Piperazine-derived fluorescent compounds and use of same in the detection, localization and / or quantification of MDR proteins

Abstract

The present invention relates to fluorescent compounds that can be used for the selective detection, localization and / or quantification of MDR proteins such as P-gp in any membrane system expressing these MDR proteins at the cellular and subcellular levels (intracellular organelles), at the tissue, organ and whole-organism levels. These compounds allow the selective labeling of MDR proteins such as P-gp.

Description

Description Title of the invention: Fluorescent compounds derived from piperazine and their use in the detection, localization and / or quantification of MDR proteins

[0001] The present invention relates to fluorescent compounds useful for the selective detection, localization, and / or quantification of MDR proteins, such as P-gp (known by the English term "permeability glycoprotein"), on any membrane system expressing these MDR proteins, at the cellular and subcellular level (intracellular organelles), as well as at the tissue, organ, and whole-organism level. These compounds enable the selective labeling of MDR proteins such as P-gp.

[0002] Multidrug resistance (MDR) proteins are membrane efflux transporters (pumps) of xenobiotics (pollutants, drugs, and / or any other molecule foreign to the cell). Highly ubiquitous and extremely conserved throughout evolution, they are expressed in the membrane systems of cells in all organisms, from prokaryotes to eukaryotes. MDR proteins belong to the ABC transporter superfamily, also known as ATP-binding cassettes.

[0003] The main MDR proteins include P-glycoprotein or P-gp which is encoded by the ABCB1 gene (originally called MDR1), MRP1 protein encoded by the ABCC1 gene, MRP2 protein encoded by the ABCC2 gene, MRP7 protein encoded by the ABCC10 gene, proteins encoded by the ABCC3-6 and ABCC11 genes, and MXR / BCRP protein encoded by the ABCG2 gene.

[0004] In humans and mammals, several healthy tissues naturally express P-gp, including the blood-brain barrier, adrenal glands, pancreas, liver and kidneys, hematopoietic cells, and stem cells, among others. The presence of P-gp has also been demonstrated in bacteria and parasites, protozoa, aquatic invertebrates, fruit flies, and plants (Arabidobsis and maize). In other eukaryotic and prokaryotic organisms (animal and plant), and in fish, gene expression of ABCB1 and other ABC transporter members such as ABCC1-5, ABCB3, ABCB11, and ABCG2 has also been observed.

[0005] MDR proteins are efflux pumps with a detoxifying role. They recognize a very wide variety of substrates, ranging from organic molecules with highly diverse physicochemical properties to heavy metals. Through this role, MDR proteins play a cellular defense function against xenobiotics.

[0006] MDR proteins are naturally expressed on the membrane systems of virtually all living organisms. This is the natural basal expression. In the presence of xenobiotics, the expression level of these proteins can increase in a dose-dependent manner. Currently, this basal or induced expression level can be quantified in vitro using protein-specific antibodies via immunocytological or immunohistological approaches. However, the use of antibodies to quantify MDR proteins has the following drawbacks: such antibodies lack membrane permeability and can diffuse poorly or not at all into cell masses (e.g.spheroids, biopsies, tissues, organisms), the method of identifying and quantifying MDR proteins using antibodies is indirect with a long (over 1 hour) and species-dependent protocol, the antibody production processes are expensive and use biological raw materials (biological processes by animal experimentation (ascites process / blood sampling) or by bioreactors (use of calf serum)), not to mention the ethical considerations that may pose a problem.

[0007] MDR proteins are also overexpressed in all tumor cells. They are responsible for the concept of "Multidrug Resistance (MDR)," or the cross-resistance of cancer cells to anticancer agents. Indeed, some tumors exhibit an inherent resistance profile linked to a high basal expression level of P-gp and / or other MDR proteins. For other tumors, the expression level of P-gp and / or other MDR proteins increases during treatment (treatment resistance). In both cases, the massive efflux of chemotherapy drugs renders the treatment ineffective. The xenobiotic efflux activity of these MDR pumps can be circumvented by using other, more specific target substrates of these pumps called "competitive inhibitors."Indeed, by combining an inhibitor with a greater affinity / specificity for the MDR pump than the chemotherapeutic molecule, the efflux activity of the pump is focused. The inhibitors primarily target the efflux of the inhibitor, rather than the chemotherapy drug. This allows the chemotherapy drug to remain within the cancer cell and exert its effect. These competitive inhibitors are preferentially recognized and transported by MDR pumps from the inside to the outside of cancer cells. The most well-known fluorescent and non-fluorescent competitive inhibitors described in the literature include cyclosporine (cyclosporin A), calcein AM, rhodamine 123, reversin, and others.

[0008] The efflux / transport activity of MDR pumps can be analyzed / demonstrated at the cellular level, preferentially using flow cytometry or fluorescence imaging. However, neither of these methods allows for the localization of MDR proteins within a cell and / or cell mass, nor for their quantification within a cell and / or cell mass.

[0009] In particular, to assess the affinity of an MDR protein for different substrates, an activity assay (efflux modality) of the protein can be applied. This requires combining two molecules: (1) a known substrate efflued by P-gp (e.g., cyclosporine A, known to be a competitive substrate of MDR proteins coupled to a fluorochrome) and a molecule whose efflux by P-gp is to be determined (a novel cyclosporine A derivative). For this type of approach, at least one of the two molecules must be fluorescent. The evaluation method is as follows: (i) both molecules enter the cell, (ii) the MDR protein efflues the preferential molecule with which it has the highest affinity, and (iii) intracellular fluorescence is measured using imaging or flow cytometry methodologies.The measured intracellular fluorescence intensity corresponds to the overall quantification of the accumulated (persistent / non-effluated) fluorescent molecule in the cell (see figure 3a below).

[0010] Other methods assess uptake, i.e., the efflux of fluorescent compounds by MDR proteins. The measured intracellular fluorescence intensity corresponds to the overall quantification of the fluorescent molecule accumulated (persistent / non-effluated) in the cell (see Figure 3b below).

[0011] Thus, the methods described above demonstrate the dynamic transporter role of MDR proteins. This involves a very rapid efflux mechanism of a fluorescent molecule from inside the cell to the outside. of the cell via MDR proteins. Consequently, none of these fluorescent molecules bind directly and permanently to MDR proteins, which prevents their direct detection, localization, and quantification. In other words, such fluorescent substrates are used to study the activity of MDR proteins (substrate efflux), and not to detect, localize, and / or quantify MDR proteins, i.e., to quantify MDR protein expression as such.

[0012] International application W02021 / 064191 describes fluorescent peptide compounds (e.g., hydroxyproline and lysine) that can be used to quantify MDR proteins as an alternative to antibodies. Such tracers are capable of permanently binding to MDR proteins.

[0013] However, there is a need for other fluorescent compounds capable of enabling the localization and / or quantification of MDR proteins in a cell and / or cell mass, regardless of the species, in a universal manner. In particular, the aim of the present invention is to provide new compounds with improved performance in terms of selectivity for MDR proteins (selective labeling), brightness, intensity of the detectable signal (detection sensitivity), and / or stability in biological media. Another aim of the invention is to provide such compounds in a simple, economical, and easily identifiable manner using known analytical techniques (e.g., high-resolution mass spectrometry, or HRMS).

[0014] The invention has as its first object a compound corresponding to the following formula (I):

[0015] [Chem 1] in which: - R. 1 represents a fluorochrome radical chosen from among the bodipy radicals, - L represents a connecting arm that is present (n = 1) or absent (n = 0), - R 2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole, - R 3 , R 4 , R 5 and R 6 Whether identical or different, they represent a hydrogen atom or an alkyl group. - R 7 and R 8 Whether identical or different, they represent a hydrogen atom or an alkyl group. - R 9 and R 10 , identical or different, represent a hydrogen atom or an alkyl group, and - Y represents an oxygen atom, a sulfur atom, or an NH radical.

[0016] The compounds (I) of the invention allow for the direct measurement of MDR protein expression, unlike existing methods for measuring MDR protein efflux activity (e.g., the "MDR Assay Kit," Abeam®). Indeed, although also based on the use of small molecules, these methods do not allow for the direct measurement of MDR protein expression. Furthermore, they are only suitable for use on cells in suspension or monolayer.

[0017] Furthermore, the compounds (I) of the invention exhibit good performance in terms of selectivity for MDR proteins (selective labeling), brightness, intensity of the detectable signal (detection sensitivity), and / or stability in biological media. Moreover, they can be prepared by organic synthesis in a simple and economical manner and are easily identifiable with known analytical techniques (e.g., high-resolution mass spectrometry "HRMS", NMR, etc.), particularly thanks to the R group 2 and thus represent an interesting alternative (i) to the use of organic compounds without aromatic groups, which are difficult to identify, and (ii) to the use of antibodies from a technical standpoint (species-dependent protocols, lengthy development), an economic standpoint (cost comparison), and an ethical standpoint, considering the methods of antibody production through animal experimentation. All these characteristics constitute a significant added value of the invention compared to the prior art.

[0018] R 3 , R 4 , R 5 and R 6

[0019] R 3 , R 4 , R 5 and R 6, identical or different, represent a hydrogen atom or an alkyl group.

[0020] The alkyl group as group R 3 , R 4 , R 5 and R 6 It can be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical can comprise from 1 to 5 carbon atoms, and preferably from 1 to 3 carbon atoms. An alkyl radical is advantageously a methyl or ethyl group.

[0021] At least one of the R groups 3 , R 4 , R 5 or R 6 is preferably a hydrogen atom, particularly preferably at least two of the R groups 3 , R 4 , R 5 or R 6 are hydrogen atoms, more particularly preferred to at least three of the R groups 3 , R 4 , R 5 or R 6are hydrogen atoms, and even more particularly preferred are the R groups 3 , R 4 , R 5 and R 6 are hydrogen atoms.

[0022] R 7 and R 8

[0023] R 7 and R 8 , identical or different, represent a hydrogen atom or an alkyl group.

[0024] The alkyl group as group R 7 and R 8 It can be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical can comprise from 1 to 5 carbon atoms, and preferably from 1 to 3 carbon atoms. An alkyl radical is advantageously a methyl or ethyl group.

[0025] At least one of the R groups 7 or R 8 is preferably a hydrogen atom, and particularly preferably the R groups 7 and R 8 are hydrogen atoms.

[0026] R 9 and R 10

[0027] R 9 and R 10 , identical or different, represent a hydrogen atom or an alkyl group.

[0028] The alkyl group as group R 9 and R 10 It can be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical can comprise from 1 to 5 carbon atoms, and preferably from 1 to 3 carbon atoms. An alkyl radical is advantageously a methyl or ethyl group.

[0029] At least one of the R groups 9 or R 10 is preferably a hydrogen atom, and particularly preferably the R groups 9 and R 10 are hydrogen atoms.

[0030] According to a particularly preferred embodiment of the invention, R 7 , R 8 , R 9 and R 10 are hydrogen atoms.

[0031] R 2

[0032] R 2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole.

[0033] In a particularly preferred way, R 2 is a quinoline or an isoquinoline.

[0034] In the compound of formula (I), the nitrogenous heteroaromatic group R 2 is specifically covalently linked to the Y radical by a carbon atom. In other words, the attachment point of the R group 2 is in particular a carbon atom.

[0035] According to a particularly preferred embodiment of the invention, compound (I) corresponds to the following formula (la) or (Ib):

[0036] [Chem 2] in which R. 1 , L, R 3 , R 4 , R 5 , R 6 , R7 , R 8 , R 9 and R 10 are as defined in the invention.

[0037] Y

[0038] Y represents an oxygen atom, a sulfur atom, or a radical NH, and preferably an oxygen atom.

[0039] Y is a divalent group.

[0040] According to a particularly preferred embodiment of the invention, the compound (I) of the invention corresponds to the following formula (lal) or (Ibl):

[0041] [Chem 3] ( ia1 ) (Ib1) in which R 1 , L, R 3 , R 4 , R 5 and R 6 are as defined in the invention.

[0042] R 1

[0043] R 1 represents a fluorochrome radical chosen from among the bodipy radicals.

[0044] In the invention, a fluorochrome radical is understood to be a chemical substance capable of emitting fluorescence after excitation. Preferably, the fluorochrome of the invention is characterized by an excitation wavelength ranging from approximately 190 to 800 nm. Preferably, the fluorochrome of the invention is characterized by an emission wavelength ranging from approximately 400 to 1100 nm.

[0045] Thanks to this fluorochromic radical, a fluorescent compound of formula (I) is obtained.

[0046] Such a fluorescent compound of formula (I) is preferably characterized by an excitation wavelength ranging from approximately 190 to 800 nm.

[0047] Such a fluorescent compound of formula (I) is preferably characterized by an emission wavelength ranging from approximately 400 to 1100 nm.

[0048] In a particularly preferred manner, the compound is characterized by a maximum difference between excitation wavelength and maximum emission wavelength of at least 2 to 10 nm approximately.

[0049] The fluorochrome radical R. 1 preferably corresponds to the following formula (II):

[0050] [Chem 4] in which: - R 11 represents a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, - R 12 , R 13 , R 14 , R 15 , R 16 , and R 17, identical or different, represent a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, - it being understood that the fluorochrome radical R 1 has an attachment point (noted *) to the ligand L when n = 1 or to the nitrogen atom of piperazine when n = 0, chosen from the carbon atom in position 1, the carbon atom in position 2, the carbon atom in position 3, the carbon atom in position 5, the carbon atom in position 6, the carbon atom in position 7, and the carbon atom in position 8.

[0051] In other words, when the attachment point is the carbon atom at position x (x = 1, 2, 3, 5, 6, 7, or 8) of the fluorochrome radical R. 1 , the group likely to be present at this position x (i.e. R. 12 , R 13, R 14 , R 15 , R 16 , R 17 , or R 11 respectively) is absent.

[0052] According to a preferred embodiment of the invention, the attachment point is the carbon atom in position 2 or 3.

[0053] R 11

[0054] R 11 represents a hydrogen atom or a group chosen from an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group.

[0055] The alkyl radical as the R group 11It can be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical can have from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more particularly preferably from 1 to 3 carbon atoms.

[0056] As examples of alkyl radicals as group R 11 We can mention the radicals methyl, ethyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, tert-pentyl, 2-methylbutyl, hexyl, n-octyl, iso-octyl, 2-ethyl-l-hexyl, 2,2,4-trimethylpentyl, nonyl, neodecanyl, decyl, dodecyl, octadecyl, behenyl, or cyclohexylmethyl, and preferably the methyl radical.

[0057] The aryl radical as the R group 11may be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, which may comprise from 1 to 20 carbon atoms, preferably from 5 to 15 carbon atoms, and particularly preferably from 5 to 10 carbon atoms.

[0058] As examples of aryl radicals as the R group. 11 Examples include phenyl, trityl, naphthalenyl, anthracenyl, and pyrenyl radicals. Among these radicals, the phenyl radical is particularly preferred.

[0059] The heteroaryl radical as group R 11may be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, comprising one or more heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom, which may comprise from 1 to 20 carbon atoms, preferably from 2 to 15 carbon atoms, and particularly preferably from 3 to 10 carbon atoms.

[0060] As examples of heteroaryl radicals as R group 11 Examples include the pyrrole and thiophene radicals. Among such radicals, the pyrrole radical is particularly preferred.

[0061] The substituents for the aryl and heteroaryl groups mentioned above can be chosen from an alkyl group comprising 1 to 2 carbon atoms and an alkoxy group comprising 1 to 2 carbon atoms.

[0062] The alkylene-aryl group as the R group 11is a group comprising an alkylene radical and an aryl radical which are directly linked by a carbon (of the alkylene radical)-carbon (of the aryl radical) covalent bond.

[0063] The alkylene radical of the alkylene-aryl group can be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical can have from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more preferably from 1 to 3 carbon atoms.

[0064] The alkylene-aryl radical is linked to position 8 of the bodipy.

[0065] The aryl radical of the alkylene-aryl group has the same definition as the aryl group as the R group 11 .

[0066] The alkenylene-aryl group as the R group 11is a group comprising an alkenylene radical (presence of at least one double bond) and an aryl radical which are directly linked by a carbon (of the alkenylene radical)-carbon (of the aryl radical) covalent bond.

[0067] The alkenylene radical of the alkenylene-aryl group can be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical can consist of 1 to 22 atoms of carbon, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more particularly preferably from 1 to 3 carbon atoms.

[0068] The alkenylene-aryl radical is linked to position 8 of the bodipy.

[0069] The aryl radical of the alkenylene-aryl group has the same definition as that of the aryl group as the R group. 11 .

[0070] The alkylene-heteroaryl group as the R group.11 is a group comprising an alkylene radical and a heteroaryl radical which are directly linked by a carbon (of the alkylene radical)-carbon (of the heteroaryl radical) covalent bond.

[0071] The alkylene radical of the alkylene-heteroaryl group has the same definition as that of the alkylene radical of the alkylene-aryl group.

[0072] The alkylene-heteroaryl radical is linked to position 8 of the bodipy.

[0073] The heteroaryl radical of the alkylene-heteroaryl group has the same definition as the aryl group as the R group 11 .

[0074] The alkenylene-heteroaryl group as the R group 11 is a group comprising an alkenylene radical (presence of at least one double bond) and a heteroaryl radical which are directly linked by a carbon (of the alkenylene radical)-carbon (of the heteroaryl radical) covalent bond.

[0075] The alkenylene radical of the alkenylene-heteroaryl group has the same definition as that of the alkenylene radical of the alkenylene-aryl group.

[0076] The alkenylene-heteroaryl radical is linked to position 8 of the bodipy.

[0077] The heteroaryl radical of the alkenylene-heteroaryl group has the same definition as that of the heteroaryl group as the R group 11 .

[0078] According to a particularly preferred embodiment of the invention, R 11 is a hydrogen atom.

[0079] R 12 , R 13 , R 14 , R 15 , R 16 , and R 17

[0080] R 12 , R 13 , R 14 , R 15 , R 16 , and R 17, identical or different, represent a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group.

[0081] The alkyl radical as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 It can be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical can have from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more particularly preferably from 1 to 3 carbon atoms.

[0082] Examples of alkyl radicals as the R group. 12 , R. 13 , R 14 , R 15 , R 16 , or R 17We can mention the radicals methyl, ethyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, tert-pentyl, 2-methylbutyl, hexyl, n-octyl, iso-octyl, 2-ethyl-l-hexyl, 2,2,4-trimethylpentyl, nonyl, neodecanyl, decyl, dodecyl, octadecyl, behenyl, or cyclohexylmethyl, and preferably the methyl radical.

[0083] The aryl radical as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 may be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, which may comprise from 1 to 20 carbon atoms, preferably from 5 to 15 carbon atoms, and particularly preferably from 5 to 10 carbon atoms.

[0084] As examples of aryl radicals as group R 12 , R 13 , R 14 , R 15 , R 16 , or R17 Examples include phenyl, trityl, naphthalenyl, anthracenyl, and pyrenyl radicals. Among these radicals, the phenyl radical is particularly preferred.

[0085] The heteroaryl radical as group R 12 , R 13 , R 14 , R 15 , R 16 , or R 17 may be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, comprising one or more heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom, which may comprise from 1 to 20 carbon atoms, preferably from 2 to 15 carbon atoms, and particularly preferably from 3 to 10 carbon atoms.

[0086] As examples of heteroaryl radicals as R group 11 Examples include the pyrrole and thiophene radicals. Among such radicals, the pyrrole radical is particularly preferred.

[0087] The substituents for the aryl and heteroaryl groups mentioned above can be chosen from an alkyl group comprising 1 to 2 carbon atoms and an alkoxy group comprising 1 to 2 carbon atoms.

[0088] The alkylene-aryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 is a group comprising an alkylene radical and an aryl radical which are directly linked by a carbon (of the alkylene radical)-carbon (of the aryl radical) covalent bond.

[0089] The alkylene radical of the alkylene-aryl group can be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical can have from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more preferably from 1 to 3 carbon atoms.

[0090] The alkylene-aryl radical is linked to position 1, 2, 3, 5, 6, or 7 of the body depending on the R group. 12 , R. 13 , R 14 , R 15 , R 16 , or R 17 respectively.

[0091] The aryl radical of the alkylene-aryl group has the same definition as the aryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 .

[0092] The alkenylene-aryl group as the R group 11is a group comprising an alkenylene radical (presence of at least one double bond) and an aryl radical which are directly linked by a carbon (of the alkenylene radical)-carbon (of the aryl radical) covalent bond.

[0093] The alkenylene radical of the alkenylene-aryl group can be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical can contain from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more particularly preferably from 1 to 3 carbon atoms.

[0094] The alkenylene-aryl radical is linked to position 1, 2, 3, 5, 6, or 7 of the body, depending on the R group. 12 , R 13 , R 14 , R 15 , R 16 , or R 17 respectively.

[0095] The aryl radical of the alkenylene-aryl group has the same definition as that of the aryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 .

[0096] The alkylene-heteroaryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 is a group comprising an alkylene radical and a heteroaryl radical which are directly linked by a carbon (of the alkylene radical)-carbon (of the heteroaryl radical) covalent bond.

[0097] The alkylene radical of the alkylene-heteroaryl group has the same definition as that of the alkylene radical of the alkylene-aryl group.

[0098] The alkylene-heteroaryl radical is linked to position 1, 2, 3, 5, 6, or 7 of the body, depending on the R group. 12 , R 13 , R 14 , R 15 , R 16 , or R 17 respectively.

[0099] The heteroaryl radical of the alkylene-heteroaryl group has the same definition as the heteroaryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 .

[0100] The alkenylene-heteroaryl group as the R group. 12 , R. 13 , R 14 , R 15 , R 16 , or R 17 is a group comprising an alkenylene radical (presence of at least one double bond) and a heteroaryl radical which are directly linked by a carbon (of the alkenylene radical)-carbon (of the heteroaryl radical) covalent bond.

[0101] The alkenylene radical of the alkenylene-heteroaryl group has the same definition as that of the alkenylene radical of the alkenylene-aryl group.

[0102] The alkenylene-heteroaryl radical is linked to position 1, 2, 3, 5, 6, or 7 of the body, depending on the R group. 12 , R 13 , R 14 , R 15 , R16 , or R 17 respectively.

[0103] The heteroaryl radical of the alkenylene-heteroaryl group has the same definition as that of the heteroaryl group as the R group 12 , R 13 , R 14 , R 15 , R 16 , or R 17 .

[0104] According to a preferred embodiment of the invention, - R 12 and R 13 represent hydrogen atoms, the point of attachment is the carbon atom in position 3, R 16 represents a hydrogen atom, and R 15 and R 17 , identical or different, represent a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, or - R 12 and R 14represent alkyl groups, the point of attachment is the carbon atom in position 2, R 16 represents a hydrogen atom, and R 15 and R 17 , identical or different, represent a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group.

[0105] According to a particularly preferred embodiment of the invention, - R 12 and R 13 represent hydrogen atoms, the point of attachment is the carbon atom in position 3, R 16 represents a hydrogen atom, R 15 represents a hydrogen atom or an alkyl group, and R 17represents a group selected from an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, and preferably also an alkyl group, an aryl group, an alkenylene-aryl group, a heteroaryl group, and an alkenylene-heteroaryl group, or - R 12 and R 14 represent alkyl groups, the point of attachment is the carbon atom in position 2, R 16 is a hydrogen atom, R 15 represents a hydrogen atom or an alkyl group, and R 17 represents a group chosen from an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, and preferably also an alkyl group, an aryl group, an alkenylene-aryl group, a heteroaryl group, and an alkenylene-heteroaryl group.

[0106] The radical bodipy preferably corresponds to any one of the following formulas (II):

[0107] [Chem 5]

[0108] L

[0109] L is a connecting arm present (n = 1) or absent (n = 0).

[0110] When n = 1, the linking arm allows piperazine to be covalently linked with the bodipy radical.

[0111] When the linking arm is absent, piperazine is directly linked to one of the attachment points of the bodipy radical and preferably the carbon atom in position 2 or 3 of the bodipy radical of formula (II).

[0112] The bonding arm can be a bonding function chosen from among -(CH2)m- with m > 1, -(CH2-CH2-O)p-CH2-CH2- with p > 1, -(CH2-CH2-NH)q-CH2-CH2- with q > 1, phenylene, alkylene-phenylene, alkylene-phenylene-alkylene, phenylene-alkylene, alkenylene-phenylene, alkenylene-phenylene-alkenylene, phenylene-alkenylene, alkylene-phenylene-alkenylene, alkenylene-phenylene-alkylene, and a radical corresponding to the following formula (III): -*C(=O)-(L 1 )r-(F)s-(L 2 )t- (III) in which: - F is a divalent bonding function present (s = 1) or absent (s = 0), said divalent bonding function F being chosen from an oxygen atom, - C(=O), -S(=O)2-, -NH-C(=O)-, and -NH-C(=O)-CH2-O-, - L 1 and L 2, identical or different, are independently present (r = 1 and / or t = 1) or absent (r = 0 and / or t = 0), and are chosen from - (CH2)m'- with m' > 1, -(CH2-CH2-O)p'-CH2-CH2- with p' > 1, -(CH2-CH2-NH)q'- CH2-CH2- with q' > 1, phenylene, alkylene-phenylene, alkylene-phenylene-alkylene, phenylene-alkylene, alkenylene-phenylene, alkenylene-phenylene-alkenylene, phenylene-alkenylene, alkylene-phenylene-alkenylene, alkenylene-phenylene-alkylene, -* denotes the point of attachment of the (III) bonding arm to the nitrogen atom of piperazine.

[0113] m, m', p, p', q, q' are integers. Preferably, m < 10, m' < 10, p < 10, p' < 10, q < 10, q' < 10, particularly preferred m < 5, m' < 5, p < 5, p' < 5, q < 5, q' < 5, and even more particularly preferred m < 3, m' < 3, p < 3, p' < 3, q ​​< 3, q' < 3.

[0114] The alkylene radical can be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical can contain from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more preferably from 1 to 3 carbon atoms.

[0115] The alkenylene radical can be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical can contain from 1 to 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and even more particularly preferably from 1 to 3 carbon atoms.

[0116] The connecting arm corresponding to formula (III) is preferred.

[0117] According to a preferred embodiment of the invention, the connecting arm is a radical corresponding to formula (III) in which: - L 1 is -(CH2)m'-, s = 0, t = 0, - L 1 is -(CH2)m'-, s = 1 and F = -NH-C(=O)-CH2-O-, and t = 1 and L 2 is phenylene-alkenylene, - L 1 is -(CH2)m'-, s = 1 and F = an oxygen atom, and t = 1 and L 2 is phenylene-alkenylene or phenylene.

[0118] In the polycycles of the invention, the cycles can be joined, of the spiral type or bridged or condensed.

[0119] Compounds (I) include compounds as such but also their enantiomers, diastereomers, pharmaceutically acceptable salts, solvates and hydrates.

[0120] Pharmaceutically acceptable addition salts can be addition salts with bases such as sodium salts, potassium salts, and calcium salts, which can be obtained using the corresponding alkali and alkaline earth metal hydroxides as bases. Other types of pharmaceutically acceptable addition salts with bases include salts with amines, notably glucamine, N-methylglucamine, N,N-dimethylglucamine, ethanolamine, morpholine, N-methylmorpholine, and lysine.

[0121] Pharmaceutically acceptable addition salts may be addition salts with mineral or organic acids and preferably pharmaceutically acceptable acids such as hydrochloric, phosphoric, fumaric, citric, oxalic, sulfuric, ascorbic, tartaric, maleic, mandelic, methanesulfonic, lactobionic, gluconic, glucaric, succinic, sulfonic or hydroxypropanesulfonic acids.

[0122] The present invention also relates to a composition containing at least one compound of formula (I) conforming to the first object of the invention, and a liquid medium, for example a solvent such as an organic solvent in particular dimethyl sulfoxide, ethanol, a saline solution, such as for example a phosphate buffer at physiological pH.

[0123] The compounds (I) of the invention find applications in the following fields: - Research field * An alternative to antibodies for targeting MDR proteins. (detection and direct measurement of their expression level) for isolated cells, in monolayers, or on cell clusters / masses (3D cultures, tissues, etc.), or whole organisms * Complementarity with existing methods for measuring MDR protein activity. * Targeting of ubiquitous MDR proteins in all types of prokaryotic, eukaryotic and unicellular or multicellular parasite cells. * Targeting MDR proteins in artificial tumors - Clinical field * Targeting MDR proteins in tumor biopsies * Monitoring the effectiveness of a treatment and / or optimizing the choice of chemotherapy treatments - Environmental field * Environmental pollution biomarker * Bioassays of environmental pollution * Targeting of MDR proteins in whole organisms.

[0124] The second object of the invention is the use of a compound of formula (I) as defined in the first object as a marker of MDR proteins, and in particular for the detection, localization and / or quantification of MDR proteins.

[0125] MDR proteins are preferentially selected from among P-gp, MRP1, MRP2, MRP7, proteins encoded by the ABCC3-6 and ABCC11 genes, and MXR / BCRP. MDR proteins are particularly preferred over P-gp proteins.

[0126] Due to the presence of the R group 2 and the bodipy radical linked by a piperazine, it is possible to precisely explore the localization of MDR proteins and quantify them. The compounds (I) conforming to the first objective are stable and do not interfere with biological processes, which should allow the preservation of the biological properties of MDR proteins.

[0127] The invention has as its third object the use of a compound of formula (I) as defined in the first object in the field of health, and in particular as a diagnostic agent or imaging agent, in vitro or ex vivo.

[0128] Use in the health field, and in particular as a diagnostic or imaging agent, can also be implemented on a dead or fixed organism.

[0129] Due to the significant role of MDR proteins in chemoresistance mechanisms, their detection and quantification in tumor cells before and during treatment are highly relevant. They allow for the estimation of treatment efficacy, both preclinically during in vitro modeling of anticancer drugs and clinically on patient biopsies. Currently, the determination of MDR protein expression levels in biopsies during treatment is not used. The described invention is an innovative tool applicable to this research, thus enabling the optimization of treatments.

[0130] The fourth object of the invention is the use of a compound of formula (I) as defined in the first object in the environmental field, in particular as a biomonitoring tool and / or as a tool for detecting a contaminated environment, for example by xenobiotics (pollutants, industrial discharges, domestic discharges including all types of contaminants such as herbicides, pesticides, and pharmaceuticals, among others), particularly in an aquatic environment. The compounds (I) of the invention are particularly effective for labeling red blood cells from trout and trout fry.

[0131] The use of a compound of formula (I) as defined in the first object in the field of the environment, in particular as a tool for biomonitoring and / or detection of a contaminated environment, for example by xenobiotics, especially of an aquatic environment, is implemented preferentially in vitro or ex vivo or on a dead or fixed organism.

[0132] The invention relates as a fifth object an in vitro or ex vivo method for the detection, localization and / or quantification of MDR proteins, characterized in that it comprises at least the following steps: i) contacting a sample in a liquid medium with a compound (I) conforming to the first object of the invention, ii) allowing the sample to be incubated with the compound (I), such that the compound (I) binds to the MDR proteins when they are present, iii) removing the compound (I) not bound to MDR proteins, to form a sample labeled with compound (I), iv) detect, locate and / or quantify compound (I) bound to MDR proteins.

[0133] The sample preferentially contains cells, especially in suspension, in cell clusters or in the form of cell extract, of prokaryotic or eukaryotic type of animal or plant type, unicellular and multicellular, in the living or fixed state; or a whole organism consisting of cells, tissues and organs, in the living state, such as yeasts, daphnia, corbicula, gammarus, or bees.

[0134] By "cells" we mean whole cells and elements of cellular origin, such as organelles, the cell membrane (plasma membrane), endoplasmic membranes for eukaryotes; membrane, wall, capsule for prokaryotes.

[0135] By "suspended cells" we mean that the sample contains isolated cells maintained in a liquid medium.

[0136] By "cell cluster" we mean that the sample is a group of cohesive and aggregated cells, capable of forming a tissue.

[0137] By "cell extract" we mean a cell sample containing elements of cellular origin, for example organelles, and maintained in a living or fixed state.

[0138] For the purposes of this invention, the fixed state refers to the fixation of biological samples, which consists of freezing cellular structures in a state that allows for subsequent use by exposing them to a chemical agent such as paraformaldehyde. This fixation necessarily results in the cessation of all metabolic activity.

[0139] The method can be used independently for the detection, localization, and quantification of MDR proteins expressed in cells. It can also be used to perform two of these functions (detection and localization; detection and quantification; localization and quantification), or all three. The method according to the invention does not allow for the measurement of MDR protein activity but can be combined with a method capable of measuring said activity.

[0140] The sample may include, but is not limited to, cell extracts, cell cultures (e.g., cells in suspension, cells adhered to a culture medium, monolayer or multilayer cell culture, 3D cell culture, etc.) A spheroid, a biological tissue or tissue fragment, or a unicellular or multicellular organism. The sample may include, but is not limited to, patient cancer cells, a patient cancer biopsy, cancer cell lines, or an artificial tumor.

[0141] Step i)

[0142] According to the method of the invention, a sample is brought into contact in a liquid medium with a compound (I) conforming to the first object of the invention.

[0143] The sample is preferably in a liquid medium or comprises a liquid phase before contact with the compound (I) according to the first object of the invention.

[0144] Advantageously, the sample is maintained, cultured, or preserved in a liquid medium suitable for its preservation. This means that the sample is placed in a liquid medium compatible with the proper preservation of cells, tissues, etc., or is already in a suitable liquid medium. In the case of cells, tissues, biopsies, multicellular organisms, etc., the cells present or constituent are preferably placed or present in a preservation or culture medium.

[0145] The compound (I) according to the first object of the invention is capable of penetrating cells and diffusing within the sample and binding to the accessible portion of MDR proteins. The compound (I) according to the first object of the invention is further detectable by means of the bodipy radical. This detection / localization / quantification is based on fluorescence emission.

[0146] The compound (I) according to the first object of the invention is added to the sample in a sufficient quantity to perform the desired action, namely detection, localization, and / or quantification. In particular, a known quantity of this compound (I) according to the first object of the invention is added.

[0147] Preferably, compound (I) according to the first object of the invention is used at a concentration of approximately 1 nM to 100 mM, and particularly preferably from 100 nM to 10 pM, for approximately 1 cell to approximately 10 9 cells.

[0148] Step ii)

[0149] The sample is allowed to incubate with compound (I), so that compound (I) binds to MDR proteins when they are present.

[0150] The sample is incubated with compound (I), which binds to MDR proteins present in the cells. Incubation is carried out for a specific duration and at a temperature that maintains the cells in good condition.

[0151] The incubation of the sample with compound (I) is of sufficient duration to allow its binding to the cell extracts, its penetration into the cells, its diffusion within the cell clusters, and its penetration into the cells of these clusters. Generally, the incubation time can range from approximately 1 minute to approximately 24 hours.

[0152] This incubation can be carried out at a temperature that maintains the cells, tissues, etc., in a good state of preservation to allow the method to be performed. This temperature will advantageously be from approximately 0°C to approximately 39°C for cells of human or animal origin.

[0153] Step iii)

[0154] The compound (I) not bound to MDR proteins is removed, to form a sample labeled with compound (I).

[0155] After incubation, the non-MDR protein-bound compound (I) is removed. This removal may involve aspirating the liquid medium and recovering the cells (cell pellet or adherent cells) or the cell mass (tissue, biopsy, etc.). This removal can be achieved by centrifugation, precipitation, or filtration, particularly for a cell suspension, or by filtering or flowing the liquid medium, leaving the cell mass or adherent cells.

[0156] The method may also include washing or rinsing the cells with a liquid medium suitable for preserving cells, tissues, etc.

[0157] Preferably, washing leads to an efflux of compound (I) which has entered the cells but has not bound to an MDR protein.

[0158] Washing refers to placing the cells in a liquid suitable for sample preservation, called the "washing medium." The washing medium may be replaced one or more times during this period. A sufficient number of washes are performed to adequately remove the free, unbound compound (I).

[0159] Step iv)

[0160] We can then proceed to the detection, localization, and / or quantification of the compound (I) bound to MDR proteins. We can also combine these actions, in particular detection and quantification or localization and quantification or detection and localization.

[0161] According to different modalities, which can be combined, using compound (I): - The presence of MDR proteins is detected in the sample. - The binding of compound (I) to an intracellular MDR protein is detected. - We localize the presence of MDR proteins in the sample and within the cells, that is to say we determine (i) the location of the cells expressing the MDR proteins detected in the sample, in particular in a 3D culture, a tissue, a biopsy, a multicellular organism, and possibly (ii) the location of the MDR proteins at the cellular level. - We quantify the level of expression of MDR proteins in the sample. - Different levels of expression of MDR proteins are determined (i) in the sample, including in a 3D culture, tissue, biopsy or multicellular organism and possibly (ii) within each cell. - The expression levels of MDR proteins are compared in cell or tissue samples (biopsies) taken from the same patient at different times. This determines the patient's response to treatment, for example, the tumor's response to cancer treatment or the emergence of resistance to drug treatment, for example, cancer treatment.

[0162] In the latter case, the method involves processing multiple cell or tissue samples (biopsies) from the same patient over time (for example, during drug treatment, such as cancer treatment) using the method according to the invention. The method used for clinical monitoring of a patient includes comparing samples taken at different times. It allows for the detection of the emergence of MDR protein expression, as well as increases, stagnation, or regressions in the expression of these MDR proteins over the observation period.

[0163] Detection, localization, and / or quantification uses a fluorescence measurement method. The quantification or expression level of an MDR protein, such as P-gp, is obtained primarily through the measured fluorescence intensity. This allows for comparison of samples with each other or with reference values.

[0164] The invention has as its sixth object a method for the in vitro or ex vivo diagnosis of an overexpression of MDR proteins in a sample, characterized in that the method comprises the following steps: a) the addition to the sample of a compound (I) in accordance with the first object, b) the incubation of the sample with said compound (I), so that the compound (I) binds to the MDR proteins when they are present, c) the removal of the compound (I) not bound to MDR proteins, to form a sample labeled with compound (I), d) the measurement of a fluorescence value F(I) of the sample labeled with compound (I), and e) the comparison with at least one fluorescence value of a reference sample F(0).

[0165] Thus, the invention is based on the development of easily detectable and quantifiable fluorescent markers specifically targeting active or inactive MDR proteins, particularly applicable in vitro or ex vivo. MDR proteins are highly ubiquitous and expressed in the membrane systems of cells and intracellular organelles (such as lysosomes) of all organisms. The developed compounds (I) have the ability to penetrate / diffuse into cells and cell clusters. They allow for the detection of expression and direct global quantification of MDR proteins under conditions where cells constitute a single layer and also when they are composed of multiple layers, such as floating cell cultures, monolayers (2D), three-dimensional (3D) cell cultures, tissue fragments, or multicellular organisms, among others.The invention includes the use of these compounds (I), capable of penetrating a cell or diffusing into cell masses and penetrating cells, for membrane labeling and / or the localization and quantification of MDR protein expression (e.g., P-gp protein). These compounds can be used on various biological models, both living and non-living, including in fixed states, and thus offer a broader range of applications compared to current tools such as antibodies. In particular, compounds (I) can specifically detect MDR proteins such as P-gp at the membrane systems of intracellular organelles such as lysosomes. They are therefore particularly useful for... the labeling of MDR proteins, such as Pg-P, at the level of the membrane systems of intracellular organelles such as lysosomes.

[0166] The invention will now be described in more detail with the aid of examples and embodiments taken by way of non-limiting examples and referring to the attached drawings. Brief description of the drawings

[0167] [Fig. 1] Figure 1 shows the cellular distribution of a compound (I) of the invention on Triple Negative breast cancer cell lines by fluorescence imaging. [Fig. 2] Figure 2 shows the cellular distribution of compounds (I) of the invention on Triple Negative breast cancer cell lines by fluorescence imaging. [Fig. 3] Figure 3 shows the mode of operation of the state-of-the-art compounds as competitive substrates to demonstrate the efflux activity of MDR proteins and the mode of operation of the compounds (I) of the invention as non-competitive substrates to localize and quantify MDR proteins. [Fig. 4] Figure 4 shows the cellular distribution of a compound (I) of the invention in a gamma. [Fig. 5] Figure 5 shows the location of the labeling by a compound (I) of the invention on a brown trout red blood cell. [Fig. 6] Figure 6 shows the cellular labeling by a compound (I) of the invention of brown trout, from different streams of the Massif Central. [Fig. 7] Figure 7 shows the specific labeling of trout red blood cell lysosomes by a compound (I) of the invention [Fig. 8] Figure 8 shows the labeling of P-gp using a prior art comparative compound and a compound (I) of the invention on trout red blood cells [Fig. 9] Figure 9 shows an overall quantification of P-gp with a prior art comparative compound and a compound (I) of the invention on trout red blood cells, at 50 different stations [Fig. 10] Figure 10 shows the detection by a compound (I) of the invention of an increase in the level of expression of Pgp of a cell line exposed to river water.

[0168] Examples

[0169] Characterizations Tl

[0170] The analyses by mass spectrometry (HR.MS) were carried out with an instrument sold under the trade name "Q-Exactive" by the company Thermo Scientific™.

[0171] High-performance liquid chromatography with UV detection and diode array detection (HPLC-UV-DAD) analyses were carried out with an instrument sold under the trade name Prominence by the company Shimadzu equipped with a "Zorbax" column.

[0172] Spectral characterizations were performed by recording the excitation and emission spectra of the compounds in solution in ethanol on a "Cytation 10" fluorescence reader from Biotek Instrument.

[0173] Example 1: Synthesis of compound (Ial-1)

[0174] The compound (Ial-1) was obtained according to the following reaction scheme:

[0175] [Chem 6]

[0176] First step:

[0177] 10.0 g of 4-quinolinol 1 were diluted in 100 mL of dimethylformamide (DMF). Then, 3.58 g of sodium hydride and 20.0 g of ( / ?)-(-)-glycidyl tosylate were added. The resulting reaction mixture was stirred for 12 h at 25°C. A crude reaction mixture was obtained and purified by liquid chromatography on silica gel (eluent: 60% v / v acetone in hexane) to obtain the compound (Æ)-4-(oxiran-2-ylmethoxy)quinoline 2 with a yield of 4%.

[0178] HR.MS: m / z calculated for C12H12NO2, 202.0863 [M + H] + ; found, 202,0861.

[0179] Second step:

[0180] 0.47 g of the compound (Δ)-4-(oxiran-2-ylmethoxy)quinoline 2 obtained in the previous step was contacted with 0.44 g of N-boc-piperazine in 30 mL of isopropanol. The resulting reaction mixture was stirred for 24 h at 70°C. A crude reaction product was obtained by vacuum evaporation to produce the compound tert-butyl (Δ)-4-(2-hydroxy-3-(quinolin-4-yloxy)propyl)piperazine-l-carboxylate 3 in quantitative yield.

[0181] HR.MS: m / z calculated for C21H30N3O4, 388.2231[M + H] + ; found, 388,2218.

[0182] Third step:

[0183] 970 g of the compound tert-butyl(Φ)-4-(2-hydroxy-3-(quinolin-4-yloxy)propyl)piperazine-l-carboxylate 3 obtained in the previous step was placed in an acidic medium, i.e., in 25 mL of a 4N hydrochloric acid solution in 1,4-dioxane. The resulting reaction mixture was stirred for 3 h at 20°C. A crude reaction product was obtained by vacuum evaporation to obtain the compound (Φ)-l-(piperazin-l-yl)-3-(quinolin-4-yloxy)propan-2-ol 4 with a quantitative yield.

[0184] HR.MS: m / z calculated for C16H22N3O2, 288.1707[M + H] + ; found, 288,1695.

[0185] Fourth step:

[0186] 7.4 mg (2 equivalents) of compound (Æ)-l-(piperazin-l-yl)-3- The (quinolin-4-yloxy)propan-2-ol 4 obtained in the previous step were contacted with 5.0 mg (1 equivalent) of the commercial fluorochrome "BODIPY-FL-NHS" (CAS No. 146616-66-2) in 500 µl of DMF and in the presence of 2 equivalents of triethylamine. The resulting reaction mixture was stirred for 24 h at 20°C. A crude reaction mixture was obtained and purified by preparative thin-layer chromatography (eluent: dichloromethane / methanol 90 / 10 by volume) to obtain compound (Ial-1) with a yield of 40% (purity of 85%, HPLC 510 nm, retention time of 12.1 min).

[0187] HR.MS: m / z calculated for CsoHssNsC 1 ^, 562.2795 [M + H] + ; found, 562,2797.

[0188] The excitation and emission maxima are 505 nm and 513 nm respectively.

[0189] Example 2: Synthesis of compound (Ial-2)

[0190] The compound (Ial-2) was obtained according to the following reaction scheme:

[0191] [Chem 7]

[0192] The first, second and third steps to form compound 4 are identical to those described in example 1.

[0193] Fourth step:

[0194] 4.5 mg (1 equivalent) of compound (Æ)-l-(piperazin-l-yl)-3- (Quinolin-4-yloxy)propan-2-ol 4 were contacted with 10.0 mg (1 equivalent) of the commercial fluorochrome "BODIPY-650 / 665-X" (CAS No. 235439-04-0) in 500 µl of DMF and in the presence of 1 equivalent of triethylamine. The resulting reaction mixture was stirred for 72 h at 20°C. A crude reaction mixture was obtained and purified by preparative thin-layer chromatography (eluent: dichloromethane / methanol 95 / 5 by volume) to obtain the compound (Ial-2) with a yield of 10% (90% purity, HPLC 645 nm, retention time 13.5 min).

[0195] HR.MS: m / z calculated for C45H49N7O9 11 BF2, 816,3851 [M + H] +; found, 816,3837

[0196] The excitation and emission maxima are 649 nm and 669 nm respectively.

[0197] Example 3: Synthesis of compound (Ial-3)

[0198] The compound (Ial-3) was obtained according to the following reaction scheme:

[0199] [Chem 8]

[0200] The first, second and third steps to form compound 4 are identical to those described in example 1.

[0201] Fourth step:

[0202] 6.2 mg (1 equivalent) of compound (Æ)-l-(piperazin-l-yl)-3- (Quinolin-4-yloxy)propan-2-ol 4 were contacted with 10 mg (1 equivalent) of the commercial fluorochrome "BODIPY-564-570-NHS" (CAS No. 150173-90-3) in 500 µl of DMF and in the presence of 1 equivalent of triethylamine. The resulting reaction mixture was stirred for 48 h at 20°C. A crude reaction mixture was obtained and purified by preparative liquid chromatography to obtain the compound (Ial-3) with a yield of 10% (92% purity, HPLC 565 nm, retention time 13.5 min).

[0203] HR.MS: m / z calculated for C36H37N5O3 11 BF2, 636,2952 [M + H] + ; found, 636,2956.

[0204] The excitation and emission maxima are 562 nm and 575 nm respectively.

[0205] Example 4: Synthesis of compound (Ibl-1)

[0206] The compound (Ibl-1) was obtained according to the following reaction scheme:

[0207] [Chem 9]

[0208] First step:

[0209] 10.0 g of 5-hydroxyquinoline 5 were diluted in 100 mL of dimethylformamide (DMF). Then, 3.58 g of sodium hydride and 20.0 g of (R)-(-)-glycidyl tosylate were added. The resulting reaction mixture was stirred for 12 h at 25°C. A crude reaction mixture was obtained and purified by liquid chromatography on silica gel (eluent: 50% v / v acetone in hexane) to obtain the compound (R)-5-(oxiran-2-ylmethoxy)quinoline 6 with a yield of 19%.

[0210] HRMS: m / z calculated for C12H12NO2, 202.0863 [M + H] + ; found, 202.0861.

[0211] Second step:

[0212] 0.44 g of compound (R)-5-(oxiran-2-ylmethoxy)quinoline 6 obtained in the previous step was contacted with 0.40 g of N-boc-piperazine in 30 mL of isopropanol solution. The resulting reaction mixture was stirred for 24 h at 70°C. A crude reaction product was obtained by vacuum evaporation to produce the compound tert-butyl (R)-4-(2-hydroxy-3-(quinolin-5-yloxy)propyl)piperazine-l-carboxylate 7 in quantitative yield.

[0213] HRMS: m / z calculated for C21H30N3O4, 388.2231[M + H] + ; found, 388,2227.

[0214] Third step:

[0215] 830 mg of the compound tert-butyl(R)-4-(2-hydroxy-3-(quinolin-5-yloxy)propyl)piperazine-l-carboxylate 7 obtained in the previous step was placed in an acidic medium, i.e., in 25 mL of a 4N hydrochloric acid solution in 1,4-dioxane. The resulting reaction mixture was stirred for 1 hour at 20°C. A crude reaction mixture was obtained by evaporation under vacuum for obtain the compound (R)-l-(piperazin-l-yl)-3-(quinolin-5-yloxy)propan-2-ol 8 with a quantitative yield.

[0216] HRMS: m / z calculated for C16H22N3O2, 288.1707 [M + H] + ; found, 288,1700.

[0217] Fourth step:

[0218] 7.4 mg (2 equivalents) of compound (R)-l-(piperazin-l-yl)-3- The (quinolin-5-yloxy)propan-2-ol 8 obtained in the previous step were contacted with 5.0 mg (1 equivalent) of the commercial fluorochrome "BODIPY-FL-NHS" (CAS No. 146616-66-2) in 500 µl of DMF and in the presence of 2 equivalents of triethylamine. The resulting reaction mixture was stirred for 48 h at 20°C. A crude reaction mixture was obtained and purified by preparative thin-layer chromatography (eluent: dichloromethane / methanol 90 / 10 by volume) to obtain compound (Ibl-1) with a yield of 40% (purity of 85%, HPLC 510 nm, retention time of 12.1 min).

[0219] HRMS: m / z calculated for CsoHssNsC 1 ^, 562.2795 [M + H] + ; found, 562,2786.

[0220] The excitation and emission maxima are 505 nm and 513 nm respectively.

[0221] Example 5: Use of the compound (Ibl-1) as a marking tool

[0222] Cell labeling experiments were performed on two Triple Negative Breast Cancer cell lines (SUM1315 and DU4475) exhibiting different levels of P-gp expression.

[0223] 5.1 Preparation of cell samples

[0224] For the adherent cell line SUM1315

[0225] The SUM1315 cell line was obtained from Asterand and was cultured in Ham F12 medium (marketed by Gibco) supplemented with 5% by volume of Fetal Bovine Serum, 20 pg / ml of gentamicin (marketed by Panpharma), 10 mM of Hepes buffer (marketed by Sigma), 4 pg / ml of insulin (marketed by Novo Nordisk) and 10 ng / ml of Epidermal Growth Factor well known by the acronym EGF (marketed by Sigma).

[0226] The cells of this SUM1315 cell line are seeded one day before labeling in 8-well microplates (commercially available from Ibidi). under the name "Ibitreat Blades") at a rate of 50,000 cells per well in 200 µl of the recommended culture medium. The cells are then maintained under normal culture conditions (37°C and 5% by volume CO2).

[0227] For the DU4475 suspension-cultured cell line

[0228] The DU4475 cell line was obtained from ATCC (“HTB-123”) and was cultured in R.PMI1640 glutamine-free medium (marketed by Gibco) supplemented with 10% by volume of Fetal Bovine Serum, 20 pg / ml of gentamicin (marketed by Panpharma), and 2 mM of L-glutamine (marketed by Gibco).

[0229] Cells from this DU4475 cell line are subjected to a Malassez counting chamber on the day of labeling using erythrosine (commercially available from Thermo Scientific). A cell suspension volume corresponding to 800,000 cells is collected and transferred to a 2 ml tube.

[0230] 5.2 Cell fixation

[0231] The cells prepared in point 4.1 above are fixed by contact for 10 minutes with a phosphate-buffered saline solution marketed by Sigma (well known by the acronym PBS solution) comprising 4% by volume of paraformaldehyde (marketed by Sigma).

[0232] 5.3 Cell washing

[0233] The cells are washed three times with a PBS solution maintained at room temperature (i.e. close to 20°C).

[0234] 5.4 Cell Incubation

[0235] The cells are incubated for 1 hour at 4°C with the compound (Ibl-1) as prepared in example 4 at a concentration of 1 pM.

[0236] 5.5 Cell washing

[0237] The cells are washed three times with a PBS solution maintained at 4°C.

[0238] For the DU4475 line cultured in suspension, centrifugation steps are also carried out between the different washes in order to remove the supernatant medium while preserving the cells.

[0239] 5.6 Observations

[0240] Cell samples are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".

[0241] For SUM1315 cell line seeded in microplates, cells maintained in PBS are directly imaged in their culture wells.

[0242] For the DU4475 cell line suspension, the cells are mounted between a microscope slide (commercially available under the reference “Superfrost Plus” by Fisher) and a 24 x 24 mm coverslip (marketed by Thermo Scientific).

[0243] The cells are imaged at 40X magnification with Bright Field and Fluorescent filters (GFP filter for the anglicism "green fluorescence protein") (excitation: 469 / 35; emission: 525 / 39).

[0244] The average fluorescence intensity on the cells is quantified using the image analysis software marketed under the reference "Gen5 3.14" by the company Agilent on about fifteen different microscopy fields.

[0245] Figure 1 shows the cellular distribution of the compound (Ibl-1) on Triple Negative breast cancer cell lines, namely on the DU4475 cell line (Figure 1a) and on the SUM1315 cell line (Figure 1b), by fluorescence imaging.

[0246] From the acquired images, the average fluorescence intensities were calculated and are presented in the graph in Figure 1c (as a mean ± standard deviation, *****p<0.00001).

[0247] A high labeling intensity was observed, with the DU4475 cell line showing a significantly higher labeling intensity than the SUM1315 cell line (ratio of 2.65). This ratio confirms the feasibility of using the compounds of the invention as markers to reliably and rapidly quantify P-gp compared to antibodies.

[0248] Furthermore, it is noted that a ratio of the same order of magnitude (2.88) is found between the 2 lines by immunolabeling (anti-P-gp immunofluorescence, Goisnard A. et al. Cancers, 2021, 13, 1-19).

[0249] Example 6: Use of compounds (Ial-1), (Ial-2), (Ial-3) and (Ibl-1) as a specific labeling tool for P-gp

[0250] 6.1 Preparation of cell samples

[0251] The SUM1315 cell line was obtained from Asterand and cultured in Ham F12 medium (marketed by Gibco) supplemented with 5% by volume of fetal bovine serum, 20 pg / ml of gentamicin (marketed by Panpharma), and 10 mM of HEPES buffer. (marketed by Sigma), 4 pg / ml of insulin (marketed by Novo Nordisk) and 10 ng / ml of Epidermal Growth Factor well known by the acronym EGF (marketed by Sigma).

[0252] The cells of this SUM1315 cell line are seeded in 8-well microplates (marketed by Ibidi under the name "Ibitreat Slides") at a rate of 50,000 cells per well in 200 µl of the recommended culture medium. The cells are then maintained under normal culture conditions (37°C and 5% by volume CO2).

[0253] 6.2 Treatment with small RNAs interfering with P-gp synthesis

[0254] The following day, the cells are treated with small RNAs interfering with P-gp synthesis or P-gp siRNAs (commercially available from Invitrogen) at a concentration of 50 nM. The cells are then maintained for 72 hours under normal culture conditions (37°C and 5% by volume of CO2).

[0255] 6.3 Cell fixation

[0256] The cells treated in point 5.3 above are fixed by contact for 10 minutes with a PBS solution comprising 4% by volume of paraformaldehyde (marketed by Sigma).

[0257] 6.4 Cell washing

[0258] The cells are washed three times with a PBS solution maintained at room temperature (i.e. close to 20°C).

[0259] 6.5 Cell Incubation

[0260] The cells are incubated for 1 hour at 4°C with a compound (Ial-1), (Ial-2), (Ial-3) or (Ibl-1) as prepared in examples 1 to 4 respectively at a concentration of 1 pM.

[0261] 6.6 Cell washing

[0262] The cells are washed three times with a PBS solution maintained at 4°C.

[0263] 6.7 Observations

[0264] Cell samples are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".

[0265] Cells maintained in PBS are directly imaged in their culture wells.

[0266] The cells are imaged at 40X magnification in Bright Field and with suitable Fluo filters (GFP filter for "green fluorescence protein", excitation: 469 / 35, emission: 525 / 39; RFP filter for "red fluorescence protein", excitation: 531 / 40, emission: 593 / 40; CY5 filter, excitation: 628 / 40, emission: 685 / 40).

[0267] The average fluorescence intensity on the cells is quantified using the image analysis software marketed under the reference "Gen5 3.14" by the company Agilent on about fifteen different microscopy fields.

[0268] Figure 2 shows the cellular distribution of compounds (Ial-1), (Ial-2), (Ial-3) and (Ibl-1) on the SUM1315 cell line without treatment step 6.2 (Figure 2a) and on the SUM1315 cell line with treatment (Figure 2b), by fluorescence imaging.

[0269] From the acquired images, the average fluorescence intensities were calculated and are presented in the graph in Figure 2c (as a mean ± standard deviation, *****p<0.00001).

[0270] We observe a non-existent labeling intensity for the treated DU4475 line (i.e., P-gp is silenced) compared to the significantly higher labeling intensity obtained for the untreated SUM1315 line, which demonstrates the specificity of the compounds of the invention with respect to P-gp and their usefulness for measuring the expression level of P-gp on cultured cells.

[0271] Figure 3 illustrates the mode of operation of the prior art compounds as competitive substrates for demonstrating the efflux activity of MDR proteins (Figures 3a and 3b) and the mode of operation of the compounds (I) of the invention as non-competitive substrates for localizing and quantifying MDR proteins (Figure 3c). In particular, Figure 3a implements a non-fluorescent competitive substrate 1 and a fluorescent competitive substrate 2. To evaluate the affinity of an MDR protein for different substrates, a known substrate effluvated by P-gp (e.g., cyclosporine A, known to be a competitive substrate of MDR proteins coupled to a fluorochrome) and a molecule whose P-gp efflux is to be determined (a novel cyclosporine A derivative) are combined.The measured intracellular fluorescence intensity (grey spherical area) corresponds to the overall quantification of the accumulated (persistent / non-efferent) fluorescent molecule in the cell. Figure 3b. assesses uptake, i.e., the efflux of a fluorescent compound. Here again, the measured intracellular fluorescence intensity (gray spherical area) corresponds to the overall quantification of the accumulated (persistent / non-effluated) fluorescent molecule in the cell (see Figure 3b below). The substrates in Figures 3a and 3b are not used to detect, localize, and / or quantify MDR proteins, i.e., to quantify MDR protein expression as such. Conversely, Figure 3c shows the selective binding of a compound (I) of the invention to the MDR protein (in the manner of an antibody to its antigen) and is not an efflux substrate.

[0272] Example 7: Use of the compound (Ibl-1) as a biomonitoring tool (biomarker)

[0273] This example was carried out with Gammarus fossa rum freshwater shrimp from different streams in the Massif Central.

[0274] 7.1 Attachment of gammarus

[0275] The gammarus are fixed by contact for 10 minutes with a phosphate-buffered saline solution marketed by Sigma (well known by the acronym PBS solution) comprising 4% by volume of paraformaldehyde (marketed by Sigma).

[0276] 7.2 Washing of amphipods

[0277] The amphipods are washed twice with water.

[0278] 7.3 Incubation of Gammarus

[0279] The gammarus are incubated for 30 minutes at 37°C in a 5% by volume trypsin solution (Thermo Fisher) for the permeabilization of the gammarus cuticle.

[0280] 7.4 Incubation of Gammarus

[0281] The gammarus are incubated for 90 minutes at 4°C with the compound (Ibl-1) as prepared in example 4 at a concentration of 1 pM.

[0282] 7.5 Cell washing

[0283] The gammarus are washed three times with water (30 minutes for each wash).

[0284] 7.6 Observations

[0285] Gammarus are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".

[0286] Gammarus are imaged at 1.5X magnification with Bright Field and Fluorescent filters (GFP filter for "green fluorescence protein") (excitation: 469 / 35; emission: 525 / 39).

[0287] The average fluorescence intensity on the gammarus is quantified using the image analysis software marketed under the reference "Gen5 3.14" by the company Agilent on about fifteen different microscopy fields.

[0288] Figure 4 shows the cellular distribution of the compound (Ibl-1) in a gamma (scale bar 2 cm). This example demonstrates the possibility of using the biomarker on an entire organism.

[0289] Example 8: Use of the compound (Ibl-1) as a biomonitoring tool (tracer)

[0290] 8.1 Preparation of cell samples

[0291] 6 µl of red blood cells, from brown trout blood samples, are rinsed in 1 ml of a PBS solution in a 2 ml microtube, then centrifuged at 200 rpm for 2 minutes.

[0292] 8.2 Cell Incubation

[0293] The cell pellet obtained after centrifugation is incubated for 1 hour at room temperature with 1 ml of a solution of compound (Ibl-1) as prepared in example 4 at a concentration of 1 pM.

[0294] 8.3 Cell washing

[0295] The cells are washed twice with a PBS solution maintained at room temperature for 30 minutes. Between the washes, centrifugation steps are also performed to remove the supernatant while preserving the cells.

[0296] 8.4 Observations

[0297] The cell pellet obtained after centrifugation is resuspended in 1 ml of PBS solution maintained at room temperature. The cell samples are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".

[0298] With the cells in suspension, the cells are mounted between a microscope slide (marketed under the reference "Superfrost Plus" by Fisher) and a 24 x 24 mm coverslip (marketed by Thermo Scientific).

[0299] The cells are imaged at 40X magnification with Bright Field and Fluorescent filters (GFP filter for "green fluorescence protein") (excitation: 469 / 35; emission: 525 / 39) (scale bar 10 pm).

[0300] Figure 5 shows the localization of the (Ibl-1) compound labeling on a brown trout red blood cell. Figure 5a shows the red blood cell under bright-field microscopy and Figure 5b under fluorescence microscopy.

[0301] The location of the labeling is mainly observed at the level of the nuclear membrane as well as on the membrane of intracellular organelles (lysosomes).

[0302] In parallel, fluorescence intensity is measured by flow cytometry with a device sold under the commercial name "BD Accuri C6+ flow" by BD Biosciences equipped with a blue excitation laser (488 nm) and a green filter (533 / 30nm), then quantified using data analysis software sold under the commercial name "FlowJo" by BD Biosciences.

[0303] Figure 6 shows the cellular labeling with the compound (Ibl-1) of brown trout from different streams in the Massif Central.

[0304] The level of expression of P-gp (in fluorescence units UF) was obtained as a function of the locations of the trout (different watercourses).

[0305] Fluorescence intensities are shown in Figure 6 as mean ± standard deviation (in grey). A solid circle represents the mean fluorescence for an individual.

[0306] Examples 5, 7 and 8 confirm the universality of compounds of formula (I) (tracers) for detecting and quantifying the P-gp biomarker in the context of biomonitoring for example.

[0307] Example 9: Performance of a compound of the invention

[0308] Red blood cells from trout fry were co-labeled with a compound of the invention, Ibl-1, as prepared in Example 4 above, and a commercial lysosome-specific marker called "Lysotracker." Specifically, live trout red blood cells were in contact with 1 pM of compound Ibl-1 for 1 hour and then with 50 nM of the commercial marker "Lysotracker" for 30 minutes. The resulting trout red blood cells were then washed three times with PBS buffer.

[0309] Figure 7 shows images acquired with a Cytation3 MV plate reader (Agilent, M = 40X, scale bar = 30 pm) in bright field (Figure 7a) and with GFP (green fluorescence protein, excitation: 469 / 35; emission: 525 / 39) fluorescence filters to detect the compound of the invention Ibl-1 (Figure 7b) and CY5 (which corresponds to a known type of cyanine, excitation: 628 / 40, emission: 685 / 40) to detect the lysotracker (Figure 7c). Figure 7d shows the application of both filters. This demonstrates that the compound of the invention specifically labels P-gp at the level of lysosomes. The compounds of the invention allow the detection, localization, and / or quantification of intracellular MDR proteins.

[0310] Example 10: Comparison of the performance of a compound of the invention with respect to a compound of the prior art

[0311] P-gp is expressed in all membrane systems within the cell, including on certain intracellular organelles such as lysosomes. Lysosomes play an important role in sequestering xenobiotics (from the cytoplasm into the lysosomes) to protect the cell against external aggressions. Quantifying P-gp across all membrane systems is an important element in measuring the degree of cellular resistance. In this Example 10, the effectiveness of labeling with a compound (I) according to the invention (which corresponds to compound Ibl-1 as prepared in Example 4 above) and a prior art comparator compound as described in international application W02021 / 064191 (which corresponds to compound 3 of international application W02021 / 064191) for the detection and quantification of P-gp on red blood cells from trout is investigated.

[0312] The tagging protocol used in this example is as follows:

[0313] Live red blood cells were labeled with 1 pM of the compound of the invention or of a comparator compound and washed 3 times with PBS. Images were acquired with a "Cytation3 MV" plate reader (Agilent, M = 60X, scale = 25 pm) in bright field (figures 8a and 8c) and with the GFP fluorescence filter (figures 8b and 8d).

[0314] Figure 8b shows the labeling of P-gp using the prior art comparator compound on trout red blood cells. The nuclear membrane is indicated by arrow 1, and lysosomes are indicated by arrow 2.

[0315] Figure 8d below shows the labeling of P-gp with compound (I) of the invention on trout red blood cells. The nuclear membrane is indicated by arrow 1, and the lysosomes are indicated by arrow 2.

[0316] The results show that the prior art comparative compound preferentially labels the nuclear membrane and weakly the lysosomes (Figure 8b), and the compound of the invention shows significant labeling for both the nuclear membrane and the lysosomes (Figure 8d).

[0317] Figure 9 shows an overall quantification of P-gp (fluorescence intensity in arbitrary units) with the comparator compound (Figure 9a) and with the compound of the invention (Figure 9b) on trout red blood cells at 50 different stations. Live red blood cells were labeled with 1 pM of the compound and washed 3 times with PBS buffer. P-gp was quantified by flow cytometry (BD Accuri C6+, BD biosciences, equipped with a blue excitation laser (488 nm) and a green filter (533 / 30 nm)).

[0318] The results of the P-gp quantification show less variability with the comparator compound than with the compound of the invention. The compound of the invention allows for better quantification of overall P-gp (nuclear membrane + lysosomes) and therefore more sensitively and effectively reflects the variability of environmental pollution between each site studied. The compounds of the invention are thus suitable for the sensitive detection and overall quantification of P-gp in environmental models.

[0319] Example 11: Use of a compound of the invention as a marker of river water pollution

[0320] Breast cancer cell line "T47D kb lue" is seeded in 24-well plates in 1 ml of "L15" medium which is The recommended culture medium is used. After 24 hours, the cells are maintained under normal culture conditions (37°C and 5% by volume CO2) in their culture medium, or exposed to river water after the culture medium has been removed and the cells have been rinsed. After another 24 hours, the culture medium or river water is removed, and the cells are rinsed with PBS. The cells are fixed by contact for 10 minutes with a PBS solution containing 4% by volume paraformaldehyde and then washed three times with a PBS solution maintained at room temperature (i.e., close to 20°C). The cells are incubated for 1 hour at 4°C with compound Ial-1 as prepared in Example 1 at a concentration of 1 pM. The cells are washed three times with a PBS solution maintained at 4°C. Cell samples are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".Cells maintained in PBS were imaged directly in their culture wells. The cells were imaged with a suitable Fluo filter (GFP filter, for "green fluorescence protein," excitation: 469 / 35, emission: 525 / 39). The average fluorescence intensity on the cells was quantified using the image analysis software marketed under the reference "Gen5 3.14" by Agilent on approximately fifteen different microscopy fields. Figure 10 shows the cellular distribution of the compound Ial-1 on the human breast cancer cell line "T47D kb lue" after culture in the reference medium "L15" (Figure 10a) and after 24 h of exposure to polluted river water (Figure 10b). From the acquired images, the average fluorescence intensities were measured and are presented on the histogram in Figure 10 (as a mean ± standard deviation), which highlights the extreme pollution of the river water.

Claims

Demands 1. Compound corresponding to the following formula (I): [Chem 10] in which: - R. 1 is a fluorochrome radical chosen from among the bodipy radicals, - L is a connecting arm present (n = 1) or absent (n = 0), - R 2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole, - R 3 , R 4 , R 5 and R 6 , identical or different, represent a hydrogen atom or an alkyl group, - R 7 and R 8 , identical or different, represent a hydrogen atom or an alkyl group, - R 9 and R 10 , identical or different, represent a hydrogen atom or an alkyl group, and - Y represents an oxygen atom, a sulfur atom, or an NH radical.

2. Compound according to claim 1, characterized in that n = 1 and the connecting arm L corresponds to the following formula (III): -*C(=O)-(L 1 )r-(F)s-(L 2 )t- (III) in which: - F is a divalent bonding function present (s = 1) or absent (s = 0), said divalent bonding function F being chosen from an oxygen atom, - C(=O), -S(=O)2-, -NH-C(=O)-, and -NH-C(=O)-CH2-O-, - L 1 and L 2, identical or different, are independently present (r = 1 and / or t = 1) or absent (r = 0 and / or t = 0), and are chosen from - (CH2)m'- with m' > 1, -(CH2-CH2-O)p'-CH2-CH2- with p' > 1, -(CH2-CH2-NH)q'- CH2-CH2- with q' > 1, phenylene, alkylene-phenylene, alkylene-phenylene-alkylene, phenylene-alkylene, alkenylene-phenylene, alkenylene-phenylene-alkenylene, phenylene-alkenylene, alkylene-phenylene-alkenylene, alkenylene-phenylene-alkylene, -* denotes the point of attachment of the (III) bonding arm to the nitrogen atom of piperazine.

3. Compound according to claim 1 or 2, characterized in that the fluorochrome radical R. 1 corresponds to the following formula (II): [Chem 11] in which: - R 11represents a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, - R 12 , R 13 , R 14 , R 15 , R 16 , and R 17 , identical or different, represent a hydrogen atom or a group chosen from among an alkyl group, an aryl group, an alkylene-aryl group, an alkenylene-aryl group, a heteroaryl group, an alkylene-heteroaryl group, and an alkenylene-heteroaryl group, - it being understood that the fluorochrome radical R 1has an attachment point (noted *) to the ligand L when n = 1 or to the nitrogen atom of piperazine when n = 0, chosen from the carbon atom in position 1, the carbon atom in position 2, the carbon atom in position 3, the carbon atom in position 5, the carbon atom in position 6, the carbon atom in position 7, and the carbon atom in position 8.

4. Composed according to any one of the preceding claims, characterized in that R 3 , R 4 , R 5 and R 6 are hydrogen atoms.

5. Compound according to any one of the preceding claims, characterized in that R 7 , R 8 , R 9 and R 10 are hydrogen atoms.

6. A compound according to any one of the preceding claims, characterized in that it corresponds to the following formula (1) or (11): [Chem 12] in which R. 1 , L, R 3, R 4 , R 5 and R 6 are as defined previously.

7. Use of a compound of formula (I) as defined according to any one of the preceding claims, as a marker for MDR (multidrug resistant) proteins, and in particular for the detection, localization and / or quantification of MDR proteins.

8. Use according to claim 7, characterized in that the MDR proteins are P-gp proteins.

9. Use of a compound of formula (I) as defined according to any one of claims 1 to 6, in the field of health, and in particular as a diagnostic agent or imaging agent, in vitro or ex vivo.

10. Use of a compound of formula (I) as defined according to any one of claims 1 to 6, in the field of the environment, in particular as a biomonitoring tool and / or as a tool for detecting a contaminated environment.

11. In vitro or ex vivo method for the detection, localization and / or quantification of MDR proteins, characterized in that it comprises at least the following steps: i) bringing a sample into contact in a liquid medium with a compound (I) as defined according to any one of claims 1 to 6, ii) allowing the sample to be incubated with the compound (I), so that the compound (I) binds to the MDR proteins when they are present, iii) removing the compound (I) not bound to MDR proteins, to form a sample labeled with compound (I), iv) detecting, localizing and / or quantifying the compound (I) bound to MDR proteins.

12. Method for in vitro or ex vivo diagnosis of an overexpression of MDR proteins in a sample, characterized in that the method comprises the following steps: a) adding to the sample a compound (I) as defined according to any one of claims 1 to 6, b) incubating the sample with said compound (I), such that the compound (I) binds to MDR proteins when present, c) removing the compound (I) not bound to MDR proteins, to form a sample labeled with compound (I), d) measuring a fluorescence value F(I) of the sample labeled with compound (I), and e) comparing with at least one fluorescence value of a reference sample F(0).

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