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

Abstract

The present invention relates to fluorescent compounds useful for the selective detection, localization, and / or quantification of MDR proteins such as P-gp 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. (Shortened figure: Figure 1)

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 allow for the selective labeling of MDR proteins such as P-gp.

[0002] MDR (multidrug resistance) 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, such as the blood-brain barrier, the adrenal gland, the pancreas, the liver and kidney, 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 or prokaryotic organisms (animal or plant) and in fish, gene expression of ABCB1 and also of other members of ABC transporters such as ABCC1-5, ABCB3, ABCB11, and ABCG2 has also been demonstrated.

[0005] MDR proteins are efflux pumps with a detoxifying role. They recognize a very wide variety of substrates ranging from organic molecules with highly varied physicochemical properties to heavy metals. Because of this role, MDR proteins play a role at the cellular level in defense 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 specific antibodies for each protein by immunocytological or immunohistological approaches. However, the use of antibodies to quantify MDR proteins has the following drawbacks: such antibodies lack membrane permeability, and they cannot diffuse, or only minimally, within 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 / blood sampling process) 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 chemotherapeutic agents 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 higher affinity / specificity for the MDR pump than the chemotherapy drug, the pump's efflux activity is primarily focused on the inhibitor's efflux rather than that of the chemotherapy drug. In this way, the chemotherapy drug remains within the cancer cell and can act. These competitive inhibitors are preferentially recognized and transported by MDR pumps from the inside to the outside of cancer cells. The best-known fluorescent and non-fluorescent competitive inhibitors described in the literature include cyclosporine (cyclosporin A), calcein AM, rhodamine 123, reversin, etc.

[0008] The efflux / transport activity of MDR pumps can be analyzed / demonstrated at the cellular level, preferably by flow cytometry or by Fluorescence imaging. However, none 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 evaluate 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 new 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 [Fig.6] a 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 [Fig. 6] b below).

[0011] Thus, the methods described above express the dynamic transporter role of MDR proteins. This involves a very rapid efflux mechanism of a fluorescent molecule from inside the cell to outside the cell via MDR proteins. Consequently, none of these fluorescent molecules binds directly to MDR proteins permanently, 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 WO2021 / 064191 describes fluorescent peptide compounds that can be used to quantify MDR proteins as a replacement for 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 protein selectivity. MDR (selective labeling), brightness, detection intensity, and / or stability in biological media. Another objective 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 analysis "HRMS").

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

[0015] [Chem.l] in which: - R1 represents a fluorochrome radical chosen from among the bodipy radicals, - L represents a connecting arm that is present (n = 1) or absent (n = 0), - R2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole, - R3, R4, R5 and R6, whether identical or different, represent a hydrogen atom or an alkyl group, - R7 and R8, whether identical or different, represent a hydrogen atom or an alkyl group, - R9 and R10, whether 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 the direct measurement of MDR protein expression, unlike existing methods for measuring MDR protein efflux activity (e.g., "MDR Assay Kit," Abcam®). Indeed, although also based on the use of small molecules, these methods do not allow 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, detection intensity, and / or stability in biological media. Moreover, they can be prepared by simple and economical organic synthesis and are easily identifiable by NMR, particularly thanks to the R2 group, and thus represent an attractive 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 by animal experimentation. All these characteristics constitute a significant added value of the invention compared to the prior art.

[0018] R3, R4, R5 and R6

[0019] R3, R4, R5 and R6, whether identical or different, represent a hydrogen atom or a alkyl group.

[0020] The alkyl group, as R3, R4, R5, and R6, may be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical may 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 groups R3, R4, R5 or R6 is preferably a hydrogen atom, particularly preferably at least two of the groups R3, R4, R5 or R6 are hydrogen atoms, more particularly preferably at least three of the groups R3, R4, R5 or R6 are hydrogen atoms, and even more particularly preferably the groups R3, R4, R5 and R6 are hydrogen atoms.

[0022] R7 and R8

[0023] R7 and R8, identical or different, represent a hydrogen atom or an alkyl group.

[0024] The alkyl group, as R7 and R8, may be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical may 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 groups R7 or R8 is preferably a hydrogen atom, and particularly preferably groups R7 and R8 are hydrogen atoms.

[0026] R9 and R10

[0027] R9 and R10, identical or different, represent a hydrogen atom or an alkyl group.

[0028] The alkyl group, as R9 and R10 groups, may be linear or branched, cyclic or non-cyclic. The alkyl radical is preferably linear and non-cyclic. The alkyl radical may 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 groups R9 or R10 is preferably a hydrogen atom, and particularly preferably groups R9 and R10 are hydrogen atoms.

[0030] According to a particularly preferred embodiment of the invention, R7, R8, R9 and R10 are hydrogen atoms.

[0031] R2

[0032] R2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole.

[0033] In a particularly preferred manner, R2 is a quinoline or an isoquinoline.

[0034] In the compound of formula (I), the nitrogenous heteroaromatic group R2 is in particular covalently linked to the radical Y by a carbon atom. In other words, the attachment point of the R2 group is in particular a carbon atom.

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

[0036] [Chem.2] (there) (lb) in which R1, L, R3, R4, R5, R6, R7, R8, R9 and R10 are as defined in the invention.

[0037] Y

[0038] Y represents an oxygen atom, a sulfur atom, or an NH radical, 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] d31) (ibl) in which R1, L, R3, R4, R5 and R6 are as defined in the invention.

[0042] R1

[0043] R1 represents a fluorochrome radical chosen from among the bodipy radicals.

[0044] In the invention, a fluorochromic radical is understood to be a chemical substance capable of emitting fluorescence light 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 excitation wavelength-maximum emission wavelength of at least 2 to approximately 10 nm.

[0049] The fluorochrome radical R1 preferably corresponds to the following formula (II):

[0050] [Chem.4] in which: - R11 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; - R12, R13, R14, R15, R16, and R17, identical or different, represent 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. - it being understood that the fluorochrome radical R1 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 R1, the group likely to be present at this position x (i.e. R12, R13, R14, R15, R16, R17, or R11 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] R11

[0054] R11 represents a hydrogen atom or 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.

[0055] The alkyl radical as group R11 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 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] Examples of alkyl radicals as group R11 include 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 group R11 can be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, which can comprise from 1 to 20 carbon atoms, preferably from 5 to 15 carbon atoms, and particularly preferably from 5 to 10 carbon atoms.

[0058] Examples of aryl radicals as R11 groups include, in particular, phenyl, trityl, naphthalenyl, anthracenyl, and pyrenyl radicals. Among such radicals, the phenyl radical is particularly preferred.

[0059] The heteroaryl radical as group R11 can 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] Examples of heteroaryl radicals as R11 groups include, in particular, the pyrrole and thiophene radicals. Among such radicals, the pyrrole radical is particularly preferred.

[0061] The substituents of 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 group R11 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.

[0063] The alkylene radical of the alkylene-aryl group may be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical may consist of 1 to 22 carbon atoms, preferably 1 to 10 atoms. of carbon, particularly preferably from 1 to 5 carbon atoms, and more particularly 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 R11 group.

[0066] The alkenylene-aryl group as group R11 is 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 may be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical may comprise 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.

[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 group R11.

[0070] The alkylene-heteroaryl group as group R11 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 that of the aryl group as the R11 group.

[0074] The alkenylene-heteroaryl group as group R11 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 group R11.

[0078] According to a particularly preferred embodiment of the invention, R11 is a hydrogen atom.

[0079] R12, R13, R14, R15, R16, and R17

[0080] R12, R13, R14, R15, R16, and R17, whether identical or different, represent an atom hydrogen or 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.

[0081] The alkyl radical as group R12, R13, R14, R15, R16, or R17 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 22 carbon atoms, preferably from 1 to 10 carbon atoms, particularly preferably from 1 to 5 carbon atoms, and most particularly preferably from 1 to 3 carbon atoms.

[0082] Examples of alkyl radicals as R12, R13, R14, R15, R16, or R17 groups may be mentioned, such as methyl, ethyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, 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 group R12, R13, R14, R15, R16, or R17 can be a monocyclic or polycyclic aromatic hydrocarbon group, optionally substituted by one or more substituents, which can comprise from 1 to 20 carbon atoms, preferably from 5 to 15 carbon atoms, and particularly preferably from 5 to 10 carbon atoms.

[0084] Examples of aryl radicals as R12, R13, R14, R15, R16, or R17 groups include, in particular, the phenyl, trityl, naphthalenyl, anthracenyl, and pyrenyl radicals. Among such radicals, the phenyl radical is particularly preferred.

[0085] The heteroaryl radical as group R12, R13, R14, R15, R16, or R17 can 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] Examples of heteroaryl radicals as R11 groups include, in particular, the pyrrole and thiophene radicals. Among such radicals, the pyrrole radical is particularly preferred.

[0087] The substituents of 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 group R12, R13, R14, R15, R16, or R17 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 may be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical may consist of 1 to 22 carbon atoms, preferably 1 to 10 carbon atoms, most preferably 1 to 5 carbon atoms, and most preferably 1 to 3 carbon atoms.

[0090] The alkylene-aryl radical is linked to position 1, 2, 3, 5, 6, or 7 of the body according to group R12, R13, R14, R15, R16, or R17 respectively.

[0091] The aryl radical of the alkylene-aryl group has the same definition as the aryl group as group R12, R13, R14, R15, R16, or R17.

[0092] The alkenylene-aryl group as group R11 is 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 may be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical may comprise 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 according to group R12, R13, R14, R15, R16, or R17 respectively.

[0095] The aryl radical of the alkenylene-aryl group has the same definition as that of the aryl group as group R12, R13, R14, R15, R16, or R17.

[0096] The alkylene-heteroaryl group as group R12, R13, R14, R15, R16, or R17 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 bodipy according to group R12, R13, R14, R15, R16, or R17 respectively.

[0099] The heteroaryl radical of the alkylene-heteroaryl group has the same definition as that of the heteroaryl group as group R12, R13, R14, R15, R16, or R17.

[0100] The alkenylene-heteroaryl group as group R12, R13, R14, R15, R16, or R17 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 bodipy according to group R12, R13, R14, R15, R16, or R17 respectively.

[0103] The heteroaryl radical of the alkenylene-heteroaryl group has the same definition as that of the heteroaryl group as group R12, R13, R14, R15, R16, or R17.

[0104] According to a preferred embodiment of the invention, - R12 and R13 represent hydrogen atoms, the point of attachment is the carbon atom in position 3, R16 represents a hydrogen atom, and R15 and R17, identical or different, represent 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, or - R12 and R14 represent alkyl groups, the point of attachment is the carbon atom in position 2, R16 represents a hydrogen atom, and R15 and R17, identical or different, represent 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.

[0105] According to a particularly preferred embodiment of the invention, - R12 and R13 represent hydrogen atoms, the point of attachment is the carbon atom in position 3, R16 represents a hydrogen atom, R15 represents a hydrogen atom or an alkyl group, and R17 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, or - R12 and R14 represent alkyl groups, the point of attachment is the carbon atom in position 2, R16 is a hydrogen atom, R15 represents a hydrogen atom or an alkyl group, and R17 represents 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,and preferably, another 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 the piperazine to be covalently linked with the bodipy radical.

[0111] When the linking arm is absent, the 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 -(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)-(L1)r-(F)s-(L2)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-, - L1 and L2, 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] The alkylene radical may be linear or branched, cyclic or non-cyclic. The alkylene radical is preferably linear and non-cyclic. The alkylene radical may consist of 1 to 22 carbon atoms, preferably 1 to 10 carbon atoms, particularly preferably 1 to 5 carbon atoms, and even more particularly preferably 1 to 3 carbon atoms.

[0114] The alkenylene radical may be linear or branched, cyclic or non-cyclic. The alkenylene radical is preferably linear and non-cyclic. The alkenylene radical may comprise 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.

[0115] The connecting arm corresponding to formula (III) is preferred.

[0116] According to a preferred embodiment of the invention, the connecting arm is a radical corresponding to formula (III) in which: - L1 is -(CH2)m'-, s = 0, t = 0, - L1 is -(CH2)m'-, s = 1 and F = -NH-C(=O)-CH2-O-, and t = 1 and L2 is phenylene-alkenylene, - L1 is -(CH2)m'-, s = 1 and F = an oxygen atom, and t = 1 and L2 is phenylene-alkenylene or phenylene.

[0117] In the polycycles of the invention, the cycles can be joined, of the spiral type or bridged or condensed.

[0118] Compounds (I) include compounds as such but also their enantiomers, diastereomers, pharmaceutically acceptable salts, solvates and hydrates.

[0119] Pharmaceutically acceptable addition salts may be addition salts with bases such as sodium salts, potassium salts, calcium salts, which may be obtained using alkali metal hydroxides and Alkaline earth metals correspond as bases. Other types of addition salts with pharmaceutically acceptable bases include salts with amines, notably glucamine, N-methylglucamine, N,N-dimethylglucamine, ethanolamine, morpholine, N-methylmorpholine, or lysine.

[0120] 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.

[0121] 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.

[0122] 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) in isolated cells, 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.

[0123] The invention has as its second object 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.

[0124] MDR proteins are preferably selected from P-gp, MRP1, MRP2, MRP7, proteins encoded by genes ABCC3-6 and ABCC11, and MXR / BCRP. MDR proteins are particularly preferred over P-gp proteins.

[0125] Due to the presence of the R2 group and the bodipy radical linked by a piperazine, it is possible to precisely explore the localization of MDR proteins and to quantify them. The compounds (I) conforming to the first object are stable and do not interfere with biological processes, which should allow the preservation of the biological properties of the MDR proteins.

[0126] 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.

[0127] Use in the health field, and in particular as a diagnostic agent or imaging agent, can also be implemented on a dead or fixed organism.

[0128] Due to the strong involvement of MDR proteins in chemoresistance mechanisms, their detection and quantification in tumor cells before and during treatment are highly relevant. They allow for an estimation of treatment efficacy, both preclinically during anticancer drug screening using in vitro modeling and clinically on patient biopsies. The determination of MDR protein expression levels is not currently used on biopsies during treatment. The described invention is an innovative tool applicable to this research, thus enabling the optimization of treatments.

[0129] The invention has as its fourth object the use of a compound of formula (I) as defined in the first object in the field of the environment, in particular as a biomonitoring tool and / or as a tool for detecting an environment contaminated, for example by xenobiotics (pollutants, industrial discharges, domestic discharges including all varieties of contaminants such as herbicides, pesticides, medicines, among others), in particular of an aquatic environment.

[0130] 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, in particular of an aquatic environment, is implemented preferably in vitro or ex vivo or on a dead or fixed organism.

[0131] 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) to bring a sample into contact in a liquid medium with a compound (I) conforming to the first object of the invention, ii) allow the sample to incubate with compound (I), so that compound (I) binds to the MDR proteins when they are present, iii) eliminate compound (I) not bound to MDR proteins, to form a sample labeled with compound (I), iv) detect, localize and / or quantify compound (I) bound to MDR proteins.

[0132] The sample preferentially contains cells, in particular 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.

[0133] 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.

[0134] By "suspended cells" is meant that the sample contains isolated cells maintained in a liquid medium.

[0135] By "cell cluster" we mean that the sample is a set of cohesive and aggregated cells, capable of forming a tissue.

[0136] By "cell extract" is meant a cell sample containing elements of cellular origin, for example organelles, and maintained in a living or fixed state.

[0137] For the purposes of the present invention, the fixed state corresponds to the fixation of biological samples, which consists of freezing cellular structures in a state allowing for subsequent use by exposing them to a chemical agent such as paraformaldehyde. This fixation necessarily results in the cessation of all metabolic activity.

[0138] 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.

[0139] The sample may include, but is not limited to, cell extracts, cell cultures (e.g., cells in suspension, cells adhered to a culture medium, a monolayer or multilayer cell culture, a 3D cell culture, a spheroid, or a biological tissue or tissue fragment, or a unicellular or multicellular organism. The sample may, in particular, include patient cancer cells, a patient cancer biopsy, cancer cell lines, or an artificial tumor.

[0140] Step i)

[0141] 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.

[0142] 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.

[0143] 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 constituting them are preferably placed or present in a preservation or culture medium.

[0144] 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 / quantification is based on fluorescence emission.

[0145] 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.

[0146] Preferably, the compound (I) according to the first object of the invention is used at a rate of about 1 nM to 100 mM, and particularly preferably from 100 nM to 10 pM, for about 1 cell to about 109 cells.

[0147] Step ii)

[0148] The sample is allowed to be incubated with compound (I), so that compound (I) binds to MDR proteins when they are present.

[0149] The sample is incubated with compound (I) which binds to the MDR proteins present in the cells. The incubation is carried out for a specific duration and at a temperature that maintains the cells in good condition.

[0150] 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.

[0151] 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.

[0152] Step iii)

[0153] The compound (I) not bound to MDR proteins is eliminated, to form a sample labeled with compound (I).

[0154] After incubation, the compound (I) not bound to the MDR proteins is removed. This removal may include aspirating the liquid medium and recovering the cells (cell pellet or adherent cells) or the cell mass (tissue, biopsy, etc.). This removal may be carried out 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.

[0155] The method may further include washing or rinsing the cells with a liquid medium suitable for preserving cells, tissues, etc.

[0156] Preferably, washing leads to an efflux of compound (I) having entered the cells but not having bound to an MDR protein.

[0157] Washing refers in particular to placing the cells in a liquid compatible with the preservation of the sample, called the "washing medium," possibly replacing the washing medium one or more times during this period. A sufficient number of washes are performed to adequately remove the free, unbound compound (I).

[0158] Step iv)

[0159] The compound (I) bound to MDR proteins can then be detected, localized, and / or quantified. These actions can also be combined, in particular detection and quantification, or localization and quantification, or detection and localization.

[0160] 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. The patient's response to treatment is determined, for example, the response of the tumor to an anticancer treatment or the appearance of resistance to a drug treatment, for example anticancer.

[0161] In the latter case, the method comprises processing as many cell or tissue samples (biopsies) from the same patient over time (for example, during drug treatment, for example, anticancer treatment) as there are samples according to the invention. The method used for clinical monitoring in a patient includes comparing samples taken at different times. The appearance of MDR protein expression, or the increase, stagnation, or regression of MDR protein expression over the observation period, can be detected.

[0162] Detection, localization, and / or quantification uses a fluorescence measurement method. The quantification or expression level of an MDR protein, for example P-gp, is obtained, in particular, by measuring the fluorescence intensity. It is then possible to compare samples with each other or with reference values.

[0163] The sixth object of the invention is 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) conforming to the first objective, 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 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) comparison with at least one fluorescence value from a reference sample F(0).

[0164] 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 of all organisms. The compounds (I) developed 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 labeling. membranes and / or the localization and quantification of MDR protein expression (e.g., P-gp protein). These can be used on various biological models, in the living state, in the non-living state, particularly in the fixed state, and therefore have a wider range of applications compared to current tools such as antibodies.

[0165] The invention will now be described in more detail with the aid of examples and embodiments taken by way of non-limiting examples and with reference to the accompanying drawings. Brief description of the drawings

[0166] [Fig.1] Fig.1 shows the cellular distribution of a compound (I) of the invention on Triple Negative breast cancer cell lines by fluorescence imaging. [Fig.2] Fig.2 shows the cellular distribution of compounds (I) of the invention on Triple Negative breast cancer cell lines by fluorescence imaging. [Fig.3] Fig.3 shows the cellular distribution of a compound (I) of the invention in a gammarus. [Fig.4] Fig.4 shows the location of the labeling by a compound (I) of the invention on a brown trout red blood cell. [Fig.5] Fig.5 expresses cellular labeling by a compound (I) of the invention of brown trout, from different streams of the Massif Central. [Fig.6] Fig.6 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.

[0167] Examples

[0168] Characterizations

[0169] The analyses by high-resolution mass spectrometry (HRMS) were carried out with an instrument sold under the trade name "Q-Exactive" by Thermo Scientific™.

[0170] 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.

[0171] Spectral characterizations were carried out by recording the excitation and emission spectra of the compounds in solution in ethanol on a “Cytation 10” fluorescence reader from Biotek Instrument.

[0172] Example 1: synthesis of compound (Ial-1)

[0173] The compound (Ial-1) was obtained according to the following reaction scheme:

[0174] [Chem.6]

[0175] First step:

[0176] 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 (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: 60% v / v acetone in hexane) to obtain the compound (R)-4-(oxiran-2-ylmethoxyquinoline 2) with a yield of 4%.

[0177] HRMS: m / z calculated for Ci2Hi2NO2, 202.0863 [M + H]+; found, 202.0861.

[0178] Second step:

[0179] 0.47 g of compound (R)-4-(oxiran-2-yhnethoxy)quinoline 2 obtained in the step The previous mixture 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(R)-4-(2-hydroxy-3-(quinolin-4-yloxy)propyl)piperazine-l-carboxylate 3 in quantitative yield.

[0180] HRMS: m / z calculated for C2iH30N3O4, 388.2231[M + H]+; found, 388.2218.

[0181] Third step:

[0182] 970 g of tert-butyl(R)-4-(2-hydroxy-3-(quinolin-4-yloxy)propyl)piped compound The razine-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 (R)-l-(piperazin-l-yl)-3-(quinolin-4-yloxy)propan-2-ol 4 in quantitative yield.

[0183] HRMS: m / z calculated for C16H22N3O2, 288.1707[M + H]+; found, 288.1695.

[0184] Fourth step:

[0185] 7.4 mg (2 equivalents) of compound (R)-l-(piperazin-l-yl)-3-(quinolin-4- yloxy)propan-2-ol 4 obtained in the previous step were brought into contact with 5.0 mg (1 equivalent) of 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 12.1 min).

[0186] HRMS: m / z calculated for C3OH35N5OH310BF2, 562.2795 [M + H]+; found, 562.2797.

[0187] The excitation and emission maxima are 505 nm and 513 nm respectively.

[0188] Example 2: Synthesis of compound (Ial-2)

[0189] The compound (Ial-2) was obtained according to the following reaction scheme:

[0190] [Chem.7]

[0191] The first, second and third steps for forming compound 4 are identical to those described in example 1.

[0192] Fourth step:

[0193] 4.5 mg (1 equivalent) of compound (R)-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 compound (Ial-2) with a yield of 10% (90% purity, HPLC 645 nm, retention time 13.5 min).

[0194] HRMS: m / z calculated for C45H49N7O911BF2, 816.3851 [M + H]+; found, 816.3837

[0195] The excitation and emission maxima are 649 nm and 669 nm respectively.

[0196] Example 3: synthesis of compound (Ial-3)

[0197] The compound (Ial-3) was obtained according to the following reaction scheme:

[0198] [Chem.8] 12 3 4

[0199] The first, second and third steps for forming compound 4 are identical to those described in example 1.

[0200] Fourth step:

[0201] 6.2 mg (1 equivalent) of compound (7?)-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 compound (Ial-3) with a yield of 10% (purity of 92%, HPLC 565 nm, retention time of 13.5 min).

[0202] HRMS: m / z calculated for C36H37N5O311BF2, 636.2952 [M + H]+; found, 636.2956.

[0203] The excitation and emission maxima are 562 nm and 575 nm respectively.

[0204] Example 4: Synthesis of compound (Ibl-1)

[0205] The compound (Ibl-1) was obtained according to the following reaction scheme:

[0206] [Chem.9]

[0208] 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 silica gel liquid chromatography (eluent: 50% v / v acetone in hexane) to obtain the compound (R)-5-(oxiran-2-ylmethoxy)quinoline 6 with a yield of 19%.

[0209] HRMS: m / z calculated for Ci2Hi2NO2, 202.0863 [M + H]+; found, 202.0861.

[0210] Second step:

[0211] 0.44 g of compound (R)-5-(oxiran-2-ylmethoxy)quinoline 6 obtained in the step The previous mixture was contacted with 0.40 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(R)-4-(2-hydroxy-3-(quinolin-5-yloxy)propyl)piperazine-l-carboxylate with a quantitative yield.

[0212] HRMS: m / z calculated for C2iH30N3O4, 388.2231[M + H]+; found, 388.2227.

[0213] Third step:

[0214] 830 mg of tert-butyl(R)-4-(2-hydroxy-3-(quinolin-5-yloxy)propyl)piped compound The razin-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 h at 20°C. A crude reaction mixture was obtained by vacuum evaporation to obtain the compound (R)-l-(piperazin-l-yl)-3-(quinolin-5-yloxy)propan-2-ol 8 with a quantitative yield.

[0215] HRMS: m / z calculated for Ci6H22N3O2, 288.1707 [M + H]+; found, 288.1700.

[0216] Fourth step:

[0217] 7.4 mg (2 equivalents) of compound (R)-l-(piperazin-l-yl)-3-(quinolin-5- The yloxy)propan-2-ol 8 obtained in the previous step was 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).

[0218] HRMS: m / z calculated for C30H35N5O310BF2, 562.2795 [M + H]+; found, 562.2786.

[0219] The excitation and emission maxima are 505 nm and 513 nm respectively.

[0220] Example 5: Use of the compound (Ibl-1) as a labeling tool

[0221] Cell labeling experiments were performed on two Triple Negative Breast Cancer cell lines (SUM1315 and DU4475) exhibiting different levels of P-gp expression.

[0222] 5.1 Preparation of cell samples

[0223] For the adherent cell line SUM1315

[0224] The SUM 1315 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).

[0225] Cells of this SUM1315 cell line are inoculated one day before labeling into 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).

[0226] For the DU4475 suspension-cultured cell line

[0227] The DU4475 cell line was obtained from ATCC (“HTB-123”) and was cultured in glutamine-free RPMI1640 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).

[0228] 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 volume of cell suspension corresponding to 800,000 cells is withdrawn and transferred into a 2 ml tube.

[0229] 5.2 Cell fixation

[0230] 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).

[0231] 5.3 Cell washing

[0232] The cells are washed three times with a PBS solution maintained at room temperature (i.e. close to 20°C).

[0233] 5.4 Cell Incubation

[0234] 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.

[0235] 5.5 Cell washing

[0236] The cells are washed three times with a solution of PBS maintained at 4°C.

[0237] For the DU4475 line cultured in suspension, centrifugation steps are further carried out between the different washes in order to be able to remove the supernatant medium while preserving the cells.

[0238] 5.6 Observations

[0239] Cell samples are observed using the plate reader marketed by Agilent under the reference “Cytation C10-MV”.

[0240] For SUM1315 cell line seeded in microplates, cells maintained in PBS are directly imaged in their culture wells.

[0241] For DU4475 cell 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).

[0242] 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).

[0243] 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.

[0244] Fig. 1 shows the cellular distribution of the compound (Ibl-1) on Triple Negative breast cancer cell lines, namely on the DU4475 cell line (Fig. 1 a) and on the SUM1315 cell line (Fig. 1 b), by fluorescence imaging.

[0245] From the acquired images, the mean fluorescence intensities were calculated and are presented in the graph in [Fig.1]c (in the form of a mean ± standard deviation, *****p<0.00001).

[0246] A significant labeling intensity is observed, with the labeling intensity for the DU4475 cell line being significantly higher than that measured for the SUM1315 cell line (ratio of 2.65). This ratio confirms the possibility of using the compounds of the invention as markers to quantify P-gp reliably and more rapidly than with antibodies.

[0247] 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).

[0248] Example 6: Use of compounds (Ial-1), (Ial-2), (Ial-3) and (Ibl-1) as a specific labeling tool for P-gp

[0249] 6.1 Preparation of cell samples

[0250] 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).

[0251] 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).

[0252] 6.2 Treatment with small RNAs interfering with P-gp synthesis

[0253] The next day, the cells are treated with small interfering RNAs with the synthesis of P-gp or P-gp siRNAs (marketed by 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).

[0254] 6.3 Cell fixation

[0255] The cells treated in point 5.3 above are fixed by contacting them for 10 minutes with a PBS solution comprising 4% by volume of paraformaldehyde (marketed by Sigma).

[0256] 6.4 Cell washing

[0257] The cells are washed three times with a PBS solution maintained at room temperature (i.e. close to 20°C).

[0258] 6.5 Cell Incubation

[0259] 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.

[0260] 6.6 Cell washing

[0261] The cells are washed three times with a PBS solution maintained at 4°C.

[0262] 6.7 Observations

[0263] Cell samples are observed using the plate reader marketed by Agilent under the reference “Cytation C10-MV”.

[0264] Cells maintained in PBS are directly imaged in their culture wells.

[0265] 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).

[0266] 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.

[0267] [Fig.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 ([Fig.2] a) and on the SUM1315 cell line with treatment ([Fig.2] b), by fluorescence imaging.

[0268] From the acquired images, the mean fluorescence intensities were calculated and are presented in the graph in [Fig.2] c (in the form of a mean ± standard deviation, *****p<0.00001).

[0269] No labeling intensity is observed for the treated DU4475 cell line (i.e., P-gp is silenced) compared to the significantly higher labeling intensity obtained for the untreated SUM1315 cell line, which demonstrates the specificity of the compounds of the invention with respect to P-gp and their usefulness for measuring the level of P-gp expression on cultured cells.

[0270] Example 7: Use of the compound (Ibl-1) as a biomonitoring tool (biomarker)

[0271] This example was carried out with freshwater shrimp (Gammarus fossarum) from different streams in the Massif Central.

[0272] 7.1 Attachment of gammarus

[0273] 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).

[0274] 7.2 Washing of amphipods

[0275] The gammarus are washed twice with water.

[0276] 7.3 Incubation of Gammarus

[0277] 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.

[0278] 7.4 Incubation of Gammarus

[0279] Gammarus are incubated for 90 minutes at 4°C with compound (Ibl-1) as prepared in Example 4 at a concentration of 1 pM.

[0280] 7.5 Cell washing

[0281] The gammarus are washed three times with water (30 minutes for each wash).

[0282] 7.6 Observations

[0283] Gammarus are observed using the plate reader marketed by Agilent under the reference “Cytation C10-MV”.

[0284] 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).

[0285] 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.

[0286] Figure 3 shows the cellular distribution of the compound (Ibl-l) in a gamma (scale bar 2 cm). This example demonstrates the possibility of using the biomarker on a whole organism.

[0287] Example 8: Use of the compound (Ibl-1) as a biomonitoring tool (tracer)

[0288] 8.1 Preparation of cell samples

[0289] 6 pl of red blood cells, from blood samples of brown trout, are rinsed in 1 ml of a PBS solution in a 2 ml microtube, then centrifuged at 200 revolutions per minute for 2 minutes.

[0290] 8.2 Cell Incubation

[0291] 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.

[0292] 8.3 Cell washing

[0293] The cells are washed twice with a PBS solution maintained at room temperature for 30 minutes. Between the different washes, centrifugation steps are also carried out in order to remove the supernatant medium while preserving the cells.

[0294] 8.4 Observations

[0295] The cell pellet obtained after centrifugation is resuspended in 1 ml of a PBS solution maintained at room temperature. The cell samples are observed using the plate reader marketed by Agilent under the reference "Cytation C10-MV".

[0296] 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).

[0297] 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).

[0298] Figure 4 shows the localization of the labeling by the compound (Ibl-1) on a brown trout red blood cell. Figure 4a shows the red blood cell under bright-field microscopy and Figure 4b under fluorescence microscopy.

[0299] 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).

[0300] In parallel, the fluorescence intensity is measured by flow cytometry with an instrument sold under the trade 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 trade name "FlowJo" by BD Biosciences.

[0301] The [Fig.5] expresses cellular labeling with the compound (Ibl-1) from brown trout, from different streams of the Massif Central.

[0302] The level of expression of P-gp (in fluorescence units UF) was obtained as a function of the locations of the trout (different streams).

[0303] Fluorescence intensities are shown in [Fig. 5] as mean ± standard deviation (in grey). A solid circle represents the mean fluorescence for an individual.

[0304] 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.

Claims

Demands

1. Compound corresponding to the following formula (I): [Chem. 10] in which: - R1 is a fluorochrome radical chosen from among the bodipy radicals, - L is a connecting arm present (n = 1) or absent (n = 0), - R2 is a nitrogenous heteroaromatic group selected from quinoline, isoquinoline, quinoxaline, pyridine, pyrimidine, pyrazine, pyridazine, purine, and indole, - R3, R4, R5 and R6, whether identical or different, represent a hydrogen atom or an alkyl group, - R7 and R8, whether identical or different, represent a hydrogen atom or an alkyl group, - R9 and R10, whether 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)-(L1)r-(F)s-(L2)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 a

3. oxygen atony, -C(=O), -S(=O)2-, -NH-C(=O)-, and -NH-C(=O)-CH 2-O-, - L1 and L2, 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-CH 2- 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. Compound according to claim 1 or 2, characterized in that the fluorochrome radical R1 corresponds to the following formula (II): [Chem. 11] in which: - R11 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, - R12, R13, R14, R15, R16, and R17, whether 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 R1 has an attachment point (denoted *) 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

4.

5.

6. 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. A compound according to any one of the preceding claims, characterized in that R3, R4, R5, and R6 are hydrogen atoms. A compound according to any one of the preceding claims, characterized in that R7, R8, R9, and R10 are hydrogen atoms. A compound according to any one of the preceding claims, characterized in that it conforms to the following formula (11a) or (11b): [Chem. 12] (ibl)

7.

8.

9. in which R1, L, R3, R4, R5 and R6 are as defined previously. 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. Use according to claim 7, characterized in that the MDR proteins are P-gp proteins. 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. 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) 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 the in vitro or ex vivo diagnosis of MDR protein overexpression 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).

Images