducted a dynamic study of dephosphorylation by performing 1P NMR analysis. Enzyme digestion test of ProAMFA1 by alkaline phosphatase An aliquot of 2.3 mg of ProAMFA1 was dissolved in 200 μL of D2O and 300 μL of 50 mM Tris buffer pH 7.4. The initial analysis of the sample placed in the NMR tube was carried out at 37 °C using a Bruker AVANCE 400 MHz spectrophotometer with the following parameters: 162 MHz, ns = 16, D1 = 2 s, pulse width; 15°, at 310 K. An aliquot of 2.3 mg of alkaline phosphatase isolated from human placenta was dissolved in 100 μL of 50 mM Tris pH 7.4 and added to the NMR tube. The parameters shown were used for the kinetic study of the enzymatic digestion. After integration of the different signals observed by NMR, the kinetic conversion of ProAMFA1 to AMFA1 was established. The results obtained are shown in Figure 10. As shown by the series of spectra in this figure, a decrease in the ProAMFA1 phosphate signal supporting the appearance of inorganic phosphate (formed during the hydrolysis reaction by phosphatase) is observed in the presence of phosphatase. On the other hand, the 31 P phosphonate signal from ProAMFA1 disappears supporting the 31 P phosphonate signal from AMFA1. The formation of AMFA1 was confirmed by the addition of a known amount of AMFA1 leading to an increase in the AMFA1-specific signal. As part of the tests, a ProAMFA1 sample with 87% purity was used. This by-product is ProAMFA1-oxime (ProAMFA1ox) resulting from the reaction between ProAMFA1 and acetone. ProAMFA1ox presents the following structural formula:

Chemical formula

[0111] Example 4: Biological evaluation of conjugate(II) Myo-ProAMFA1 and Infli-ProAMFA1: Cytotoxicity and affinity for CI-M6PR

[0112] Cytotoxicity evaluation of conjugate Myo-ProAMFA1 The cytotoxicity of the conjugate Myo-ProAMFA1 and conjugate Myo-AMFA1 of the present invention was evaluated against human MCF-7 breast cancer cells (Figure 11) and healthy human fibroblasts (Figure 12) compared to the lysosomal enzyme Myo alone, using the same protocol as described in Example 3. The results illustrated in Figures 11 and 12 demonstrate that Myo-ProAMFA1 and Myo-AMFA1 conjugates do not exhibit significant cytotoxicity (100% ± 10%) in selected cell models compared to Myozyme® or untreated control cells.

[0113] Affinity evaluation of the conjugate Infli-ProAMFA1 on the CI-M6PR. The conjugated Infli-ProAMFA1 and conjugated Infli-AMFA1 of the present invention were evaluated for their affinity to the CI-M6PR receptor using the same protocol as described in Example 3. The results obtained are illustrated in Figure 13 and Table 8 below. Table 8 shows the concentrations (IC) of M6P and Infli-AMFA1 required to achieve 50% interaction with CI-M6PR. 50 ) indicates. The concentrations of infliximab and Infli-ProAMFA1 could not be determined within the tested concentration range, and 10 -5 It's higher than M.

[0114] [Table 8]

[0115] Affinity testing of the Infli-ProAMFA1 conjugate against CI-M6PR showed that the Infli-ProAMFA1 conjugate exhibited very low affinity for CI-M6PR (Figure 13 and Table 8). This affinity was comparable to that of the infliximab antibody alone, while the Infli-AMFA1 conjugate showed high affinity for CI-M6PR. The very low affinity of the Infli-ProAMFA1 conjugate is consistent with the results obtained with ProAMFA1 alone.

[0116] Example 5: Evaluation of activation of conjugate (II) Myo-ProAMFA1 by alkaline phosphatase

[0117] Activation of conjugated Myo-ProAMFA1 to conjugated Myo-AMFA1 by alkaline phosphatase To investigate its therapeutic potential and activation capabilities, we studied the conversion of Myo-ProAMFA1 conjugates with a P1=phosphate group to Myo-AMFA1 conjugates with a P1=H group. Dephosphorylation kinetics studies were conducted by incubating Myo-ProAMFA1 in the presence of alkaline phosphatase isolated from human placenta, according to the protocol described below. Alkaline phosphatase digestion protocol for Myo-AMFA1 and Myo-ProAMFA1 conjugates Myo-ProAMFA1 conjugate is incubated with alkaline phosphatase at 37°C. After 1 hour, 24 hours, and 96 hours, the reaction is stopped on ice, and samples are prepared for Western blotting analysis using SDS-PAGE polyacrylamide gel and compared with the enzyme alone (Myozyme®) and the conjugated Myo-AMFA1. After transfer to the membrane, the fraction of Myo-ProAMFA1 converted to Myo-AMFA1 is characterized using a specific antibody directed against AMFA1, followed by a peroxidase-conjugated anti-rabbit IgG secondary antibody. The membrane is then revealed using a chemiluminescent substrate. To check the amount of enzyme present on the membrane, the enzyme is recognized using a rabbit antibody specific to the above enzyme (anti-Myo), followed by a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody, and then detected chemiluminescently. The results obtained are shown in Figure 14. Over time, treatment of the Myo-ProAMFA1 conjugate with phosphatase induced an increase in the intensity of the 110 kDa signal corresponding to Myo-AMFA1, which is recognized by the anti-AMFA1 antibody. This result indicates that in the presence of alkaline phosphatase, the Myo-ProAMFA1 conjugate can be dephosphorylated to Myo-AMFA1.

[0118] Example 6: Evaluation of activation of conjugate (II) Infli-ProAMFA1 by phosphatase and human serum.

[0119] Western blot evaluation of activation of conjugated Infli-ProAMFA1 by alkaline phosphatase and acid phosphatase. Conjugate Infli-ProAMFA1 is incubated at 37°C with human placental alkaline phosphatase 8 UI / ml or potato (solanum tuberosum) acid phosphatase 3 UI / ml. After 16, 24, 48, or 72 hours, the reaction is stopped on ice and analyzed by Western blotting as described above for Myo-ProAMFA1 (Figure 14). Samples are marked with anti-AMFA1 antibody (to recognize AMFA1) and anti-human IgG antibody (to verify that the amount of antibody deposited is equivalent). The results obtained are shown in Figure 15. Over time, treatment of the Infli-ProAMFA1 conjugate with phosphatase induced an increase in the signal intensity of 50 kDa corresponding to Infli-AMFA1 recognized by the anti-AMFA1 antibody. This result indicates that the Infli-ProAMFA1 conjugate can be dephosphorylated to Infli-AMFA1 in the presence of alkaline phosphatase and acid phosphatase.

[0120] Affinity of Infli-ProAMFA1 to CI-M6PR after activation by alkaline phosphatase The affinity of the Infli-ProAMFA1 conjugate to CI-M6PR (0.5 μg / ml), which was adsorbed onto a 96-well microplate, was fixed with antibody for 90 minutes. The affinity was then analyzed by ELISA before and after digestion with alkaline phosphatase (8 units / ml) at 37°C. The retained antibody was quantified using a secondary anti-human IgG antibody conjugated to peroxidase and a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Absorbance was read at 650 nm. The results presented in Figure 16 show that the action of phosphatase is 2.10 with respect to digestion / activation of the Infli-ProAMFA1 conjugate (i.e., conjugate Infli-AMFA1). -7This allows affinity to be achieved at concentrations as low as M, while demonstrating that affinity for Infli-ProAMFA1 is undetectable. This result is the first to show that activation of Pro-AMFA1 on the antibody surface to AMFA1 by alkaline phosphatase can generate affinity for CI-M6PR, and that this affinity is much higher than that of M6P, as shown in Figure 13.

[0121] Western blot evaluation of the activation of conjugate Infli-ProAMFA1 to conjugate Infli-AMFA1 in human serum. The Infli-ProAMFA1 conjugate was incubated at 37°C in the presence of human serum (40 μl). After 4, 16, and 24 hours, the reaction was stopped on ice, and the samples were analyzed by Western blotting to clearly show anti-AMFA1 antibody and anti-human IgG antibody, as described above with respect to Figure 15. The results presented in Figure 17 show that, over time, treatment of the Infli-ProAMFA1 conjugate with human serum induces an increase in the 50 kDa signal intensity corresponding to the Infli-AMFA1 conjugate recognized by the anti-AMFA1 antibody. This result suggests that the Infli-ProAMFA1 conjugate can be dephosphorylated into Infli-AMFA1 by phosphatases present in human serum.

[0122] Affinity of activated Infli-ProAMFA1 to CI-M6PR in human serum The affinity of the Infli-ProAMFA1 conjugate to CI-M6PR before and after digestion with human serum (40 μl) at 37°C for 24 hours was measured according to the same protocol as described in Example 3. The results shown in Figure 18 indicate that the action of human serum-derived phosphatase is 10% effective with respect to the digested Infli-ProAMFA1 conjugate, i.e., the Infli-AMFA1 conjugate. -7 This allows for the detection of affinity at low concentrations, around M, while demonstrating that the affinity of Infli-ProAMFA1 is undetectable.

[0123] Example 7: Evaluation of phosphatase-mediated activation of conjugate (II) Cetux-ProAMFA1

[0124] Coupling of AMFA1 or ProAMFA1 with cetuximab Analysis using MALDI-TOF mass spectrometry allowed us to estimate the number of ProAMFA1 or AMFA1 molecules coupled to the cetuximab antibody to be approximately 7. The integrity of the product of interest (cetuximab) after coupling with AMFA1 or ProAMFA1 was verified by Coomassie blue staining after electrophoresis on a 12% SDS-polyacrylamide gel. The results illustrated in Figure 19 show that the coupling did not cause degradation, as the same enzyme-specific bands were observed at 50 kDa and 25 kDa before and after coupling.

[0125] Western blot evaluation of activation of Cetux-ProAMFA1 conjugates by alkaline phosphatase and acid phosphatase. Cetux-ProAMFA1 Conjugate The sample is incubated at 37°C with human placenta-derived alkaline phosphatase 8 UI / ml or potato-derived acid phosphatase 3 UI / ml. After 24, 48, 72, or 96 hours, the reaction is stopped on ice and analyzed by Western blotting for Myo-ProAMFA1 as described above (Figure 14). The sample is detected with anti-AMFA1 antibody and anti-human IgG antibody. The results obtained are shown in Figure 20. Over time, treatment of the Cetux-ProAMFA1 conjugate with phosphatase induced an increase in the signal intensity of 50 kDa corresponding to the Cetux-AMFA1 conjugate recognized by the anti-AMFA1 antibody. This result indicates that the Cetux-ProAMFA1 conjugate can be dephosphorylated to Cetux-AMFA1 in the presence of alkaline phosphatase and acid phosphatase.

[0126] Example 8: Intracellular relocation of Infli-ProAMFA1 and Cetux-ProAMFA1 conjugate (II) after phosphatase activation.

[0127] Intracellular relocation of the Infli-ProAMFA1 conjugate in Hela cells in advance Infli-ProAMFA1 coupled to the AlexaFluor488 fluorescent dye ConjugateThe cells are then incubated for 48 hours in the presence of human serum, or not, for phosphatase activation. Two batches of antibody (0.75 mg / ml) are then incubated with human HeLa cells derived from uterine cancer for 5 hours. Cell fluorescence is observed by flow cytometry. The results illustrated in Figure 21 show a 2.7-fold increase in conjugate internalization due to ProAMFA1 digestion by serum phosphatase.

[0128] Intracellular relocation of the conjugate Cetux-ProAMFA1 in Hela cells in advance Cetux-ProAMFA1 coupled to the AlexaFluor488 fluorescent dye Conjugate The cells are then incubated for 24 hours in the presence of human serum, or not, for phosphatase activation. Two batches of antibody (0.75 μg / ml) are then incubated with human HeLa cells derived from uterine cancer for 18 hours. Cell fluorescence is measured by flow cytometry. The results illustrated in Figure 22 show a twofold increase in conjugate internalization after ProAMFA1 digestion by serum phosphatase.

[0129] Example 9: Affinity between the conjugate (II) Infli-ProAMFA1 and infliximab antibody against TNFα antigen.

[0130] Antibody affinity to the antigen was quantified by ELISA. TNFα antigen, pre-adsorbed onto a microplate, and the antibody were incubated at 37°C for 1 hour. The antibody retained on the plate was quantified by adding o-phenylenediamine hydrochloride (OPD) substrate using a secondary anti-human IgG antibody coupled with peroxidase. Absorbance was read at 450 nm. The results illustrated in Figure 23 demonstrate that coupling infliximab with ProAMFA1 does not modify the affinity of the infliximab antibody to its antigen. Modification of the antibody by implanting several Pro-AMFAs does not alter the three-dimensional structure of the antibody, thereby preventing its recognition of the antigen.

[0131] This disclosure is not limited to the above-described examples provided for illustrative purposes only, but includes all modifications that can be anticipated by those skilled in the art in the context of seeking protection.

[0132] List of cited documents Patent document For all intents and purposes, the following patent documents are cited: - patcit1:EP 2 448 600 B1; - patcit2:EP 3 350 192 B1; - patcit3: Application FR2302097 submitted on 07 / 03 / 2023

[0133] Non-patent literature For all intents and purposes, the following non-patent documents are cited: - nplcit1:Vidil C. et al., Eur. J. Org. Chem., 1999, 447; - nplcit2:Jeanjean A. et al., Bioorg. Med. Chem. Lett., 2008, 18, 6240; - nplcit3:El Cheikh K. et al., Angew. Chem. Int. Ed., 2016, 55, 14774; - nplcit4: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 S. 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. Soft. Med., 2019, 23, 6499; - nplcit9:Daurat M. et al., Front. Immunol., 2024, 15, 1273280; - nplcit10:Gauthier J. et al., Biomed. Pharmacother., 2024, 175, 116707; - nplcit11:Gauthier C. et al., J. Control. Release, 2023, 358, 465; - nplcit12:Vaillant O. et al., Angewandte Chemie, 2015, 54, 5952; - nplcit13:Bouffard E. et al., Int. J. Mol. Sci., 2019, 20, 2809; - nplcit14:Daurat M. et al., Biomater. Sci., 2020, 8, 3678; - nplcit15:Melsheimer R. et al., Biologics. 2019, 13, 139; - nplcit16:Baselga J. et al., J. Clin. Oncol., 2005, 23, 5560; - nplcit17:Galizia G. et al., Oncogene. 2007, 26, 3654.

Claims

1. The following general formula (I) 【Chemistry 1】 During the ceremony: The dotted line indicates a connection that may or may not exist; X is -CH 2 -P(O)(OZ) 2 ; -CH 2 -CO 2 Z; -CH(CO 2 Z) 2 ; -CH(P(O)(OZ) 2 ) 2 ; -CHF-CO 2 Z; -CHF-P(O)(OZ) 2 ; -CF 2 -CO 2 Z; -CF 2 -P(O)(OZ) 2 ; -CH(CO 2 Z)(P(O)(OZ) 2 ) is shown, where the bond indicated by the dotted line does not exist; or X is =CH-CO 2 Z; = CH - P(O)(OZ) 2 ;=CF-CO 2 Z; = CF - P(O)(OZ) 2 This indicates that, in this case, the connection shown by the dotted line exists; Z is independent of each other, H; Na; K or NH 4 ; indicates P1, P2, and P3 are independently H; P(O)(OZ) 2 -S(O) 2 (OZ) is shown; Z is as previously defined, and the condition is that at least one of P1, P2, and P3 is not a hydrogen atom; Alternatively, P2 and P3 together form an acetonide group. 【Chemistry 2】 Forming; A is -O-; -S-; -NH-; -CH 2 - Indicates more selected divalent radicals; L is -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n -, where n is an integer in the range of 0 to 6; -H; -NH 2 ;-(CH 2 ) n1 -CH=CH 2 or -(CH 2 ) n1 -C≡CH, where n 1 This represents an integer in the range of 0 to 4, and in each of these cases, L 1 This means non-existence; Saturated linear or branched divalent hydrocarbon radicals having 1 to 30 carbon atoms; unsaturated linear or branched divalent hydrocarbon radicals having 2 to 30 carbon atoms; A saturated or unsaturated divalent hydrocarbon radical as defined above, wherein one or more of the saturated or unsaturated hydrocarbon radicals are -CH 2 -, -CH=CH- and / or -C≡C- groups, independently of each other -O-; -NH-; -S-; -CO-NH-; -NH-CO-O- group; and / or Table 1 below Table 1 The saturated or unsaturated divalent hydrocarbon radicals are substituted by a ring system or heterocyclic system derived from those listed above. Show; L 1 teeth, -O-NH 2 ; 【Transformation 3】 In the formula, R 1 is C 1 ~C 5 It exhibits alkyl, preferably ethyl; 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 A more selected system of rings or heterocyclic rings; -O-N=C(CH 3 ) 2 ; -(CH 2 ) n1 -CH=CH 2 ;-(CH 2 ) n1 -C≡CH;-(CH 2 ) n1 -N 3 ;-(CH 2 ) n1 -SH;-(CH 2 ) n1 -NH 2 ; -(CH 2 ) n1 -N=C=O; -(CH 2 ) n1 -N=C=S;-(CH 2 ) n1 -NHR 1 ;-(CH 2 ) n1 -A 1 -NH 2 ;-(CH 2 ) n1 -A 1 -NHR 1 ; -(CH 2 ) n1 -NHCO-CH 2 Hal;-(CH 2 ) n1 -COZ 1 ;-(CH 2 ) n1 -A 1 COZ 1 ;-(CH 2 ) n1 -O-NH 2 ; -(CH 2 ) n1 -CO-NH-NH 2 ; wherein n 1 and R 1 are as defined above; A 1 is -O-; -NH-; Hal is Cl; Br or I; Z 1 is -OH; -OR 1 ; -NHR 1 ; -NH-NH 2 ; -NH-NHR 1 ; and R 1 is as defined above; Halogen selected from F, Cl, Br, or I This indicates A proligand characterized by having the following characteristics.

2. A proligand according to claim 1: X is -CH 2 -P(O)(OZ) 2 ;-CH 2 -CO 2 Z; -CH(CO 2 Z) 2 ;-CH(P(O)(OZ) 2 ) 2 ;-CHF-CO 2 Z;-CHF-P(O)(OZ) 2 ;-CF 2 -CO 2 Z;-CF 2 -P(O)(OZ) 2 -CH(CO 2 Z)(P(O)(OZ) 2 ); =CH-CO 2 Let Z be shown; Z is as defined above; P1, P2, and P3 are independent of each other, and are either H or P(O)(OZ) 2 This indicates that; Z is as previously defined, and at least one of P1, P2, and P3 is different from a hydrogen atom; or P2 and P3 together form an acetonide group of formula “Chemical Formula 2” as defined in claim 1; A is -O-; -S-; -CH 2 - indicates a divalent radical; L, -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n -, wherein the formula, n is as defined in claim 1; -H; -NH 2 ;-(CH 2 ) n1 -CH=CH 2 or -(CH 2 ) n1 -C≡CH, where n 1 L is as defined in claim 1, and in each of these cases, 1 This means non-existence; Saturated linear or branched divalent hydrocarbon radicals having 1 to 30 carbon atoms; unsaturated linear or branched divalent hydrocarbon radicals having 2 to 30 carbon atoms Show; L 1 but, -O-NH 2 ; -O-N=C(CH 3 ) 2 ; A ring system or heterocyclic ring system selected from "Chemical Formula 3"; "Chemical Formula 4"; and "Chemical Formula 5"; -(CH 2 ) n1 -CH=CH 2 ;-(CH 2 ) n1 -C≡CH;-(CH 2 ) n1 -N 3 ;-(CH 2 ) n1 -SH;-(CH 2 ) n1 -NH 2 ;-(CH 2 ) n1 -N=C=O;-(CH 2 ) n1 -N=C=S;-(CH 2 ) n1 -NHCO-CH 2 Hal; in the formula, n 1 And Hal is as defined in claim 1. Halogen selected from Cl, Br, or I This indicates The proligand characterized by a point.

3. A proligand according to claim 1 or 2: X is -CH 2 -P(O)(OZ) 2 ;-CH 2 -CO 2 Z; -CH(CO 2 Z) 2 ;-CH(P(O)(OZ) 2 ) 2 Thereafter, where Z is as defined in claim 1 or 2, preferably Z is equal to H; P1, P2, and P3 are independently H or P(O)(OZ) 2 The formula is such that Z is as previously defined, and at least one of P1, P2, and P3 is not a hydrogen atom; or P2 and P3 together form an acetonide group of formula “Chemical Formula 2” as defined in claim 1; A represents the oxygen atom -O-; L is -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n - indicates that in the formula, n is an integer in the range of 0 to 6, preferably n is equal to 0; L 1 is -O-NH 2 -ON=C(CH 3 ) 2 ; A substituent of formula "Chemical Formula 3" or "Chemical Formula 4" as defined in claim 1. The proligand characterized by a point.

4. A proligand according to any one of claims 1 to 3: X is -CH 2 -P(O)(OZ) 2 This indicates that Z is as defined in claim 3; P1, P2, and P3 are as defined in claim 3; A, L and L 1 However, as defined in claim 3. The proligand characterized by a point.

5. A proligand according to any one of claims 1 to 4: X is as defined in claim 4; A, L and L 1 This is as defined in claim 4; P1, P2, and P3 are independently H or P(O)(OZ) 2 The formula shows that Z is as defined in claim 4, and at least one of P1, P2, and P3 is different from a hydrogen atom. The proligand characterized by a point.

6. A proligand according to claim 5: X is -CH 2 -P(O)(OZ) 2 This shows that Z=H; P1 is P(O)(OZ) 2 This shows that Z=H, and P2 and P3=H; A represents an oxygen atom; L is -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n - indicates that n is equal to 0; L 1 -O-NH 2 This indicates The proligand characterized by a point.

7. A proligand according to any one of claims 1 to 4: X is -CH 2 -P(O)(OZ) 2 This shows that Z=H; P1 is H, and together P2 and P3 form an acetonide group of formula “Chemical Formula 2” as defined in claim 1; A represents an oxygen atom; L is -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n - indicates that n is equal to 0; L 1 -O-NH 2 Or -ON=C(CH 3 ) 2 This indicates The proligand characterized by a point.

8. The following general formula (II): 【Chemistry 9】 During the ceremony, n 2 is an integer in the range of 1 to 1000, preferably 1 to 20, more preferably 1 to 10. P1, P2, P3, X, A, and L are as defined in any of claims 1 to 7, L' 1 L 1 Y 1 A substituent L as defined in any of claims 1 to 7 when involved in covalent bonding with 1 Show, Y 1 This refers to product Y of interest, selected from the group comprising proteins, particularly antibodies and lysosomal enzymes, nanoparticles, protein activators or inhibitors, cytotoxic compounds, and markers for medical imaging. Y 1 is L' 1 and n 2 Forms individual covalent bonds (multiple bonds are possible). A conjugate characterized by having the following features.

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

10. A conjugate according to claim 8 or 9; X is -CH 2 -P(O)(OZ) 2 This shows that Z=H; P1 is P(O)(OZ) 2 It represents a cetonide group of formula "Chemical Formula 2" as defined in claim 1, where Z=H and P2 and P3=H; or P1 represents H and P2 and P3 together form an acetonide group of formula "Chemical Formula 2" as defined in claim 1; A represents an oxygen atom; L is -(CH 2 ) 2 -(O-CH 2 -CH 2 ) n - indicates that n is equal to 0; L' 1 This indicates radical-ON= The conjugate characterized by a point.

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

12. The conjugate for use according to claim 11, wherein the conjugate is of a type suitable for oral, parenteral, intravenous, intramuscular, or subcutaneous administration.

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