Methods for labeling eukaryotic cells from multicellular organism as well as for treating and / or diagnosing cancer using modified monosaccharide compounds

Abstract

To meet a constant need to find and develop new candidates, especially for labeling and detecting eukaryotic cells from multicellular organisms, to provide methods for identifying or isolating cancer cells, and to provide methods of diagnosis or cell therapy, particularly in the cancer field.SOLUTION: The present invention relates to modified monosaccharide compounds implemented in methods for labeling and / or detecting an eukaryotic cell from a multicellular organism. It also relates to such modified monosaccharide compounds implemented in methods for identifying or isolating cancer cells, diagnosing a cancer or for cell therapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of medicine, in particular oncology. The present invention relates to modified monosaccharide compounds implemented in a method for labeling and / or detecting and / or targeting eukaryotic cells from multicellular organisms. The present invention also relates to such modified monosaccharide compounds implemented in a method for identifying or isolating cancer cells, diagnosing cancer, or cell therapy. [Background technology]

[0002] Carbohydrates are important as signaling molecules and for cell recognition events. Indeed, carbohydrates can undergo multivalent interactions with carbohydrate recognition proteins (CRPs) and can be used as biological probes. Thus, carbohydrates offer numerous opportunities in disease diagnosis and therapy. Thus, the development of carbohydrate-based bioactive compounds and sensors has become an active research area. An effective and modular synthetic approach to prepare functional carbohydrate derivatives is click chemistry. Several carbohydrate derivatives prepared by CuAAC (Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition) click chemistry for therapy and diagnosis have been reported in a review by He et al. (Carbohydrate Research 429 (2016) pp. 1-22). Copper-free click chemistry using strain-promoted azide-alkyne cycloaddition has also been described for cancer cells. Certain sugars that display clickable groups (e.g., triacetyl N-azidoacetylmannosamine (Ac3ManNAz) analogs) are metabolized by cancer cells due to their specific overexpression of the enzymatically cleavable sugar analogs, and thus are specifically incorporated into the cancer cell membrane.

[0003] Even though various carbohydrate / monosaccharide click chemistry methods have been described for therapeutics and diagnostics, these click chemistry methods and the compounds used therein appear to be cytotoxic and / or lack selectivity for certain cells, such as selectivity for cancer cells. The cytotoxicity of these compounds means that they use low, non-toxic concentrations, which reduces their effectiveness.

[0004] WO2013 / 1077559 describes a particular compound, a modified monosaccharide compound such as 8-azido-3,8-dideoxy-D-manno-octulosonic acid (also referred to herein as "KDO-N3"), in a method for specifically labeling live microorganisms. The labelled live microorganisms were limited to unicellular prokaryotic microorganisms (bacteria).

[0005] WO2016 / 177712 also describes a specific compound, modified monosaccharide compounds such as 5-azido-5-deoxy-D-arabinofuranose (also referred to herein as "Ara-N3"), in a method for specifically labeling living microorganisms. The labeled living microorganisms were limited to unicellular prokaryotic microorganisms (bacteria) and unicellular eukaryotic microorganisms (yeast, fungi, and amoebae).

[0006] However, so far it has not been explained where and how said monosaccharide compounds are assimilated by the microbial cell membrane. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2013 / 1077559 [Patent Document 2] WO2016 / 177712 [Non-patent literature]

[0008] [Non-Patent Document 1] Carbohydrate Research 429(2016) pages 1~22 [Non-Patent Document 2] 2006, Annals of Oncology, 17, pp. 735-749. Summary of the Invention [Problem to be solved by the invention]

[0009] There is a constant need to find and develop new candidates for labeling and detecting eukaryotic cells, especially from multicellular organisms, to provide methods for identifying or isolating cancer cells, and to provide methods for diagnosis or cell therapy, especially in the cancer field. [Means for solving the problem]

[0010] The present invention relates to monosaccharide compounds of formula (I) or their precursors:

[0011] [ka]

[0012] where X is a reactive group that allows for the covalent attachment of a further compound, such as a label or an anticancer drug or a particle containing an anticancer drug, via a click chemistry reaction. The formed conjugates can thus be implemented in methods for labeling or detecting eukaryotic cells from multicellular organisms, in methods for identifying or isolating cancer cells, or in methods for treating cancer.

[0013] More specifically, the inventors have used eukaryotic cells from multicellular organisms to produce monosaccharide compounds of formula (I), in particular Ara-N. 3、They found that uptake of the monosaccharide compounds of formula (I) and their precursors occurs in tumor eukaryotic cells (particularly in bladder, blood, skin, pancreas, brain, liver, kidney, lung, muscle, lymphocyte, prostate, stomach, breast cancer compared to non-cancer cells) that is different from and more significant than non-cancer eukaryotic cells. Thus, the monosaccharide compounds of formula (I) and their precursors can also be used as cancer probes and markers, which can be useful for identifying, isolating, or targeting cancer cells and / or for diagnosing cancer in a subject.

[0014] Accordingly, an aspect of the present invention is a method, preferably an in vitro method, for labeling or detecting or targeting a eukaryotic cell from a multicellular organism, comprising the steps of: a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound or a precursor thereof; b) contacting the sample of step (a) with a compound having a first reactive group, optionally in the presence of copper; and c) optionally detecting the compound of step (b) bound to the monosaccharide of step (a) to detect eukaryotic cells. wherein the at least one modified monosaccharide compound has the following formula (I):

[0015] [ka]

[0016] wherein X is a second reactive group, and the first reactive group and the second reactive group are capable of reacting with each other in a click chemistry reaction to provide a compound of step (b) attached to said monosaccharide. In a preferred embodiment, the first reactive group is an alkyne group and the second reactive group X is an azide group (-N3).

[0017] A further aspect of the present invention is a kit for carrying out a method for labelling, detecting or targeting a eukaryotic cell as defined herein, comprising: a modified monosaccharide compound of formula (I) or a precursor thereof, preferably a precursor of formula (I′), and - a compound having a first reactive group A kit comprising:

[0018] A further aspect of the invention is a method for identifying or isolating cancer cells or for diagnosing cancer in a subject, comprising the step of carrying out a method of labelling, detecting or targeting eukaryotic cells from a multicellular organism as defined herein from said subject, preferably in a biological sample from said subject.

[0019] A further aspect of the present invention is a composition comprising a eukaryotic cell presenting at its surface at least one modified monosaccharide compound of formula (I) as defined herein or a precursor thereof, preferably a precursor of formula (I'), operably linked or not to an anti-cancer drug or to a particle comprising at least one anti-cancer drug. Another aspect is a pharmaceutical composition comprising such a cell. Another aspect is such a pharmaceutical composition for use in the treatment of cancer, in particular by cell-based therapies.

[0020] Another aspect is such a pharmaceutical composition for use in the diagnosis of cancer.

[0021] Another aspect of the present invention is a method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of a composition as defined herein comprising a eukaryotic cell displaying on its surface at least one modified monosaccharide compound of formula (I) as defined herein or a precursor thereof, preferably a precursor of formula (I'), operably linked to an anti-cancer drug or to a particle comprising at least one anti-cancer drug.

[0022] A further aspect of the present invention is the use of a modified monosaccharide compound as defined herein or a precursor thereof, optionally together with a compound carrying a first reactive group, for medical imaging or diagnosis, preferably for the diagnosis of cancer. [Brief description of the drawings]

[0023] [Figure 1] FIG. 13 is a graph showing the ex vivo tumor to skeletal muscle fluorescence signal ratio (tumor / muscle ratio) obtained using Panc-1 tumor-bearing mice iv injected or not with Ara-N3 and fluorophore. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition According to the present invention, the following terms have the following meanings:

[0025] A "multicellular organism" includes any organism that includes more than one cell. The multicellular organism may be, for example, of plant or animal origin or origin, preferably a mammal, more preferably a human.

[0026] As used herein, the terms "patient" and "subject" may be used interchangeably and include both humans and animals, and more specifically, humans.

[0027] A "eukaryotic cell from a multicellular organism" is a cell that, unlike prokaryotes, has a nucleus within a membrane and is derived from a multicellular organism, such as an animal or plant cell. Animal and plant cells are the most familiar eukaryotic cells from multicellular organisms. In the context of this invention, "eukaryotic cells" includes cancer cells or normal cells (or non-tumor and tumor cells).

[0028] Cancer cells are cells that divide continuously, form solid tumors, or flood the blood with abnormal cells. Thus, cancer cells can be solid tumors or hematopoietic cancers such as lymphoma or leukemia.

[0029] The term "cancer" or "tumor" as used herein refers to the presence of cells with typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. The term refers to any type of malignant tumor (primary or metastatic). Exemplary cancers are solid or hematopoietic cancers, such as breast, brain, stomach, liver, skin, prostate, pancreas, esophagus, sarcoma, ovarian, endometrial, bladder, cervical, rectal, colon, kidney, lung, or ORL cancer, childhood tumors (neuroblastoma, glioblastoma multiforme), lymphoma, carcinoma, glioblastoma, hepatoblastoma, leukemia, myeloma, seminoma, Hodgkin's, or malignant hematological disorders.

[0030] The present invention provides a method for labeling or detecting or targeting a eukaryotic cell from a multicellular organism, comprising the steps of: a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound or a precursor thereof; b) contacting the sample of step (a) with a compound having a first reactive group, optionally in the presence of copper; and c) optionally detecting the compound of step (b) bound to the monosaccharide of step (a). wherein the at least one modified monosaccharide compound has the following formula (I):

[0031] [ka]

[0032] wherein X is a second reactive group, and the first reactive group and the second reactive group are capable of reacting with each other in a click chemistry reaction. The reaction between the first reactive group and the second reactive group allows the compound of step (b) to be attached to the monosaccharide of formula (I).

[0033] Click chemistry is a method known to those skilled in the art for attaching a probe or substrate of interest to a specific biomolecule, such as the modified monosaccharide compounds according to the invention. Azide-alkyne cycloaddition is a well-known so-called click chemistry reaction in which an azide group reacts with an alkyne group in the presence or absence of a copper catalyst to give a triazole. The alkyne group can be strained or unstrained.

[0034] Such azide-alkyne cycloadditions can be carried out under copper catalyzed conditions in the presence of a ligand, preferably a tris-triazole ligand such as TGTA (tris((l-(-D-glucopyranosyl)-l[l,2,3]-triazol-4-yl)methyl)amine) or TBTA (tris-[(l-benzyl-ll,2,3-triazol-4-yl)methyl]amine). Other suitable ligands that are frequently used include tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 2-(4-((bis((l-tert-butyl-l 1,2,3-triazol-4-yl)methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)ethanesulfonic acid (BTTES), tris((l-(((ethyl)carboxymethyl)-(l,2,3-triazol-4-yl))methyl)amine, bathophenanthroline disulfonate, or tris(2-benzimidazolylmethyl)amines.

[0035] Alternatively, the azide-alkyne cycloaddition can be carried out in the absence of copper when a strained alkyne such as azadibenzocyclooctyne (ADIBO, DIBAC, or DBCO) or tetramethoxydibenzocyclooctyne (TMDIBO) is used. Other suitable strained alkynes frequently used for copper-free reactions include cyclooctyne (OCT), aryl-lesscyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctyne (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa-dibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne.

[0036] Other reactive groups and other reactions are possible in click chemistry, such as: Staudinger ligation (first reactive group=azide, and second reactive group=phosphine), copper-free click chemistry (first reactive group=azide, and second reactive group=strained alkyne (inner ring alkyne)), carbonyl condensation (first reactive group=aldehyde or ketone, and second reactive group=hydrazide or oxyamine), thiol-ene click chemistry (first reactive group=thiol, and second reactive group=alkene), nitrile-oxide-ene click chemistry (first reactive group=nitrile oxide or aldehyde, oxime, or hydroximoyl chloride or chlororoxime, and second reactive group=hydrazide or oxyamine). = alkene or alkyne), nitrile imine-ene click chemistry (first reactive group = nitrile imine or aldehyde, hydrazone, or hydrazonoyl chloride or chlorohydrazone, and second reactive group = alkene or alkyne), inverse electron demand Diels-Alder ligation (first reactive group = alkene, and second reactive group = tetrazine), isonitrile-tetrazine click chemistry (first reactive group = isonitrile, and second reactive group = tetrazine), Suzuki-Miyaura coupling (first reactive group = aryl halide, and second reactive group = aryl boronate), His-tag (first reactive group = oligo-histidine, and second reactive group = nickel complex or nickel ligand).

[0037] Monosaccharide compounds and their precursors According to the present invention, the modified monosaccharide compound of formula (I) comprises a reactive group X (second reactive group) suitable for reaction in a click chemistry reaction, preferably in an azide-alkyne cycloaddition. Thus, X includes any reactive group capable of reacting with a further reactive group by a click chemistry reaction, such as the reactive groups defined above. In a particular embodiment, X includes any group consisting of or carrying an azide group (-N3) and groups consisting of or carrying a strained or unstrained alkyne group (-C≡C-). In a preferred embodiment, X is an azide group (-N3).

[0038] In one particular embodiment of the present invention, the modified monosaccharide compounds of formula (I) include their diastereoisomers. In a further particular embodiment, the modified monosaccharide compounds of formula (I) have the following formula:

[0039] [ka]

[0040] is selected from the group consisting of:

[0041] In a preferred embodiment, said at least one modified monosaccharide compound of formula (I) is in particular of the following formula:

[0042] [ka]

[0043] and 5-azido-5-deoxy-D-arabinofuranose (Ara-N3),

[0044] In a further particular embodiment of the invention, at least one modified monosaccharide compound of formula (I) is a precursor of said modified monosaccharide compound. More specifically, said precursor is a precursor of formula (I'):

[0045] [ka]

[0046] wherein X is a second reactive group as defined above, preferably an azide group (-N3), and the first reactive group and the second reactive group are capable of reacting with each other in a click chemistry reaction.

[0047] In a further particular embodiment, the precursor of formula (I') includes its diastereoisomers. In a further particular embodiment, the precursor of formula (I') has the following formula:

[0048] [ka]

[0049] is selected from the group consisting of:

[0050] In a preferred embodiment, the precursor of formula (I') has the following formula:

[0051] [ka]

[0052] and 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose having the formula:

[0053] The modified monosaccharide compound of formula (I) or its precursors can be used according to the present invention at any concentration since it is not toxic to cells. According to a particular embodiment, the concentration of the monosaccharide compound of formula (I) of the present invention or its precursors can vary from 10 μM to 100 mM, preferably from 1 mM to 50 mM, more preferably from 1 mM to 20 mM.

[0054] Compound having a first reactive group The compound having a first reactive group includes or is a directly detectable moiety, or includes or is an indirectly detectable moiety. Without being bound by any theory, the cell is linked to the compound having a first reactive group by a click reaction with the second reactive group of the monosaccharide of step (a) that is taken up by the cell membrane in step (a). The detectable moiety (or label) is a moiety that can be detected by techniques known to those skilled in the art, such as fluorescence, colorimetry, or luminescence. The imaging technique can thus be fluorescence, magnetic resonance, or computed tomography.

[0055] According to certain embodiments, the compound may comprise or be a detectable moiety, i.e. a moiety that is composed of or has a detectable substance (or label), i.e. a substance that can be detected by techniques known to those skilled in the art, such as fluorescence, colorimetry, or luminescence.

[0056] According to another embodiment, said compound having a first reactive group comprises or is an indirectly detectable moiety which is a first ligand (or more specifically a first binding protein having said first reactive group), and the detection and / or immobilization in step (c) can be performed by contacting said eukaryotic cell bound to said first ligand (or more specifically a first binding protein) with a second ligand (or second binding protein) which specifically reacts or binds to said first ligand (or more specifically a first binding protein), as described in more detail below.

[0057] More specifically, the compound is a first ligand carrying the first reactive group, preferably biotin, and in step c) the eukaryotic cells bound to the first ligand are detected by reaction of the eukaryotic cells with an antibody or another protein specific for the first ligand, the antibody carrying a detectable substance or moiety, preferably a fluorescent or luminescent molecule or an enzyme.

[0058] The detectable substance or moiety may be selected from among dyes, radiolabels, and affinity tags. In particular, the dye may be selected from the group consisting of fluorescent, luminescent, or phosphorescent dyes, preferably dansyl, fluorescein, acridine, rhodamine, coumarin, BODIPY, and cyanine dyes. More specifically, the fluorescent dye may be selected from among dyes sold by Thermo Fisher, such as Alexa Fluor dyes, Pacific dyes, or Texas Red, or cyanine 3, 5, and 7 dyes sold by other vendors. In particular, azide-bearing dyes for CuAAC include Alexa Fluor® 488, 55, 594, and 647, and TAMRA (tetramethylrhodamine). In a second embodiment, the detectable substance or moiety (or label) may be an affinity tag. Such affinity tags may be selected, for example, from the group consisting of biotin, His-tag, Flag-tag, strep-tag, sugar, lipid, sterol, PEG-linker, and cofactor. In a particular embodiment, the detectable substance is a biotinylated label. Biotin linked to azide is commercially available (Biotin azide). In a preferred embodiment, the label is a fluorescent label.

[0059] According to the present invention, the compound having a first reactive group comprises a first reactive group that is complementary to a second reactive group X as defined above, for reacting with each other in a click chemistry reaction, preferably an azide-alkyne cycloaddition. In one particular embodiment, the first reactive group of the compound comprises any group consisting of or having an azide group (-N3) and a group consisting of or having a strained or unstrained alkyne group (-C≡C-).

[0060] In the above list of groups involved in the reaction, the first reactive group and the second reactive group X can be interchanged. All the above chemical reactions result in a covalent bond. For example, if X is an azide group (-N3) or a group having an azide group, then the first reactive group is an alkyne or a group having an alkyne group. If X is an alkyne or a group having an alkyne group, then the first reactive group is an azide group (-N3) or a group having an azide group. In a preferred embodiment of the present invention, the second reactive group X is an azide group (-N3) and the first reactive group is an alkyne group (-C≡C-).

[0061] Methods of Labeling, Detecting, or Targeting Step a) according to the method for labeling, detecting or targeting eukaryotic cells from a multicellular organism comprises contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound of formula (I) or a precursor thereof comprising a reactive or functional group X. Such contacting step a) allows the incorporation of at least one modified monosaccharide compound of formula (I) or a precursor thereof into the membrane of said eukaryotic cells from a multicellular organism, more particularly at the surface of said cells. Such a process may correspond to the uptake of at least one modified monosaccharide compound of formula (I) by said eukaryotic cells. Thus, said cells present at their surface or membrane at least one modified monosaccharide compound of formula (I).

[0062] Step b) comprises contacting the sample of step (a) (wherein at least one modified monosaccharide compound of formula (I) or a precursor thereof is incorporated into the membrane of a eukaryotic cell) with a compound comprising a first reactive group as defined above. Such step b) allows a click chemistry reaction to take place between the first reactive group of the compound and the second reactive group X of at least one modified monosaccharide compound of formula (I) or a precursor thereof, preferably a precursor of formula (I'), thereby obtaining a eukaryotic cell from the associated multicellular organism, which is or can be subsequently labeled or targeted (as detailed above).

[0063] One preferred embodiment of the present invention is a method for labeling, detecting, or targeting a eukaryotic cell from a multicellular organism, comprising the steps of: a) contacting a sample containing eukaryotic cells with Ara-N3; and b) contacting said sample with a compound comprising a first reactive group, optionally in the presence of copper; The method includes:

[0064] The skilled person knows how to carry out step (a) or (b). According to a particular embodiment, said steps (a) and / or (b) are carried out in a culture or incubation medium that allows the growth of the sample comprising eukaryotic cells, preferably specific for the growth of said eukaryotic cells.

[0065] More specifically, the culture conditions (including time and cell culture medium) of step (a) or (b) are configured such that eukaryotic cells are labeled, detected or targeted. The cell culture medium may be supplemented with any compound that enhances or stimulates cell doubling and / or uptake of the modified monosaccharide compound of formula (I) at the surface or membrane of the cell.

[0066] According to a particular embodiment, the duration of step (a) allows the incorporation of at least one monosaccharide compound of formula (I) or a precursor thereof into the membrane of said eukaryotic cell. More particularly, the duration of step (a) is at least the doubling time of the eukaryotic cell to be labeled, detected or targeted. More particularly, the duration of step (a) is less than 5 times the doubling time of the eukaryotic cell to be labeled, detected or targeted. According to a particular embodiment, the duration of step (a) corresponds to one or two doubling times of the eukaryotic cell to be labeled, detected or targeted.

[0067] According to one particular embodiment, steps (a) and / or (b) are carried out using a reactant and / or a catalyst for effecting the reaction of the first reactive group with the second reactive group.

[0068] It is important to note that, as illustrated by the examples, the monosaccharide compounds of formula (I) or their precursors exhibit low or no toxicity to cells, and therefore the amount of use thereof can be varied in a wide range, such amount being determined by the skilled artisan to be sufficient to label, identify or detect eukaryotic cells.

[0069] The method can be carried out with any sample, typically a biological sample of a subject, such as a body fluid, such as a sample of blood, plasma, serum, urine, cerebrospinal fluid, or a sample from a tissue of a subject, or a part thereof. The invention can be carried out with a sample from any subject, including any human patient with or suspected of having cancer. The method is typically carried out on a blood, serum, or plasma sample, or a sample derived therefrom, such as a pre-treated blood sample. The sample may be processed (e.g., diluted, concentrated, isolated, partially purified, frozen, etc.) prior to being used in the invention. According to a particular embodiment, each sample used in step (a) of the method comprises a cell population or preferably individual cells, preferably obtained by cell sorting, in particular by flow cytometry. When the method is carried out in vivo, the method can be carried out on the whole body of a subject or on a part thereof. In that context, the monosaccharide compound of formula (I) or a precursor thereof and optionally a compound having a first reactive group can be administered enterally (including orally) or parenterally (including intravenously or intramuscularly).

[0070] For detecting eukaryotic cells from the associated multicellular organism, the method further comprises a step c) which comprises detecting the compound of step (b) bound to the monosaccharide of step (a).

[0071] Advantageously, the invention comprises a further step (c) of detecting eukaryotic cells in detecting whether they are associated with the compound bearing a first reactive group of step (b) and / or in immobilizing said eukaryotic cells associated with the compound bearing a first reactive group on a solid substrate, wherein said compound bearing a first reactive group is a moiety or molecule comprising a detectable substance or a moiety or molecule capable of reacting with or binding to a detectable substance, or preferably said compound bearing a first reactive group is a first molecule capable of reacting with or binding to a second molecule and / or a solid substrate, preferably said second molecule comprises a detectable substance and / or said second molecule is bound or capable of binding to said solid substrate.

[0072] The invention thus makes it possible to label and to count or detect eukaryotic cells from multicellular organisms, optionally immobilized on a solid support, in particular immobilized on a solid support composed of magnetic beads carrying said first reactive groups, as well as to enrich and / or separate eukaryotic cells.

[0073] More specifically, said compound having a first reactive group is a first molecule capable of reacting with or binding to a second molecule and / or a solid substrate, preferably said second molecule comprises a detectable substance, and the method comprises a step c) of detecting said eukaryotic cell in finding out whether said eukaryotic cell contains said detectable molecule or moiety bound to said eukaryotic cell.

[0074] According to the methods of the present invention, in the absence of labeling or detection, the sample taken can be determined to be free of eukaryotic cells from a multicellular organism.

[0075] The detecting step c) can be carried out in a liquid medium or on a solid substrate.

[0076] Preferably, the method for labelling or detecting or targeting a eukaryotic cell from a multicellular organism is an in vitro method.

[0077] According to a particular embodiment, the method of the invention may further comprise one or more washing steps.

[0078] According to a particular embodiment, the method according to the invention can be carried out on one or several samples at the same time, for example using a microwell plate, which typically has 6, 12, 24, 48, 96, 384 or 1536 sample wells. According to said particular embodiment, each sample well used in step (a) of the method preferably contains a cell population or preferably individual cells, more preferably obtained by cell sorting, in particular by flow cytometry.

[0079] Thus, a further object of the present invention is the in vitro use of at least one modified monosaccharide compound of formula (I) or a precursor thereof, preferably a precursor of formula (I'), preferably together with a compound comprising a first reactive group, optionally together with a second molecule and / or a solid substrate, preferably said second molecule comprising a detectable substance, for labelling or detecting eukaryotic cells from a multicellular organism.

[0080] A further object of the present invention is a kit for carrying out the method for labelling or detecting eukaryotic cells from a multicellular organism as defined herein, comprising: a modified monosaccharide compound of formula (I) or a precursor thereof, preferably of formula (I′), as defined above, and - a compound having a first reactive group, as defined above A kit comprising:

[0081] According to a particular embodiment, the kit may further comprise a second molecule and / or a solid substrate as defined above, preferably said second molecule or solid substrate comprising a detectable substance and the compound bearing a first reactive group is a first molecule capable of reacting with or binding to the second molecule and / or the solid substrate.

[0082] A further object is the use of a kit as defined above for carrying out a method for labelling or detecting eukaryotic cells from a multicellular organism as defined herein.

[0083] In a particular embodiment, the eukaryotic cell from the multicellular organism is a susceptible cell that is a cancer or tumor cell. Thus, the methods and kits according to the invention are useful for identifying cancer or tumor cells by detection of the label.

[0084] Methods for identifying or isolating cancer cells or diagnosing cancer The present invention further relates to a method for identifying or isolating cancer cells or diagnosing cancer in a subject, preferably an in vitro or ex vivo method, comprising carrying out a method for labeling or detecting eukaryotic cells as defined herein in said subject or in a biological sample from said subject. In a preferred embodiment, the method for identifying or isolating cancer cells or diagnosing cancer in a subject further comprises the step of detecting the label and optionally comparing the label to a reference level.

[0085] The biological sample from the subject is as defined above, preferably the sample is a sample from a human patient having or suspected of having cancer. According to a particular embodiment, each sample used in step (a) comprises a cell population or preferably individual cells, preferably obtained by cell sorting, in particular by flow cytometry.

[0086] The sample is more particularly suspected of containing cancer cells.

[0087] Examples of such samples include bodily fluids, such as blood, plasma, saliva, urine, and semen samples, as well as biopsy, organ, tissue, or cell samples. The sample may be processed prior to its use.

[0088] The cancer cells identified or isolated according to the present invention can be of any type. The cancer cells may be obtained from solid tumors or hematopoietic cancers. Cancer cells include circulating or non-circulating tumor cells. Circulating tumor cells (CTCs) are cells that are shed from primary tumors into the vasculature or lymphatics and circulate in the blood throughout the body. CTCs then seed the growth of additional tumors in distant organs (tumor metastasis), which is the mechanism responsible for most cancer-related deaths.

[0089] The detection and analysis of cancer cells according to the present invention can aid in early prognosis of patients and determine appropriate targeted treatment. The ability to monitor disease progression over time can facilitate appropriate modification of patient therapy, potentially improving the prognosis and quality of life of patients. When circulating tumor cells are detected and analyzed, the method allows early detection of cancer and especially tumor metastasis. In that respect, the sample according to the present invention is blood. Blood tests are easy and safe to perform, and multiple samples can be taken over time. An important aspect of the ability to predict future progression of disease is that the need for surgery is (at least temporarily) eliminated if CTC counts are not repeatedly low and increasing. Thus, the obvious benefits of avoiding surgery include avoiding the risks associated with the tumorigenicity inherent in cancer surgery. For this reason, a technology such as the present invention that has the necessary sensitivity and reproducibility for the detection of CTCs in patients with metastatic disease is of great interest.

[0090] The phrase "detecting a label" as used herein may include visualization or detection of the presence of eukaryotic cells from a labeled multicellular organism, or even measurement of such a label. Measuring such a label, such as fluorescence, allows for the detection, identification, or isolation of cancer cells, optionally by comparing the label to a reference level.

[0091] It has been found herein that the uptake of modified monosaccharide compounds of formula (I) or precursors thereof, preferably precursors of formula (I'), is different in cancer cells compared to non-cancerous cells, more specifically is higher compared to non-cancerous cells (e.g., a reference level or control sample). As shown in the examples, the fluorescence intensity is higher in cancer cells compared to non-cancerous cells (e.g., after 1 or 2 cell doubling times).

[0092] Accordingly, the present invention provides a method of identifying or isolating cancer cells or diagnosing cancer in a subject, comprising the steps of: - carrying out a method for labelling eukaryotic cells as described herein on a sample from said subject; - after carrying out said labelling method, detecting the label and optionally comparing the label with a reference level, and then - Identifying cancer cells or diagnosing cancer based on the measurement of the marker. The present invention relates to a method comprising the steps of:

[0093] The method for identifying or isolating cancer cells or diagnosing cancer can advantageously be carried out within the time of one cell cycle.Preferably, detection of the label is carried out between 10 and 40 hours, more preferably between 16 and 24, 25 or 36 hours, after carrying out said labeling method, more particularly after carrying out step (a) as detailed above.

[0094] The method may optionally include a comparison with a reference level, more specifically a comparison with a control sample or reference. The reference level may be the intensity of a label (such as fluorescence) measured in normal cells (e.g. non-cancer cells) and / or known cancer cells. Preferably, the reference cells are closest to the cells to be examined, preferably from the same cell line, same organ, and / or same type. The method may include a prior step of providing a tumor sample and a histologically matched normal tissue from the subject.

[0095] In certain embodiments, a cancer cell is identified or diagnosed as cancer when the measured value of the label of the sample is higher than the measured value of the label of a control sample that is a non-cancer sample. By "higher measured value" is meant that the label ratio of the sample to the non-cancer sample is greater than 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3.0. More specifically, the label ratio of the sample to the non-cancer sample is greater than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3.0.

[0096] According to a particular embodiment, the cancer to be diagnosed is selected from among rectal cancer, colorectal cancer, gastric cancer, head and neck cancer, thyroid cancer, cervical cancer, uterine cancer, breast cancer, ovarian cancer, brain cancer, lung cancer, skin cancer, bladder cancer, blood cancer, kidney cancer, liver cancer, prostate cancer, multiple myeloma, and endometrial cancer. More specifically, the cancer to be diagnosed is selected from the group consisting of bladder, blood, skin, pancreas, brain, liver, kidney, lung, muscle, lymphocyte, prostate, stomach, and breast cancer. According to a more particular embodiment, the cancer to be diagnosed is selected from the group consisting of bladder, blood, colon, stomach, breast, lung, skin, and pancreatic cancer.

[0097] According to a particular embodiment, the cancer cells to be identified or isolated are cancer cells from the cancers listed above.

[0098] Pharmaceutical compositions and uses thereof The present invention relates to a composition comprising a eukaryotic cell presenting on its surface at least one modified monosaccharide compound of formula (I) or a precursor thereof, preferably a precursor of formula (I'), as defined above, operatively linked or not to an anti-cancer drug or to a particle comprising at least one anti-cancer drug.

[0099] In one particular embodiment, cells presenting on their surface at least one modified monosaccharide compound of formula (I) or a precursor thereof as defined above, operably linked or not linked to an anti-cancer drug or to a particle comprising at least one anti-cancer drug, can be prepared as described above in the method of the invention.

[0100] The composition according to the present invention can be prepared by the following steps: a) contacting a composition comprising eukaryotic cells from a multicellular organism with at least one modified monosaccharide compound or a precursor thereof as defined above, and b) contacting the composition of step (a) with a compound having a first reactive group, and optionally an operably linked anti-cancer drug or a particle comprising at least one anti-cancer drug, optionally in the presence of copper; It can be prepared by a method comprising:

[0101] The steps are similar to those detailed above: step a) allows the cell to present, on its surface or membrane, at least one modified monosaccharide compound of formula (I) or a precursor thereof; step b) allows a click chemistry reaction to occur between a first reactive group of a compound and a second reactive group X of at least one modified monosaccharide compound of formula (I) or a precursor thereof, preferably a precursor of formula (I').

[0102] According to one embodiment, the compound having a first reactive group also comprises or can be an operably linked anti-cancer drug or a particle comprising at least one anti-cancer drug bound to said compound, and step (b) allows for providing a eukaryotic cell from the multicellular organism associated with the anti-cancer drug or with a particle comprising the anti-cancer drug.

[0103] Thus, the method allows the preparation of a conjugate in which a modified monosaccharide compound of formula (I) or a precursor thereof of formula (I') is operatively linked to an anti-cancer drug or to a particle comprising an anti-cancer drug by a click chemistry reaction as detailed above, and a eukaryotic cell presents said conjugate on its surface. In one particular embodiment, the conjugate is prepared by a click chemistry reaction between an anti-cancer drug comprising an alkyne group or a particle presenting an alkyne group on its surface and a modified monosaccharide compound of formula (I) or a precursor thereof of formula (I') comprising an azide group.

[0104] According to another embodiment, the compound having a first reactive group is a first ligand (or more specifically a first binding protein) capable of reacting or binding to a second ligand (or more specifically a second binding protein) that is an anti-cancer drug. According to such an embodiment, the method allows the preparation of a conjugate in which the modified monosaccharide compound of formula (I) or their precursor of formula (I') is linked to a first ligand capable of reacting or binding to a second ligand that is an anti-cancer drug by a click chemistry reaction as detailed above, and the eukaryotic cell presents said conjugate on its surface. In a particular embodiment, the conjugate is prepared by a click chemistry reaction as detailed above.

[0105] As used herein, an "anti-cancer agent" refers to any agent commonly used in cancer therapy, such as an anti-tumor drug. In a preferred embodiment, the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, anti-cancer antibodies, hormonal therapy agents, immunotherapy agents, and kinase inhibitors.

[0106] The term "operably linked anti-cancer drug" refers to an anti-cancer drug that is linked, preferably covalently, while still being capable of exerting its therapeutic effect.

[0107] The particle containing at least one anticancer drug is an anticancer drug-containing particle, preferably a nanoparticle, having the first reactive group defined above.The particle can be, for example, bicyclo[6.1.0]nonyne-modified glycol chitosan nanoparticle (BCN-CNP).CNP is known to be highly compatible and can encapsulate various drugs, and is widely used for drug delivery.

[0108] Chemotherapy may also include inhibitors of topoisomerase I or II, DNA crosslinking agents, DNA alkylating agents, antimetabolites, and / or mitotic spindle inhibitors.

[0109] Topoisomerase I and / or II inhibitors include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin, and mitoxantrone.Topoisomerase I and II inhibitors include, but are not limited to, intoplicin.

[0110] DNA crosslinking agents include, but are not limited to, cisplatin, carboplatin, and oxaliplatin.

[0111] Antimetabolites block the enzymes responsible for nucleic acid synthesis or become incorporated into DNA, thereby producing incorrect genetic code and leading to apoptosis. Non-exhaustive examples of these include folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, more specifically, but not limited to, methotrexate, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, 5-fluorouracil, gemcitabine, and capecitabine.

[0112] Alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, metal salts, and triazenes, non-exhaustive examples of which include uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, fotemustine, cisplatin, carboplatin, oxaliplatin, thiotepa, streptozocin, dacarbazine, and temozolomide.

[0113] Spindle inhibitors include paclitaxel, docetaxel, vinorelbine, larotaxel (also known as XRP9881; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol-Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also known as IDN 5109, BAY 59-8862, or SB-T-101131; Bristol-Meyer-Squibb), RPR 109881A (Bristol-Meyer-Squibb), RPR These include, but are not limited to, 116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel (also known as CT-2103, PPX, paclitaxel poliglumex, paclitaxel polyglutamate, or Xyotax™), ABRAXANE® (also known as nab-paclitaxel; ABRAXIS BIOSCIENCE), tesetaxel (also known as DJ-927), IDN 5390 (INDENA), taxoplexin (also known as docosahexaenoic acid-paclitaxel; PROTARGA), DHA-paclitaxel (also known as Taxoprexin®), and MAC-321 (WYETH). See also the review by Hennenfent & Govindan (2006, Annals of Oncology, 17, 735-749).

[0114] The immune checkpoint inhibitor may be selected from the group consisting of anti-CTLA-4 (cytotoxic T-lymphocyte antigen 4) therapy such as ipilimumab, PD-1 (programmed cell death protein 1) inhibitors such as nivolumab, pembrolizumab, or BGB-A317, PDL1 (programmed cell death ligand) inhibitors such as atezolizumab, avelumab, or durvalumab, LAG-3 (lymphocyte activation gene 3) inhibitors such as BMS-986016, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) inhibitors, TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibitors, BLTA (B- and T-lymphocyte attenuator) inhibitors, IDO1 inhibitors such as epacadostat, or combinations thereof.

[0115] Hormonal therapy agents include, for example, Tamoxifen, Fairston, Arimidex, Aromasin, Femara, Zoladex / Lupron, Megase, and Halotestin.

[0116] The eukaryotic cells from a multicellular organism of the composition according to the invention are preferably isolated non-cancer cells. The non-cancer cells are preferably isolated multipotent stem cells. The multipotent stem cells are preferably mesenchymal stem cells (MSCs). MSCs are found throughout the body, preferably in adipose tissue, bone marrow, tissues supporting organs, but also in bone, cartilage, and muscle, etc. The eukaryotic cells according to the invention may be T cells.

[0117] Eukaryotic cells according to the present invention are either allogeneic or, preferably, autologous (ie, from the subject or patient himself) cells.

[0118] According to a particular embodiment, the composition comprises MSCs presenting at their surface at least one modified monosaccharide compound of formula (I) as defined above or a precursor thereof. Such a composition may be useful for example for the identification of cancer cells or tumors or for the diagnosis of cancer, by exploiting the property of the monosaccharide to be detected by a compound comprising a first reactive group, directly or indirectly, as detailed above. Such a composition may also be useful in therapeutic methods for monitoring hematopoietic transplantation.

[0119] According to another particular embodiment, the composition comprises MSCs bound to at least one modified monosaccharide compound of formula (I) or a precursor thereof as defined above, which is operatively linked to an anti-cancer drug or to a particle containing at least one anti-cancer drug. Such a composition may also be useful in a therapeutic method, particularly for the treatment of cancer.

[0120] The composition of the present invention is preferably a pharmaceutical composition.

[0121] The pharmaceutical compositions contemplated herein include a pharma- ceutically acceptable carrier in addition to the cells exhibiting the anti-cancer agent as detailed above. The term "pharmaceutical acceptable carrier" includes any carrier (e.g., support, substance, solvent, etc.) that does not interfere with the effectiveness of the biological activity of the cells, and means that the carrier is not toxic to the host to which it is administered. For example, for parenteral administration, the active compound may be formulated in a unit dosage form for injection in a vehicle such as saline, dextrose solution, serum albumin, and Ringer's solution. The pharmaceutical composition may be formulated as a solution in a pharma- ceutically compatible solvent, or as an emulsion, suspension, or dispersion in a suitable pharmaceutical solvent or vehicle, or as a pill, tablet, or capsule containing a solid vehicle in a manner known in the art. Formulations suitable for parenteral administration conveniently include a sterile oily or aqueous preparation of the active ingredient, preferably isotonic with the blood of the recipient.

[0122] The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The pharmaceutical compositions are advantageously administered by injection or intravenous infusion of a suitable sterile solution. Methods for safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art, and their administration is described in the standard literature.

[0123] The pharmaceutical composition of the present invention can be used in cancer therapy, more specifically, in cancer cell therapy.

[0124] A preferred embodiment of the invention is a pharmaceutical composition as defined herein for use in the treatment of cancer as defined above.

[0125] A further preferred embodiment is a method of treating cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition as defined herein.

[0126] A further preferred embodiment is the use of a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of cancer.

[0127] Imaging and Diagnostics As detailed above, the modified monosaccharide compounds of formula (I) or their precursors of formula (I') are suitable for forming detectable entities bearing a label, preferably a fluorescent label, in particular by click chemistry reactions.

[0128] Thus, the present invention relates to the use of the modified monosaccharide compounds of formula (I) or precursors thereof of formula (I') disclosed herein as a research tool for detecting eukaryotic cells from multicellular organisms, and more particularly for identifying or isolating cancer cells. The present invention further relates to the modified monosaccharide compounds of formula (I) or precursors thereof of formula (I') disclosed herein for medical imaging or diagnosis, preferably for the diagnosis of cancer.

[0129] Further aspects and advantages of the present invention are illustrated in the following examples, which should be considered as illustrative and not limiting. EXAMPLES

[0130] Example 1 Compound synthesis Materials and Methods: Thin layer chromatography was performed on a Merck 60 F254 with detection by UV and / or by baking with sulfuric acid or KMnO4 or phosphomolybdic acid solutions. For flash column chromatography, silica gel 60 40-63 mm was used.

[0131] NMR spectra were obtained on a Bruker Avance 300 or 500 MHz spectrometer using residual protonated solvent as an internal standard. Chemical shifts δ are given in parts per million (ppm) and coupling constants are reported in Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), double doublet (dd), double doublet (ddd). Splitting patterns that could not be interpreted or easily visualized are designated as multiplets (m).

[0132] Mass spectra were acquired on a Waters LCT Premier XE (ToF) with detection by electrospray ionization in positive (ESI+) or negative (ESI-) mode.

[0133] IR-FT spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Characteristic absorptions are in cm -1 Report in.

[0134] Specific rotations were measured at 20° C. and 589 nm in a 10 cm cell using an Anton Paar MCP 300 polarimeter at 20° C.

[0135] All biological and chemical reagents were of analytical or cell culture grade, obtained from commercial sources, and used without further purification.

[0136] Ara-N3 and its precursor VI were prepared by the following procedure:

[0137] [ka]

[0138] It was synthesized according to

[0139] 2:3,4:5-Diisopropylidene-D-arabinose O-methyloxime (Compound II) To a solution of D-(-)-arabinose (4.00 g, 26.6 mmol, 1.0 equiv) in dry pyridine (90 ml) was added methoxyamine hydrochloride (2.72 g, 32.0 mmol, 1.2 equiv) and the mixture was left at room temperature for 15 h. The solvent was removed under reduced pressure and the residue was coevaporated with toluene three times. The residue was resuspended in 2,2-dimethoxypropane (100 ml) and 7-toluenesulfonic acid (1.01 g, 5.33 mmol, 0.2 equiv) was added and the suspension was heated to reflux for 4 h and then stirred at room temperature for a further 15 h. The reaction mixture was filtered through Celite® and the solvent was evaporated. The residue was dissolved in ethyl acetate (200 ml) and washed with saturated aqueous NaCl (2 x 150 mL). Purification by silica flash column chromatography (cyclohexane / ethyl acetate, 9:1) affords a mixture of isomeric 2:3,4:5-diisopropylidene-D-arabinose O-methyloximes (IIE / IIZ) and an unknown impurity (NMR ratio, 6:1:0.4, 5.83 g) as a colorless oil. This mixture was used in the next step without further purification. A second flash column chromatography (dichloromethane / MTBE 98:2) afforded a pure aliquot (2E) which was characterized.

[0140] Isomer (IIE): Rf (CH2Cl2 / MTBE 98:2): 0.35. IR (cm -1 ): 2987 , 2939 , 2900 , 2821 , 1631 , 1456 , 1381 , 1371 , 1241 , 1212 , 1150 , 1065 , 1038 , 887 , 842 . 1 H-NMR (500 MHz, CDCl3) δ: 7.35 (d,1H,J 1,2 6.3 Hz, H-1); 4.46 (dd, 1H, J 2,3 7.1, J 1,2 6.3 Hz, H-2); 4.13 (ddd, 1H, J 3,4 6.9, J 4,5a 6.1, J 4,5b 4.8 Hz, H-4); 4.08 (dd, 1H, J.S 5a,5b 8.5, J 4,5a 6.1 Hz, H-5a); 3.97 (d, 1H, J 2,3 7.1, J 3,4 6.9 Hz, H-3); 3.94 (dd, 1H, J 5a,5b 8.5, J 4,5b 4.8 Hz, H-5b); 3.85 (s, 3H, CH3-O); 1.40 (s, 3H, CH3-C); 1.38 (s, 6H, 2 CH3-C); 1.32 (s, 3H, CH3-C). 13 C-NMR (125 MHz, CDCl3) δ: 147.8 (C-1); 110.8 (C-6); 110.0 (C-7); 79.4(C-3); 76.7 (C-2); 76.6 (C-4); 67.1 (C-5); 62.1 (CH3-O); 27.1, 27.0, 26.9, 25.4 (4 CH3-C). HRMS (ESI + ): [M+H] + (C 12 H 22 NO5 + ) Specification m / z: 260.1492, range: 260.1502.

[0141] 2: 3-Isopropylidene-D-arabinose O-methyloxime (Compound III) 2:3,4:5-diisopropylidene-D-arabinose O-methyloxime (II) (IIE / IIZ) and impurities (1.50 g) in 80% (v / v) aqueous acetic acid (30 ml) were heated on a rotary evaporator at 40° C. at 200 mbar pressure. After 2.5 h, the solvent was removed under reduced pressure and the residue was coevaporated with toluene. After silica gel flash column chromatography (cyclohexane / ethyl acetate 1:1), a mixture of isomers of 2:3-isopropylidene-D-arabinose O-methyloxime (IIIE / IIIZ) (NMR ratio 4:1, 876 mg, 58% over three steps) was obtained as a colorless oil. The purity by NMR was >95%.

[0142] Rf (cyclohexane / ethyl acetate 1:1): 0.24. IR(cm -1 ):3409, 2939, 1373, 1216, 1040, 885. HRMS(ESI + ):[M+H] + (C9H 18 No.5 + ) Calculated m / z: 220.1179, Found: 220.1184.

[0143] Isomer (IIIE): 1 H-NMR (500 MHz, CDCl3) δ: 7.44 (d, 1H, J 1,2 5.5 Hz, H-1); 4.56 (dd, 1H, J 2,3 7.4, J 1,2 5.5 Hz, H-2); 4.07 (dd, 1H, J 2,3 7.4, J 3,4 5.6 Hz, H-3); 4.13 (ddd, 1H, J 3,4 5.6, J 4,5 5.1, J 4,54.7 Hz, H-4); 3.84 (s, 3H, CH3-O); 3.72-3.68 (m, 2H, 2 H-5); 1.42 (s, 3H, CH3-C); 1.38 (s, 3H, CH3-C). 13 C-NMR (125 MHz, CDCl3) δ: 149.1 (C-1); 110.3 (C-6); 79.4 (C-3); 75.0 (C-2); 71.6 (C-4); 63.4 (C-5); 62.2 (CH3-O); 26.9, 26.7 (2 CH3-C).

[0144] Heterosexual body (IIIZ): 1 H-NMR (500 MHz, CDCl3) δ: 6.86 (d, 1H, J 1,2 5.9 Hz, H-1); 4.95 (dd, 1H, J 2,3 7.7, J 1,2 5.9 Hz, H-2); 3.92 (s, 3H, CH3-O); 3.87 (dd, 1H, J 2,3 7.7, J 3,4 6.9 Hz, H-3); 3.82-3.75 (m, 2H, H-4, H-5a); 3.72-3.68 (m, 1H, H-5b); 1.40 (2s, 6H, 2 CH3-C). 13 C-NMR (125 MHz, CDCl3) δ: 151.0 (C-1); 110.9 (C-6); 80.4(C-3); 72.9 (C-2); 72.7 (C-4); 63.5 (C-5); 62.8 (CH3-O); 27.0, 26.5 (2 CH3-C).

[0145] 2:3-イソプロピリデン-5-O-メタンスルホニル-D-アラビノースO-メチルオキシム (Compound IV) To a solution of 2:3-isopropylidene-D-arabinose O-methyloxime (III) (IIIE / IIIZ) (100 mg, 0.46 mmol, 1.0 equiv.) in dry pyridine (2.0 ml) at -20°C, mesyl chloride (0.10 ml, 1.37 mMol, 3.0 equiv.) was added and the reaction mixture was stirred at -20°C for 1.5 h. After quenching the reaction with CH3OH (0.3 ml), the solvent was removed under vacuum. The resulting residue was purified by silica flash column chromatography (cyclohexane / ethyl acetate 6:4) to give a mixture of 2:3-isopropylidene-5-O-methanesulfonyl-D-arabinose O-methyloxime (IVE / IVZ) (NMR ratio, 4:1, 110 mg, 81%) as a colorless oil. Aliquots of the pure (IVE) isomer were obtained and characterized by flash column chromatography (dichloromethane / diethyl ether 9:1) and were >95% pure by NMR.

[0146] Rf (cyclohexane / ethyl acetate 6:4): 0.24. IR(cm -1 ):3500, 2989, 2941, 2824, 1631, 1458, 1350, 1215, 1170, 1067, 1033, 959, 887, 863, 833. HRMS(ESI + ):[M+H] + (C 10 H 20 NO7S + ) Calculated m / z: 298.0955, Found: 298.0947.

[0147] Isomers (IVE): Rf (cyclohexane / ethyl acetate 6:4): 0.20. 1 H-NMR (500 MHz, CDCl3) δ: 7.42 (d, 1H, J 1,2 5.6 Hz, H-1); 4.56 (dd, 1H, J 2,3 6.8, J 1,2 5.6 Hz, H-2); 4.41 (dd, 1H, J 5a,5b 11.0, J4,5a 2.7 Hz, H-5a); 4.28 (dd, 1H, J 5a,5b 11.0, J 4,5b 5.7 Hz, H-5b); 4.03 (ddd, 1H, J 3,4 7.0, J 4,5b 5.7, J 4,5a 2.7 Hz, H-4); 4.01 (dd, 1H, J 2,3 6.8, J 3,4 7.0 Hz, H-3); 3.85 (s, 3H, CH3-O); 3.06 (s, 3H, CH3-S); 1.41 (s, 3H, CH3-C); 1.39 (s, 3H, CH3-C). 13 C-NMR (125 MHz, CDCl3) δ: 148.2 (C-1); 110.9 (C-6); 77.9 (C-3); 76.2 (C-2); 70.9 (C-5); 70.8 (C-4); 62.3 (CH3-O); 37.8 (CH3-S); 27.0, 26.9 (2 CH3-C).

[0148] Isomer (IVZ): 1 H-NMR (300 MHz, CDCl3) δ: 6.87 (d, 1H, J 1,2 5.9 Hz, H-1); 4.96 (dd, 1H, J 2,3 7.3, J 1,2 5.9 Hz, H-2); 4.45 (dd, 1H, J 5a,5b 11.4, J 4,5a 2.4 Hz, H-5a); 4.29 (dd, 1H, J 5a,5b 11.4, J 4,5b 7.9 Hz, H-5b); 3.98 (ddd, 1H, J 4,5b 7.9, J 4,3 7.5, J 4,5a 2.4 Hz, H-4); 3.93 (s, 3H, CH3-O); 3.84 (dd, 1H, J 3,4 7.5, J 3,27.3 Hz, H-3); 3.06 (s, 3H, CH3-S); 1.40 (s, 3H, CH3-C); 1.39 (s, 3H, CH3-C). 13 C-NMR (75 MHz, CDCl3) δ: 150.7 (C-1); 111.2 (C-6); 79.1 (C-3); 72.9 (C-2); 71.3 (C-5); 70.9 (C-4); 62.9 (CH3-O); 37.9 (CH3-S); 27.0, 26.6 (2 CH3-C).

[0149] 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloxime (Compound V): To a solution of (IVE / IVZ) (810 mg, 2.72 mmol, 1.0 equiv) in N,N-dimethylformamide (30.0 mL, 0.10 M) was added sodium azide (531 mg, 8.17 mmol, 3.0 equiv) and the reaction mixture was heated at 80° C. for 15 h. The solvent was then removed under reduced pressure and the residue was purified by flash column chromatography (cyclohexane / ethyl acetate 9:1) to give a mixture of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloximes (VE / VZ) (NMR ratio 7:3, 637 mg, 96%) as a yellowish oil. A fraction of (VE) was isolated for characterization by flash column chromatography (dichloromethane / MTBE 97:3). The purity by NMR was >95%.

[0150] Rf (cyclohexane / ethyl acetate 8:2): 0.30. IR(cm -1 ):3458, 2989, 2939, 2823, 2100, 1630, 1443, 1373, 1213, 1164, 1066, 1036, 885, 865. HRMS(ESI + ):[M+H] + (C9H 17 N4O4 + ) Calculated m / z: 245.1245, measured: 245.1250.

[0151] Opposite sex (VE): Rf(ジクロロメタン / MTBE 97:3):0.23. 1 H-NMR (500 MHz, CDCl3) δ: 7.42 (d, 1H, J 1,2 5.8 Hz, H-1); 4.54 (dd, 1H, J 2,3 7.2, J 1,2 5.8 Hz, H-2); 3.98 (dd, 1H, J 2,3 7.2, J 3,4 6.4 Hz, H-3); 3.92 (dddd, 1H, J 3,4 6.4, J 4,5b 6.2, J 4,OH 3.9, J 4,5a 3.8 Hz, H-4); 3.85 (s, 3H, CH3-O); 3.46 (dd, 1H, J 5a,5b 12.5, J 4,5a 3.8 Hz, H-5a); 3.42 (dd, 1H, J 5a,5b 12.5, J 4,5b 6.2 Hz, H-5b); 2.61 (d, 1H, J 4,OH 3.9 Hz, OH); 1.41 (s, 3H, CH3-C); 1.39 (s, 3H, CH3-C). 13 C-NMR (125 MHz, CDCl3) δ: 148.4 (C-1); 110.6 (C-6); 78.8 (C-3); 75.8 (C-2); 71.5 (C-4); 62.3 (CH3-O); 53.7 (C-5); 27.0, 26.8 (2 CH3-C).

[0152] Heterosexual (VZ): 1 H-NMR (500 MHz, CDCl3) δ: 6.86 (d, 1H, J 1,2 6.1 Hz, H-1); 4.94 (dd, 1H, J 2,3 7.2, J 1,26.1 Hz, H-2); 3.93 (s, 3H, CH3-O); 3.87 (ddd, 1H, J 3,4 7.5, J 4,5b 6.4, J 4,5a 2.8 Hz, H-4); 3.82 (dd, 1H, J 3,4 7.5, J 2,3 7.2 Hz, H-3); 3.47 (dd, 1H, J 5a,5b 12.8, J 4,5a 2.8 Hz, H-5a); 3.39 (dd, 1H, J 5a,5b 12.8, J 4,5b 6.4 Hz, H-5b); 1.40 (s, 3H, CH3-C); 1.38 (s, 3H, CH3-C). 13 C-NMR (75 MHz, CDCl3) δ: 150.8 (C-1); 111.0 (C-6); 80.0 (C-3); 72.9 (C- 2); 72.5 (C-4); 62.8 (CH3-O); 53.5 (C-5); 26.9, 26.5 (2 CH3-C).

[0153] 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose (Compound VI) To a solution of (VE / VZ) (820 mg, 3.36 mmol, 1.0 equiv) in 80% (v / v) aqueous acetic acid (120 mL) was added formaldehyde (0.8 mL) and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and co-evaporated with toluene to ensure complete elimination of acetic acid. The crude compound 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (VI) (682 mg) was obtained.

[0154] colorless oil Rf (cyclohexane / ethyl acetate 7:3): 0.56. IR (cm -1 ): 3408, 2988, 2936, 2100, 1733, 1440, 1373, 1238, 1213, 1164, 1063, 863. 1H-NMR (500 MHz, CDCl3) δ: 9.79 (d, 1H, J 1,2 1.2 Hz, H-1); 4.41 (dd, 1H, J 2,3 6.4, J 1,2 1.2 Hz, H-2); 4.04 (dd, 1H, J 2,3 6.4, J 3,4 6.1 Hz, H-3); 3.90 (ddd, 1H, J 4,5b 6.4, J 3,4 6.1, J 4,5a 3.4 Hz, H-4); 3.51 (dd, 1H, J 5a,5b 12.8, J 4,5a 3.4 Hz, H-5a); 3.43 (dd, 1H, J 5a,5b 12.8, J 4,5b 6.4 Hz, H-5b); 1.47 (s, 3H, CH3-C); 1.37 (s, 3H, CH3-C). HRMS (ESI + ): [2M+Na] + (C 16 H 26 N6NaO8 + ) Calculated m / z: 453.1704, Found: 453.1726.

[0155] 5-Azido-5-deoxy-D-arabinofuranose (Compound VII, Ara-N3) To a solution of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (VI) (100 mg) in a CHCl / HO mixture (20:1, 21 mL) was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 1 h. The solvent was then evaporated and the crude residue was resuspended in water and lyophilized. After silica flash column chromatography (dichloromethane / methanol 92:8), compound 5-azido-5-deoxy-D-arabinofuranose or Ara-N3 (VII) (50 mg, 58% over two steps from compound (V)) was obtained as a colorless oil as a mixture with the / β anomer (NMR ratio, 55:45). The purity by NMR was >95%.

[0156] Rf(dichloromethane / methanol 92:8): 0.28. IR(cm -1 ):3367, 2106, 1281 1040. HRMS(ESI + ):[M+H-N2] + (C5H10NO4 + ) Calculated m / z: 148.0604, observed: 148.0610.

[0157] Anomer alpha (VIIα): 1 H-NMR (500 MHz, D2O) δ: 5.24 (d, 1H, J 1,2 2.9 Hz, H-1); 4.17 (ddd, 1H, J 3,4 6.4, J 4,5b 5.8, J 4,5a 3.5 Hz, H-4); 4.01 (dd, 1H, J 2,3 4.6, J 1,2 2.9 Hz, H-2); 3.97 (dd, 1H, J 3,4 6.4, J 3,2 4.6 Hz, H-3); 3.64 (dd, 1H, J 5a,5b 13.6, J 4,5a 3.5 Hz, H-5a); 3.44 (dd, 1H, J 5a,5b 13.6, J 4,5b 5.8 Hz, H-5b). 13 C-NMR (125 MHz, D2O) δ: 101.0 (C-1); 81.3 (C-4); 81.2 (C-2); 76.3 (C- 3); 51.5 (C-5).

[0158] Anomer beta (VIIβ): 1 H-NMR (500 MHz, D2O) δ: 5.28 (br d, 1H, J 1,2 3.1 Hz, H-1); 4.10-4.05 (m, 2H, H-2, H-3); 3.89 (ddd, 1H, J3,4 7.1, J 4,5b 6.5, J 4,5a 3.5 Hz, H-4); 3.59 (dd, 1H, J 5a,5b 13.3, J 4,5a 3.5 Hz, H-5a); 3.42 (dd, 1H, J 5a,5b 13.3, J 4,5b 6.5 Hz, H- 5b). 13 C-NMR (125 MHz, D2O) δ: 95.2 (C-1); 79.6 (C-4); 75.8 (C-2); 74.7 (C-3); 52.6 (C-5).

[0159] Example 2 Labeling of bladder non-tumor and tumor eukaryotic cells Materials and Methods: Cell culture: Human urothelial cells (SV-HUC-1) and human bladder carcinoma (T24) were purchased from ATCC (Manassas, Van, USA). These cell lines were grown in RPMI 1640 (Lonza Biowhttaker) containing glutamine 1 medium supplemented with 10% fetal bovine serum (FBS) (VWR international SAS). The culture medium was changed every 2 days. Cell passage was performed using Tryp-LE express 1× (Gibco). Cell viability was assessed using the trypan blue exclusion test.

[0160] Cell exposure to Ara-N3 probe: SV-HUC-1 and T24 cells were cultured in a 24-well plate at 5 x 10 4The cells were seeded at a density of 1000 cells / well. The cells were then incubated at 37°C, 5% CO2 for 24 hours. After that, the culture medium in each well was discarded and the attached cells were washed twice with phosphate-buffered saline (PBS) (Lonza Biowhittaker) before being exposed to the Ara-N3 probe (10 mM). The probe was added to culture medium containing various FBS concentrations (3% and 10%). The cells were then incubated again at 37°C, 5% CO2 for 24 hours. Control cells were cultured using 5% or 10% FBS-supplemented culture medium without Ara-N3.

[0161] Anti-biotin antibody labeling: Fluorescent incorporation of the Ara-N3 probe was visualized by copper-free click chemistry using sulfo-DBCO-biotin (1 mM) followed by labeling with mouse anti-biotin Alexa Fluor 488 antibody conjugate (0.62 mg / ml stock, Jackson ImmunoResearch, 1 / 10 dilution). Click reactions were performed as follows: at 24 h of incubation, the culture medium was removed and the attached cells were washed twice with PBS. The cells were detached using Tryp-LE, washed twice with PBS, and centrifuged at 13000 g for 2 min. The cell pellet was resuspended in 10 μL of sulfo-DBCO-biotin and incubated at 37° C. in the dark for 30 min. The cells were then washed twice with PBS, centrifuged, and the cell pellet was resuspended in 10 μL of mouse anti-biotin antibody solution before being incubated at room temperature in the dark. The cells were then washed twice with PBS and a small spot (5 μL) of the cell suspension was placed on a polysine® adhesive slide (VWR international SAS) and placed in the dark until dry (20 min). The spot was fixed with 4% paraformaldehyde for 20 min in the dark at room temperature. The slide was then washed twice with PBS, covered with a square cover slip (VWR international SAS) using glycerol mounting medium (Dako), and stored at 4° C. in the dark.

[0162] Fluorescence microscopy: Fluorescence acquisition was recorded using an Olympus Microscopy IX83 (Olympus Life Sciences) equipped with a 60X / 1.3 digital aperture / 1.4 SRI (silicone refractive index) objective and a Hamamatsu Orca Flash 4 LT camera with a calibrated pixel size of 109 nm / pixel. Excitation light was generated by an X-CITE 120 LED using an AT180 / 30X excitation filter, and the signal was monitored using an AT535 / 40m emission filter. The green light exposure time was 600 ms, and the white light exposure time was determined by DIC to be 340 ms. The size of the acquisition area analyzed by cellSens Dimension V1.16 software varied depending on the number of cells contained in the spot.

[0163] Image processing and fluorescence quantification: Images were processed using the counting and measuring units of cellSens Dimension V1.16 software. Background noise was removed as follows: first, an ROI was defined on the background of the image and the average pixel intensity was calculated. The value thus obtained was subtracted from all pixels of the image. The fluorescence intensity of each cell was determined from a threshold set at a value of 3030. All pixels with an intensity below 3030 were not counted.

[0164] result:

[0165] [Table 1]

[0166] [Table 2]

[0167] The results in Table 1 and Table 2 show that after 24 hours, the percentage of labeled cells was higher in T24 cells compared to SV-HUC1 cells, demonstrating a higher uptake of the Ara-N3 probe in neoplastic bladder cells. More specifically, the intensity / μm higher than 500,000 was observed. 2 At 3% FBS, the percentage of labeled cells with low intensity was 21.76% for SV-HUC1 and 66.81% for T24, whereas at 10% FBS, the percentage was 14.62% for SV-HUC1 and 79.42% for T24, whereas the percentage was the opposite. The overall intensity distribution is different between normal and cancer cells.

[0168] Example 3 Labeling of non-cancerous and cancerous eukaryotic cells Materials and Methods: The same experiment as that in Example 2 was carried out using other cancer cells and corresponding non-cancer cells. The culture medium was adapted to the test cells and supplemented with 10% fetal bovine serum (FBS). The Ara-N3 probe (10 mM) was added to the culture medium with FBS concentration (10%). The incubation time was matched to the 1st or 2nd doubling time of the test cells (see table below).

[0169] The cancer / non-cancer ratio of the fluorescent signal was calculated based on the fluorescence intensity obtained with Ara-N3 tested on cancer cells and corresponding non-cancer cells.

[0170] result: The results are shown below (Table 3) and demonstrate that the fluorescence intensity in the tested cancer cell lines was higher (ratio higher than 1) compared to the corresponding non-cancer cell lines.

[0171] [Table 3A]

[0172] [Table 3B]

[0173] [Table 3C]

[0174] Similar experiments were performed using other human cancer cells, more specifically, the following human cancer cells (Table 4), compared with the corresponding non-cancerous cells:

[0175] [Table 4]

[0176] Results similar to those in Table 3 are expected, i.e., higher fluorescence intensities (specifically ratios greater than 1) for the tested cancer cell lines compared to the corresponding non-cancer cell lines.

[0177] Example 4 Comparison of labeling of Ara-N3 and KDO-N3 KDO-N3 (8-azido-3,8-dideoxy-D-manno-octulosonic acid) is described in patent application WO2013 / 107759 as a useful tool for labelling bacteria.

[0178] Ara-N3 and KDO-N3 were assayed under the same conditions as in Example 2 in other human cell lines: HeLa, Raw, and macrophages.

[0179] The cell line was not labeled with KDO-N3 (no fluorescence intensity was observed), whereas the cell line was labeled with Ara-N3.

[0180] Example 5 Non-toxicity of Ara-N3 probe to cells (in vitro / in vivo) A- In vitro experiments Materials and Methods The Ara-N3 probe was prepared as detailed above.

[0181] The cell lines (detailed in the table below) were prepared by Inserm / Aquiderm Transfer Cells. Cell culture and cytotoxicity testing of the cell lines was performed in the laboratory of INSERM U1035, also by Aquiderm Transfer Cells.

[0182] Isolation of peripheral blood mononuclear cells (monocytes / lymphocytes) Human mononuclear cells were isolated from peripheral blood from two different healthy donors, according to the agreement established by the Etablissement FranCais du Sang d'Aquitaine. The technique used was Ficoll density gradient isolation. Briefly, to separate the various blood elements, blood diluted with phosphate-buffered saline solution (PBS) was placed on a sucrose solution (Ficoll, Eurobio) and centrifuged. Between the ficoll and the plasma, a ring of cells was settled, composed of monocytes and lymphocytes. They were harvested and washed in PBS for counting on Malassez cells.

[0183] Human CD14+ monocytes were isolated from lymphocytes by magnetic beads (Miltenyi Biotech) bound to anti-CD14 antibodies. The monocytes were incubated in PBS buffer for 15 minutes in the presence of beads at a concentration determined according to cell number. The cells were then washed with PBS and then loaded onto a column mounted on a strong magnet. CD14+ cells were bound to the anti-CD14 magnetic balls and may remain bound to the column. After washing, the column was removed from the magnetic support and the cells were released by washing the column in PBS buffer.

[0184] Monocyte numbers were determined by counting on Malassez cells.

[0185] cell culture Cells were cultured in one of the following different culture media in an oven at 37°C, 5% CO2, and 95% humidity: - RPMI 1640 medium supplemented with 10% complement-depleted FCS (fetal calf serum) and the antibiotic penicillin (100 IU / ml) and streptavidin (100 μg / ml) for pairing with monocytes / THP1. - DMEM medium supplemented with 10% complement-depleted FCS (fetal calf serum) and the antibiotic penicillin (100 IU / ml) and streptavidin (100 μg / ml) that binds Hacat / A431.

[0186] Culture medium was changed every 2 days throughout the study.

[0187] When the cultures reached 70-80% confluence, the adherent cells were detached using 10% trypsin-EDTA, and cell viability was assessed by lipan blue exclusion.

[0188] Contact with Ara-N3 probe All cells were cultured at 1 x 10 for MTS proliferation assay. 4 Cells / well density is 96 wells, 5*10 for Annexin V test / IP test 4 The cells were seeded in either 24-well culture plates at a density of 1000 cells / well. The cells were then incubated at 37°C, 5% CO2 for 24 hours. After 24 hours, the culture medium in all wells was replaced and the cells attached to the bottom of the wells were rinsed twice with PBS (Eurobio), or centrifuged and the cells in suspension were washed twice with PBS, and then exposed to various concentrations of Ara-N3 probes at 1, 10, 50, and 100 mM. The cells were incubated at 37°C, 5% CO2 for 48 hours.

[0189] Control cells were cultured with capture culture medium without the Ara-N3 probe.

[0190] Annexin V / IP cytotoxicity assay This test requires the use of the Annexin V-APC kit (Biolegend) to detect apoptosis / necrosis and utilizes the ability of Annexin V to bind to membrane phosphatidylserine (PS) and the property of propidium iodide (PI) to intercalate into DNA.

[0191] Indeed, living cells express PS on the inner layer of their membrane bilayer. Therefore, PS is less accessible to annexin V-APC. Similarly, cells maintain the integrity of their DNA away from the nucleus, which also makes the DNA less accessible to IP. When a cell enters apoptosis (programmed cell death), a series of events occurs. Among these events is the inversion of the layers of the cytoplasmic membrane. PS, which was exposed on the inner membrane, is found on the outer membrane of vesicles formed by the breakdown of the lipid bilayer. Therefore, PS can fix annexin V-APC molecules and emit fluorescence at 660 nm, which can be detected by flow cytometry.

[0192] During the process of apoptosis, the cell then undergoes necrosis. Disintegration of the nuclear membrane and release of DNA into the culture medium occurs. PI can intercalate between the bases of DNA and emit a second fluorescence at 370-550 nm that is also detectable by flow cytometry.

[0193] Briefly, cells were incubated with various concentrations of Ara-N3 probe for various times (24 and 48 h) and then harvested, trypsinized (for adherent cells) and washed with PBS. The cells were counted in trypan blue on Malassez cells and then incubated in labeling buffer (100 μL) in the presence of Annexin V-APC (5 μL) and IP (10 μL) for 15 min in the dark at room temperature. After adding 100 μL of labeling buffer, the cells were then directly analyzed by cytometry.

[0194] The non-toxic concentrations of Ara-N3 for the tested cells are summarized in Table 5 below.

[0195] B- In vivo experiments Materials and Methods The Ara-N3 probe was prepared as detailed above.

[0196] Ten female NMRI nude mice (6 weeks old, Janvier, Le Genest-Saint Isle, France) were included in the assay. Three were intravenously injected with 50 mg / mouse / injection of Ara-NThree for a cumulative dose of 200 mg every other day for 8 days (4 injections), and another three mice were treated with 50 mg of Ara-Nthree for a cumulative dose of 400 mg in addition to a daily intake of 8 ml of water for 8 days. As a control group, four tumor-bearing mice were left untreated.

[0197] At the end of treatment, mice were euthanized and dissected: heart, lung, brain, uterus, ovaries, kidneys, skeletal muscle, spleen, pancreas, abdominal fat, liver, skin, stomach, colon, cecum, small intestine, mesenteric lymph nodes, axillary lymph nodes, brachial lymph nodes, and whole blood were collected and frozen for analysis.

[0198] result Addition of Ara-N3 to drinking water (up to 6.25 mg / mL) did not alter water intake in mice.

[0199] Treatment with Ara-N3 either in the drinking water or intravenously did not induce any weight loss.

[0200] [Table 5]

[0201] Example 6 Specific labeling of Ara-N3 probes to tumor cell lines (in vivo) Materials and Methods All animal experiments were performed in accordance with the European Economic Community institutional guidelines for the use of laboratory animals (EU Directive 2010 / 63 / EU) and were approved by the French Ministry of Higher Education and Research under the reference number APAFIS#8854-2017031314338357.

[0202] Tumor xenografts subcutaneously Female NMRI nude mice (6 weeks old, Janvier, Le Genest-Saint Isle, France) were injected subcutaneously in the flank with 8 × 10 6 Panc-1 cells were injected.

[0203] Ara-N3 treatment by intravenous infusion: A cumulative dose of 20 mg Ara-N3 was evaluated.

[0204] Ten days after subcutaneous injection of tumor cells, Panc-1 tumor-bearing mice were treated with 1.8 mg / mouse / injection of Ara-N3 every other day for 21 days (11 injections) for a cumulative dose of 20 mg each (n=3 mice per dose).

[0205] Untreated: As a control group, three tumor-bearing mice per cell line were untreated (Ctr).

[0206] Contrast administration DBCO-IRDye800CW Mice treated by i.v. - 24 hours after the last treatment injection, two Panc-1 tumor-bearing mice per treatment condition were intravenously injected with 100 μg DBCO-IRDye800CW (Ara-N3+Fluo).

[0207] Control mice - One mouse bearing an untreated Panc-1 tumor was injected intravenously with 100 μg of DBCO-IRDye800CW (Ctr+Fluo).

[0208] Fluorescence imaging data analysis (ex vivo) Mice were euthanized and dissected 48 hours after injection of DBCO-IRDye800CW.

[0209] Ex vivo fluorescence imaging was performed on skeletal muscle and tumors.

[0210] Semi-quantitative data was obtained from the fluorescence images by manually defining a region of interest (ROI) adjusted on the tumor area to be quantified. The total fluorescence signal measured in the ROI is divided by the number of pixels in the ROI. Thus, data is expressed as relative fluorescence units (RLU) per pixel on skeletal muscle and tumor. Then, the fluorescence signal ratio between tumor and skeletal muscle is calculated (tumor / muscle ratio).

[0211] The results are shown in Figure 1.

Claims

[Claim 1] 1. An in vitro method for labeling or detecting or targeting a eukaryotic cell from a multicellular organism, comprising: a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound or a precursor thereof; b) contacting the sample of step (a) with a compound having a first reactive group, optionally in the presence of copper; and c) optionally detecting the compound of step (b) bound to the monosaccharide of step (a) to detect eukaryotic cells. wherein the at least one modified monosaccharide compound has the following formula (I): 【Chemistry 1】 wherein X is a second reactive group, and the first reactive group and the second reactive group are capable of reacting with each other in a click chemistry reaction.

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