Novel VASH inhibitors, their complexes, and their use as drugs or research tools

Abstract

The present invention relates to a compound of formula (I), wherein X, R1, R2 and R3 are as defined above. [Formula 1] JPEG2024532604000127.jpg39170 or a pharma- ceutically acceptable salt and / or solvate thereof, complexes thereof and their use as drugs or research tools.

Description

[Technical field]

[0001] The present invention relates to novel VASH inhibitors, which are particularly useful for the prevention and / or treatment of VASH peptidase-associated disorders and / or as research tools. The present invention also relates to conjugates of said VASH inhibitors with biomolecules and their use as drugs or research tools. [Background technology]

[0002] Microtubules (MTs) are the major type of cytoskeletal element, especially abundant in neuronal cells. They are formed by polymerization of heterodimers of α- and β-tubulin. Within cells, MTs have the ability to expand and contract, a phenomenon called dynamic instability. This dynamic behavior of MTs allows them to support numerous cellular processes, including intracellular transport, cell motility, cell division and cell morphogenesis. The functional diversity of microtubules is due to the many different post-translational modifications (PTMs) that affect tubulin. Known PTMs include acetylation, polyglutamylation, polyglycylation, phosphorylation, and tyrosination / detyrosination cycles. These modifications affect microtubule dynamics, organization, and interactions with other cellular compounds and cell compartments.

[0003] MT dysfunction is associated with neurodevelopmental disorders and neurodegeneration. Well-known dysfunctions are associated with mutations in either tubulin, a component of MTs, or in MT-associated proteins (MAPs) that regulate MT function. Binding of many MAPs is mediated by the C-terminal tails of α- and β-tubulin, which protrude from the surface of MTs and are known to be highly subject to various post-translational modifications. The most abundant tubulin modification in neuronal cells is polyglutamylation, which generates multiple glutamic acid side chains of different lengths at the C-terminus of α- and β-tubulin. Negatively charged glutamic acids are added to the C-terminal tails, which provide binding sites for various MT-associated proteins (MAPs) and molecular motors. Thus, polyglutamylation has been shown to regulate the activity or binding of numerous MT-interacting proteins. Early studies suggested that the binding of neuronal MAPs, such as tau, a key player in Alzheimer's disease, is regulated by MT polyglutamylation. Furthermore, recent reports have shown that the activity of MT-severing enzymes, such as spastin and katanin, which play important roles in axon growth and plasticity, is also controlled by this modification. Furthermore, polyglutamylation is thought to regulate the speed and processivity of the KIF1A kinesin motor protein, which is involved in synaptic vesicle delivery and thus regulates synaptic transmission. Taken together, tubulin polyglutamylation controls a variety of important neuronal processes. Polyglutamylation is reversible and is catalyzed by enzymes that belong to the tubulin tyrosine ligase-like (TTLL) family. In contrast, the reverse enzyme is a member of the cytosolic carboxypeptidase (CCP) family. Thus, the overall level of tubulin polyglutamylation in a particular cell is established as a result of the competition between both activities.

[0004] The first tubulin modification discovered was detyrosination, which is specific for α-tubulin and consists of the removal of the C-terminal tyrosine residues of MTs. The tyrosination / detyrosination cycle is involved in microtubule regulation, especially in neurons, muscle cells and dividing cells. Thus, alterations in microtubule detyrosination have been implicated in disorders such as neurodegenerative diseases, impaired nerve regeneration, cancer, muscular dystrophies, heart diseases, vascular disorders, retinal degeneration, infertility or ciliopathy.

[0005] Although detyrosination was discovered about half a century ago, the enzymes involved in detyrosination formation, with tubulin carboxypeptidase (TCP) activity, have only recently been described (Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456; Aillaud C. et al. Science, 2017, 358(6369:1448-1453)). The vasohibin family of VASH1 and VASH2 are the enzymes catalyzing this modification. Recent studies in particular have led to the identification of reversible and irreversible inhibitors of the protein enzyme activity, which are of particular interest for the treatment of the above mentioned disorders as well as neurodegenerative disorders, including but not limited to tauopathies (e.g. Alzheimer's disease, frontotemporal dementia, corticobasal dementia, etc.) (see WO 2019 / 016259).

[0006] By employing a high affinity purification step using custom designed inhibitors compatible with click chemistry, it was possible to identify VASH as a tubulin detyrosinase (Aillaud, C. et al., Science. 2017, 358(6369):1448-1453). The most efficient of these inhibitors is called the compound EPO-Y (Aillaud et al. 2017) and consists of an active group called trans-epoxysuccinic acid (TES) ethyl ester linked to a tyrosine residue, represented as follows:

[0007] [ka]

[0008] EPO-Y (also called Epo-Y) is an irreversible VASH inhibitor that was initially considered a promising candidate for the treatment of disorders involving altered microtubule detyrosination. However, this compound has a modest IC of approximately 15 μM in cellulo. 50 In light of possible biomedical applications, an ideal inhibitor requires high specificity and potency against VASH. Small, highly electrophilic epoxide molecules such as EPO-Y may interact with free thiols present in other proteins and cysteine ​​protease active sites, especially at high concentrations (μM range). In addition, they may react with nucleophilic lysine residues in tyrosine kinase active sites, resulting in loss of specificity. Of note, the only other reported detyrosination inhibitor previously identified via cell-based screening, called parthenolide (Fonrose et al. Cancer Res 2007;67 (7) :3371-8), does not inhibit VASH in vitro even at very high concentrations (Hotta T.et al., Curr.Biol., 2021, 31 (4) :900-907). This suggests that the observed in cell effect of parthenolide is not mediated through a direct inhibition of VASH.

[0009] Therefore, there is a need to improve the specificity, potency and bioavailability of existing VASH inhibitors to provide suitable candidates useful for the treatment of disorders involving defects in microtubule detyrosination.In this invention, we describe in celluar, highly efficient, low nanomolar to picomolar VASH inhibitors as potential drugs for the treatment of neurodegenerative diseases. Summary of the Invention

[0010] Using medicinal chemistry to generate libraries of compounds and measure efficacy in ideal predictive in vitro systems, and bioinformatics to model and understand the structural features that account for the diversity of biological activity of compounds, the inventors have thus developed novel VASH inhibitors with greatly enhanced in vitro and / or in cell activity compared to EPO-Y.

[0011] Thus, the present invention relates to a compound of formula (I) [ka] or a pharma- ceutically acceptable salt and / or solvate thereof, During the ceremony, X, [ka] and R 1 is O-C1-C6 alkyl, O-C2-C6 alkenyl, NR 1a R 1b or [ka] and R 1a is H or C1-C6 alkyl; R 1b is OH or C1-C6 alkyl, said alkyl being optionally substituted with C(O)OH, C(O)O-C1-C6 alkyl or aryl; R is OR 2 or NH-S(O)2-R 9 and R 2 is H, a C1-C6 aliphatic chain, aryl, heteroaryl, or C1-C6 alkyl-aryl, wherein up to four methylene units of said aliphatic chain are optionally replaced with O, C(O), NH, or N-C1-C6 alkyl, and wherein said aliphatic chain, aryl, heteroaryl, or alkyl-aryl are optionally substituted; R 3is OH, O-C1-C6 aliphatic chain, O-aryl, O-C1-C6 alkyl-aryl, O-heteroaryl, O-C1-C6 alkyl-heteroaryl or NHOH, wherein up to four methylene units of said aliphatic chain are optionally replaced with O, C(O), NH or N-C1-C6 alkyl, and wherein said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl or alkyl-aryl are optionally substituted; R 4 But H or C1-C 12 an aliphatic chain in which up to four methylene units are optionally replaced by O, C(O), NH or N-C1-C6 alkyl, 12 the aliphatic chain is optionally substituted; Y is -(CH2) m -or [ka] and R 5 is OH, O-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, O-C1-C6 alkyl-aryl, O-C1-C6 alkyl-heteroaryl, C(O)OH, C(O)O-C1-C6 alkyl, C(O)NHOH, C(O)NH2, C(O)NH-C1-C6 alkyl, C(O)NH-O-C1-C6 alkyl, NH-C1-C6 alkyl, N(C1-C6 alkyl)2, NH-C(O)-C1-C6 alkyl or NH-S(O)2-R 9 wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl or alkyl-heteroaryl is optionally substituted; R 6 is OH, O-C1-C6 aliphatic chain, NH-OH or NH-CH(R 7 )-(CH2) n -R 8 wherein the aliphatic chain is optionally substituted; R 7is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C1-C6 alkyl-aryl or C1-C6 alkyl-heteroaryl; R 8 is C(O)NH2, C(O)NH-C1-C6 alkyl, aryl, heteroaryl, SH, NH2 or S-C1-C6 alkyl, and m and n are each independently an integer ranging from 0 to 6; R 9 is a C1-C6 aliphatic chain or aryl, said aliphatic chain or aryl being optionally substituted; However, if X is [ka] Less than or equal to R 3 When is OH, R 1 is not O-C1-C6 alkyl Compound or Pharmaceutically Acceptable Salt and / or Solvate thereof Regarding.

[0012] According to another aspect, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I) as defined above and at least one pharma- ceutically acceptable excipient.

[0013] According to another aspect, the present invention also relates to a compound of formula (I) or a pharmaceutical composition as defined above, for use as a medicament.

[0014] The present invention also relates to the compounds of formula (I) for use as research tools.

[0015] According to another aspect, the present invention also relates to a conjugate comprising a compound of formula (I) bound to a biomolecule.

[0016] According to another aspect, the present invention relates to a conjugate as defined above for use as a drug or research tool. [Brief description of the drawings]

[0017] [Figure 1] In cellulo VASH-mediated tubulin detyrosination assay. Effect of increasing concentrations of Epo-Y on tubulin detyrosination in human CHL-1 cells (a melanoma-derived cell line). [Diagram 2] The putative inhibitor parthenolide does not inhibit VASH-mediated tubulin detyrosination. (A) Chemical structures of Epo-Y and the putative detyrosinase inhibitor parthenolide (PTL). (B) In vitro detyrosination assay with recombinant VASH1 protein. DeTyr, detyrosination. (C) Effect of increasing concentrations of Epo-Y and PTL on tubulin detyrosination in human CHL-1 cells. [Diagram 3] The newly developed inhibitors target VASH1 and VASH2 and peptidase activity. Both VASH1- and VASH2-dependent tubulin detyrosination activities are inhibited by the newly designed inhibitors using an in vitro detyrosination assay. [Figure 4-1] A cell-permeable, potent inhibitor of VASH peptidase activity. (A) Chemical structure of LV-43 (outside the scope of the invention). (B) In vitro detyrosination assay with recombinant VASH1 protein. (C) In cellulo detyrosination assay with recombinant VASH1 protein. (D) In ​​cellulo detyrosination assay with CHL-1 cells. (E) Comparison of dose-response curves obtained in vitro with Epo-Y and LV-80. (F) Quantification of the effect of increasing concentrations of Epo-Y and LV-80 on tubulin detyrosination in human CHL-1 cells. (G) Chemical structure of LV-80 and (H) molecular docking to VASH1 showing the putative binding mode (hydrogen bonds). [Figure 4-2]A cell-permeable, potent inhibitor of VASH peptidase activity. (A) Chemical structure of LV-43 (outside the scope of the invention). (B) In vitro detyrosination assay with recombinant VASH1 protein. (C) In cellulo detyrosination assay with recombinant VASH1 protein. (D) In ​​cellulo detyrosination assay with CHL-1 cells. (E) Comparison of dose-response curves obtained in vitro with Epo-Y and LV-80. (F) Quantification of the effect of increasing concentrations of Epo-Y and LV-80 on tubulin detyrosination in human CHL-1 cells. (G) Chemical structure of LV-80 and (H) molecular docking to VASH1 showing the putative binding mode (hydrogen bonds). [Diagram 5] Complete inhibition of VASH-mediated tubulin detyrosination in cells. Analysis of tubulin detyrosination levels in human CHL-1 cells incubated with LV-80 or vehicle (DMSO) for 24 h and comparison with VASH1 / 2 knockout cells (2KOs) (disclosed in Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456). Vinculin, loading control. DeTyr, detyrosination. [Figure 6-1] Favorable safety profile of VASH enzyme inhibitors. (A) Cell viability assay using Taxol (paclitaxel) and parthenolide as references. (B) Measurement of cell metabolism and extracellular acidification rate (ECAR) after 24 h incubation of CHL-1 cells with vehicle (DMSO) or the VASH-specific inhibitor LV-80. Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live CHL-1 cells using a Seahorse XF Analyzer after 24 h treatment. OCR rate is a key indicator of mitochondrial respiration and provides a systems-level view of cellular metabolic function in cultured cells. [Figure 6-2]Favorable safety profile of VASH enzyme inhibitors. (A) Cell viability assay using Taxol (paclitaxel) and parthenolide as references. (B) Measurement of cell metabolism and extracellular acidification rate (ECAR) after 24 h incubation of CHL-1 cells with vehicle (DMSO) or the VASH-specific inhibitor LV-80. Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live CHL-1 cells using a Seahorse XF Analyzer after 24 h treatment. OCR rate is a key indicator of mitochondrial respiration and provides a systems-level view of cellular metabolic function in cultured cells. [Figure 7] Newly developed VASH inhibitors are specific for VASH enzymatic activity. (A, B) Analysis of cytosolic carboxypeptidase 1 (CCP1) (A) and CCP5 (B)-mediated tubulin deglutamylation in the absence or presence of the VASH inhibitor LV-80 (50 μM). PolyE, antibody recognizing long glutamic acid chains; GT335, antibody against branch-point glutamic acid residues. (C) Specificity of LV-80. BzlSA, peptidomimetic benzyl succinic acid. [Figure 8] The newly developed biotinylated version as a research tool. (A) Effect of increasing concentrations of biotinylated LV-80 on tubulin detyrosination in human CHL-1 cells. (B) Pull-down assay of endogenous VASH with biotinylated LV-80. (C) Pull-down assay of endogenous VASH from human brain protein lysate with biotinylated LV-80. [Figure 9]Significant reduction in tubulin detyrosination in primary cortical neurons treated with VASH inhibitors. (A) Analysis of lysates of mouse primary cortical neurons incubated with vehicle (DMSO) or the VASH-specific inhibitor LV-80. DeTyr, detyrosinated tubulin; TyrTub, tyrosinated tubulin; PolyE, antibody recognizing long glutamic acid chains; GT335, antibody against branch point glutamic acid residues. (B) Quantification of tubulin tyrosination. p values ​​were calculated by multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001). (C) Transcript levels of genes associated with detyrosination status analyzed by qPCR. (D) Transcript levels of neuronal differentiation markers after incubation with vehicle (DMSO) or inhibitors. (E) Transcript levels of glial markers after 7 days of incubation with vehicle (DMSO) or inhibitors. [Figure 10] Detyrosination renders tubulin susceptible to glutamylation. (A) Tubulin glutamylation levels, (B) and tubulin acetylation levels in mouse primary cortical neurons incubated with vehicle (DMSO) or the VASH-specific inhibitor LV-80. p values ​​were calculated by multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001). (C) Crosstalk between tubulin detyrosination and glutamylation. (D) Analysis of Drosophila total protein extracts. [Figure 11]Tubulin detyrosination affects tau protein binding in primary cortical neurons. (A) Analysis of mouse primary cortical neurons incubated for 3 days with vehicle (DMSO) or the VASH-specific inhibitor LV-80 after 5 days of differentiation. DeTyr, detyrosinated tubulin; TyrTub, tyrosinated tubulin; PolyE, antibody recognizing long glutamic acid chains; GT335, antibody against branch-point glutamic acid residues. (B, C) Quantification of tubulin detyrosination and tyrosination levels (B) and tubulin glutamylation levels (C) in mouse primary cortical neurons incubated for 3 days with vehicle (DMSO) or the VASH-specific inhibitor LV-80 after 5 days of differentiation as in (A). p values ​​were calculated by multiple t-test (n=3, *<0.05, **<0.01, ***<0.001). (D) Colocalization of tau protein and tubulin in mouse primary cortical neurons incubated with vehicle (DMSO) or the VASH-specific inhibitor LV-80. (E) Automated quantification of tau immunofluorescence staining in tubulin-positive areas (n>150). p-values ​​were calculated by multiple t-test (***<0.001). (F) Quantification of tubulin in mouse primary cortical neurons incubated with vehicle (DMSO) or the VASH-specific inhibitor LV-80. Automated quantification of immunofluorescence signal in positive areas (n>150). p-values ​​were calculated by multiple t-test (ns; not significant). [Figure 12] A. The newly developed prodrugs (LV-104 and LV-111) show highly efficient inhibition of VASH-dependent detyrosination in human CHL-1 cells. B. [Figure 13] In vitro and in cell assays of VASH inhibitors. A. This study aimed to compare the importance of R3 modifications in direct VASH inhibition. Concentrations of the indicated compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-80, LV-104 and IBMT11. B. CHL-1 cells were treated with increasing concentrations of LV-80, LV-104 and IBMT11 prior to VASH activity assessment using the cell-based assay. [Figure 14] Bioanalysis of cell lysates after treatment shows conversion by cell permeation. CHL-1 cells were treated with 25uM LV-104 for 1 hour. Culture supernatants were collected immediately after compound addition (initial) and 1 hour later (supernatant). Cells were washed, lysed, and processed for LC / MS reading (cells) to detect LV-104 and its bioconverted form, IBMT11. [Figure 15] In vitro and in cell assays of VASH inhibitors. A. The indicated concentrations of compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-1, LV-80, IBMT11 and IBMT23. B. CHL-1 cells were treated with increasing concentrations of LV-1, LV-80, LV-104 and IBMT23 prior to VASH activity assessment using the cell-based assay. [Figure 16] In vitro and in cell assays of VASH inhibitors. A. The indicated concentrations of compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-1, LV-80, IBMT11 and IBMT34, and the sodium salt of LV80. B. CHL-1 cells were treated with increasing concentrations of LV-80, LV-104, IBMT23 and IBMT34 prior to VASH activity assessment using the cell-based assay. [Figure 17] In vitro and in cell assays of VASH inhibitors. A. The indicated concentrations of compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-1, LV-80, IBMT11, IBMT28, and IBMT28hydro (designated IBMT28sapo). B. CHL-1 cells were treated with increasing concentrations of LV-80, LV-104, and IBMT28 prior to VASH activity assessment using the cell-based assay. [Figure 18]In Vitro Assay for VASH Inhibitors. The indicated concentrations of compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-1, LV-80, and IBMT38hydro (designated IBMT38sapo). [Figure 19] In vitro assays for VASH inhibitors. The indicated concentrations of compounds were tested for VASH activity using a standardized in vitro detyrosination ELISA-based assay with LV-1 / EpoY, LV-80, and SD-139 (reported in Aillaud, C. et al. Science, 2017, 358, p.1448-1453). A. Assay with the indicated compounds; B. After saponification of the tert-butyl groups of SD-139, before inhibition assay at the indicated concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition The term "stereoisomer" as used in the present invention means configurational stereoisomers, more specifically optical isomers.

[0019] In the present invention, optical isomers arise in particular from the different spatial positions of the substituents attached to X. The carbon or nitrogen atom of the X group to which the substituent is attached is therefore a chiral or asymmetric centre. Optical isomers that are not mirror images of one another are therefore called "diastereoisomers" and optical isomers that are non-superimposable mirror images are called "enantiomers".

[0020] An equimolar mixture of the two enantiomers of a chiral compound is called a racemic mixture or racemate.

[0021] In the context of the present invention, depending on the position of the substituents attached to the X group, the compounds of the present invention may be in the configuration (S,S), the configuration (R,R), the configuration (S,R) or the diastereoisomers in the configuration (R,S), as shown below.

[0022] [ka]

[0023] If the position of the substituents is not specified in a compound, said compound corresponds to any one of the diastereoisomers mentioned above or to a mixture of said diastereoisomers.

[0024] In the present invention, the term "pharmaceutical acceptable" is intended to mean something that is useful for the preparation of a pharmaceutical composition, and that is generally safe and non-toxic for pharmaceutical use.

[0025] The term "pharmacologically acceptable salts and / or solvates" is intended, in the framework of the present invention, to mean salts and / or solvates of compounds which are pharma- ceutically acceptable as defined above and which possess the pharmacological activity of the corresponding compounds.

[0026] Pharmaceutically acceptable salts include (1) Acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with organic acids, such as acetic acid, benzenesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphtoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, succinic acid, dibenzoyl-L25 tartrate, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, and trifluoroacetic acid; and (2) Base addition salts formed when an acid proton present in a compound is replaced with a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or is coordinated with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0027] Acceptable solvates for the therapeutic use of the compounds of the invention include conventional solvates, such as those formed during the final steps of the preparation of the compounds of the invention due to the presence of solvents. By way of example, mention may be made of solvates due to the presence of water (these solvates are also called hydrates) or ethanol.

[0028] As used herein, the term "halogen" means a fluorine, bromine, chlorine or iodine atom.

[0029] "C x -C y The term "aliphatic chain" means a linear or branched hydrocarbon chain which is fully saturated or contains one or more unsaturations but is not aromatic, comprising x to y carbon atoms, in particular 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. According to the present invention, the term "aliphatic chain" encompasses substituted or unsubstituted, linear or branched alkyl, alkenyl or alkynyl groups.

[0030] As used herein, the term "C1-C6 alkyl" means a straight or branched monovalent saturated hydrocarbon chain containing from 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

[0031] As used herein, the term "C2-C6 alkenyl" refers to a straight or branched monovalent unsaturated hydrocarbon chain containing 2 to 6 carbon atoms and comprising at least one double bond, including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0032] As used herein, the term "C2-C6 alkynyl" refers to a straight or branched monovalent unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one triple bond, including, but not limited to, ethynyl, propynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0033] As used herein, the term "aryl" refers to an aromatic hydrocarbon, preferably comprising 6 to 12 carbon atoms, and comprising one or more fused rings, such as, but not limited to, a phenyl or naphthyl group. Advantageously, it is a phenyl group.

[0034] As used herein, the term "heteroaryl" refers to an aromatic group comprising one or several, in particular one or two, condensed hydrocarbon cycles, in which one or several, in particular one to four, advantageously one or two carbon atoms are replaced by a heteroatom selected from sulfur, oxygen and nitrogen atoms, preferably oxygen and nitrogen atoms, respectively. It may be furyl, thienyl, pyrrolyl, pyridyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, quinoxalyl or indyl.

[0035] As used herein, the term "C1-C6-alkylaryl" or "C1-C6 alkylheteroaryl" refers to an alkyl group as defined above, substituted with an aryl group as defined above or a heteroaryl group as defined above, respectively. Advantageously, "C1-C6-alkylaryl" is a benzyl group.

[0036] In the context of the present invention, "optionally substituted" means that the group in question is, in particular, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkene, C2-C6 alkyne, aryl, N3, oxo, NR a R b , C.O.R. c , CO2R d ,CONR e R f , OR g , N + R h R i R j , CN and NO2 (where R a ~R j means optionally substituted with one or more substituents which may be selected independently from each other from H, C1-C6 alkyl or aryl, preferably H or C1-C6 alkyl).

[0037] The term "C1-C6 haloalkyl" refers to a C1-C6 alkyl chain as defined above, in which one or more hydrogen atoms are replaced by a halogen atom selected from fluorine, chlorine, bromine or iodine, preferably a fluorine atom. For example, it is a CF3 group.

[0038] In the context of the present invention, "unsaturated" means that the hydrocarbon chain contains one or more unsaturations, ie advantageously one double bond C=C or triple bond C≡C.

[0039] The term "pharmaceutical composition" means, in the framework of the present invention, a composition having prophylactic and therapeutic properties.

[0040] The term "peptide coupling" refers to a chemical reaction between an amine function and a carboxylic acid function. Advantageously, the peptide coupling is carried out using N-hydroxy-succinimide (NHS), N-hydroxy-benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt), N-hydroxysulfosuccinimide (sulfo diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI), hexafluorophosphate-2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium (HBTU), tetrafluoroborate-2, optionally with an additive or base such as NHS), dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA) or N-methylmorpholine (NMM); The reaction will be carried out in the presence of a coupling agent such as -(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium (TBTU), hexafluorophosphate-O-(7-azobenzotriazol-1yl)-1,1,3,3-tetramethyluronium (HATU), (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) or propylphosphonic anhydride.

[0041] As used herein, the term "VASH (enzyme)" refers to tubulin carboxypeptidase enzyme (TCPase) involved in microtubule detyrosination, which is associated with neurodegenerative disorders, psychiatric disorders, neurological disorders, ciliopathy, cancer, and muscular dystrophies.

[0042] The term "biomolecule" refers to a molecule having biological properties. In the context of the present invention, it refers to a biomarker such as a protein, peptide, photolabeling agent, or E3 ubiquitin ligase recruiter (e.g., but not limited to, thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, PROTAC6, ZNL-02-096, or d9A-2).

[0043] The term "PROTAC" as used in the present invention is an abbreviation of PROteolysis Targeting Chimeras. A "PROTAC E3 ligase recruiter" is a complex comprising two ligands connected by a linker, which is useful as a tool for selective and complete protein degradation. One of the ligands is intended to bind to the protein to be degraded, while the other should target the ubiquitin ligase. As a result, the PROTAC E3 ligase recruiter can bring the protein to be degraded and the ligase into close proximity, resulting in (poly)ubiquitination of the target protein, i.e., the addition of one or more small (8.6 kDa) regulatory proteins, ubiquitin, to the substrate protein. The (poly)ubiquitin tag is added to the protein to be degraded, sending it for degradation by the proteasome. Unlike "conventional" inhibitors, the PROTAC E3 ligase recruiter induces selective protein degradation, thereby suppressing all biological functions. In the context of the present invention, the protein to be degraded is the VASH enzyme. Thus, the ligand of the PROTAC E3 ligase recruiter intended to bind to the VASH enzyme is a fragment of the compound of formula (I), and the ligand intended to target the ubiquitin ligase is typically thalidomide.

[0044] The term "prodrug" refers to a typically pharmacologically inactive or less active derivative of an active drug that undergoes biotransformation in cellulo or in vivo, releasing the active drug by chemical or enzymatic cleavage. By "pharmacologically inactive or less active derivative", in the context of the present invention, it is understood that the prodrug has no relevant activity in terms of inhibiting the VASH active site in an in vitro setting. However, it is active in in cellulo and in vivo assays, since it is capable of the necessary transformation to provide the active drug in such tests. Prodrugs can offer many advantages over the parent drug, such as increased cell permeability, solubility, increased stability, improved bioavailability, reduced side effects, and better selectivity. Activation of prodrugs involves a number of enzymes, including, but not limited to, oxidoreductases such as CYP450 and DT-diaphorase, and hydrolases such as carboxylesterase and β-glucuronidase.

[0045] In the context of the present invention, the prodrug compounds as defined in this disclosure may be inactive in vitro. However, the prodrug compounds have higher cell permeability. After permeation into the cells, the prodrug compounds are hydrolyzed to obtain the corresponding active drugs, i.e., potent VASH inhibitors.

[0046] Compounds of formula (I) The compounds according to the invention may be in the form of a stereoisomer or a mixture of stereoisomers, such as a mixture of enantiomers or diastereoisomers, in particular a racemic mixture. According to a particular embodiment, the compound of formula (I) is in the form of one diastereoisomeric configuration (S,S), (R,R), (S,R) or (R,S), as defined above.

[0047] Formula (I), as defined herein, encompasses both active compounds, ie drugs, and their prodrugs.

[0048] In particular, the term "prodrug" according to the present invention refers to3 means a compound of formula (I) where is not OH.

[0049] Preferably, the prodrugs according to the invention are 3 is an O-C1-C6 aliphatic chain, O-aryl, O-C1-C6 alkyl-aryl, O-heteroaryl, O-C1-C6 alkyl-heteroaryl or NHOH, up to four methylene units of said aliphatic chain are optionally replaced with O, C(O), NH or N-C1-C6 alkyl, and said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl or alkyl-aryl are optionally substituted. 3 is especially an O-C1-C6 aliphatic chain such as O-C1-C6 alkyl, especially O-ethyl, or O-C1-C6 alkyl-aryl, such as O-benzyl.

[0050] In the context of the present invention, the term "drug" means 3 means a compound of formula (I) wherein

[0051] Typically, R 3 forms an ester group with the adjacent carboxyl group. When the prodrug is converted to a drug, the adjacent carboxyl groups form an ester group with R 3 The ester formed by is typically hydrolyzed in the corresponding carboxylic acid.

[0052] In the context of the present invention, a drug differs from the corresponding prodrug in that an ester group present in the prodrug is converted to the corresponding carboxylic acid in the drug. Preferably, the prodrug is R 3 Thus, typically, the drug corresponding to a given prodrug comprises only one ester group formed by R 3 Except for the group, the prodrug has the same formula as the prodrug above. In other words, in a given prodrug and its drug, typically the substituent R 1, X and R are each the same. Alternatively, the prodrug may comprise two or more ester groups. In such cases, all of the ester groups are converted to the carboxylic acid of the corresponding drug.

[0053] Preferably, the compound of formula (I) is in the form of a diastereoisomeric compound having the configuration (S,S) and has the following formula (IA): [ka] is equivalent to.

[0054] In a more preferred embodiment, the compound of formula (IA) is the following enantiomer (I-A'): [ka] is equivalent to.

[0055] In a preferred embodiment, X is [ka] and preferably [ka] and R 1 , R 2 and R 3 is as defined in this disclosure.

[0056] In another particular embodiment, X is [ka] and preferably [ka] and R 1 , R 2 and R 3 is as defined in this disclosure.

[0057] According to this embodiment, R 4is, inter alia, H or C1-C 12 an aliphatic chain in which up to four methylene units are optionally replaced by O, C(O), NH or N-C1-C6 alkyl, 12 The aliphatic chain is optionally substituted with one or more OH groups. In particular, R 4 is H, COOH, C(O)-C1-C6 alkyl, (CH2) p -C(O)-C1-C6 alkyl, C(O)-(CH2) p -C(O)-C1-C6 alkyl, C(O)NHOH, (CH2) p -C(O)-NHOH and C(O)-(CH2) p -C(O)-NHOH, p is an integer from 1 to 4, and said C1-C6 alkyl is especially methyl or ethyl, preferably ethyl.

[0058] In another particular embodiment, X is: [ka] Preferably, [ka] And R 1 , R 2 and R 3 is as defined in this disclosure.

[0059] According to a preferred embodiment, R is OR 2 It is.

[0060] According to some embodiments, R 2 is H, a C1-C6 aliphatic chain such as C1-C6 alkyl, or a C1-C6 alkyl-aryl, said aliphatic chain or alkyl-aryl being optionally substituted. 2 is H, optionally substituted C-C alkyl, or optionally substituted C-C alkyl-aryl, such as benzyl. 2When is C1-C6 alkyl, such as methyl or ethyl, it is unsubstituted or may be substituted with, inter alia, one or more halogen, OH, NH2, N(C1-C6 alkyl)2 or N + (C1-C6 alkyl)3, especially N + More preferably, R 2 is H, C1-C6 alkyl or benzyl. Even more preferably, R 2 is a C1-C6 alkyl such as ethyl or benzyl.

[0061] According to some embodiments, R 2 is preferably not H. In such embodiments, therefore, R 2 is preferably a C1-C6 aliphatic chain, such as C1-C6 alkyl, or C1-C6 alkyl-aryl, said aliphatic chain or alkyl-aryl being optionally substituted, more preferably R 2 is a C1-C6 alkyl such as ethyl or benzyl.

[0062] According to another embodiment, R is NH-S(O)-R 9 and R 9 is a C1-C6 aliphatic chain or aryl, said aliphatic chain or aryl being optionally substituted. 9 is C1-C6 alkyl, such as methyl or ethyl, or optionally substituted aryl. R 9 When is aryl, especially phenyl, it is preferably unsubstituted or substituted by OH, or C1-C6 alkyl, such as methyl or ethyl.

[0063] According to some other embodiments, R 3 is OH, O-C1-C6 aliphatic chain, O-C1-C6 alkyl-aryl, or NHOH, said aliphatic chain being optionally substituted. 3is OH, optionally substituted O-C1-C6 alkyl, O-C1-C6 alkenyl such as O-allyl, O-C1-C6 alkyl-aryl such as O-benzyl, or NHOH. 3 is O-C1-C6 alkyl, such as O-methyl or O-ethyl, said alkyl is in particular unsubstituted or substituted with one or more halogen, OH, NH2, NH-C1-C6 alkyl, N(C1-C6 alkyl)2 or N + (C1-C6 alkyl)3, especially N + More preferably, R 3 is OH or O-C1-C6 alkyl, in particular O-ethyl.

[0064] According to some embodiments, R 1 is O-C1-C6 alkyl or O-C2-C6 alkenyl, provided that R 3 When is OH, R 1 is not O-C1-C6 alkyl. 1 is O-methyl, O-ethyl or O-allyl, with the proviso that R 3 When is OH, R 1 R is not O-C1-C6 alkyl. 1 is O-C1-C6 alkyl, R 3 is preferably unsubstituted or is substituted with one or more halogen, OH, NH, NH-C1-C6 alkyl, N(C1-C6 alkyl)2 or N + (C1-C6 alkyl)3, especially N + More preferably, R is O-C1-C6 alkyl, such as O-methyl or O-ethyl, substituted with (CH3)3. 1 is O-C1-C6 alkyl, especially O-ethyl, R 3 is O-ethyl. 1 When is O-C1-C6 alkyl, R 2 is preferably not H.

[0065] According to another embodiment, R 1 is NR1a R 1b and R 1a is H or C1-C6 alkyl such as methyl or ethyl, R 1b is OH or C1-C6 alkyl, said alkyl being optionally substituted with C(O)OH, C(O)O-C1-C6 alkyl or aryl. In such embodiments, R 1 can be NHOH, NH-C1-C6 alkyl or N(C1-C6 alkyl), said alkyl such as methyl, ethyl, n-propyl or n-butyl being unsubstituted or substituted with C(O)OH, C(O)-C1-C6 alkyl, especially C(O)-methyl or C(O)-ethyl, or aryl such as phenyl.

[0066] According to a preferred embodiment, R 1 teeth [ka] and preferably [ka] It is.

[0067] Compounds of formula (I) are compounds of the formula [ka] wherein R, R 3 , R 5 , R 6 , X and Y are as defined in this disclosure.

[0068] Preferably, the compound of formula (I) has the following configuration: a ) Compound [ka] where R, R 3 , R 5 , R 6 , X and Y are as defined in this disclosure.

[0069] More preferably, the compound of formula (I) has the following configuration: a ) Compound [ka] where R, R 3 , R 5 , R 6 , X and Y are as defined in this disclosure.

[0070] Y is especially [ka] It can be.

[0071] In such an embodiment, R 5 is preferably OH, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C1-C6 alkyl-aryl, O-C1-C6 alkyl-heteroaryl, C(O)OH, C(O)O-C1-C6 alkyl, C(O)NHOH, C(O)NH2, C(O)NH-C1-C6 alkyl, C(O)NH-O-C1-C6 alkyl, NH-C1-C6 alkyl, N(C1-C6 alkyl)2, NH-C(O)-C1-C6 alkyl or NH-S(O)2-R 9 wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl or alkyl-heteroaryl is optionally substituted; R 9 is as defined above. More preferably, R 5 is O-C1-C6 alkyl, such as OH, O-methyl or O-ethyl, O-C1-C6 alkyl-aryl, such as O-benzyl, C(O)NH-O-C1-C6 alkyl, such as C(O)-NH-O-tert-butyl, or NH(S(O)2-R 9 and R 9 is preferably unsubstituted or substituted with OH or C1-C6 alkyl, such as methyl or ethyl. In particular, R 5is O-C1-C6 alkyl, especially O-ethyl.

[0072] Alternatively, Y is -(CH2) m -, where m is an integer from 0 to 6, preferably from 0 to 4. More preferably, m is 1 or 2, and even more preferably, m is 2. Y can be -(CH2) m In embodiments where R 5 is preferably C(O)OH, C(O)O-C1-C6 alkyl or C(O)NHOH, said alkyl being optionally substituted. More preferably, R 5 is C(O)OH, C(O)OEt or C(O)NHOH, in particular COOH.

[0073] According to some embodiments, R 6 is OH or an O-C1-C6 aliphatic chain such as O-methyl, O-ethyl or O-allyl, and in particular Y is [ka] In particular, O-ethyl.

[0074] According to another particular embodiment, R 6 is NH-CH(R 7 )-(CH2) n -R 8 In particular, Y is -(CH2) m In particular, when R is -, it is -(CH)-. In such embodiments, R 7 is preferably H, C1-C6 alkyl, C1-C6 alkyl-aryl such as benzyl, or C1-C6 alkyl-heteroaryl such as a CH2-indole group. 8 is preferably C(O)NH, aryl such as phenyl, heteroaryl such as indole, SH, or S-C1-C6 alkyl such as SC(CH3)3, and n is preferably 0, 1, 2, or 3. 7 is C1-C6 alkyl, R 8is C(O)NH2 and n is 0. In some other preferred embodiments, R 7 is H and R 8 is phenyl and n is 1, 2 or 3, preferably 1.

[0075] According to a preferred embodiment, R 3 When is OH, R 1 teeth [ka] and preferably [ka] It is.

[0076] In a preferred embodiment, the compound of formula (I) is a compound of formula (I-A′a) as defined above, wherein X, R and R 3 is defined above, and R 1 teeth [ka] and preferably [ka] and -Y is -(CH2) m -, m is as defined above, in particular m is 2, R 5 is C(O)OH, C(O)OEt or C(O)NHOH, R 6 is NH-CH(R 7 )-(CH2) n -R 8 and R 7 is H, C1-C6 alkyl, C1-C6 alkyl-aryl such as benzyl, or C1-C6 alkyl-heteroaryl such as a CH2-indole group; R 8is C(O)NH2, aryl such as phenyl, heteroaryl such as indole, SH, or S-C1-C6 alkyl such as SC(CH3)3, where n is 0, 1, 2 or 3; or -Y is [ka] and R 5 is OH, O-C1-C6 alkyl, such as O-methyl or O-ethyl, O-C1-C6 alkyl-aryl, such as O-benzyl, and R 6 is OH or an O-C1-C6 aliphatic chain such as O-methyl, O-ethyl or O-allyl.

[0077] According to another preferred embodiment, the compound of formula (I) is of formula (I-A'), wherein X is [ka] and R 1 is O-C1-C6 alkyl, in particular O-ethyl, and R is OR 2 and R 2 is preferably H or C1-C6 alkyl, in particular ethyl, and R 3 is OH or O-C1-C6 alkyl, in particular O-ethyl.

[0078] In a more preferred embodiment, the compound of formula (I) has the formula (I-A' a ) wherein X is [ka] and R is OR 2 and R 2 is preferably H, C1-C6 alkyl, in particular ethyl, or benzyl, and R 3 is OH or O-C1-C6 alkyl, in particular O-ethyl, and R 1 teeth [ka] and preferably [ka] and During the ceremony, -Y is -(CH2) m -, especially -(CH2)2-, R 5 is C(O)NHOH, and R 6 is NH-CH2-(CH2) n -R 8 and R 8 is aryl, such as phenyl, and n is 1, 2 or 3, especially 1; or -Y is [ka] and R 5 and R 6 is independently OH or an O-C1-C6 aliphatic chain such as O-methyl, O-ethyl or O-allyl, especially O-ethyl.

[0079] In another more preferred embodiment, the compound of formula (I) is of formula (I-A'), wherein X is [ka] and R 1 is NR 1a R 1b and R 1a is preferably H, R 1b is preferably C1-C6 alkyl, such as methyl, ethyl, n-propyl or n-butyl, said alkyl being substituted with C(O)-methyl or C(O)-ethyl, and R is OR 2 and R 2 is preferably C1-C6 alkyl, in particular ethyl, R 3 is OH or O-C1-C6 alkyl, in particular O-ethyl.

[0080] According to a particular embodiment, the compound of formula (I) is of the following formula (I-bis): [ka] or a pharma- ceutically acceptable salt and / or solvate thereof; During the ceremony X is [ka] and R 1 is O-C1-C6 alkyl, O-C2-C6 alkenyl, NH-OH, or [ka] and R 2 is H, a C1-C6 aliphatic chain, an aryl, a heteroaryl, or a C1-C6 alkyl-aryl, wherein up to four methylene units of said aliphatic chain are optionally replaced with O, C(O), NH, or N-C1-C6 alkyl, and wherein said aliphatic chain, aryl, heteroaryl, or alkyl-aryl are optionally substituted; R 3 is OH, O-C1-C6 aliphatic chain, O-aryl, O-C1-C6 alkyl-aryl, O-heteroaryl, O-C1-C6 alkyl-heteroaryl or NHOH, wherein up to four methylene units of said aliphatic chain are optionally replaced with O, C(O), NH or N-C1-C6 alkyl, and wherein said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl or alkyl-aryl are optionally substituted; R 4 is H or C1-C 12 an aliphatic chain in which up to four methylene units are optionally replaced by O, C(O), NH or N-C1-C6 alkyl, 12 the aliphatic chain is optionally substituted; Y is -(CH2) m -or [ka] and R 5is OH, O-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, O-C1-C6 alkyl-aryl, O-C1-C6 alkyl-heteroaryl, C(O)OH, C(O)O-C1-C6 alkyl or C(O)NHOH, wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl or alkyl-heteroaryl is optionally substituted; R 6 is OH, O-C1-C6 alkyl, NH-OH or NH-CH(R 7 )-(CH2) n -R 8 wherein the alkyl is optionally substituted; R 7 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C1-C6 alkyl-aryl or C1-C6 alkyl-heteroaryl; R 8 is C(O)NH2, C(O)NH-C1-C6 alkyl, aryl, heteroaryl, SH, NH2 or S-C1-C6 alkyl, and m and n are each independently an integer ranging from 0 to 6. However, if X is [ka] and R 3 When is OH, R 1 is not O-C1-C6 alkyl.

[0081] In particular, compounds of formula (I) [ka] isn't it.

[0082] Preferably, the compound of formula (I) is [ka] This is described in William R. Roush et al. Synthesis, 1999, p.1500-1504.

[0083] Preferably, the compound of formula (I) is [ka] This is described as compound SD-139 in Aillaud, C. et al. Science, 2017, 358, p.1448-1453, Supplementary material.

[0084] According to a particular embodiment, the compound of formula (I) is an amino acid or peptide-based VASH inhibitor. By "amino acid or peptide-based VASH inhibitor" according to the present invention is meant a VASH inhibitor containing a peptide moiety consisting of 1-20 amino acids, where the amino acid or the most C-terminal amino acid is selected from Y and F, which is capable of at least partially targeting and inhibiting the activity of a protein having tubulin carboxypeptidase activity, thereby inhibiting microtubule detyrosination.

[0085] The term "amino acid moiety" means one amino acid, and the term "peptide moiety" means a moiety containing at least 2 amino acids and up to 20 amino acids. When a peptide moiety comprises two or more amino acids, said amino acids are linked together by peptide bonds, whether or not they are chemically modified.

[0086] According to a preferred embodiment, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] and the pharma- ceutically acceptable salts and / or solvates thereof.

[0087] In a particular embodiment, the compounds of formula (I) according to the invention are selected from the group defined above and in particular consisting of [ka] [ka] [ka] is a prodrug of choice among

[0088] In a particular embodiment, the compounds of formula (I) according to the invention are selected from the group defined above and in particular consisting of [ka] [ka] [ka] [ka] It is the drug of choice among

[0089] In a particular preferred embodiment, the compound of formula (I) according to the invention is selected in the group consisting of compounds LV-80 to LV-124 and their complexes.

[0090] In a particular embodiment, the compound of formula (I) according to the invention is LV-80 or a conjugate thereof.

[0091] In a particular embodiment, the compound of formula (I) according to the invention is LV-91 or a conjugate thereof.

[0092] In a particular embodiment, the compound of formula (I) according to the invention is LV-104 or a conjugate thereof. LV-104 may be a prodrug of the corresponding active drug IBMT11.

[0093] In a particular embodiment, the compound of formula (I) according to the invention is LV-111 or a conjugate thereof. LV-111 may be a prodrug of the corresponding active drug.

[0094] In a particular embodiment, the compound of formula (I) according to the invention is IBMT23 or a conjugate thereof. IBMT23 may be a prodrug of the corresponding active drug.

[0095] Process for the preparation of compounds of formula (I) The compound of formula (I) above, or a pharma- ceutically acceptable salt and / or solvate thereof, can be prepared by the following steps: (a) A compound of formula (II) [ka] with a compound of formula (III) [ka] (In the formula, R Z is R as defined above. 1 or OH) To react with (b) optionally, R is OH; Z R defined above 1 Converting to The method can be obtained by a process comprising the steps of:

[0096] Compounds of formula (III) can be obtained according to methods well known to those skilled in the art.

[0097] The reaction between compounds of formula (II) and formula (III) is especially a peptide coupling as defined above.

[0098] The compounds of formula (II) are especially [ka] It is.

[0099] Optionally, additional steps of protection / deprotection and / or functionalization well known to those skilled in the art may be carried out before or after the reaction between compounds of formula (II) and formula (III) to obtain compounds of formula (I) bearing suitable substituents as described above.

[0100] In particular, R Z is OH, the compound obtained in step (a) is [ka] After peptide coupling with R 1 but [ka] This gives a compound of formula (I)

[0101] The peptide coupling or couplings carried out in the process for preparing the compounds of formula (I) are in particular achieved in the presence of PyAOP as a coupling agent. Preferably, the base DIEA is also used.

[0102] Peptide coupling can be carried out on a solid support, inter alia, by using a resin with an amine or acid moiety attached as one of the reagents. Such methods are well known to those skilled in the art.

[0103] Conjugates Comprising Fragments of Compounds of Formula (I) Linked to Biomolecules The present invention also relates to a complex comprising a fragment of a compound of formula (I) above bound to a biomolecule (e.g., a peptide, a protein, a biomarker (e.g., a photolabeling agent such as rhodamine, a cyanine derivative or fluorescein), an affinity probe such as biotin, or an E3 ubiquitin ligase recruiter (including, but not limited to, thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, PROTAC6, ZNL-02-096 or d9A-2)).

[0104] The term "fragment of a compound of formula (I)" refers to a compound of formula (I) that is modified at one end, for example by attachment to a biomolecule via a linker. Thus, typically, R 1 The groups are modified to allow such attachment. For example, in the conjugates according to the invention, the fragments of the compounds of formula (I) may be [ka] means, During the ceremony [ka] is a single bond between the fragment and the rest of the complex, R, R 3 , X, Y and R 5 is as defined in this disclosure.

[0105] According to a particular embodiment, the conjugate according to the invention has the following formula (I'): [ka] or a pharma- ceutically acceptable salt and / or solvate thereof; During the ceremony B is a biomolecule (e.g., a peptide, a protein, a biomarker (e.g., a photolabeling agent), or an E3 ligase recruiter (e.g., thalidomide)); L is a linker, R, R 3 , X, Y and R 5 is defined above.

[0106] According to a preferred embodiment, the conjugate of formula (I') is a conjugate of formula (I'-A) having the following configuration: [ka] It is.

[0107] In particular, the conjugate of formula (I') has the following configuration: a ) complex [ka] It is.

[0108] Formula (I'), in particular formula (I'-A a In the complex of [ka] It is.

[0109] R is preferably OR 2 and R 2 is advantageously H or C1-C6 alkyl, in particular ethyl or benzyl. 3 is preferably OH or O-C1-C6 alkyl, in particular O-ethyl.

[0110] Y is -(CH2) m -or [ka] It can be.

[0111] Y is -(CH2) m -, especially -(CH2)2-, R 5 is preferably C(O)NHOH, R 6 is preferably NH-CH2-(CH2) n -R 8 and R 8is advantageously aryl, such as phenyl, and n is advantageously 1, 2 or 3, especially 1. [ka] When R 5 is preferably OH or O-C1-C6 alkyl, such as O-methyl or O-ethyl, in particular O-ethyl.

[0112] According to some embodiments, the linker L is C1-C 12 It corresponds to a divalent radical derived from an aliphatic chain, in which one or more methylene units (s) are replaced by an arylene or a structural linker selected from the fragments -O-, -S-, -C(=O)-, -SO2- and -N(C1-C6 alkyl)-, said aliphatic chain being unsubstituted or substituted with one or more radicals selected from halogen, OH, C1-C6 alkyl and / or C1-C6 alkylaryl groups, such as benzyl groups, e.g. benzyl groups.

[0113] Preferably, the biomolecule is an E3 ligase recruiter, in particular thalidomide, and thus the conjugate is a PROTAC E3 ligase recruiter.

[0114] Thus, in a preferred embodiment, the complex comprises a PROTAC E3 ligase recruiter of formula (II′): [ka] where X, Y, L, R 2 , R 3 and R 5 is defined above.

[0115] For example, PROTAC E3 ligase recruiters [ka] where X is as defined above, and in particular X is [ka] It is.

[0116] In another embodiment, the biomolecule is an affinity probe and the complex of formula (I') [ka] wherein the compound of formula (I) is conjugated to biotin.

[0117] In another embodiment, the biomolecule is a photolabeling agent and the conjugate of formula (I') is [ka] wherein the compound of formula (I) is linked to rhodamine.

[0118] Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising at least one pharma- ceutically acceptable excipient and at least one compound of formula (I) above or a pharma- ceutically acceptable salt and / or solvate thereof.

[0119] The present invention also relates to a pharmaceutical composition comprising at least one of the above conjugates, such as a conjugate of formula (I'), or a pharma- ceutically acceptable salt and / or solvate thereof, and at least one pharma- ceutically acceptable excipient.

[0120] The pharmaceutical compositions of the present invention may be intended for oral or parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraocular, intravitreal, topical, or sublingual administration), preferably oral or intravenous. The active ingredient may be administered in a unitary form for administration to animals, preferably mammals, including humans, in admixture with a conventional pharmaceutical carrier.

[0121] For oral administration, the pharmaceutical composition may be in solid or liquid form (solution or suspension).

[0122] The solid composition can be in the form of tablets, gelatin capsules, powders, granules, etc. In tablets, the active ingredient can be mixed with gelatin and pharmaceutical vehicle(s), such as starch, lactose, magnesium stearate, talc, gum arabic, and then compressed. The tablets may be further coated, especially with sucrose or other suitable materials, or may be treated so that they have a sustained or delayed activity. In powders or granules, the active ingredient can be mixed or granulated with dispersing, wetting or suspending agents, and flavoring or sweetening agents. In gelatin capsules, the active ingredient can be introduced into soft or hard gelatin capsules in the form of powder or granules as described above, or in the form of a liquid composition as described below.

[0123] The liquid composition can contain the active ingredient together with a sweetener, a flavoring agent, and a suitable coloring agent in a solvent such as water. Furthermore, the liquid composition can also be obtained by suspending or dissolving the above-mentioned powder or granules in a liquid such as water, juice, milk, etc. For example, it can be a syrup or elixir.

[0124] For parenteral administration, the composition may be in the form of an aqueous suspension or solution, which may contain suspending agents and / or wetting agents. The composition is advantageously sterile. It may be in the form of an isotonic solution (especially compared to blood).

[0125] use Therapeutic Use The compounds of formula (I), pharma- ceutically acceptable salts and / or solvates, or pharmaceutical compositions according to the invention act as VASH inhibitors, meaning that they are able to inhibit the peptidase activity of VASH, which catalyzes the detyrosination of microtubules, and whose unregulated, and in particular abnormally increased, activity leads to severe disorders as defined below.

[0126] X, [ka] Compounds of formula (I), wherein:

[0127] X, [ka] Compounds of formula (I), wherein: are inter alia reversible VASH inhibitors, meaning that VASH peptidase enzyme activity can be restored.

[0128] The present invention relates to the compounds of formula (I) according to the invention or their pharma- ceutically acceptable salts and / or solvates, for use as medicaments, in particular for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0129] In other words, the present invention relates to the use of a compound of formula (I) according to the invention or a pharma- ceutically acceptable salt and / or solvate thereof, in particular for the manufacture of a medicament intended for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0130] In other words, the present invention relates to the use of a compound of formula (I) according to the present invention or a pharma- ceutically acceptable salt and / or solvate thereof for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0131] In other words, the present invention relates to a method for preventing and / or treating disorders associated with VASH peptidase activity, comprising administering to a person in need thereof an effective amount of a compound of formula (I) according to the present invention or a pharma- ceutically acceptable salt and / or solvate thereof.

[0132] According to another aspect, the present invention relates to a pharmaceutical composition according to the invention for use as a medicament, in particular for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0133] In other words, the present invention relates to the use of a pharmaceutical composition according to the invention, especially for the manufacture of a medicament intended for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0134] In other words, the present invention relates to the use of the pharmaceutical composition according to the present invention for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0135] In other words, the present invention relates to a method for preventing and / or treating disorders associated with VASH peptidase activity, comprising administering to a person in need thereof an effective amount of a pharmaceutical composition according to the present invention.

[0136] According to another aspect, the present invention relates to a conjugate according to the invention for use as a medicament, in particular for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0137] In other words, the present invention relates to the use of a conjugate according to the invention, in particular for the manufacture of a medicament intended for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0138] In other words, the present invention relates to the use of the conjugate according to the present invention for the prevention and / or treatment of disorders associated with VASH peptidase activity.

[0139] In other words, the present invention relates to a method for preventing and / or treating disorders associated with VASH peptidase activity, comprising administering to a person in need thereof an effective amount of a conjugate according to the present invention.

[0140] A preferred embodiment relates to a PROTAC E3 ligase recruiter according to the invention, especially of formula (I″) as defined above, for use as a drug, in particular for the prevention and / or treatment of disorders associated with VASH peptidase activity. Such a PROTAC E3 ligase recruiter is able to selectively target the VASH enzyme and induce its total degradation by the proteasome.

[0141] X, [ka] The complex of formula (I″),

[0142] X, [ka] The complex of formula (I″),

[0143] According to the present invention, the disorder associated with VASH peptidase activity is preferably selected from fibrosis, cancer and tubulin carboxypeptidase-associated diseases.

[0144] In particular, disorders associated with VASH peptidase activity are disorders involving detyrosination and / or polyglutamylation of microtubules, and are selected from neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, glaucoma, psychiatric disorders, neurological disorders, cancers such as colon cancer and neuroblastoma, muscular dystrophies, infertility, retinal degenerations, Purkinje cell diseases, infantile onset degenerations, male infertility and ciliopathy, in particular neurodegenerative diseases, cancers, and muscular dystrophies.

[0145] In particular, VASH peptidase activity-associated disorders are tubulin carboxypeptidase-associated diseases, including, but not limited to, disorders involving altered microtubule detyrosination.

[0146] According to certain embodiments, the disorder involving altered microtubule detyrosination may be selected from neurodegenerative diseases such as Alzheimer's or Parkinson's disease, glaucoma, psychiatric and neurological disorders, cancers such as colon cancer and neuroblastoma, muscular dystrophies, infertility, retinal degenerations and ciliopathy, in particular neurodegenerative diseases, cancers and muscular dystrophies.

[0147] In another aspect, the disorder associated with VASH peptidase activity is a tubulin carboxypeptidase-associated disease, including disorders involving polyglutamylation.

[0148] According to certain embodiments, the disorder involving polyglutamylation may be selected from neurodegeneration, neurodevelopmental disorders, Purkinje cell diseases, infantile onset degenerations, ciliopathy, male infertility, cancer, respiratory disorders, retinal degenerations, bleeding disorders, non-Mendelian inheritance disorders.

[0149] Use as a research tool According to another aspect, the present invention relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: - In vitro and / or in cell screening assays to identify novel VASH inhibitors and / or quantify their inhibitor efficiency, - an in vitro method for studying the role of tubulin detyrosination in disorders associated with VASH peptidase activity, in particular their onset, aggressiveness and progression; - an in vitro diagnostic method for identifying or monitoring a VASH peptidase activity-associated disorder involving abnormalities in microtubule detyrosination and / or polyglutamylation, - and associated kits for carrying out the above screening assays and methods. The present invention relates to a compound of formula (I) according to the present invention or a conjugate thereof for use as a research tool for research and development activities selected in the group consisting of:

[0150] These research and development activities are illustrative, but not limiting, examples of uses of the compounds of formula (I) above or conjugates thereof.

[0151] The compound of formula (I) or a conjugate thereof is used as a positive control in the above in vitro or in celluar screening assays or methods.

[0152] The compound of formula (I) above can be conjugated to, for example, but not limited to, a fluorescent dye, a UV-sensitive dye, HRP, alkaline phosphatase, or biotin.

[0153] In a particular embodiment, the compound of formula (I) according to the present invention or its conjugate is used in an in vitro screening assay for VASH inhibitors (primary screening assay) in which the compound of formula (I) is used as a positive control.

[0154] As an example, the screening assay is an immunoassay in which a compound of formula (I) or a conjugate thereof is coated onto plates as a positive control.

[0155] The present invention also relates to an in vitro kit assay for the primary screening assay of VASH inhibitors comprising a compound of formula (I) or a conjugate thereof as a positive control.

[0156] In another particular embodiment, the compound of formula (I) according to the present invention or its conjugate is used in an in cellulo screening assay for VASH inhibitors (secondary screening assay) in which the compound of formula (I) or its conjugate is used as a positive control.

[0157] The present invention also relates to an inCelluloKit assay for secondary screening assay of VASH inhibitors comprising a compound of formula (I) or a conjugate thereof as a positive control.

[0158] In a preferred embodiment, the InCelluloKit assay comprises a CHL cell line and a compound of formula (I) as a positive control.

[0159] In another particular embodiment, the compound of formula (I) according to the present invention or its conjugate is used as a tool to better understand the role of tubulin detyrosination, in particular in tubulin detyrosination-related disorders, their development, malignancy, progression, etc. (e.g. cancer).

[0160] Therefore, the present invention also comprises the steps of: (i) contacting (a) a substrate for VASH1 or VASH2 enzyme, comprising an amino acid sequence having at least the last four amino acid residues of the C-terminal sequence of α-tubulin and / or a microtubule-associated protein (MAP) and a tyrosine (Y) as the most C-terminal amino acid residue, and (b) isolated or recombinant VAHS1 or VASH2, in the presence or absence of a test compound (negative control) or in the presence of a compound of formula (I) according to the present invention or a complex thereof (positive control) under conditions of cleavage and release of the C-terminal free tyrosine (detyrosination); (ii) using reagents to detect and measure signals associated with cleavage and release of the C-terminal free tyrosine; (iii) measuring and comparing the level of C-terminal free tyrosine in the presence and absence of the test compound (negative control) and in the presence of a compound of formula (I) according to the present invention or a complex thereof (positive control); and (iv) selecting a compound that reduces the level of C-terminal free tyrosine in the presence of a test compound (VASH inhibitor); (v) Optionally, classifying the inhibitory efficiency of said VASH inhibitor compound by comparing the level of C-terminal free tyrosine in the presence of a compound of formula (I) according to the invention or its conjugate (positive control). The present invention relates to an in vitro screening assay for identifying VASH inhibitors, comprising:

[0161] In certain embodiments, the substrate for the VASH1 or VASH2 enzyme is selected from the group consisting of purified recombinant α-tubulin and recombinant engineered telokin as disclosed in WO 2020 / 012002.

[0162] In certain embodiments, step (ii) of detecting the C-terminal free tyrosine employs a colorimetric assay that uses tyrosinase.

[0163] In another particular embodiment, the in vitro screening assay is an immunoassay, wherein step (ii) comprises: - adding an effective amount of a specific labeled antibody raised against detyrosinated α-tubulin (dTyr-Ab) under conditions favoring the formation of an antibody-cleaved substrate complex; and - a means for revealing a labeling signal, Or alternatively, - adding an effective amount of a primary specific antibody raised against detyrosinated α-tubulin (dTyr-Ab) under conditions favoring the formation of an antibody-cleaved substrate complex; - adding an effective amount of a secondary labeled antibody specific for the primary antibody under conditions favoring the formation of a primary antibody-cleaved substrate-labeled secondary antibody complex; and - Means for revealing labeling signals and the reaction occurs in the soluble (fluid) or solid phase.

[0164] In particular, the antibody is labelled with a marker selected in the group consisting of enzymes, fluorescent compounds and fluorophores, (chemi)luminescent compounds and radioactive elements, preferably with an enzyme, more preferably with a peroxidase.

[0165] In particular, the immunoassay is an enzyme-, fluoro-, luminescence- or radioimmunoassay, preferably a dot blot or an enzyme-linked immunoassay (ELISA).

[0166] In certain embodiments, the in vitro screening immunoassay for identifying VASH inhibitors is performed in a solid phase: - a substrate for VASH1 or VASH2 is coated on a solid support, in particular a membrane or a microplate, and the VASH1 or VASH2 enzyme, the test compound(s) and the cleaved substrate, in particular an antibody raised against detyrosinated α-tubulin (dTyr-Ab) (optionally labeled or combined with a labeled secondary antibody raised against the primary antibody) are added to the reaction solution, or Alternatively, an antibody raised against the cleaved substrate, in particular against detyrosinated α-tubulin (dTyr-Ab), is coated onto a solid support, in particular a membrane or a microplate, and the VASH1 or VASH2 enzyme, said VASH1 or VASH2 substrate, the test compound(s) and a second labeled antibody raised against the cleaved substrate are added to the reaction solution, or Alternatively, the VASH1 or VASH2 enzyme is coated onto a solid support, in particular a membrane or microplate, and the VASH1 or VASH2 substrate, the test compound(s) and the cleaved substrate, in particular an antibody raised against detyrosinated α-tubulin (dTyr-Ab) (optionally labelled or combined with a labelled secondary antibody raised against the primary antibody) are added to the reaction solution.

[0167] The present invention also provides a kit for performing an in vitro screening immunoassay for identifying VASH inhibitors as disclosed above, comprising: (i) a VASH1 or VASH2 enzyme substrate, preferably a recombinant α-tubulin or an artificial telokin as disclosed in WO 2020 / 012002; (ii) a VASH1 or VASH2 enzyme, preferably isolated or recombinant VASH1 or VASH2; (iii) an antibody raised against the cleaved substrate, preferably detyrosinated α-tubulin (dTyr-Ab), in particular against detyrosinated α-tubulin SF9 (dTyr-Ab SF9), conjugated to horseradish peroxidase (HRP), and optionally a secondary antibody raised against the cleaved substrate antibody, preferably against the detyrosinated α-tubulin antibody; (iv) a negative control comprising non-tyrosinated, isolated or recombinant α-tubulin or telokin; (v) a compound of formula (I) according to the present invention or a conjugate thereof as a positive control; (vi) a fluid container or alternatively a solid support, preferably a microplate, for coating or pre-coating either the VASH1 substrate or the VASH2 substrate, the VASH1 enzyme or the VASH2 enzyme; (vii) a reagent that allows contact of said substrate with the VASH1 or VASH2 enzyme under reaction conditions for substrate cleavage, preferably detyrosination; (viii) reagents for detecting and measuring the level of substrate cleavage, preferably detyrosination; and (ix) Optional notice of use The present invention relates to a kit comprising:

[0168] In another particular embodiment, the compound of formula (I) according to the invention or its conjugate is used in an in cellulo method for quantifying VASH peptidase activity.

[0169] The present invention also provides an in cellulos screening assay for identifying VASH inhibitors, comprising: (i) culturing CHL-1 cells and seeding them into multi-well culture dishes; (ii) treating CHL-1 cells with taxol in the absence (negative control) or presence of a putative VASH inhibitor to be tested or in the presence of a compound of formula (I) according to the invention or a conjugate thereof (positive control); (iii) detecting detyrosination activity in the protein sample using a specific labeled antibody (dTyr-Ab) raised against detyrosinated α-tubulin under conditions favoring antibody-cleavage substrate complex formation and a means for revealing the labeling signal; (iv) measuring and comparing the level of taxol-induced detyrosination in the presence and absence of a test compound (negative control) and in the presence of a compound of formula (I) according to the present invention or its complex (positive control); (v) selecting compounds (VASH inhibitors) in which the level of taxol-induced detyrosination is reduced in the presence of the test compound; (vi) Optionally, classifying the inhibitory efficiency of said VASH inhibitor compounds by comparing the reduction in taxol-induced detyrosination levels in the presence of a compound of formula (I) according to the invention or its conjugate (positive control). The present invention relates to an assay comprising:

[0170] The present invention also provides a kit for performing an in cellulos screening assay to identify VASH inhibitors, comprising: (i) CHL-1 cells, (ii) taxol, (iii) antibodies raised against detyrosinated α-tubulin (dTyr-Ab); (iv) a compound of formula (I) according to the present invention or a conjugate thereof as a positive control; (v) a fluid container or alternatively a solid support, preferably a microplate, for coating or pre-coating CHL-1 cells; (vi) a reagent that allows contact of the taxol-pretreated CHL-1 cells with a test compound or a positive control under detyrosination reaction conditions; (vii) Reagents for detecting and measuring the reduction in taxol-induced detyrosination levels, and (viii) Optional Notice of Use The present invention relates to a kit comprising:

[0171] In another particular embodiment, the compound of formula (I) according to the invention or its conjugate is used in an in vitro diagnostic method for detecting disorders associated with VASH peptidase activity.

[0172] Thus, the present invention also relates to kit assays for in vitro diagnostic methods in which a compound of formula (I) or a conjugate thereof is used as a positive control. EXAMPLES

[0173] 1) Synthesis Abbreviation: Aad: α-aminoadipic acid All: Allyl group BOP: Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate DCM: dichloromethane DIEA: N,N-diisopropylethylamine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate NMP: N-methyl-2-pyrrolidone PyAOP: 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RM: reaction mixture RT: Room temperature (18℃~25℃) SPPS: Solid Phase Peptide Synthesis TES: Trans-epoxysuccinic acid TFA: Trifluoroacetic acid THF: tetrahydrofuran TiS: Triisopropylsilane Tyr: Tyrosine

[0174] 1.1) Material Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) were all obtained from Iris Biotech GmbH. Piperidine, N,N-diisopropylethylamine (DIEA), TFA, triisopropylsilane (TIS), dichloromethane (DCM), 1,2-dichloroethane (DCE), N,N-dimethylformamide (DMF), N-methylpyrrolidinone (NMP) and ethyl ether (Et2O) were obtained from Sigma-Aldrich. AmphiSpheres40 RAM 0.38 mmol / g 75–150 μm resin was purchased from Agilent Technologies. Solvents used for HPLC and LC / MS were HPLC grade.

[0175] SPPS Procedure Peptide synthesis was performed using standard SPPS protocols. Each synthesis was carried out using Fmoc-Rink amide AmphiSpheres40 resin (0.37 mmol / g). Fmoc-protected amino acid (4 equiv.), HATU (4 equiv.) and DIEA (6 equiv.) were added to a syringe reactor and the mixture was stirred at room temperature for 1 h. After each coupling reaction, the peptide-resin was subjected to two 5 min deprotection cycles with DMF / piperidine 80 / 20 (vol / vol) solution.

[0176] Compound purification All crude compounds were purified by preparative HPLC (Waters4000 instrument) on a C18 reversed-phase column (C18 Deltapak column, 100 mm × 40 mm, 15 μm, 100 Å) with a mixture of H2O + 0.1% TFA and CH3CN + 0.1% TFA in gradient mode at a flow rate of 50 mL / min with UV detection at 214 nm. Fractions containing pure product were collected and lyophilized.

[0177] LC / MS analysis Samples were prepared in acetonitrile / water (50 / 50 vol / vol) mixtures containing 0.1% TFA. The LC / MS system consisted of a Waters Alliance2690 HPLC and a Micromass (Manchester, UK) ZQ spectrometer (electrospray ionization mode, ESI+). All analyses were performed using a C18 Chromolith Flash25×4.6 mm column. A flow rate of 3 ml / min was used with 0–100% acetonitrile, followed by a gradient of H2O+0.1% HCOOH and CH3CN+0.1% HCOOH mixtures over 5 min in gradient mode with UV detection at 214 nm. Positive ion electrospray mass spectra were obtained with a solvent flow rate of 100–200 μl / min. Nitrogen was used as both the nebulizing and drying gas. Data were acquired in scan mode in the range 200–1700 m / z with 0.1 s intervals. Ten scans were summed to obtain the final spectrum. Retention times are given in minutes. Solvents used for HPLC and LC / MS were of HPLC grade.

[0178] 1.2) Synthesis and Characterization 1.2.1) Synthesis of compounds of formula (I) according to the invention Synthesis of compound 1

[0179] [ka]

[0180] Boc-Tyr(Bn)-OH (2 g, 5.4 mmol, FLUKA AG) was dissolved in 3 ml of anhydrous DMF, followed by Cs2CO3 (1.8 g, 5.4 mmol). The RM was stirred at RT for 5 min, after which allyl bromide (696.6 μl, 8.1 mmol) was added. After reacting at RT for 2 h, the RM was diluted with brine and EtOAc, then extracted twice. The recollected organic phase was then washed twice with 1 M KHSO4 solution, twice with saturated NaHCO3 solution, twice with brine, then the organic phase was dried over MgSO4, filtered and concentrated to give Boc-Tyr(Bn)-OAll (2.45 g, yield: 110%, t R =2.19, MS(ESI+):m / z=412.3[M+H]+ ) was obtained. The product was used directly in the next step without further purification.

[0181] Boc-Tyr(Bn)-OAll (2.22 g, 5.4 mmol) was dissolved in 15 ml of DCM, followed by 15 ml of TFA / TIS / H2O 95 / 2.5 / 2.5 (v / v / v). The RM was then stirred at RT for 1 h 10 min. The RM was concentrated to dryness and the residual oil was precipitated with cold ether. The precipitate was filtered and dried under vacuum to give compound 1 as a white solid. The product was used as is without further purification. Estimated mass: 2.37g Mass obtained: 2.83g Yield: Quantitative, some impurities present t R =1.38, MS(ESI+):m / z=312.2[M+H] +

[0182] Synthesis of compound 2 [ka]

[0183] (2S,3S)-Oxirane-2,3-dicarboxylic acid (66 mg, 0.5 mmol) was dissolved in 3 ml of NMP, followed by compound 1 (212 mg, 0.5 mmol) and DIEA (374 μl, 2 mmol). The RM was stirred and 260 mg (0.5 mmol) of PyAOP was added. The RM was stirred at room temperature for 20 min, then poured into 50 ml of cold water and kept in ice until a precipitate appeared. The precipitate was removed by filtration and the pH of the filtrate was adjusted to 3 with 1 M HCl solution. The filtrate was then extracted three times with EtOAc, the organic phase was washed three times with 1 M HCl solution and once with brine, dried over MgSO4, and evaporated to dryness to give 100 mg of compound 2 as a clear oil. The product was used as is without further purification. Estimated mass: 212.5 mg Mass obtained: 100 mg Yield: 47% HPLC purity=95%, t R =3.33, MS(ESI+):m / z=426.2[M+H] + , (expected m / z=426.16)

[0184] Synthesis of compound 3 [ka]

[0185] Fmoc-Aad(OtBu)-OH (1 g, 2.28 mmol) was dissolved in 5 ml of NMP, followed by 2-phenylethan-1-amine (316 μl, 2.5 mmol) and DIEA (1.175 ml, 6.83 mmol). The RM was stirred and PyAOP (1.18 g, 2.28 mmol) was added. The RM was stirred at RT for 15 min. Then 50 ml of EtOAc was poured in and washed once with a water / brine 50 / 50 (v / v) mixture, three times with a saturated NaHCO3 solution, three times with a 1 M HCl solution, once with brine, dried over MgSO4, and concentrated to dryness to give 1.24 g of compound 3 as a clear oil. The compound was used without further purification. Expected mass: 1.235g Mass obtained: 1.24g Yield: quantitative HPLC purity=95%, t R =4.28, MS(ESI+):m / z=543.3[M+H] +

[0186] Synthesis of compound 4 [ka]

[0187] Compound 3 (542 mg, 1 mmol) was dissolved in 5 ml of NMP, followed by the addition of diethylamine (208 μl, 2 mmol). The RM was then stirred at RT for 1 h. The RM was then poured into 50 ml of water and the pH was adjusted to 11 with 1 M NaOH solution. The aqueous phase was then extracted three times with EtOAc and the combined organic phases were washed with brine and washed with Na2S Drying over O4 and concentration to dryness gave 294 mg of compound 4 as a clear oil. The product was used without further purification. Estimated mass: 320mg Mass obtained: 294 mg Yield: 92% t R =2.28, MS(ESI+):m / z=321.2[M+H]+

[0188] Synthesis of intermediate A [ka]

[0189] Compound 4 tert-butyl (S)-5-amino-6-oxo-6-(phenethylamino)hexanoate (131 mg, 0.41 mmol) and compound 2 HO-TES-Tyr(OBn)-OAll (174 mg, 0.41 mmol) were dissolved in DMF, followed by the addition of DIEA (139 μl, 0.82 mmol) and BOP (173 mg, 0.41 mmol). After 2 h, the product was isolated by extraction. The resulting white powder was then dried overnight at 0.2 mbar to give 260 mg of intermediate A as a white solid. Expected mass: 260mg / Obtained mass: 300mg / Crude yield: 86% t R =2.18, MS(ESI+):m / z=728.4[M+H] +

[0190] Synthesis of compound 5 [ka]

[0191] (+,-)-trans-Oxirane-2,3-dicarboxylic acid (200 mg, 1.52 mmol, TCI chemicals) was dissolved in 5 ml of NMP, followed by HCl.H-Tyr(OtBu)-OtBu (500 mg, 1.52 mmol, Iris Biotech GmbH) and DIEA (1.340 ml, 8 mmol). PyAOP (792 mg, 1.52 mmol) was then added and the reaction mixture (RM) was stirred at room temperature (RT) for 80 min. The RM was then poured into 180 ml of cold water. The resulting precipitate was removed by filtration. The pH of the filtrate was then adjusted to 3 with 1 M KHSO4 solution and extracted three times with EtOAc. The organic phase was collected again, washed with brine, dried over Na2SO4, and concentrated to dryness to give 433 mg of compound 5 as a clear oil. Estimated mass: 618mg Mass obtained: 433 mg Yield: 70% t R =3.4, MS(ESI+):m / z=408.4[M+H] +

[0192] Synthesis of LV-80 [ka]

[0193] Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM followed by 15 ml of TFA / TiS / H2O. The RM was then stirred at RT for 30 min. The RM was concentrated to dryness and the residual oil was precipitated with cold EtOEt. The precipitate was filtered and dried under reduced pressure. The precipitate was then dissolved in 1 ml of dry DMF and Pd 0 The allyl ester was removed by treatment with tetrakis (5.15 mg, 0.0045 mmol) and PhSiH3 (37 μl, 0.3 mmol). After 20 min, the RM was dissolved in 20 ml of EtOAc, washed once with 1 M HCl solution, once with brine, and concentrated to dryness. The resulting precipitate was then purified by preparative HPLC to give 41 mg of LV-80 as a white solid. Expected mass: 91mg / Obtained mass: 41mg / Crude yield: 45% t R =3.10, MS(ESI+):m / z=632.4[M+H] +

[0194] Synthesis of LV-86 [ka]

[0195] Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM, followed by 15 ml of TFA / TiS / H2O. The RM was then stirred at RT for 30 min. The RM was concentrated to dryness and the residual oil was precipitated with cold Et2O. The precipitate was filtered and dried under reduced pressure to give Intermediate B.

[0196] Intermediate B (15 mg, 0.022 mmol) was dissolved in NMP, followed by the addition of cesium carbonate (7.15 mg, 0.022 mmol) and iodoethane (2.34 μl, 0.029 mmol). The RM was then stirred at RT for 1 h. The RM was then poured into 30 ml of water. The aqueous phase was extracted 3 times with EtOAc. The recombined organic phase was washed 3 times with saturated NaHCO3, 3 times with KHSO4 1M, then with brine, dried over Na2SO4, and evaporated to dryness to give 17 mg of a white solid. The white solid was then dissolved in 1 ml of dry DMF and Pd 0 The allyl ester was removed by treatment with tetrakis (1 mg, 0.00087 mmol), PhSiH3 (3 μl, 0.024 mmol). After 20 min, the RM was dissolved in 20 ml of EtOAc, washed once with 1 M KHSO4 solution, once with brine, and concentrated to dryness. The resulting precipitate was then purified by preparative HPLC to give 8.1 mg of LV-86 as a white solid. Expected mass: 15.8mg / Obtained mass: 8.1mg / Crude yield: 51% t R = 3.49, MS (ESI +): m / z = 660 [M + H] + 11H NMR (500 MHz, DMSO) δ 8.70 (s, 1H), 8.60 (s, 1H), 8.18 (t, J = 5.5, 1H), 7.44 (d, J = 7.5, 2H), 7.38 (t, J = 7.4, 2H), 7.32 (t, J = 7.3, 1H), 7.27 (t, J = 7.5, 2H), 7.21 - 7.16 (m, 2H), 7.14 (d, J = 8.3, 2H), 6.93 (d, J = 8.2, 2H), 5.05 (s, 2H), 4.38 (d, J = 4.6, 1H), 4.23 (dd, J = 13.6, 7.6, 1H), 4.04 (q, J = 7.1, 2H), 3.57 (d, J = 5.1, 2H), 3.22 (td, J = 13.0, 6.9, 2H), 3.02 (dd, J = 13.8, 4.4, 1H), 2.84 (dd, J = 13.8, 9.4, 1H), 2.69 (t, J = 7.2, 2H), 2.24 (t, J = 7.2, 2H), 1.57 (m, J = 6.7, 1H), 1.50 (m, 1H), 1.45 (m, J = 16.4, 8.0, 3H), 1.17 (t, J = 7.1, 3H) 13 13C NMR (126 MHz, DMSO) δ 172.63, 172.50, 170.67, 165.65, 165.49, 157.11, 139.33, 137.20, 130.23, 129.57, 128.73, 128.46, 128.32, 127.85, 127.79, 126.15, 114.52, 69.15, 59.80, 53.95, 52.58, 52.46, 52.35, 50.63, 35.77, 35.04, 33.09, 31.52, 20.80, 14.17

[0197] Synthesis of LV-87

Chem.

[0198] Intermediate B (10 mg, 0.015 mmol) was dissolved in anhydrous THF. N-Methylmorpholine (3,82 μL, 0.030 mmol) was added to the solution. The reaction mixture was then cooled to −15° C. and stirred for 5 min. Isobutyl chloroformate (1,94 μL, 0.015 mmol) was added and the mixture was stirred at −15° C. for 10 min. O-Tritylhydroxylamine (8.25 mg, 0.030 mmol) was added and stirring was continued at −15° C. for 15 min and at room temperature for 30 min. The mixture was then poured into EtOAc (20 mL), washed with 1M KSHO4, a saturated solution of NaHCO3, dried over MgSO4, and concentrated to dryness to give 14 mg of intermediate C as a white powder. Expected mass: 14mg / Obtained mass: 18mg / Crude yield: quantitative

[0199] Intermediate C (13.92 mg, 0.015 mmol) was dissolved in 10 ml of DCM, followed by 10 ml of TFA / TiS / HO. The RM was then stirred at RT for 30 min. The RM was concentrated to dryness and the residual oil was precipitated with cold EtOEt. The precipitate was filtered and dried under reduced pressure. The precipitate was then dissolved in 0.5 ml of dry DMF and Pd 0 The allyl ester was removed by treatment with tetrakis (1 mg, 0.00087 mmol), PhSiH3 (2.2 μl, 0.0175 mmol). After 20 min, the RM was dissolved in 20 ml of EtOAc, washed once with 1 M HCl solution, once with brine, and concentrated to dryness. The resulting precipitate was then purified by preparative HPLC to give 2 mg of LV-87 as a white solid. Expected mass: 11 mg / Obtained mass: 2 mg / Crude yield: 17% t R =2.93, MS(ESI+):m / z=647.3[M+H] + 11H NMR (500 MHz, DMSO) δ 8.80 (d, J = 8.0, 1H), 8.70 (s, 1H), 8.61 (d, J = 8.1, 1H), 8.17 (t, J = 5.6, 1H), 7.44 (d, J = 7.2, 2), 7.38 (d, J = 14.8, 2H), 7.32 (t, J = 7.3, 1H), 7.29 - 7.26 (m, 2H), 7.20 - 7.17 (m, 3H), 7.15 (d, J = 8.6, 2H), 6.94 (d, J = 8.7, 2H), 5.06 (s, 2H), 4.44 - 4.38 (m, 1H), 4.26 - 4.19 (m, 1H), 3.58 (s, 2H), 3.26 - 3.20 (m, 2H), 3.02 (dd, J = 13.8, 4.6, 1H), 2.84 (dd, J = 13.9, 9.4, 1H), 2.72 - 2.67 (m, 2H), 1.90 (dd, J = 13.0, 6.6, 2H), 1.60 - 1.53 (m, 1H), 1.49 - 1.43 (m, 2H), 1.42 - 1.37 (m, 1H) 13 13C NMR (500 MHz, DMSO) δ 172.50, 168.75, 165.75, 165.45, 157.14, 139.32, 137.18, 130.20, 128.71, 128.45, 128.35, 127.84, 127.78, 126.15, 114.43, 69.14, 53.82, 52.53, 52.37, 48.50, 35.07, 31.98, 31.79, 30.14, 21.63

[0200] Synthesis of LV-81

Chem.

[0201] The H-Aad(tBu)Phe-sequence was synthesized via SPPS on 0.5 mmol / g cystamine-Trt resin (CA). The solid-supported sequence was then coupled to the compound HO-TES-Tyr(OBn)-OAll (95 mg, 0.225 mmol) via an amide bond via activation with PyAOP (95 mg, 0.225 mmol) and DIEA (77 μl, 0.450 mmol) at RT overnight. After 2 h, Pd tetrakis (8.66 mg, 0.008 mmol) and PhSiH3 were added, leading to deprotection of the OAll ester. The LV-80 thiolated version was cleaved from the resin by a 2-h extended treatment with TFA / DCM / H2O / TiS (50 / 50 / 2.5 / 2.5). The filtrate was then concentrated to dryness to give a brown oil, which was precipitated with cold Et2O to give a white solid, which was isolated by filtration. The compound was then purified by preparative HPLC to give 32 mg of LV-81 as a white solid. Expected mass: 55mg / Obtained mass: 32mg / Crude yield: 58%

[0202] Synthesis of intermediate D [ka]

[0203] HCl.H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-oxirane-2,3-dicarboxylic acid (80.8 mg, 0.611 mmol) were dissolved in 1.5 mL of NMP, followed by the addition of DIEA (1.628 mmol, 374 μL). The mixture was stirred and HATU (309.5 mg, 0.814 mmol) was added. The mixture was stirred at room temperature for 20 min. The reaction mixture was poured into a Na2CO3 1M solution and extracted three times with 30 mL of diethyl ether. The aqueous phase was acidified with KHSO4 1M and the resulting solution was extracted three times with 30 mL of ethyl acetate. The organic layers were combined, washed with brine, then dried over MgSO4, filtered and concentrated under reduced pressure to give intermediate D as a yellow oil. The product was used as is without further purification. Expected mass: 131 mg / Obtained mass: 104 mg / Yield: 79.3% HPLC purity=98%, t R =1.2 min, MS(ESI+): m / z=324.3[M+H]+, (expected m / z=324.31)

[0204] Synthesis of LV-111 [ka]

[0205] Cs2CO3 (314 mg, 0.966 mmol) was added to intermediate D (104 mg, 0.321 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (77.6 μL, 0,966 mmol) was added to the solution and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured into 30 mL of water and extracted three times with 30 mL of ethyl acetate, then washed three times with a solution of NaHCO3 1 M and three times with a solution of KHSO4 1 M. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give the desired product as white crystals. The product was then purified by preparative chromatography to give 51.5 mg of LV-111 as a white solid. Expected mass: 121.66 mg / Obtained mass: 51.5 mg / Yield: 42% HPLC purity=98%, t R =2.1 min, MS(ESI+):m / z=380.2[M+H] + , (expected m / z=380.16) 1 H NMR(500 MHz, DMSO)δ 8.78(d, J = 7.9, 1H), 7.07(d, J = 8.3, 2H), 6.78(d, J = 8.2, 2H), 4.42(dd, J = 14.2, 8.4, 1H), 4.17 - 3.89(m, 6H), 3.61(s, 1H), 3.40(s, 1H), 2.93(dd, J = 13.8, 5.7, 1H), 2.83(dd, J = 13.7, 9.2, 1H), 1.29 - 1.05(m, 9H) 13CNMR(126 MHz, DMSO)δ 171.40,167.43, 165.49, 157.81, 130.66, 128.90, 114.60, 63.32, 62.06, 61.24, 54.20, 53.06, 51.69, 36.17, 15.14, 14.34 HRMS(microTOF-Q):C 19 H 25 NO7[M+H] + Theoretical value: 380.1631, measured value: 380.1704

[0206] Synthesis of LV-101 [ka]

[0207] HCl.H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-oxirane-2,3-dicarboxylic acid (27 mg, 0.204 mmol) were dissolved in 1.5 mL of NMP followed by DIEA (1.628 mmol, 374 μL). The mixture was stirred for 5 min and HATU (154.7 mg, 0.407 mmol) was added. The mixture was stirred at room temperature for 20 min. The solution was extracted three times with ethyl acetate. The organic layer was washed three times with KHSO4 1M and NaHCO3 1M, then once with brine, dried over MgSO4, filtered and concentrated under reduced pressure to give the desired product as a white powder. HPLC purity=98%, t R =1.42 min, MS(ESI+):m / z=515.20[M+H] + , (expected m / z=515.20)

[0208] Synthesis of LV-104 [ka]

[0209] Cs2CO3 (126.8 mg, 0.389 mmol) was added to LV-101 (100 mg, 0.195 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (47.0 μL, 0.585 mmol) was added to the solution and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured into 30 mL of water and extracted three times with 30 mL of ethyl acetate, then washed three times with a solution of NaHCO3 1 M and three times with a solution of KHSO4 1 M. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give the desired product as a white powder. The product was then purified by preparative chromatography to give 75.6 mg of LV-104 as a white powder. Expected mass: 111.15 mg / Obtained mass: 75.6 mg / Yield: 68% HPLC purity=99%, t R =3.81 min, MS(ESI+):m / z=571.3[M+H] + , (expected m / z=571.26) 1 H NMR(500 MHz, DMSO)δ 8.87(d, J = 7.7, 2H), 7.07(d, J = 8.2, 4H), 6.78(d, J = 8.1, 4H), 4.38(dd, J = 14.2, 8.3, 2H), 4.02(q, J = 7.1, 4H), 3.93(q, J = 6.9, 4H), 3.45(s, 2H), 2.92(dd, J = 13.8, 5.7, 2H), 2.82(dd, J = 13.8, 9.2, 2H), 1.25(t, J = 6.9, 6H), 1.08(t, J = 7.1, 6H) 13 CNMR(126 MHz, DMSO)δ 170.97, 165.50, 157.29, 130.25, 128.36, 114.14, 62.70, 60.62, 53.79, 52.17, 35.51, 14.62, 13.91 HRMS(microTOF-Q):C 30 H 38 N2O9[M+H]+ theoretical value: 571.6390, measured value: 571.2650

[0210] Synthesis of intermediate E Carbonyldiimidazole CDI (113.6 mg; 1.2 equiv.) was dissolved in minimal DMF and the RM was placed at -10°C. Boc-NH-NH2 (92.5 mg; 1.2 equiv.) was also dissolved in minimal DMF and added to the RM, then stirred at -10°C for 30 min. After this, TFA.HTyr(OEt)OEt (203.6 mg; 1 equiv.) was also dissolved in minimal DMF and added to the RM, followed by Et3N (168 μL). The RM was then stirred at room temperature for 2 h 50 min. In the work-up step, distilled water and AcOEt were added to the RM and placed in a separatory funnel. The organic phase was then washed with HCl 1M (x2), saturated NaHCO3 (x2) and brine (x2). The resulting solution was then dried over MgSO4, filtered, and concentrated to give an oil (154.3 mg; yield: 56%). This resulting oil was dissolved in a TFA / TIS / H2O (95 / 2.5 / 2.5) solution and stirred at room temperature for 30 min, then concentrated to dryness to give intermediate E as a crude oil. The compound was used without further purification.

[0211] Synthesis of LV-119 [ka]

[0212] CDI (63.3 mg; 1.2 equiv.) was dissolved in minimal DMF and the RM was placed at -10°C. Intermediate E (159.7 mg; 1.2 equiv.) was also dissolved in minimal DMF and added to the RM, then stirred at -10°C for 30 min. The pH was adjusted to 8 with a few drops of Et3N. After this, TFA.HTyr(OEt)OEt (115.9 mg; 1 equiv.) was also dissolved in minimal DMF and added to the RM, followed by Et3N (102.2 μL; 2.2 equiv.). The RM was then stirred at room temperature for 3 h. In the work-up step, distilled water and AcOEt were added to the RM and placed in a separatory funnel. The organic phase was then washed with HCl 1M (x2), saturated NaHCO3 (x2) and brine (x2). The resulting solution was then dried over MgSO4, filtered, and concentrated to give an oil.

[0213] The crude product was then purified by HPLC and lyophilized to give very bright white crystals (43.9 mg; yield: 24%; purity: >96%). 1 H NMR(500MHz, CDCl3)δ 6.99(d,J = 8.5, 4H), 6.75(d,J = 8.6, 4H), 6.50(s, 2H), 6.05(d,J = 8.1, 2H), 4.60(dd,J = 14.3, 6.6, 2H), 4.16 - 4.04(m, 4H), 3.92(q,J = 7.0, 4H), 3.03 - 2.89(m, 4H), 1.38 - 1.28(m, 6H), 1.18(t,J = 7.1, 6H)

[0214] Synthesis of intermediate F [ka]

[0215] Boc-5-Ava-OH (1 g, 4.6 mmol) was dissolved in anhydrous DMF (30 ml) and EtOH (1.3 ml, 23 mmol) was added. DMAP (56.4 mg, 0.46 mmol), NMM (605 μl, 5.5 mmol), OxymaPure (653.7 mg, 4.6 mmol) and EDCi (1.1 g, 5.98 mmol) were then added in that order. The RM was stirred at RT for 2 h 05 min. Three AcOEt extractions were performed. The organic layer was then washed three times each with NAHCO3 (sat), KHSO4 (1 M) and brine. The organic layer was then dried over MgSO4, filtered and concentrated to dryness. Expected mass: 1.13 g; Obtained mass: 966.3 mg; Yield: 86%

[0216] Synthesis of intermediate G [ka]

[0217] Intermediate F (100 mg, 0.41 mmol) was dissolved in DCM (700 μl) followed by TFA / TIS / H2O 95 / 2.5 / 2.5 (v / v / v) (663 μl). The RM was then stirred at RT for 1 h and concentrated to dryness. Expected mass: 106.2 mg; Obtained mass: 286.02 mg; Crude yield: 269%

[0218] Synthesis of IBMT23 [ka]

[0219] Intermediate G (106.2 mg, 0.41 mmol) and IBMT7 (49.2 mg, 0.14 mmol) were dissolved in anhydrous DMF (911 μl). DMAP (1.2 mg, 0.01 mmol), NMM (127.6 μl, 1.16 mmol), OxymaPure (19.9 mg, 0.14 mmol) and EDCi (34.5 mg, 0.18 mmol) were then added in that order. The RM was stirred overnight at RT. Three AcOEt extractions were performed. The organic layer was then washed three times each with NAHCO3 (sat), KHSO4 (1M), and brine. The organic layer was then dried over MgSO4, filtered, and concentrated to dryness to give 47.03 mg of the desired product. The crude product was purified by reverse phase preparative HPLC. Expected mass: 66.9 mg; Obtained mass: 33.4 mg; Yield: 50%; Purity: 100% (MS(ESI+): m / z=479.0 [M+H] + ; time = 3.12 min) (expected m / z = 479.55)

[0220] Synthesis of intermediate H [ka]

[0221] Boc-Phe(4-NH2)-OH (100 mg, 0.36 mmol) was dissolved in NMP (691 μl) and DBU (54 μl, 0.36 mmol) was added. Iodoethane (29 μl, 0.36 mmol) was preheated to 0° C. and then the reaction mixture was added dropwise. After stirring at RT for 30 min, H2O and AcOEt were added and then poured into a separatory funnel. Three AcOEt extractions were performed and then the organic layer was washed three times each with KHSO4 (1M) and brine. The organic layer was then dried over MgSO4, filtered and concentrated to dryness. Expected mass: 110.9 mg; Obtained mass: 67.5 mg; Crude yield: 61%

[0222] Synthesis of intermediate I [ka]

[0223] Intermediate H (47.9 mg, 0.16 mmol) was dissolved in anhydrous DMF (139 μl) and pyridine (78 μl, 0.96 mmol) was added. Ms-Cl (37 μl, 0.48 mmol) was then added and the RM was stirred at RT for 1 h. H2O and AcOEt were added and then poured into a separatory funnel. Three AcOEt extractions were performed. The organic layer was then washed three times each with NaHCO3 (sat), KHSO4 (1M), and brine. The organic layer was then dried over MgSO4, filtered, and concentrated to dryness. Expected mass: 61.8 mg; Obtained mass: 19.8 mg; Crude yield: 32%

[0224] Synthesis of intermediate J [ka]

[0225] Intermediate I (19.8 mg, 0.05 mmol) was dissolved in DCM (200 μl) followed by TFA / TIS / H2O 95 / 2.5 / 2.5 (v / v / v) (122 μl). The RM was then stirred at RT for 1 h and concentrated to dryness. Expected mass: 20 mg; Obtained mass: 24.4 mg; Crude yield: 122%

[0226] Synthesis of IBMT35 [ka]

[0227] Intermediate J (20 mg, 0.05 mmol) was dissolved in NMP (240 μl) followed by (2S,3S)-trans-oxirane-2,3-dicarboxylic acid (6.6 mg, 0.05 mmol) and DIEA (61.2 μl, 0.35 mmol). The RM was stirred and HATU (19.1 mg, 0.05 mmol) was added. The RM was stirred at room temperature for 3 h. The RM was then extracted 3 times with EtOAc and the organic phase was washed 3 times with HCl (1M), once with brine, dried over MgSO4, filtered and evaporated to dryness to give 18 mg of a mixture of 1 and 2. The crude product was purified by reverse phase preparative HPLC. (IBMT35) Expected mass: 16.7 mg; Obtained mass: 3.2 mg; Yield: 19%; Purity: 98% (MS(ESI+): m / z=669.0 [M+H] + ; time = 2.64 min) (expected m / z = 668.7)

[0228] Synthesis of intermediate K [ka]

[0229] Boc-Phe(4-NH2)-OH (50 mg, 0.18 mmol) was suspended in a solution of NaOH (1M) and the RM was placed at 0 °C. Ts-Cl was then added and the RM was stirred at 0 °C for 2 h. Acidification was performed with HCl (1M) to pH 2-3 and the RM was extracted three times with AcOEt. The organic layer was then washed three times with KHSO4 (1M), twice with brine, dried over MgSO4, filtered and concentrated to dryness. Expected mass: 78.1 mg; Obtained mass: 76.9 mg; Yield: 98%

[0230] Synthesis of intermediate L [ka]

[0231] Intermediate K (78.1 mg, 0.18 mmol) was dissolved in anhydrous DMF (1.2 ml) and EtOH (62.6 μl, 1.08 mmol) was added. DMAP (2.5 mg, 0.02 mmol), NMM (24.2 μl, 0.22 mmol), OxymaPure (25.6 mg, 0.18 mmol) and EDCi (44 mg, 0.23 mmol) were then added in that order. The RM was stirred at RT for 40 min. Three AcOEt extractions were performed. The organic layer was then washed three times each with NAHCO3 (sat), KHSO4 (1 M) and brine. The organic layer was then dried over MgSO4, filtered and concentrated to dryness. Expected mass: 83.3 mg; Obtained mass: 69.4 mg; Yield: 83%

[0232] Synthesis of intermediate M [ka]

[0233] Intermediate L (69.4 mg, 0.15 mmol) was dissolved in DCM (400 μl) followed by TFA / TIS / HO 95 / 2.5 / 2.5 (v / v / v) (365 μl). The RM was then stirred at RT for 1 h, concentrated to dryness and precipitated in diethyl ether. Expected mass: 71.5 mg; Obtained mass: 69.8 mg; Crude yield: 98%

[0234] Synthesis of IBMT38 [ka]

[0235] Intermediate M (69.8 mg, 0.15 mmol) was dissolved in anhydrous DMF (385 μl) followed by (2S,3S)-trans-oxirane-2,3-dicarboxylic acid (13.2 mg, 0.1 mmol) and DIEA (63.8 μl, 0.38 mmol). The RM was stirred and HATU (57 mg, 0.15 mmol) was added. The RM was stirred at room temperature for 2 h 20 min. The RM was then directly purified by reverse phase preparative HPLC. (IBMT38) Expected mass: 61.5 mg; Obtained mass: 8.3 mg; Yield: 13%; Purity: 91% (MS(ESI+): m / z=821.0[M+H] + ; time = 3.64 min) (expected m / z = 820.9)

[0236] 1.2.2) Synthesis of the conjugates of formula (I') according to the invention Synthesis of LV-82 [ka]

[0237] Compound 81 (7.35 mg, 0.01 mmol) was dissolved in 800 μl of DMF, followed by the addition of biotin-maleimide (4.5 mg, 0.01 mmol) and 400 μl of PBS solution. To dissolve both reagents, the mixture was heated briefly and then stirred at RT for 1 h. The RM was then directly purified by preparative HPLC to give 7 mg of LV-82 as a white solid. Expected mass: 11.85 mg / Obtained mass: 7 mg / Yield: 59% t R =2.89, MS(ESI+):m / z=1186.2[M+H] + , 593.7[M+2H] 2+

[0238] 1.2.3) Synthesis of Comparative Example LV-43 [ka]

[0239] Fmoc-Aad(OtBu)-OH (88 mg, 0.216 mmol) was coupled via standard SPPS HATU (82 mg, 0.216 mmol) DIEA (50 μl, 0.288 mmol) activation. The RM was mixed with 200 mg of rink amide (RA) resin at 0.37 mmol / g in a solid phase synthesis reactor for 2 h. After standard washing, Fmoc protection was removed by two 10 min DMF / Pip 80 / 20 (vol / vol) treatments. In the next step, compound 5 (88 mg, 0.216 mmol) was coupled to the free amine via PyAOP (113 mg, 0.216 mmol), DIEA (545 μl, 0.63 mmol) activation. After overnight reaction, the resin was washed 3 times with DMF and 3 times with DCM, and the inhibitor was cleaved with 20 ml TFA treatment for 50 min. The product containing TFA filtrate was then evaporated to dryness, and the peptide was precipitated with EtOEt, filtered, purified by preparative HPLC, and lyophilized to give 10.7 mg of LV-43 as a white solid. Expected mass: 31.4mg Mass obtained: 10.7 mg Yield: 34% HPLC purity=100%, tR=0.84, MS(ESI+): m / z=438,20[M+H] + (Expected m / z=438.15)

[0240] 2) Biological results 2.1) Experimental model cell culture Human CHL-1 cells (melanoma-derived cell line, Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456) were routinely cultured in DMEM containing 10% (vol / vol) fetal bovine serum (FBS), 50 U / ml penicillin and 50 μg / ml streptomycin (pen / strep) and incubated at 37°C. Prior to testing, CHL-1 cells were seeded at 30% confluency in appropriate plates (96, 48, 24, 12 or 6 wells). The next day, cells were processed and collected for further analysis (immunoblotting, qPCR, immunofluorescence).

[0241] Cortical cell cultures were grown on glass coverslips or plastic plates coated with 0.1 mg / ml poly-L-lysine (Sigma) at ∼10 5 cells / cm 2 , or 2 × 10 for low-density cultures. 4 cells / cm 2 Neurons were plated in DMEM containing 10% (vol / vol) fetal bovine serum (FBS), 50 U / ml penicillin and 50 μg / ml streptomycin (pen / strep), and 1-2 hours later the medium was replaced with Neurobasal medium supplemented with 2% B27, pen / strep, 0.6% glucose and 1% Glutamax (all reagents from Life Technologies).

[0242] Drosophila Melanogaster Drosophila were reared at 25°C. yw flies were used as controls (yw is an αTub84BΔ3 mutant). All males were disrupted in laemli loading buffer, boiled, sonicated, and mounted for immunoblotting.

[0243] 2.2) Method details Protein expression and purification The method disclosed in WO 2020 / 012002 was used. Human VASH1 (hVASH1) was cloned into an expression vector with a polyhistidine tag. Bacteria were transformed and induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight or for 4 h. Bacteria were collected and disrupted using an HTU-DIGI-F press (Heinemann). Recombinant protein was purified using nickel-based affinity chromatography (IMAC) according to the manufacturer's protocol (GE Healthcare).

[0244] In vitro detyrosination assay Spodoptera frugiperda Sf9 cells were cultured, lysed, and used for tubulin purification by affinity chromatography. Microtubules were obtained using Taxol and stored until use. In vitro analysis of detyrosination activity was performed using recombinant hVASH1. Detyrosination assays were performed in the presence of 0.5 μM microtubules. The reaction was stopped by the addition of denaturing loading buffer followed by incubation at 95°C for 5 min, and samples were loaded onto SDS PAGE for immunoblot analysis.

[0245] Standardized in vitro detyrosination ELISA-based assay A standardized primary ELISA-based in vitro detyrosination assay in a 96-well format has been developed according to the method disclosed in WO 2020 / 012002. Briefly, a substrate for the VASH enzyme is made, 100 μL of the enzyme mixture (enzyme and inhibitor) is added to each well and the plate is incubated for 5 min at 37 °C. The primary (rabbit anti-detyrosinated tubulin) is incubated for 1 h and, after washing, the secondary (anti-rabbit) antibody is added to each well. The plate is incubated for 1 h at room temperature. The development consists of adding TMB (3,3',5,5'-tetramethylbenzidine) to each well and incubating for 30 min at room temperature. The color development is stopped by the addition of 0.5 M sulfuric acid. The OD is measured at 450 nm.

[0246] Deglutamylation assay and tubulin purification Deglutamylation assays were performed as previously described (Rogowski et al., Cell, 2010, 143(4):564-78).

[0247] Transfection of human cells Plasmid transfection was performed, and cells were harvested 24 h after transfection for immunoblotting and immunofluorescence analysis.

[0248] quantitative PCR Total RNA was isolated with TRIzol (Invitrogen). Reverse transcription was performed using random hexanucleotides. Quantitative PCR assays were performed using Lightcycler SYBR Green Master mix on a Lightcycler instrument (Roche). All primers used were intron-spanning. Relative amounts of target cDNA were obtained by normalization with the geometric mean of multiple internal control genes.

[0249] Immunofluorescence labeling Methanol-fixed CHL-1 cells and mouse cortical neurons were analyzed using a Zeiss Axioimager Apotome microscope following standard immunofluorescence experiments. Briefly, samples were incubated overnight with primary antibodies, washed three times with PBS, and then incubated for 1 h with the following secondary antibodies: Alexa Fluor488-conjugated goat anti-rat, goat anti-mouse, and Alexa Fluor555-conjugated goat anti-rabbit (Invitrogen). After incubation, samples were washed three times with PBS, stained with DAPI, and mounted.

[0250] Microscopy Microscopic image acquisition was performed at the Montpellier RIO Imaging facility and images were processed using OMERO.

[0251] Bioanalysis of compound penetration and transformation in human cells by LC-MS Stability of peptides in serum Human serum reaction samples contained 250 μl heat-inactivated human serum (Sigma-Aldrich) and 750 μL RPMI medium 1640 (Sigma-Aldrich). Reactions were initiated by adding 50 μL of compound (in mM stock solutions in DMSO). Measurements were performed in a shaking water bath at 37°C. 100 μl samples were taken at known time intervals and added to 200 μl acetonitrile 1‰ TFA to precipitate serum proteins. The cloudy samples were cooled to 4°C for 15 min and then centrifuged at 12,000 rpm for 10 min to pellet the precipitated serum proteins. 150 μl of clear supernatant was taken and finally peptides were analyzed by RPHPLC and LC-MS.

[0252] 2.3) Results 2.3.1 In cellulo VASH-mediated tubulin detyrosination assay We used human CHL-1 cells, in which tubulin detyrosination is exclusively catalyzed by VASH. In these cells, detyrosination is predominantly VASH-dependent (Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456). Cells were pretreated with taxol and then incubated with Epo-Y. Samples were immunoblotted with the indicated antibodies.

[0253] The use of Epo-Y to reduce taxol-induced detyrosination was analyzed using CHL-1 cells. Cells were routinely cultured in a standard humidified tissue culture incubator at 37°C in the presence of 5% CO2 and seeded in 6-well culture dishes. Cells were treated with taxol for 2 hours in the absence or presence of Epo-Y.

[0254] Cells were harvested in RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 1.0% (vol / vol) NP-40, 0.5% (wt / vol) sodium deoxycholate, 1.0 mM EDTA, pH 7.4) and total protein quantification was performed using a BCA kit (Thermo Fisher Scientific). 20 μg protein samples of whole-cell extracts were subjected to 10% SDS-PAGE, transferred to nitrocellulose, and probed with each antibody. Western blot analysis showed a marked decrease in tubulin detyrosination following taxol treatment (2 h) in CHL-1 cells. As shown in Figure 1, incubation of CHL-1 cells with taxol markedly increased tubulin detyrosination and acetylation levels, likely due to MT stabilization. Furthermore, tubulin detyrosination activity was dose-dependently inhibited by Epo-Y, a previously reported VASH inhibitor.

[0255] Therefore, this in cell screening assay using CHL-1 cells and taxol is a suitable screening assay for identifying putative inhibitors of VASH-mediated tubulin detyrosination.

[0256] 2.3.2 The putative inhibitor parthenolide does not inhibit VASH-mediated tubulin detyrosination To investigate the inhibitory effect of parthenolide (PTL) on VASH-mediated tubulin detyrosination, we used a previously developed in vitro detyrosination assay, disclosed in WO 2020 / 012002. Reactions were incubated with inhibitors, stopped, and analyzed by immunoblotting with the indicated antibodies. As shown in Figure 2, the addition of 10 μM Epo-Y to the reaction mixture containing MTs completely inhibited VASH1-mediated tubulin detyrosination (Figure 2B). Conversely, the addition of much higher PTL concentrations (50 and 100 μM) in the same assay did not result in any detectable inhibition of VASH1-dependent tubulin detyrosination (Figure 2B). To further extend the analysis, we used human CHL-1 cells (Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456), in which tubulin detyrosination is exclusively catalyzed by VASH. In these cells, detyrosination is mainly VASH-dependent (Nieuwenhuis J. et al. Science, 2017, 358(6369):1453-1456). Cells were pretreated with taxol and then incubated with Epo-Y or PTL. Samples were immunoblotted with the indicated antibodies. Incubation of CHL-1 cells with taxol markedly increased tubulin detyrosination and acetylation levels, most likely due to MT stabilization (Figure 2C). This allowed a direct comparison of Epo-Y and PTL inhibitory potencies in cells incubated with taxol. As expected, Epo-Y dose-dependently reduced tubulin detyrosination without affecting tubulin acetylation. Consistent with the in vitro data (Figure 2B), tubulin detyrosination was not inhibited by PTL even at the highest concentration used (Figure 2C). We conclude that PTL is not an inhibitor of VASH-mediated tubulin detyrosination.

[0257] 2.3.3 Newly developed inhibitors target VASH1 and VASH2 and putidase activity Figure 3 shows examples of the inhibitory efficiency of various newly designed VASH inhibitors against both VASH1- and VASH2-mediated tubulin detyrosination at concentrations ranging from 0.25 to 0.5 µM.

[0258] 2.3.4 Potent cell-permeable inhibitors of VASH peptidase activity Small, highly reactive epoxide-based molecules such as Epo-Y may interact with free thiols present in proteins other than VASH or with nucleophilic lysine residues in the active sites of tyrosine kinases. Thus, through iterative chemical variations and analysis with standardized in vitro assays, we identified a compound, named LV-43 (Figure 4A). Samples were immunoblotted with the indicated antibodies. As shown, LV-43 has a more potent in vitro inhibition of VASH1 enzyme activity than Epo-Y (Figure 4B). However, when tested in a cell-based assay involving taxol-treated CHL-1 cells, LV-43 was much less efficient than Epo-Y (Figure 4C). The discrepancy between in vitro and in cell data suggests less cell penetration of LV-43 compared to Epo-Y. Therefore, we generated a new series of compounds and appended hydrophobic aromatic functional groups for improved cell permeability and enzyme affinity. In in vitro assays, the indicated inhibitors were added to the reaction mixture, and then detyrosination was analyzed by immunoblotting with the indicated antibodies. As shown by its in vitro IC50 (28 nM for LV80 vs. 320 nM for Epo-Y), LV-80 was found to inhibit VASH1-mediated tubulin detyrosination more potently than LV-43 and Epo-Y (Figure 4D-E). Measurement of in cell IC50 using taxol-induced detyrosination assay in CHL-1 cells confirmed the greater potency of LV-80 than Epo-Y even in cells (676 nM for LV-80 vs. 3 µM for Epo-Y) (Figure 4F). Finally, using the available structural data of VASH1, molecular docking was performed to visualize the most likely binding mode of the LV-80 inhibitor, taking into account the formation of hydrogen bonds (Figure 4H). In summary, we have designed and generated specific cell-permeable VASH inhibitors with in vitro IC50s in the low nanomolar range that are suitable for functional analysis of tubulin detyrosination.

[0259] 2.3.5 Complete inhibition of VASH-mediated tubulin detyrosination in cells To test the potency of LV-80 on endogenous detyrosination activity, wild-type CHL-1 cells incubated with LV-80 were compared to double knockout CHL-1 cells (2KOs) lacking both VASH1 and VASH2. Cells were collected and analyzed by immunoblotting with the indicated antibodies. After 24 h of incubation with LV-80, the levels of detyrosination were comparable in treated CHL-1 and CHL-1 2KOs cells, as shown by Western blot analysis (Figure 5). This demonstrated the efficacy of the newly designed compound to completely inhibit in cellulo VASH-mediated detyrosination. Furthermore, tubulin detyrosination was analyzed by immunofluorescence in cells incubated with vehicle LV-80. The absence of signal for detyrosinated tubulin was confirmed in cells treated with the inhibitor (data not shown).

[0260] 2.3.6 Favorable Safety Profile of VASH Enzyme Inhibitors Next, we chose a widely used MTT-based colorimetric assay to assess potential metabolic toxicity and changes in cell viability in the presence of LV-80. The MTT-based assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) was performed 48 h after incubation with the indicated compounds to monitor cell viability.

[0261] Incubation with 0.1 μM taxol and 10 μM PTL for 24 h resulted in a significant decrease in cell viability. Conversely, incubation with a much higher concentration of LV-80 (100 μM) did not significantly affect cell viability, which was similar to that of cells incubated with vehicle (Figure 6A). Analysis of mitochondrial function using widely recognized and well-accepted assays showed no significant changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cells incubated with LV-80, highlighting the excellent safety profile of this compound (Figure 6B).

[0262] 2.3.7 Newly developed VASH inhibitors are specific for VASH enzyme activity We next tested LV-80 inhibitory activity against cytosolic carboxypeptidases (CCPs), another family of proteases that target the tubulin C-terminal tail and act as tubulin deglutamylases. The inhibitor did not affect CCP1 and CCP5 activity. Samples were immunoblotted with the indicated antibodies. When polyglutamylated brain tubulin was incubated in the presence of protein lysates derived from HEK293 cells transfected with GFP-CCP1, we observed that CCP1-dependent deglutamylation activity against long glutamic acid chains was not affected by the addition of LV-80 (Figure 7A). Similar results were observed when we used this assay to evaluate CCP5-mediated deglutamylation of glutamic acid residues (Figure 7B). Overall, these observations demonstrated LV-80 specificity for detyrosinating VASH activity. We then analyzed the specificity of LV-80 by testing its effect on the activity of carboxypeptidase A (CPA), an enzyme widely used for in vitro detyrosination of MTs and tubulin. VASH-specific inhibitors did not affect carboxypeptidase A (CPA)-dependent tubulin detyrosination. On the other hand, specific CPA inhibitors did not affect VASH activity. Indeed, the addition of 10 μM LV-80 resulted in a complete abolition of both VASH1- and VASH2-mediated detyrosination in vitro, but had no detectable effect on CPA activity (Figure 7C). Conversely, the peptidomimetic benzyl succinic acid (BzlSA) completely abolished CPA-dependent detyrosination, but not VASH1- or VASH2-mediated detyrosination.

[0263] 2.3.8 Biotinylated versions of newly developed inhibitors as research tools As detyrosination levels are particularly high in the brain, we tested whether LV-80 fused to biotin could be used to pull down VASH1 from human brain extracts. Cells were pretreated with taxol and then incubated with biotinylated LV-80. Samples were immunoblotted with the indicated antibodies. We first confirmed that biotinylated LV-80 retained its inhibitory properties against VASH1- and VASH2-mediated detyrosination in vitro (Figure 8A) and validated the pull-down methodology using wild-type and 2KOs CHL-1 as controls (Figure 8B). Pull-down experiments were performed using protein lysates from wild-type and double VASH knockout cells. HEK293 cell protein lysates were loaded as controls. Samples were immunoblotted with the indicated antibodies. We then used human brain protein extracts to efficiently pull down VASH1 (Figure 8C), demonstrating target engagement in relevant human tissues. Input, flow-through (FT) and pull-down (Pd) fractions were immunoblotted with antibodies against the indicated proteins.

[0264] 2.3.9 Significant reduction in tubulin detyrosination in primary cortical neurons treated with VASH inhibitors We decided to evaluate the functional role of tubulin detyrosination in the differentiation of primary cortical neurons purified from mouse embryonic cerebral cortical tissue. After dissociation and filtration, cells were differentiated for 7 days in the presence or absence of LV-80. Cells were collected and analyzed by immunoblotting with the indicated antibodies. Samples were analyzed by immunoblotting and relative optical density was measured in three independent assays (n=3, error bars represent SEM). p values ​​were calculated by multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001). Analysis of tubulin post-translational modification levels using specific antibodies (Figure 9A-B) showed that incubation of primary neurons with LV-80 significantly decreased tubulin detyrosination levels, accompanied by an increase in tyrosination (Figure 9B). In the presence of LV-80, tubulin detyrosination was reduced by 70% and tyrosinated tubulin was increased by 50% compared to the control (DMSO), with the minor differences reflecting the individual affinities of the antibodies. The reduced levels of tubulin detyrosination are not due to changes in VASH1 / 2 or SVBP expression as assessed by qRT-PCR (Figure 9C), but are a result of direct inhibition of the enzymatic activity of VASH. The differentiation status of neurons incubated with LV-80 or vehicle was then analyzed by qPCR quantification of a panel of neuronal differentiation and glial markers. The mRNA levels of Dcx and Neurod1, markers of early neuronal differentiation, and Syp, Map2 and Dlg4, markers of later differentiation stages, were comparable between conditions (Figure 9D). Similarly, the expression levels of Sox2 and Gfap (glial markers) were comparable in both conditions (Figure 9E).

[0265] 2.3.10 Detyrosination makes tubulin susceptible to glutamylation Samples were analyzed by immunoblotting and relative optical density was measured in three independent assays (n=3, error bars represent SEM). p values ​​were calculated using multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001).

[0266] Remarkably, acetylation levels remained unaffected in treated neurons (Figure 10B). On the other hand, polyglutamylation was significantly reduced as shown with two different polyglutamylation-specific antibodies: PolyE, which recognizes long glutamic acid chains, and GT335, which is specific for branch-point glutamic acid residues (Figure 10A). This observation suggested that a crosstalk exists between tubulin detyrosination and polyglutamylation. To test this hypothesis, we co-overexpressed wild-type or enzymatically inactive VASH2 (VASH2D) with TTLL4 or TTLL6 glutamylase in HEK293 cells. HEK293 cells were co-transfected with wild-type VASH2 or dead variants (VASH2D) and TTLL4 or TTLL6 glutamylase. Cells were collected and analyzed by immunoblotting with antibodies against the indicated modifications. Increasing tubulin detyrosination levels stimulated TTLL6, but not TTLL4, polyglutamylation activity (Figure 10C). This indicated that TTLL6 activity is sensitive to the presence of a C-terminal tyrosine residue in a-tubulin. This result indicates that detyrosination renders tubulin permissive to several polyglutamilases, revealing a crosstalk between these pathways. We next investigated whether crosstalk exists between tubulin detyrosination and polyglutamylation in Drosophila. Both modifications are particularly prominent in males, where they accumulate on sperm axons during spermatogenesis. Drosophila lacks a VASH homolog, and detyrosination is catalyzed by an unknown enzyme. To mimic detyrosination, we used flies genetically engineered to express a truncated form of the major α-tubulin isotype aTub84B (aTub84BΔ3 mutant 13 ), which lacks the last three amino acid residues, including tyrosine. Samples were collected and analyzed by immunoblotting with antibodies against the indicated modifications. aTub84BΔ3 males show a marked increase in polyglutamylation levels compared to wild-type males (Figure 10D). Thus, C-terminal amino acid residues affect polyglutamylation levels in vivo. These results were consistent with the existence of a conserved crosstalk between detyrosination and polyglutamylation.

[0267] 2.3.11 Tubulin detyrosination affects tau protein binding in primary cortical neurons To investigate the role of tubulin detyrosination in regulating the interaction of MTs with MT-associated proteins (MAPs), we first completed the differentiation of cortical neurons in the absence of VASH inhibitors for 5 days, followed by 3 days of treatment. This approach allows us to monitor the cellular distribution of tau protein independent of cell differentiation. Cells were collected and analyzed by immunoblotting with the indicated antibodies. Incubation of cortical neurons with LV-80 for 3 days efficiently reduced tubulin detyrosination levels and significantly increased tyrosinated tubulin levels (Figure 11A-B). Samples were analyzed by immunoblotting and relative optical density was measured in three independent assays (n=3, error bars represent SEM). p-values ​​were calculated by multiple t-test (n=3, *<0.05, **<0.01, ***<0.001). In cells incubated with the inhibitors, detyrosination-polyglutamylation crosstalk was confirmed, as indicated by a significant decrease in PolyE and GT355 signals (Figure 11C). Overall, tau levels were not significantly different between conditions, as confirmed by immunoblot (Figure 11A) and qRT-PCR quantification of tau mRNA (encoded by the Mapt gene) (not shown). We then stained vehicle- and LV80-treated neurons for tau and tubulin for quantitative analysis. We chose areas with specific tubulin staining and measured the immunofluorescence signal of tau protein precisely in the same areas of vehicle- and inhibitor-treated primary cortical neurons. While tubulin staining and quantification were not significantly different between conditions, the cellular distribution of tau was significantly altered in projections (Figure 11D-F), and was especially decreased in cells incubated with our VASH inhibitors (data not shown). This suggests a role for tubulin detyrosination in regulating the interaction between MTs and tau protein. Collectively, we show that inhibition of tubulin detyrosination in primary cortical neurons directly affects tau localization.

[0268] 2.3.12. Newly designed, low-nM in-cellulo VASH inhibitors As shown in FIG. 12, we have designed a set of efficient, cell-permeable, and highly potent prodrugs (including examples such as LV-104 and LV-111) with low nM IC50 in cellulo.

[0269] Compounds were added prior to Taxol treatment. VASH-mediated detyrosination was abolished in CHL-1 cells at low nM range. Quantification showed that LV-104 and LV-111 (IC50 of 8 nM and 20 nM, respectively) were much more efficient on cells than LV-80 (IC50 of 500 nM).

[0270] Two examples of prodrug compounds (LV-104 and LV-111) and direct VASH inhibitors. Both prodrugs had no effect on VASH1 and VASH2 in the context of in vitro assays.

[0271] Considering these results, we propose that targeting VASH peptidase activity represents a safe and well-tolerated mechanism for the treatment of neurodegenerative diseases such as Alzheimer's disease, but also cancer, muscular dystrophies and ciliopathy.Interestingly, these compounds have a strong effect on tubulin glutamylation and could also be used to reduce TTL-dependent modifications of tubulin, for example, in infantile-onset neurodegeneration or glaucoma.

[0272] We showed that parthenolide, a widely used inhibitor of detyrosination, does not inhibit VASH activity either in vitro or in cell. This is consistent with a recently published study (Hotta et al. Curr Biol. 2021, Volume 31, Issue 4, Pages 900-907) that showed that the effect of parthenolide on detyrosination is indirect and likely due to changes in microtubule dynamics caused by its covalent binding to tubulin. More importantly, we reported on the medicinal chemistry-based optimization of Epo-Y compounds, leading to the development of novel VASH inhibitors that are highly specific and cell-permeable. We showed that the newly developed compounds (active ingredients) have the ability to completely inhibit VASH activity and, in contrast to parthenolide, do not exhibit detectable toxicity. Furthermore, by applying VASH inhibitors to primary neuronal cell cultures, we discovered a previously unknown crosstalk between tubulin detyrosination and another important tubulin modification called polyglutamylation. The newly revealed link between these two modifications has far-reaching effects on the establishment of neuronal polarity and the localization of the main neuronal microtubule-associated protein called tau. Overall, these compounds have great potential for use in drug development and research purposes.

[0273] 2.3.13 IBMT11 in vitro and in cellulo detyrosination assays The in vitro inhibitory activity of IBMT11 was tested by adding the compounds to the reaction medium described above (see part 2.2) and then detyrosination was analyzed by immunoblotting with the indicated antibodies.

[0274] While LV-104 did not inhibit VASH in vitro, we found that IBMT-11 inhibited VASH1-mediated tubulin detyrosination more potently than Epo-Y, as indicated by the in vitro IC50 (IBMT-11, 0.6 uM vs. Epo-Y, 10 uM) (Figure 13A). These results also indicate that LV-104 has no in vitro activity.

[0275] When tested in a cell-based assay involving taxol-treated CHL-1 cells, IBMT11 was found to have weak inhibitory activity (Figure ​(Figure13B),13), suggesting that IBMT11 reduced cell penetration, whereas LV-104 produced highly efficient inhibition of VASH.

[0276] 2.3.14. Conversion of LV104 to IBMT11 in human cells Bioanalysis of the penetration and transformation of compound LV-104 was achieved in human cells using the protocol described above (see part 2.2). The results are shown in Figure 14.

[0277] It has been confirmed that the prodrug LV-104 is completely converted to IBMT11 inside human cells. Although some conversion occurs in the cell culture medium (supernatant), LV104 was not detected inside the cells (cells). These data are particularly relevant for R 3 This supports the hypothesis that the presence of less polar groups, such as esters, at the position corresponding to significantly increases their cellular penetration. Once inside the cells, the compounds are hydrolyzed, for example converting the ester to a carboxylic acid, resulting in potent VASH inhibitors.

[0278] Therefore, IBMT11 is the counterpart of the prodrug LV-104. 3 and R 6 The corresponding group is an ethyl ester in LV-104, whereas it is a carboxylic acid in IBMT11. Therefore, LV-104 is unable to exert in vitro activity, whereas IBMT-11 has poor in cellulo activity. Conversely, IBMT-11 is highly efficient in vitro, whereas LV-104 is a potent VASH inhibitor in cellulo.

[0279] 2.3.15. In Vitro and In Cellular Assays of Compounds According to the Invention a) IBMT23 IBMT23 was found to be inactive when tested in an in vitro detyrosination assay (using the protocol described above) (Figure 15.A), but had excellent in cellulo VASH inhibitory activity in human CHL1 cells (Figure 15.B). Activity was expressed in comparison to the activities of LV80, IBMT11, and EPO-Y (designated LV1) in the in vitro assay, and to the activities of LV80, LV104, and EPO-Y in the in cellulo assay. IBMT23 was found to be more potent than LV80 in cultured cells and to have similar activity to LV-104 in vivo. These results are consistent with the above hypothesis. IBMT23 has R 3 IBMT23 is a prodrug with an ethyl ester at position 1. Such prodrugs are therefore hydrolyzed inside cells to produce the corresponding carboxylic acid, which results in VASH inhibitory activity. Therefore, IBMT23 has no in vitro activity but shows excellent in cellulo activity.

[0280] b) IBMT34 IBMT34 is the sodium salt of LV80. It has been shown to have the same in vitro and in cellulo activity as the corresponding base LV-80, but has dramatically increased solubility in water or PBS solution (see Figures 16A and 16B).

[0281] c) IBMT28 and IBMT28hydro IBMT28 was found to be inactive when tested in the in vitro detyrosination assay (using the protocol described above) (Figure 17.A), but to have excellent in cellulo VASH inhibitory activity in human CHL1 cells (Figure 17.B). Activity was expressed in comparison with the activities of LV80, IBMT11, and EPO-Y (designated LV1) in the in vitro assay, and with the activities of LV80 and LV104 in the in cellulo assay. IBMT23 is found to be as potent as LV80 in vivo. These results are consistent with the above hypothesis. IBMT28 inhibits R 3IBMT28 is a prodrug with an ethyl ester at position 1. Such a prodrug is therefore hydrolyzed inside the cell to produce the corresponding carboxylic acid IBMT28hydro, which results in VASH inhibitory activity. Therefore, IBMT28 has no in vitro activity but shows excellent in vivo activity. The in vitro activity of IBMT28hydro was evaluated in Figure 17.A.

[0282] d) IBM T38 hydro IBMT38hydro was tested in the in vitro detyrosination assay as described above and was found to have as good in vitro activity as LV80 (see Figure 18).

[0283] In vitro IC 50 Calculation The following compounds were tested in the in vitro detyrosination assay as described above and had their IC 50 Calculate the IC of compound LV-80 50 The "Fold Change" column shows the IC 50 (compound) / IC 50 (LV80).

[0284] [Table 1-1] [Table 1-2]

[0285] 2.3.17. Comparison example of SD-139 [ka]

[0286] SD139 was synthesized as previously described in Aillaud, C. et al. Science, 2017, 358, p.1448-1453.

[0287] Incubation with 150 mM NaOH for 5 min and then neutralization with 150 mM HCl gave SD139hydro.

[0288] SD-139 and SD-139hydro (also called SD-139sapo) were tested in an in vitro detyrosination assay and compared to the activity of LV-80 and EPO-Y (see Figures 19.A and 19.B). Both SD-139 and SD-139sapo were found to be much less potent than LV-80 and even less potent than EPO-Y. Thus, SD-139 does not demonstrate a minimum requirement in terms of inhibition of VASH-dependent detyrosination.

Claims

1. Compounds of formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt or solvate thereof, During the ceremony X is, 【Chemistry 2】 and R １ But O-C １ -C ６ Alkyl, O-C ２ -C ６ Alkenyl, NR １ａ R １ｂ or 【Chemistry 3】 and R １ａ is H or C １ -C ６ is alkyl, R １ｂ But OH or C １ -C ６ alkyl, wherein the alkyl is C(O)OH, C(O)O—C １ -C ６ optionally substituted with alkyl or aryl; R is O-R ２ or NH—S(O) ２ -R ９ and R ２ But H, C １ -C ６ aliphatic chain, aryl, heteroaryl or C １ -C ６ alkyl-aryl, wherein up to four methylene units of the aliphatic chain are O, C(O), NH, or N—C １ -C ６ optionally substituted alkyl, wherein said aliphatic chain, aryl, heteroaryl or alkyl-aryl is optionally substituted; R ３ But, OH, O-C １ -C ６ Aliphatic chain, O-aryl, O—C １ -C ６ Alkyl-aryl, O-heteroaryl, O-C １ -C ６ alkyl-heteroaryl or NHOH, wherein up to four methylene units of the aliphatic chain are O, C(O), NH, or N—C １ -C ６ optionally substituted alkyl, wherein said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl, or alkyl-aryl is optionally substituted; R ４ is H or C １ -C １２ It is an aliphatic chain, and up to four methylene units are O, C(O), NH or N—C １ -C ６ optionally substituted with alkyl, １ -C １２ the aliphatic chain is optionally substituted; Y is -(CH ２ ) ｍ -or 【Chemistry 4】 and R ５ But, OH, O-C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, aryl, heteroaryl, O—C １ -C ６ Alkyl-aryl, O—C １ -C ６ Alkyl-heteroaryl, C(O)OH, C(O)O—C １ -C ６ Alkyl, C(O)NHOH, C(O)NH ２ , C(O)NH-C １ -C ６ Alkyl, C(O)NH—O—C １ -C ６ Alkyl, NH—C １ -C ６ Alkyl, N(C １ -C ６ alkyl) ２ ,NH-C(O)-C １ -C ６ Alkyl or NH—S(O) ２ -R ９ wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl, or alkyl-heteroaryl is optionally substituted; R ６ But, OH, O-C １ -C ６ Aliphatic chain, NH—OH or NH—CH(R ７ )-(CH ２ ) ｎ -R ８ wherein the aliphatic chain is optionally substituted; R ７ But H, C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, aryl, heteroaryl, C １ -C ６ Alkyl-aryl or C １ -C ６ alkyl-heteroaryl; R ８ But C(O)NH ２ , C(O)NH-C １ -C ６ Alkyl, aryl, heteroaryl, SH, NH ２ or S-C １ -C ６ alkyl, m and n are each independently an integer ranging from 0 to 6; R ９ But C １ -C ６ an aliphatic chain or aryl, said aliphatic chain or aryl being optionally substituted; However, X is 【Chemistry 5】 and R ３ When is OH, R １ O-C １ -C ６ is not alkyl, and the compound of formula (I) is 【Chemistry 6】 provided that it is not A compound or a pharmaceutically acceptable salt or solvate thereof.

2. R is OR ２ 2. The compound of claim 1, wherein:

3. R ２ is H, optionally substituted C １ -C ６ Alkyl or optionally substituted C １ -C ６ 3. The compound of claim 2, or a pharmaceutically acceptable salt or solvate thereof, which is alkyl-aryl.

4. R ３ But, OH, O-C １ -C ６ Aliphatic chain or O—C １ -C ６ 2. The compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the aliphatic chain is alkyl-aryl, NHOH, wherein the aliphatic chain is optionally substituted.

5. R １ But O-C １ -C ６ Alkyl, O-C ２ -C ６ alkenyl or NR １ａ R １ｂ 2. The compound of claim 1, wherein:

6. R １ but, 【Chemistry 7】 2. The compound of claim 1, wherein:

7. Y, 【Chemistry 9】 and R ５ But, OH, O-C １ -C ６ Alkyl, O-C １ -C ６ Alkyl-aryl, C(O)NH—O—C １ -C ６ Alkyl, or NH(S(O) ２ -R ９ and R ６ is OH or O—C １ -C ６ alkyl or O-aryl; 7. The compound of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

8. Y is -(CH ２ ) ｍ - and R ５ is C(O)OH, C(O)OEt or C(O)NHOH, and R ６ is NH-CH(R ７ )-(CH ２ ) ｎ -R ８ and R ７ But H, C １ -C ６ Alkyl, C １ -C ６ Alkyl-aryl, or C １ -C ６ alkyl-heteroaryl, and R ８ But C(O)NH ２ , aryl, heteroaryl, SH, or S—C １ -C ６ 7. The compound of claim 6, or a pharmaceutically acceptable salt or solvate thereof, wherein n is alkyl and n is 0, 1, 2, or 3.

9. X is, 【Chemistry 10】 2. The compound of claim 1, wherein:

10. The following formula (IA'): 【Chemistry 12】 2. The compound of claim 1, which corresponds to:

11. The following compounds: 【Chemistry 13-1】 【Chemistry 13-2】 【Chemistry 13-3】 【Chemistry 13-4】 【Chemistry 13-5】 【Chemistry 13-6】 2. The compound of claim 1 selected from: or a pharmaceutically acceptable salt and / or solvate thereof.

12. 12. A pharmaceutical composition comprising at least one compound of formula (I) as defined in any one of claims 1 to 11 or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient.

13. 13. A pharmaceutical composition according to claim 12 for use as a medicament.

14. 13. The pharmaceutical composition of claim 12 for treating a disorder associated with VASH peptidase activity.

15. The pharmaceutical composition of claim 14, wherein the VASH peptidase activity-associated disorder is a disorder involving altered detyrosination and / or polyglutamylation of microtubules.

16. The pharmaceutical composition described in claim 14, wherein the VASH peptidase activity-associated disorder is selected from the group consisting of neurodegenerative diseases, glaucoma, psychiatric disorders, neurological disorders, cancer, muscular dystrophy, infertility, retinal degeneration, Purkinje cell disease, infantile-onset degeneration, male infertility, and ciliopathy.

17. The pharmaceutical composition described in claim 14, wherein the VASH peptidase activity-associated disorder is selected from the group consisting of neurodegenerative diseases, cancer and muscular dystrophy.

18. A conjugate comprising a fragment of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as defined in any one of claims 1 to 11 bound to a biomolecule.

19. The complex of claim 18, wherein the biological molecule is selected from the group consisting of a peptide, a protein, a biomarker, an affinity probe, and an E3 ubiquitin ligase recruiter.

20. 19. The conjugate of claim 18 for use as a medicament.

21. The complex of claim 18 for use as a research tool for research and development activities.

22. The research and development activities include: - in vitro and / or in cell screening assays to identify new VASH inhibitors and / or to quantify their inhibitory efficacy; - an in vitro method for studying the role of tubulin detyrosination, in particular in the development, malignancy and progression of disorders associated with VASH peptidase activity; - in vitro diagnostic methods for identifying or monitoring disorders associated with VASH peptidase activity involving altered detyrosination and / or polyglutamylation of microtubules, and - Related kits for carrying out said screening assays and methods 22. The complex of claim 21, selected from the group consisting of:

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