Application of aza-fluorinated boron fluorescent fluorophore compounds as short-wave infrared contrast agents

Abstract

The present invention relates to the use of aza-fluoroboron fluorophore compounds as contrast agents in the optical window of 1000 nm to 1700 nm. The present invention also relates to the use of a composition comprising the fluorophore compound and a pharmaceutically acceptable excipient and / or solvent as a contrast agent in a kit comprising an injection system and the fluorophore or the composition, and to a method for identifying biological targets (e.g., healthy or tumor cells, proteins, DNA, RNA).

Description

Technical Field

[0001] The present invention belongs to the field of optical imaging technology, the field of contrast agents, and more specifically to the field of organic contrast agents capable of emitting in the wavelength range of 1000nm-1700nm corresponding to the extreme far infrared.

[0002] The present invention relates to the use of aza-fluoroboron fluorophore compounds as contrast agents in the optical window of 1000 nm to 1700 nm. The present invention also relates to the use of a composition comprising the fluorophore compound and a pharmaceutically acceptable excipient and / or solvent as a contrast agent in a kit comprising an injection system and the fluorophore or the composition, and to a method for identifying biological targets (such as healthy or tumor cells, proteins, DNA, RNA, lipids or any other animal or plant biological target part) in vitro or in vivo.

[0003] In the description, references between square brackets ([ ]) refer to the Reference List at the end of the Examples. Background Art

[0004] Optical imaging is experiencing a new boom with the advent of highly sensitive cameras with a detection range of 900 nm to 1700 nm, which corresponds to the extreme far infrared known as NIR II or SWIR (short-wave infrared). This detection range is particularly interesting in optical imaging because it theoretically allows the observation of fluorescent signals located deeper in the tissue (compared to NIR I imaging) and / or with higher resolution and sensitivity. These observations, described in the articles by Bruns et al., Carr et al., and Thimsen et al. [1], are possible due to the lower autofluorescence of tissue in this optical range.

[0005] Below 1000 nm, many organic molecules have been described as contrast agents, including cyanines, Alexa, Atto, fluorophores, DyLight, rhodamine, fluorescein, indocyanine green, and others. In addition, inorganic compounds such as QDOT can be used for optical imaging in this optical range. "Visible" compounds in the 1000 nm to 1700 nm optical range have the advantage of emitting in an optical range that improves resolution and detection depth compared to the optical range below 1000 nm.

[0006] With the advent of these new cameras and various optical tools (such as lenses) suitable for wavelengths between 1000 nm and 1700 nm, the development of suitable contrast agents became necessary.

[0007] Therefore, there is a real need to find contrast agents that can be used with these new tools, thereby greatly improving the observed image quality, making it easier to identify target cells (e.g., tumors), and expanding the detection range of multifluorescence analysis.

[0008] The applicant is credited with discovering a class of fluorophore compounds called aza-fluoroborates, which can emit light in the detection range of 1000 nm to 1700 nm alone, encapsulated or grafted to molecules of interest (such as antibodies or biological ligands) or cells of interest (such as macrophages or stem cells).

[0009] The use of contrast agents according to the invention also allows tracking the distribution of biological targets intravascularly, in vitro, in vivo or ex vivo.

[0010] Other advantages will become apparent to a person skilled in the art on reading the following examples illustrated by the accompanying drawings, which are given by way of illustration and not by way of limitation. Summary of the Invention

[0011] The present invention relates to the use of fluorophore compounds of formula I as contrast agents in the optical window ranging from 1000 nm to 1700 nm:

[0012]

[0013] in,

[0014] -R 1 、R 2 、R 3 and R 4 are the same or different, and are optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group,

[0015] -R 1 、R 2 、R 3 and R 4 At least one of them is -NR c R d group and optionally selected from halogen, -OR d , hydrazine, -CF3 and -CN substituted C5-C7 aryl groups,

[0016] -R 5 and R 6 are identical or different and represent hydrogen, halogen, a C1-C15 group containing an aldehyde, ketone, carboxylic acid or ester functional group, nitrile, -SO3Na, a vinyl group optionally substituted by a ketone, ester or aromatic group, an imine substituted by an alkyl or aromatic group, an alkyne group optionally substituted by an alkyl or aromatic group, SPh, an aromatic chalcogenide (SePh, TePh), an amide, a molecule optionally selected from halogen, -NR c Rd 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group,

[0017] - Optionally, R 3 and R 5 and / or R 4 and R 6 covalently bonded together to form a radical optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group,

[0018] -R c and R d are the same or different and represent hydrogen or a linear or branched C1-C3 alkyl chain,

[0019] -R a and R b Is the same or different, meaning:

[0020] halogen, preferably selected from the group comprising fluorine and chlorine,

[0021] C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated groups, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, preferably in the form of one or more hydrophilic functional groups selected from quaternary ammonium, sulfate, sulfonate and phosphonate functional groups

[0022] a C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, preferably in the form of one or more bioconjugated functional groups selected from amines, carboxylic acids, activated esters of the N-hydroxysuccinimide type, pentafluorophenyl, tetrafluorophenyl, squarates and more particularly diethyl ester, maleimides, thiols, isothiocyanates, isocyanates, oxadiazolylmethylsulfones, azides, substituted or unsubstituted tetrazines, triazoles, trans-cyclooctenes, cyclooctynes ​​and more particularly dibenzocyclooctynes, bicyclononynes and PPh2AuCl complexes,

[0023] a biocarrier covalently coupled via a C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more heteroatoms selected from O, N, P and / or S, preferably in the form of one or more bioconjugated functional groups as defined above,

[0024] Metal complexes for therapeutic purposes, formed from chelating agents and metals,

[0025] Radiometal complexes, formed from chelating agents and radiometals, or

[0026] Molecules with a hydrodynamic diameter of less than 10 nm, cyclic or linear peptides, antibodies, antibody fragments, nanobodies, affibodies, aptamers, short DNA or RNA sequences, sugars, polysaccharides, amino acids, vitamins, AMD3100 molecules, PSMA ligands, steroids (e.g., progesterone), fatty acids (e.g., C4-C36), polyamines (e.g., C4-C14), polyphenols, DNA bases, or caffeine derivatives.

[0027] Advantageously, the fluorophore compound may be in the form of a salt or a pharmaceutically acceptable salt.

[0028] In the context of the present invention, "optical window" means a wavelength range. Fluorophores used as contrast agents according to the present invention may emit in an optical window in the range of 1000 nm to 1700 nm, preferably 1000 nm to 1300 nm, even more preferably 1000 nm to 1100 nm.

[0029] "Biological target" means a healthy or pathological cell, an organelle, a component of an animal or plant cell (such as a protein, lipid, DNA / RNA), as well as an antibody, a component of the extracellular matrix or a component of a biological fluid.

[0030] "Contrast agent" means a molecule or substance that artificially increases contrast, thereby allowing visualization of anatomical structures (e.g., organs) or pathological structures (e.g., tumors) that are naturally low-contrast or non-contrast and therefore difficult to distinguish in their environment. In the context of the present invention, contrast agents emit in the range of 1000 nm to 1700 nm, which is in the extreme far infrared, also known as NIR II or SWIR.

[0031] "Fluoroborofluor" is a compound containing a boron-dipyrromethene unit, mainly known as a strong ultraviolet absorbing dye, with the characteristics of emitting narrow fluorescence and high quantum yield. They are all derived from 4,4-difluoro-4-boron-3a,4a-diaza-sym-indacene:

[0032]

[0033] "Azafluoroborane" refers to a fluoroborane compound containing a nitrogen atom at the 8-position:

[0034]

[0035] For the purposes of the present invention, "aliphatic" means a non-aromatic group. An aliphatic group can be cyclic. An aliphatic group can be saturated, such as hexane, or unsaturated, such as hexene and hexyne. An open-chain group (whether straight or branched) does not contain any type of ring and is therefore aliphatic. An aliphatic group can be saturated, connected by a single bond (alkanes), or unsaturated, with a double bond (alkenes) or a triple bond (alkynes). A "heteroaliphatic" group is an aliphatic group having one or more heteroatoms, the most common of which are oxygen, nitrogen, phosphorus, and sulfur.

[0036] The term "derivative" refers to a compound or molecule made from a parent compound by one or more chemical reactions.

[0037] In general, the term "substituted" or "unsubstituted" (whether or not preceded by the term "finally" or "optionally") and substituents contained in the chemical formulae of the present invention refer to the replacement of hydrogen radicals in a given structure with the specified substituent radical. When more than one position in a given structure can be substituted with more than one substituent selected from a given group, the substituents may be the same or different at each position. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds.

[0038] As used herein, the term "alkyl" refers to straight and branched chain alkyl groups. Similar conventions apply to other general terms such as "alkenyl", "alkynyl" etc. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl etc., which may also carry one or more substituents. Alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-butenyl-2-1-yl etc. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl etc.

[0039] As used herein, the term "optionally containing one or more heteroatoms" refers to a group that carries or has included a heteroatom selected from O, N, P and S in the main chain.

[0040] Generally, the term "unsaturated" as used herein refers to a group whose molecular structure contains one or more carbon-carbon double or triple bonds.

[0041] Generally, as used herein, the term "aromatic group," "aromatic or heterocyclic ring," "aryl," or "heteroaryl" refers to a monocyclic or polycyclic unsaturated, stable, substituted or unsubstituted hydrocarbon group, preferably having 3 to 14 carbon atoms, containing at least one ring that satisfies Hückel's aromaticity rule. Examples of aromatic groups are, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthrenyl, and anthracenyl.

[0042] In general, as used herein, "cyclic" refers to a 3-8 membered cyclic fragment that is substituted or unsubstituted and optionally contains one or more heteroatoms on the main chain or side chain. Examples of such heteroaryl groups include, but are not limited to, the following: pyridyl, thiazolyl, thiazolyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, benzazepinyl, thiazolyl, indolyl, dihydroindolinyl, quinolyl, isoquinolyl, benzimidazolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, triazinyl, thianthracene, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, quinolyl, phthalazinyl, naphthyl benzotriazole, benzoisoxazolyl, oxindolyl, benzoxazolinyl, benzothiophene, benzothiazolyl, isatinyl, dihydropyridinyl, pyrimidinyl, s-triazolinyl, oxazolyl, and thiofuranyl.

[0043] In general, the terms "independently" refer to the fact that the substituents, atoms or groups to which these terms refer are independently selected from the list of variables (this means that they may be the same or different) from one another.

[0044] As used herein, the term "halogen" refers to an atom selected from fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.

[0045] "Bioconjugative functional groups" are defined as chemical functional groups that allow the compounds of the present invention to be covalently linked to a molecule of interest, more specifically a biomolecule of interest, preferably a biovector. Examples of such functional groups include, but are not limited to, amines, carboxylic acids, N-hydroxysuccinimide-type activated esters, pentafluorophenyl groups, tetrafluorophenyl groups, squaric acid functional groups and more specifically diethyl ester, maleimide groups, thiols, isothiocyanates, isocyanates, oxadiazolylmethylsulfones, azides, substituted or unsubstituted tetrazines, triazoles, trans-cyclooctenes, cyclooctyne-type functional groups and more specifically dibenzocyclooctyne, bicyclononyne and PPh2AuCl complexes.

[0046] "Biocarrier" means any encapsulation system, or any ligand that allows recognition of a specific biological target, covalently coupled or not via a bioconjugated functional group.

[0047] By "chelating agent" is meant any chemical substance that has the property of permanently fixing ions, more particularly metal cations, to form a complex. This may include chelating agents of the polyamine family, whether cyclic or not.

[0048] "Radio metal" means a radioactive metal that emits, for example, gamma or beta+ radiation such as gallium-68 or fluorine-18, or beta or alpha radiation such as lutetium-177 or actinium-225.

[0049] "Metal complex for therapeutic purposes" means any complex which, by itself or after activation by external or internal stimuli, contains an atom with therapeutic properties. It can be, for example, a complex selected from the group consisting of: PR2M (M = Ru(II), Os(II), Ru(III), Au(I) or (III), Pt(II), Pt(IV), Pd(II), Ir(III), Cu(I), Cu(II)) (R = alkyl, aryl, heteroaryl, preferably triazaphosphaadamantane), carbene-M (M = Ru(II), Os(II), Ru(III), Au(I) or (III), Pt(II), Pt(IV), Ir(III), Cu(I), Cu(II)) II)), phenylpyridine-M (M=Au(III), Pt(II), Ru(II), Ir(III), Re(V), Au(II), Pt(IV), Pd(II), Ir(III), Cu(II), Ir(III), Re(V ), Re(III), Cu(II)), polypyridine (M=Au(III), Pt(II), Ru(II), Ir(III), Re(V), Re(III), Cu(II), Os(II)), SM(M=Au(I), Au(I II), Cu(I), Cu(II), Ti(IV), Zr(IV)), alkyne Au(I), dithiocarbamate-M (M=Au(I), Au(III), Cu(I), Cu(II)), quinoline-M (M=Ga, Fe), η3-arene-M (M=Ru(II), Os(II), Cr(VI), Mo(III)), metallocene-M (M=Fe(II), Fe(III), Ti(IV), Ti(III), Zr(IV), Ir(III), Rh(III) , Cr(VI), Ta(III), Os(II)), salen and salan-M (M=Au(III), Ti(IV), Zr(IV), Cu(II), Pt(II), Pd(II)), malonic acid derivatives-M (M=Pt(II), Ti(IV)), ethylenediamine-M (M=Pt(II), Pd(II), Cu(II), Au(III), Ru(II), Os(II)), benzaldimine-M (M=Ru(II), Rh(III), Ir(III)).

[0050] Similarly, "radiometallic complex" means any complex containing a radiometallic atom. The complex can be selected from DTPA, NOTA, NODAGA, DOTA, DOTAGA, p-NCS-Bn-DOTA, p-NCS-Bn-NOTA, DFO, sarcophagine, bridged The invention is composed of a chelating agent of a derivative of cyclam, salan, salen, HBED, bipyridine-type polypyridine, terpyridine, phenanthroline, phosphine or diphosphine polypyridine, carbene, aromatic hydrocarbons, cyclopentadiene, alkyne, thiolate, phenylpyridine, and phenyltriazole, and a radio metal selected from Ga68, Ga67, AlF18, In111, Zr89, Sc43, Sc44, Sm153, Cu61, Cu64, Co55, Co57, Tb152, Tb157, Ru103, Ru97, Ru95, Os191, Au198, Au199, Ti45, Pt195, Pt193, Pd100, Re186, and Re188. It may, for example, be of the DOTA or NODAGA type and a radiometal selected from Ga68 and In111 to obtain a double-peak probe detectable in PET / SPECT and optical imaging, or selected from Lu177, Y90, Ac225, Pb212, Bi212, Eb109, Yt161, Sc47, Cu67, Tb161, Os191, Pt195, Pt193, Au199, Pd103, Re186, Re188, Sm153 for therapeutic diagnostic applications; such as DOTAGA- 111 In.

[0051] As will be understood by those skilled in the art, all numbers, including those expressing amounts of ingredients, properties such as molecular weight, reaction conditions, etc., are approximate and should be understood to be modified in all instances by the term "about." These values ​​may vary depending on the properties sought by those skilled in the art utilizing the teachings described below. It should also be understood that these values ​​contain inherent variability necessarily resulting from the standard deviation found in their respective testing measurements.

[0052] Those skilled in the art will also readily recognize that when members are grouped together in the same manner, such as in a Markush group, the present invention encompasses not only the entire group listed, but also each individual member of the group and all possible subgroups of the main group. Furthermore, for all practical purposes, the present invention encompasses not only the main group, but also the main group in which one or more group members are absent. Thus, the present invention provides for explicit exclusion of one or more members of the group. Thus, the reserve condition may apply to any disclosed category or embodiment, thereby excluding one or more of the elements, types, or embodiments from these categories or embodiments, such as when used in a clear negative limitation.

[0053] As used herein, the term "isomer" refers to a compound that can exist in one or more geometric, optical, enantiomeric, diastereomeric, epimeric, atropisomeric, stereoisomeric, tautomeric, conformational, or specific anomeric forms. Examples of isomers limited to cis and trans forms; E and Z forms; C, T, and R forms; C, T, and R forms; endo and exo forms; R, S, and meso forms; D and L forms; d and l forms; (+) and (-); keto, enol, and enolate forms; cis and trans forms; cis- and anti-anisomeric forms; α and β forms; upright and equatorial forms; boat, chair, twist boat, shell, and semi-chair forms; and combinations thereof are hereinafter collectively referred to as "isomers" (or "isomer forms"). In the present disclosure, the term "isomer" does not include structural isomers or constitutional isomers that are structurally different and described by different linear formulas [2].

[0054] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R 1 、R 2 、R 3 and R 4 Same or different. 1 、R 2 、R 3 and R 4 R may independently represent a C5-C7 aryl or heteroaryl group. 1 、R 2 、R 3 and R 4 may be independently optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN are substituted by at least one group.

[0055] Fluorophore compounds may be selected from compounds of formula I, wherein R 1 、R 2 、R 3 and R 4 At least one of them is -NR c R d group and optionally selected from halogen, -OR d , hydrazine, -CF3 and -CN. Preferably, when it is a C6 aryl group, -NR c R d The group is in the para position.

[0056] Advantageously, the fluorophore compound is selected from compounds of formula I, wherein R 5 and R 6 Same or different. 5 and R6 and -S03Na, a vinyl group optionally substituted by a ketone, an ester or an aromatic group, an imine substituted by an alkyl or aromatic group, an alkyne group optionally substituted by an alkyl or aromatic group, SPh, an aromatic chalcogenide (SePh, TePh), an amide, a halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted C5-C7 aryl or heteroaryl group. Preferably, R 5 and R 6 It is hydrogen or -SO3Na.

[0057] Advantageously, the fluorophore compound is selected from compounds of formula I, wherein R c and R d Same or different. c and R d Can independently represent hydrogen or a linear or branched C1-C3 alkyl chain. Preferably, -NR c R d Selected from the group consisting of -NH2, -NMe2, -NEt2, -NPr2, preferably -NMe2.

[0058] Advantageously, R 3 and R 5 and / or R 4 and R 6 can be covalently bonded together to form a radical optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN.

[0059] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R 5 and R 6 For hydrogen.

[0060] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R 1 and R 2 Are the same or different, indicated by -NR c R d group and optionally selected from halogen, -OR d , hydrazine, -CF3 and -CN substituted C5-C7 aryl groups. Preferably, the fluorophore compound can be selected from the compounds of formula I, wherein R 1 and / or R 2 Indicates -NR c Rd The phenyl group is substituted in the para position.

[0061] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and R b may represent halogen, preferably fluorine or chlorine.

[0062] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and R b It may represent a C1-C50, preferably a C2-C30 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, preferably in the form of one or more hydrophilic functional groups selected from quaternary ammonium, sulfate, sulfonate and phosphonate functional groups.

[0063] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and R b It may represent a C1-C50, preferably a C5-C30 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, preferably in the form of one or more bioconjugated functional groups selected from amines, carboxylic acids, activated esters of the N-hydroxysuccinimide type, pentafluorophenyl, tetrafluorophenyl, squarates and more particularly diethyl ester, maleimides, thiols, isothiocyanates, isocyanates, oxadiazolylmethylsulfones, azides, substituted or unsubstituted tetrazines, triazoles, trans-cyclooctenes, cyclooctynes ​​and more particularly dibenzocyclooctynes, bicyclononynes and PPh2AuCl complexes.

[0064] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and R b May refer to a biocarrier covalently coupled via a group comprising a bioconjugated functional group as defined above.

[0065] Advantageously, the fluorophore compound is selected from compounds of formula I, wherein R a and or R b are the same or different, including 10 BSH(Na2B12 H 11 SH, or borocaltic sodium, is rich in boron (10), and is used to obtain theranostic activity in boron therapy (boron neutron capture therapy, or BNCT).

[0066] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and R bIt may represent a metal complex for therapeutic purposes, preferably selected from the following complexes: formula PR2M, wherein M is a metal selected from the group consisting of Ru(II), Os(II), Ru(III), Au(I) or (III), Pt(II), Pt(IV), Pd(II), Ir(III), Cu(I), Cu(II), and R is a C1-12 alkyl, aryl, heteroaryl, preferably triazaphosphaadamantane; carbene-M, wherein M is a metal selected from the group consisting of M=Ru(II), Os(II), Ru(III), Au(I) or (III), Pt(II), Pt(IV), Ir(III) , Cu(I), Cu(II); phenylpyridine-M, wherein M is a metal selected from the group consisting of Au(III), Pt(II), Ru(II), Ir(III), Re(V), Re(III), Cu(II); polypyridine-M, wherein M is a metal selected from the group consisting of Au(III), Pt(II), Ru(II), Ir(III), Re(V), Re(III), Cu(II), Os(II); SM, wherein M is a metal selected from the group consisting of Au(I), Au(III), Cu(I), Cu(II), Ti(IV), Zr(IV) Metal; Alkyne-Au(I); Dithiocarbamate-M, wherein M is a metal selected from the group consisting of Au(I), Au(III), Cu(I), Cu(II); Quinoline-M, wherein M is a metal selected from the group consisting of Ga, Fe; η3-arene-M, wherein M is a metal selected from the group consisting of Ru(II), Os(II), Cr(VI), Mo(III); Metallocene-M, wherein M is a metal selected from the group consisting of Fe(II), Fe(III), Ti(IV), Ti(III), Zr(IV), Ir(III), Rh(III), Cr(VI), Ta(III), Os(I I); salen and salan-M, wherein M is a metal selected from the group consisting of Au(III), Ti(IV), Zr(IV), Cu(II), Pt(II), Pd(II); malonic acid derivative-M, wherein M is a metal selected from the group consisting of Pt(II), Ti(IV); ethylenediamine-M, wherein M is a metal selected from the group consisting of Pt(II), Pd(II), Cu(II), Au(III), Ru(II), Os(II); benzaldimine-M, wherein M is a metal selected from the group consisting of Ru(II), Rh(III), Ir(III).

[0067] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Same or different. a and Rb It can represent a radio metal complex formed by a chelating agent and a radio metal. The complex can be selected from DTPA, NOTA, NODAGA, DOTA, DOTAGA, p-NCS-Bn-DOTA, p-NCS-Bn-NOTA, DFO, sarcophagine, bridged The invention is composed of a chelating agent of a derivative of cyclam, salan, salen, HBED, bipyridine, terpyridine, phenanthroline, phosphine or diphosphine, carbene, aromatic hydrocarbon, cyclopentadiene, alkyne, thiolate, phenylpyridine, phenyltriazole, and a radio metal selected from Ga68, Ga67, AlF18, In111, Zr89, Sc43, Sc44, Sm153, Cu61, Cu64, Co55, Co57, Tb152, Tb157, Ru103, Ru97, Ru95, Os191, Au198, Au199, Ti45, Pt195, Pt193, Pd100, Re186, Re188. It can, for example, be of the DOTA or NODAGA type and a radiometal selected from Ga68 and In111 to obtain a double-peak probe detectable in PET / SPECT and optical imaging, or selected from Lu177, Y90, Ac225, Pb212, Bi212, Eb109, Yt161, Sc47, Cu67, Tb161, Os191, Pt195, Pt193, Au199, Pd103, Re186, Re188, Sm153 for therapeutic diagnostic applications.

[0068] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are identical or different and are selected from small molecules (meaning molecules with a hydrodynamic diameter of less than 10 nm, such as gold clusters or metal complexes), cyclic or linear peptides (such as α v β3 integrin targeting c (RGDfK) or neuropilin targeting ATWLPPR), antibodies (e.g., anti-CD44 type), antibody fragments or nanobodies targeting membrane or intracellular receptors, affibodies, aptamers, short DNA or RNA sequences, sugars (e.g., thioglucose or peracetylated thioglucose) or polysaccharides, amino acids, vitamins (e.g., folic acid type), AMD3100 type ligands, PSMA ligands, steroids, fatty acids, polyamines (e.g., spermine, spermidine, cadaverine or putrescine type), polyphenols (e.g., resveratrol), DNA bases, caffeine derivatives, progesterone.

[0069] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different and are selected from halogen, preferably fluorine.

[0070] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different hydrophilic groups selected from the following formulas:

[0071]

[0072] In this application, the symbol represents the point of attachment of the group shown to the molecule. For example, it can be the point of attachment to B (boron atom).

[0073] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different and are selected from the group consisting of bioconjugated functional groups of the following formula:

[0074]

[0075]

[0076] -NH2 and -Si(OMe)3.

[0077] Preferably, the bioconjugated functional group can be selected from N-hydroxysuccinimide, conjugated isothiocyanate, tetrazine, diethyl squarate, maleimide, oxadiazolylmethyl sulfone, pentafluorophenyl, azide, PPh2AuCl complex and NH2 functional group.

[0078] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and / or R b Contains a covalently coupled biological carrier and has, for example, the following formula:

[0079]

[0080] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different and are selected from PPh2-Au(I), PPh2-Pt(II), PPh2-Pt(IV), and phenylpyridine-Au(III).

[0081] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different and are selected from DOTA-In(III), DOTAGA-In(III), NODAGA-Cu(II), and NODAGA-Ga(III).

[0082] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and Rb are identical or different and are selected from cyclic or linear peptides (e.g. v β3 integrin targeting c (RGDfK) or neuropilin targeting ATWLPPR), antibodies (e.g., anti-CD44 type), antibody fragments or nanobodies targeting membrane or intracellular receptors, short DNA or RNA sequences, sugars (e.g., thioglucose or peracetylated thioglucose) or polysaccharides, amino acids, vitamins (e.g., folic acid type), AMD3100 type ligands, PSMA type ligands, steroids (e.g., progesterone), fatty acids (e.g., C4-C36), polyamines (e.g., spermine, spermidine, cadaverine or putrescine type), polyphenols (e.g., resveratrol), DNA bases, caffeine derivatives (e.g., caffeine).

[0083] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b Selected from cyclic or linear peptides (α v β3 integrin targeting c (RGDfK) or neuropilin targeting ATWLPPR), antibodies (e.g., anti-CD44 type), antibody fragments targeting membrane or intracellular receptors, short DNA or RNA sequences, sugars (e.g., thioglucose or peracetylated thioglucose) or polysaccharides, amino acids, vitamins (e.g., folic acid type), AMD3100 ligands, steroids (e.g., progesterone), fatty acids (e.g., C4-C36), polyamines (e.g., spermine, spermidine, cadaverine or putrescine type), polyphenols (e.g., resveratrol), DNA bases, caffeine derivatives (e.g., caffeine).

[0084] Advantageously, the fluorophore compound may be selected from compounds of formula I, wherein R a and R b are the same or different, and are selected from the following groups:

[0085]

[0086] Advantageously, the fluorophore compound may be selected from the fluorophore compounds of formula I, wherein R 5 and R 6 H, R 1 and R 2 for And R3 and R4 are respectively

[0087] in,

[0088] -R a 、R b 、R c 、R d Having the definitions given above,

[0089] -R e and R f are the same or different, representing hydrogen, halogen, -NR c R d 、-OR d , hydrazine, -CF3 and at least one of -CN.

[0090] Advantageously, the fluorophore compound may be selected from compounds of formula I or II, wherein R c and R d is -CH3.

[0091] Advantageously, the fluorophore compound may be selected from compounds of formula II, wherein R e and R f is H or -OMe, preferably in the para position.

[0092] Advantageously, the fluorophore compound can be included in a composition that also includes a pharmaceutically acceptable excipient and / or solvent. This can be any pharmaceutically acceptable excipient that one skilled in the art can place into a composition to alter its pH, osmotic pressure, viscosity, or solubility. Examples include NaCl (0.9% in water), 5 g / L glucose solution, ppi water, or a buffered solution such as PBS or other pharmaceutically acceptable buffer.

[0093] Advantageously, the composition may have a pH of from 4 to 10, preferably from 6 to 8 or from 6.8 to 7.6. Preferably, the pH of the composition is 7.4.

[0094] Advantageously, the concentration of the fluorophore compound in the composition is in the range of 0.1 μmol / L to 1000 μmol / L, preferably 1 μmol / L to 100 μmol / L or 10 μmol / L to 40 μmol / L (these values ​​are particularly compatible with in vitro assays), or in the range of 4 nmol / kg to 300 μmol / kg, preferably 40 nmol / kg to 12.5 μmol / kg, or about 1 μmol / kg to 1.5 μmol / kg in mice (these values ​​are particularly compatible with in vivo assays).

[0095] Advantageously, the fluorophore compound can be encapsulated. In the context of the present invention, "encapsulated" means any biocompatible object that is capable of grouping together several fluorophore compounds and possibly protecting them and directing them to the target of interest, such as lipid nanoparticles, nanoformulations (especially polysaccharide-based nanoformulations), carbon nanotubes, micelles. For example, the fluorophore compound can be encapsulated in a lipophilic formulation, including liposomes as described in the publication by Gravier et al. [3], et al. [4] or hydrophobic nanodomains such as polysaccharide nanoformulations as described in the publication by Garcia et al. [5].

[0096] Advantageously, the fluorophore compound may be covalently linked to the nanoparticle. The coupling may be via an R having a bioconjugated functional group as defined above. a or R b The nanoparticles that can be covalently linked to the compound of formula I or II can be small nanoparticles with a size less than 10 kDa or lipid nanoparticles. These can be, for example, gold nanoclusters or liposomes.

[0097] The present invention also relates to a kit comprising an injection system and a composition comprising a fluorophore compound of formula I or II as defined above and a pharmaceutically acceptable excipient and / or solvent.

[0098] The present invention also relates to a method for in vitro identification of biological targets (such as healthy or tumor cells, proteins, DNA, RNA), the method comprising at least the following steps:

[0099] - labeling cells of a collected or cultured sample with a composition comprising a fluorophore compound of formula I or II as defined above,

[0100] - Fluorescence measurements in the optical window include the range of 1000nm to 1700nm, and

[0101] - Identify target cells.

[0102] Advantageously, in the in vitro identification method according to the present invention, the fluorophore concentration in the composition is in the range of 0.1 μmol / L to 1000 μmol / L, preferably 1 μmol / L to 100 μmol / L or 10 μmol / L to 40 μmol / L.

[0103] Advantageously, labelling of the sample cells is performed by injection or spraying of a composition comprising a fluorophore.

[0104] Advantageously, the fluorescence measurement in the optical window comprised in the interval of 1000 nm to 1700 nm is performed by any means known to those skilled in the art and capable of measuring said fluorescence. This can be, for example, confocal microscopy or epifluorescence flow cytometry, by fluorescence reflectance (2D or 3D) or even by optical imaging performed by optical probes for assisting surgical procedures or for readings suitable for multiwell plates.

[0105] The present invention also relates to a method for in vivo identification of biological targets (such as healthy or tumor cells, proteins, DNA, RNA), the method comprising at least the following steps:

[0106] - labeling the cells of the subject by injection or spraying with a composition comprising a fluorophore compound of formula I or II as defined above,

[0107] - Fluorescence measurements in the optical window include the range of 1000nm to 1700nm, and

[0108] - Identify target cells.

[0109] Advantageously, in the in vivo identification method according to the invention, the fluorophore concentration in the composition is in the range of 4 nmol / kg to 300 μmol / kg, preferably 40 nmol / kg to 12.5 μmol / kg, or about 1 μmol / kg to 1.5 μmol / kg in mice.

[0110] Advantageously, labelling of the sample cells is performed by injection or spraying of a composition comprising a fluorophore.

[0111] Advantageously, the fluorescence measurement in the optical window comprised within the interval 1000 nm to 1700 nm is performed by any means known to those skilled in the art and capable of measuring said fluorescence. This can be, for example, epifluorescence or confocal microscopy, flow cytometry, optical imaging by fluorescence reflectance (2D or 3D) or even by portable optical probes such as those used to assist surgery or suitable for plate readers.

[0112] The in vivo model used herein is based on mice. Advantageously, in the method according to the invention, the concentration of the fluorophore in the composition administered in vivo is in the range of 4 nmol / kg to 300 μmol / kg, preferably 40 nmol / kg to 12.5 μmol / kg, or about 1 μmol / kg to 1.5 μmol / kg in mice. Any other administration may be performed according to interspecies conversion, as described, for example, in the article by Reagan-Shaw et al. [6].

[0113] In a non-limiting manner we can mention the advantages associated with the present invention:

[0114] - Improve the quality of observed images and increase image resolution (compared to NIR imaging),

[0115] - Easier identification of tumor cells, including visualization of tumors in vivo,

[0116] - Deep tissue detection of biological targets,

[0117] - The distribution of biological targets in the tube can be monitored in vitro or in vivo,

[0118] - Increase the wavelength range that can be used for multi-label analysis,

[0119] - Expand the range of fluorescent compounds in multiple labels,

[0120] -Enable imaging in the optical range above 1000nm (called SWIR or extreme far infrared),

[0121] -Also allows combining this imaging technique with photoacoustic imaging,

[0122] - Reduces scatter and autofluorescence from tissue.

[0123] Furthermore, according to the present invention, optical contrast agents can be used alone, or coupled to a molecule of interest, or encapsulated in a nanoformulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 A mouse with a U87MG tumor in the right hind leg is shown before and 24 hours after injection of azafluoroborane (AG22). The upper image was recorded in NIR imaging: Fluo800 corresponds to excitation at 780nm and collection between 830nm-900nm. The lower image was excited at 830nm (SWIR800) and emission was collected using a 1064nm longpass filter (meaning between 1064 and 1700nm).

[0125] Figure 2 The emission spectra of compounds AG22 and AG04 are shown.

[0126] Figure 3 Shown from left to right are mice bearing a U87MG tumor in the right hind leg: before, 5 hours, and 48 hours after injection of the AG66 compound. DETAILED DESCRIPTION

[0127] Example

[0128] Example 1: Synthesis of a contrast agent according to the present invention

[0129] Materials and methods

[0130] Unless otherwise stated, reactions were carried out at standard atmospheric pressure in technical grade Carlo Erba solvents. Experiments requiring anhydrous conditions were performed under argon. Anhydrous solvents were purchased from Carlo Erba, unstabilized, and used with MB-SPS-800 (MBraun) or PureSolv-MD-5 All commercial reagents were purchased from or ACROS TOTA-Boc (boc-1-amino-4,7,10-trioxa-13-tridecylamine) was purchased from Iris Biotech. and 10B-BSH was purchased from Reaction monitoring in 0.2mm thick HPLC-MS and thin layer chromatography were performed on 60F254 silica gel plates and visualized by UV (254 nm). Industrial silica gel, 40-63μm, 230-400 mesh, Onward.

[0131] NMR spectra were recorded on a Bruker 500Avance III or Bruker 600Avance III HD equipped with a dual resonance broadband probe ( 1 H, 13 C). Chemical shifts are expressed in ppm and are relative to TMS ( 1 H, 13 C) is given, with the residual solvent signal as reference. High-resolution mass spectra were recorded on a Thermo LTQ Orbitrap XL ESI-MS spectrometer. NMR and mass analysis were performed at the Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne (PACSMUB).

[0132] HPLC-MS analysis was performed on a Thermo-Dionex Ultimate 3000 instrument (pump + autosampler at 20°C + column oven at 25°C) equipped with a diode array detector (Thermo-Dionex DAD 3000-RS) and a Phenomenex Chromatographic column (2.6 μm, C18, The characterization was performed on a simple MSQ Plus quadrupole mass spectrometer equipped with a 50x2.1 mm LC column. The gradient used for characterization was as follows (Gradient A):

[0133]

[0134] Semi-preparative HPLC purification was performed on a Shimadzu HPLC instrument equipped with two LC-20AT pumps, an SPD-20A UV / Vis detector, an FRC-10A fraction collector, a SIL-10AP sampler, and a CBM-20A control unit. The column used was a Shim-Pack GIST 5 μm C18 10 x 250 mm column, and the gradient used was as follows:

[0135]

[0136] CompoundAG22

[0137]

[0138] 18 μ L N, N-dimethyl propargylamine (156 μ mol, 2eq) is dissolved in 2mLTHF (tetrahydrofuran) under argon (shlenk glassware). Then add ethylmagnesium bromide (0.17mL, 170 μ mol, 2.2eq) and reflux the mixture 45 minutes, make it return to room temperature, then transfer to the shlenk glassware that aza-fluorine boron fluorescent precursor (50mg, 78 μ mol, 1eq) is housed via sleeve pipe. Then the mixture is refluxed under argon for 45 minutes, then by adding 2mL EtOH stopped reaction. Remove solvent by evaporation under reduced pressure. The crude product formed is dissolved among the 10mL AcOEt, then add 10mL distilled water. In separating funnel, stir and after sedimentation, organic phase is set aside, with 10mL AcOEt extracted aqueous phase twice. Merge organic phase, with the NaHCO3 aqueous solution washed twice, and at anhydrous MgSO4 top drying with 10mL twice dilution. The resulting solution was filtered and the solvent was removed by evaporation under reduced pressure.The residue was purified by silica gel column chromatography (eluent: 98 / 2 DCM / MeOH→100% MeOH) to isolate AG22 (51.1 mg, 66.3 μmole, 85%) as a purple shiny powder.

[0139] 1 H NMR (CDCl3, 500MHz) δ (ppm): 8.18 (d; 3 J = 8.9 Hz; 4H); 8.05 (d; 3 J = 9.0 Hz; 4H); 6.97 (d; 3 J = 9.0 Hz; 4H); 6.77 (d; 3 J=8.9Hz; 4H); 6.77 (s; 2H); 3.86 (s; 6H); 3.27 (s; 4H); 3.08 (s; 12H); 2.26 (s; 12H).

[0140] 13 C NMR {1H} (CDCl3, 150MHz) δ (ppm): 160.8; 156.5; 150.8; 143.0; 142.4; 132.0; 130.6; 125.6; 121.0; 115.6; 113.4; 112.0; 55.4; 48.0; 42.3; 40.2.

[0141] HR-MS(ESI)(Da): m / z C 48 H 52 BN7O2[M+H] +Calculated value: 770.42755; measured value: 770.43559.

[0142] Analytical HPLC (gradient A): Tr = 4.46 min.

[0143] Compound AG24

[0144]

[0145] AG22 (30 mg, 39 μ mol, 1 eq) is dissolved in 3 mL DCM (dichloromethane). Iodomethane (1.5 mL, in large excess) is then added, and the reaction medium is stirred at room temperature for 1 hour. The solvent is then removed under reduced pressure, and the crude product of the gained is dissolved in 10 mL H2O / DCM mixture (1 / 1). The aqueous phase is extracted with DCM (3 × 5 mL), and the organic phase of the gained is then extracted with water (8 × 10 mL) in a second step. The aqueous phase recovered is evaporated, and the formed precipitate is purified by HPLC (gradient A). The solid gained is freeze-dried to obtain pure AG24 (17.8 mg, 17.2 μ mol, 44%) as a bluish-green solid.

[0146] 1 H NMR (DMSO, 500MHz) δ (ppm): 8.24 (d; 3 J He-Hf =8.9Hz; 4H); 8.10(d; 3 J Hb-Hc =9.0Hz; 4H); 7.22(s; 2H); 7.11(d; 3 J Hb-Hc =9.0 Hz; 4H); 6.86 (d; 3 J He-Hf =8.9Hz; 4H); 4.00 (s; 4H); 3.87 (s; 6H); 3.07 (s; 12H); 2.78 (s; 18H).

[0147] HR-MS (ESI) (Da): m / z = C 50 H 58 BN7O2 2+ [M] 2+ Calculated value 399.73670; measured value 399.73869.

[0148] Analytical HPLC (gradient A): Tr = 4.49 min.

[0149] Compound AG38

[0150]

[0151] AG22 (250mg, 0.32mmol, 1eq) is dissolved in 50mL THF and 8mL H o.Add NaHCO (137mg, 1.63mmol, 5.1eq), then add 4-bromoethyl benzoic acid (144mg, 0.67mmol, 2.1eq).The reaction mixture is stirred at room temperature overnight.Then add 90mL Et o and 90mL H o, and separate organic phase and aqueous phase. Use Et o (6 × 60mL) wash aqueous phase to remove remaining trace 4-bromoethyl benzoic acid.Then by evaporating water in a rotary evaporator (35 ℃ bath), aqueous phase is reduced to 1 / 3 of its initial volume.Then add 10mL hydrochloric acid (3M).Then the content of the flask is centrifuged.Remove supernatant, precipitate is suspended in 15mL Et o and centrifuged again. This operation was repeated three times, and the resulting precipitate was then dissolved in MeOH and evaporated to dryness on a rotary evaporator (35°C bath) to afford pure AG38 (336 mg, 0.28 mmol, 85%) as a black flaky powder.

[0152] 1 H NMR (DMSO, 500MHz) δ (ppm): 8.30 (d; 3 J = 8.9 Hz; 4H); 8.12 (d; 3 J = 9.0 Hz; 4H); 7.95 (d; 3 J = 8.3 Hz; 4H); 7.43 (d; 3 J=8.3Hz; 4H); 7.28(s; 2H); 7.08(d; 3 J = 9.0 Hz; 4H); 6.88 (d; 3 J=8.9Hz; 4H); 4.24 (s; 4H); 3.96 (s; 4H); 3.74 (s; 6H); 3.08 (s; 12H); 2.76 (s; 12H).

[0153] Analytical HPLC (gradient A): Tr = 4.50 min.

[0154] Compound AG57

[0155]

[0156] AG22 (75 mg; 0.097 mmol, 1 eq) was dissolved in 60 mL of anhydrous THF in a 250 mL flask under argon. 4-Bromoethylbenzoic acid (23 mg; 0.106 mmol, 1.1 eq) was then added, and the mixture was stirred under reflux overnight. After cooling, the supernatant was removed and the formed precipitate was washed with THF (3 × 15 mL), ether (2 × 15 mL) and pentane (2 × 15 mL). All supernatants were combined and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (8:2 toluene / MeOH→100% MeOH) to isolate AG57 (20.7 mg, 0.021 mmol, 22%) as a black flaky powder.

[0157] 1 H NMR (DMSO, 500MHz) δ (ppm): 8.36 (d; 3 J = 8.9 Hz; 4H); 8.07 (d; 3 J = 9.0 Hz; 4H); 7.98 (d; 3 J = 8.3 Hz; 2H); 7.24 (d; 3 J = 8.3 Hz; 2H); 7.00 (d; 3 =9.0Hz;4H);6.96(s;2H);6.82(d; 3 J=8.9Hz; 4H); 3.84 (s; 2H); 3.75 (s; 6H); 3.39 (s; 2H); 3.25 (s; 2H); 3.06 (s; 12H); 2.58 (s; 6); 2.24 (s; 6H).

[0158] Analytical HPLC (gradient A): 4.48 min.

[0159] Compound AG46

[0160]

[0161] AG38 (250 mg, 0.209 mmol, 1 eq) was dissolved in 10 mL of anhydrous DMF (dimethylformamide) in a 100 mL flask. HBTU (208 mg, 0.548 mmol, 2.6 eq) was dissolved in 10 mL of anhydrous DMF and then added to the reaction mixture. 341 μL (1.959 mmol, 9.3 eq) of DIPEA (diisopropylethylamine) was then added, and the mixture was stirred at room temperature for 1 hour. 109.2 mg (0.618 mmol, 2.9 eq) of 2-aminoethylmaleimide hydrochloride was dissolved in 10 mL of anhydrous DMF and then added to the reaction medium, which was then stirred at room temperature overnight, evaporated to dryness, and then purified by semi-preparative HPLC (25% ACN gradient → 100% program 30 min) to isolate pure AG46 (151 mg, 0.133 mmol, 63%) as a green solid.

[0162] 1 H NMR (MeOD, 500MHz) δ (ppm): 8.36 (d; 3 J = 8.9 Hz; 4H); 8.19 (d; 3 J = 8.9 Hz; 4H); 7.72 (d; 3 =8.2Hz; 4H); 7.39(d; 3 J=8.2Hz; 4H); 7.22(s; 2H); 7.18(d; 3 J = 8.9 Hz; 4H); 7.09 (d; 3 J=8.9Hz; 4H); 6.76 (s; 4H); 4.18 (s; 4H); 3.87 (s; 4H); 3.76 (s; 6H); 3.71 (dd; 3 J = 6.3 Hz; 3 J = 4.6 Hz; 2H); 3.52 (dd; 3 J = 6.3 Hz; 3 J=4.6Hz; 2H); 3.20 (s; 12H); 2.84 (s; 12H).

[0163] 13C NMR (DMSO, 150MHz): 171.1; 165.6; 160.9; 158.6; 158.3; 158.1; 157.8; 155.9; 151.0; 142.1; 141.6; 136.0; 134.6; 132 .5;132.0;130.4;130.1;127.6;124.3;119.8;116.7;116.0;114.7;113.7;112.1;64.6;55.4;54.0;49.1;37.7;37.1.

[0164] HR-MS(ESI)(Da): m / z C 76 H 78 BN 11 O8 2+ [M] 2+ Calculated value 641.80585, measured value 641.80752.

[0165] HPLC analysis (gradient A): Tr = 4.56 min.

[0166] Compound AG49

[0167]

[0168] AG46 (120 mg; 79 μmol; 1 eq) was dissolved in 2 mL of ACN (acetonitrile) in a 10 mL flask. 42 mg (166 μmol; 2.5 eq) of BSH was then added, and the reaction was stirred at 40° C. for 48 hours. The reaction mixture was transferred to a Falcons tube and centrifuged. The supernatant was removed, and the precipitate was washed again with ACN (3×15 mL), DCM (1×15 mL), Et2O (2×15 mL), and pentane (2×15 mL). AG49 (62 mg, 41.9 μmol, 53%) was isolated as a blue precipitate.

[0169] 1 H NMR (DMSO, 500MHz) δ (ppm): 8.65-8.63 (m; 1H); 8; 48-8.42 (m; 1H); 8.33-8.30 (m; 4H); 8.13-8.11 (m; 4H); 7.82-7.7 5 (m; 4H); 7.39-7.36 (m; 4H); 7.28-7.27 (m; 2H); 7.09-7.07 (m; 4H); 7.00-6.99 (m; 1H); 6.89-6.85 (m; 4H); 4.17 (t; 3J=17.9Hz; 4H); 3.97-3.90 (m; 4H); 3.74-3.73 (m; 6H); 3.62 (dd; 3 J = 8.0 Hz; 4 J=3.1Hz; 2H); 3.59-3.55 (m; 2H); 3.54-3.48 (m; 4H); 3.07 (s; 12H); 3.03 (d; 3 J = 8.5 Hz; 1H); 2.99 (d; 3 J=8.5Hz; 1H); 2.78-2.73 (m; 13H); 1.03 (bs; 11H).

[0170] HR-MS(ESI)(Da): m / z C 76 H 90 B 10 B 12 N 11 Na 2+ O8S 2+ [M+2Na] 2+ Calculated value 746.90622, measured value 746.90811.

[0171] HPLC analysis (gradient A): Tr = 5.32 min.

[0172] Compound AG58

[0173]

[0174] AG35-3 (35 mg; 0.029 mmol; 1 eq) was dissolved in 3 mL of DMF in a 50 mL flask. 25 mg of HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole tetramethyluronium hexafluorophosphate) (0.067 mmol; 2.3 eq) dissolved in 3 mL of DMF was then introduced, followed by 40 μL of DIPEA (0.232 mmol, 8 eq). The reaction was stirred at room temperature for 30 minutes under argon. H2N-CH2-CH-(SO3 -)2 (TBA or tetrabutylammonium salt) (61 μ L, 0.030 mmol, 1.1 eq) in 3 mL of DMF was added to the reaction medium at 0.5 M, which was then stirred at room temperature for 1 hour. 14 mg (0.030 mmol, 1.1 eq) of TOTA-Boc in 3 mL of DMF was then added to the solution, which was stirred at room temperature for another hour. The reaction mixture was evaporated to dryness, dissolved in 30 mL of ACN, and then 15 mL of HCl (3 M) was added. The reaction was stirred at 40 ° C for 2 hours. The crude reaction product was evaporated to dryness and purified by semi-preparative HPLC (gradient 25% → 100%, program 40 min) and freeze-dried to give pure AG58 (7 mg, 5 μ mol, 18%) as a green powder.

[0175] 1 H NMR (DMSO, 500MHz) δ (ppm): 8.69 (t; 3 J = 4.4 Hz; 1H); 8.50 (t; 3 J = 5.5 Hz; 1H); 8.23 ​​(d; 3 J = 8.9 Hz; 4H); 8.12 (d; 3 J = 9.0 Hz; 4H); 7.94 (d; 3 J = 8.3 Hz; 2H); 7.90 (d; 3 J = 8.3 Hz; 2H); 7.63 (d; 3 J=8.3Hz; 2H); 7.60 (bs; 3H); 7.56 (d; 3 J=8.3Hz;2H);7.18(s;2H);6.98(d; 3 J = 8.9 Hz; 4H); 6.87 (d; 3 J=8.9Hz; 4H); 4.60 (s; 2H); 4.28 (s; 2H); 3.95 (t; 3 J=4.4Hz; 2H); 3.80 (s; 2H); 3.72 (t; 3 J=4.9Hz; 1H); 3.63 (s; 6H); 3.51-3.41 (m; 12H); 3.33 (dd; 3 J = 12.4 Hz; 3 J=6.6Hz; 4H); 3.07 (s; 12H); 3.06-3.01 (m; 4H); 2.85-2.82 (m; 2H); 2.72 (s; 6H); 1.79-1.73 (m; 4H); 1.60-1.55 (m; 2H).

[0176] HR-MS(ESI)(Da): m / z C 76H 93 BN 10 O 13 S2 2+ [M] 2+ Calculated value 714.32235, measured value 714.32560.

[0177] HPLC analysis (gradient A): Tr = 4.32 min.

[0178] Compound AG60

[0179]

[0180] AG57-3 (21 mg; 0.021 mmol; 1 eq) was dissolved in 2 mL of DMF. 20 mg (0.052 mmol; 2.5 eq) of HBTU was dissolved in 2 mL and added to the AG57-3 solution, followed by 32 μL (0.160 mmol; 8.6 eq) of DIPEA. The reaction mixture was stirred at 30°C under argon. After 30 minutes, 10 mg (0.024 mmol; 1.1 eq) of TOTA-Boc was added, and the solution was stirred at 30°C for 1.5 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: 100% DCM → 50 / 50 DCM / MeOH) to isolate the TOTA intermediate as a black solid.

[0181] Then, in the 25mL flask under argon, previous intermediate product is dissolved in 4mL anhydrous DCM.Iodomethane (1mL, a large amount of excess) is added, and the reaction is stirred at room temperature for 1 hour.Solvent is removed under reduced pressure, and gained residue is dissolved in 20mL ACN.10mL HCl aqueous solution (3M) is added, and the reaction is stirred at 40 DEG C for 2 hours.Reaction medium is evaporated to dryness and purified by semi-preparative HPLC (20% ACN gradient → 100%, 30min), then lyophilized overnight to separate the pure AG60 (4mg, 2.4 μmol, 14%) in blue solid.

[0182] 1 H NMR (ACN, 500MHz) δ (ppm): 8.21-8.19 (m; 2H); 8.18-8.15 (m; 2H); 8.11-8.08 (m; 4H); 7.89 (t; 3 J=8.6Hz; 2H); 7.86 (s; 1H); 7.52 (bs; 3H); 7.36 (d; 3 J = 8.0 Hz; 1H); 7.32 (d; 3J=8.1Hz; 1H); 7.04-7.01 (m; 4H); 7.00 (d; 3 J=8.7Hz; 2H); 6.89-6.86 (m; 4H); 4.01 (s; 1H); 3.86 (s; 1H); 3.85 (s; 1H); 3.81 (s; 1H); 3.80 (s; 3H); 3.76 (s; 3H); 3.66 (q; J=5.5Hz; 2H); 3.60-3.58 (m; 4H); 3.57-3.55 (m; 6H); 3.53 (t; 3 J=5.8Hz;2H);3.44(quin; 3 J=6.4Hz; 3H) 3.35 (s; 1H); 3.08 (s; 12H); 2.83 (s; 5H); 2.70 (s; 3H); 2.66 (s; 3H); 2.60 (s; 3H); 1.90-1.85 (m; 3H); 1.83-1.78 (m; 2H).

[0183] HR-MS(ESI)(Da): m / z C 67 H 85 BN9O6 3+ [M] 3+ Calculated value 374.22331, measured value 374.22292.

[0184] HPLC analysis (gradient A): Tr = 4.18 min.

[0185] Compound AG47

[0186]

[0187] In a 10 mL flask, 2-aminoethylmaleimide hydrochloride (10.3 mg; 58 μmol; 1 eq) was dissolved in 2 mL of ACN. Then, 10 B-BSH (12.3 mg; 58 μmol; 1 eq) was added and the reaction was stirred at room temperature for 1 hour. The crude reaction product was evaporated to dryness to isolate AG47 (23 mg; 58 μmol; 1 eq) as a white solid.

[0188] Compound AG66

[0189]

[0190] In a 100 mL flask, AG38 (200 mg; 178 μmol; 1 eq) was dissolved in 16 mL of DMF. HBTU (158 mg; 406 μmol; 2.3 eq) was dissolved in 16 mL and added to the reaction, followed by DIPEA (248 μL; 800 μmol; 8 eq). The reaction was stirred at room temperature for 30 minutes under argon. AG47 (72 mg; 186 μmol; 1.05 eq) was dissolved in 16 mL of DMF and added to the reaction medium. The reaction was stirred at room temperature for 1 hour. TOTA-Boc (78 mg; 186 μmol; 1.05 eq) was dissolved in 16 mL of DMF before addition. After 1 hour, the contents of the flask were transferred to a separatory funnel, and 100 mL of DCM and 50 mL of HO were added. The two phases were separated, and the aqueous phase was extracted with DCM (3×50 mL). The organic phases were combined, washed with brine (1×100 mL) and evaporated to dryness. The crude product was dissolved in 40 mL ACN and 15 mL HCl (3 M) was added. The mixture was stirred at 40° C. for 2 hours, the crude product was evaporated to dryness, and then purified by semi-preparative HPLC (gradient A). The product was purified by HPLC in Cl - The HPLC-MS / MS was run on an ion exchange resin (IRA 410) to isolate AG66 (33 mg; 21 μmol; 12%) as a green precipitate.

[0191] 1 H NMR (ACN-d3 / D2O, 600MHz, 343K) δ (ppm): 8.19 (d, 3 J=8.2Hz;4H);8.07(d 3 J=8.3Hz,4H);7.68-7.64(m,4H);7.35-7.22(m;10H);7.11(s,2H);7.01-6.99 (m,4H);4.10(s;2H);3.95(s;2H);3.78(s,2H);3.65-3.63(m,8H),3.55-3.52( m,12H);3.50-3.48(m,3H);3.42-3.38(m,2H),3.35-3.32(m,2H),3.04-3.02(m ,2H);2.99-2.96(m,1H);2.92-2.88(m,1H);2.74(s,6H);2.66(s,6H),1.85(p, 3 J=5.9Hz,2H);1.80-1.76(m,2H);1.21(bs;11H).

[0192] 13C NMR(ACN-d3 / D2 O, 125MHz, 343K) δ (ppm): 28.1; 30.3; 30.4; 38.9; 39.4; 39.8; 41.1; 41.1; 43.3; 43.4; 45.3; 51.7; 56.4; 56.6; 57.4; 67.4; 67.5; 70.1; 70.3; 71.1; 71.2; 71.2; 71.2; 71.3; 71.4; 88.2; 11 5.9;119.1;125.8;129.4;129.6;129.6;129.9;131.3;131.4;132.5;132.6;134.1;134.3;134.4;137.7;138.1;138.2;142.3;144.5;147.9;159.2;163.3;169.6;169.6;180.4;181.9.

[0193] 11 B NMR (ACN-d3 / D2O, 193MHz, 343K): -9.39 (bs, aza-BODIPY); -14.59 (bs, BSH); -16.20 (s, BSH); -19.10 (bs, BSH).

[0194] 10 B NMR (ACN-d3 / D2O, 64MHz, 343K): -9.08 (bs, aza-BODIPY); -16.27 (s, BSH); -17.28 (s, BSH).

[0195] HR-MS(ESI)(Da):m / z C 80 H 107 11 B 10 B 12 N 11 O9S + [M] + Calculated value 1528.96140, measured value 1528.96362.

[0196] HPLC analysis (gradient A): Tr = 4.53 min.

[0197] Example 2: Small Animal Fluorescence Imaging Process

[0198] In accordance with current ethical recommendations, five-week-old female NMRI Nu / Nu mice were housed in groups of five with ad libitum access to food and water and provided with day / night lighting from 12:00 to 12:00.

[0199] At 6 weeks of age, U87MG tumor cells (3 million / 100 μL) were injected subcutaneously into the right lower limb: the animals were sedated by gas anesthesia during the injection. The animals were returned to their cages during the development of the tumor (this meant approximately 3 weeks).

[0200] When the tumor reaches about 100 mm 3 When the animal is 100 μg / mouse or larger, a solution containing the AG22 compound (25 μg / mouse to 50 μg / mouse) is injected intravenously into the tail vein of the animal under gas anesthesia. The animal is then awakened during the external imaging phase. For each imaging session, the animal is placed under gas anesthesia, imaging is performed, and then the animal is returned to its cage.

[0201] Imaging was performed under 830 nm excitation, and the fluorescence signal was then collected using a long-pass filter between 1064 nm and 1700 nm.

[0202] We observed ( Figure 1 ) Mouse with a U87MG tumor in the right hind leg. The upper image was recorded using NIR imaging. Fluo800 corresponds to excitation at 780 nm and collection between 830 nm and 900 nm. The lower image was excited at 830 nm (SWIR800) and emission was collected using a 1064 nm longpass filter.

[0203] The experiment was repeated with compound AG66.

[0204] We observed mice with U87MG tumors on their right hind legs from left to right: before, 5 hours, and 48 hours after injection of AG66 compound.

[0205] Thus, the compounds according to the invention can be observed in the wavelength range from 1000 nm to 1700 nm: these wavelengths facilitate in-depth detection with better resolution.

[0206] The compounds according to the invention also allow the delivery of small compounds, such as boron complexes.

[0207] References

[0208] [1] Bruns et al., Nat Biomed Eng. 2017; doi:10.1038 / s41551-017-0056 / Carr et al., Proc Natl Acad Sci US A. 2018 115(17):4465-70 / Thimsen et al., Nanophotonics 2017; 6(5):1043-1054).

[0209] [2] PAC, 1996, 68, 2193 (Basic terminology of stereochemistry (IUPAC Recommendations 1996)), page 2205.

[0210] [3] Gravier et al., Mol Pharm. 2014; doi:10.1021 / mp500329z., p. 3134.

[0211] [4] et al., Nanomedicine. 2013; doi:10.1016 / j.nano.2012.08.005, p. 376.

[0212] [5]Garcia et al., Biomater Sci. 2018; doi:10.1039 / c8bm00396c, p. 1755.

[0213] [6] Reagan-Shaw et al., FASEB J, 2007, vol. 22, p. 660, doi:10.1096 / fj.07-9574LSF.

Claims

1. Use of the fluorophore compound of formula I as a contrast agent in the optical window ranging from 1000 nm to 1300 nm, in -R 1 、R 2 Are the same or different, indicated by -NR c R d a C6 aryl group substituted in the para position, -R 3 and R 4 are the same or different, and are optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group, -R 5 and R 6 are the same or different and represent hydrogen, halogen, a C1-C15 group containing an aldehyde, ketone, carboxylic acid or ester functional group, nitrile, -SO3Na, a vinyl group optionally substituted by a ketone, ester or aromatic group, an imine substituted by an alkyl or aromatic group, an alkyne group optionally substituted by an alkyl or aromatic group, an aromatic chalcogenide, an amide, a halogen optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group, - Optionally, R 3 and R 5 and / or R 4 and R 6 covalently bonded together to form a radical optionally selected from halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN substituted with a C5-C7 aryl or heteroaryl group, -R c and R d are the same or different and represent hydrogen or a linear or branched C1-C3 alkyl chain, -R a and R b Is the same or different, meaning: Halogen, a C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, a biocarrier covalently coupled via a C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally containing one or more aromatic or heteroaromatic groups, optionally containing one or more heteroatoms selected from O, N, P and / or S, ●Metal complexes formed by chelating agents and metals for therapeutic purposes, Molecules with a hydrodynamic diameter of less than 10 nm, Cyclic or linear peptides, antibodies, aptamers, short DNA or RNA sequences, Sugars, amino acids, vitamins, AMD3100-type molecules, PSMA ligands, steroids, fatty acids, polyamines, polyphenols, DNA bases, or caffeine derivatives.

2. The method according to claim 1, wherein the fluorophore compound is selected from the group consisting of compounds of formula I, wherein R 5 and R 6 For hydrogen.

3. The use according to any one of the preceding claims, wherein the fluorophore compound is selected from the compounds of formula I, wherein R a and R b are the same or different, selected from halogen, a hydrophilic group selected from the following formula: , A group selected from the group consisting of bioconjugated functional groups of the formula: Selected from the group comprising biological carriers, Selected from PPh2-Au(I), PPh2-Pt(II), PPh2-Pt(IV), phenylpyridine-Au(III), Selected from DOTA-In(III), DOTAGA-In(III), NODAGA-Cu(II), NODAGA-Ga(III), or Selected from α v β3 integrin targeting c(RGDfK), neuropilin targeting ATWLPPR, anti-CD44, thioglucose, peracetylated thioglucose, folic acid, AMD3100 type ligand, PSMA ligand, spermine, spermidine, cadaverine, putrescine, resveratrol, DNA bases, caffeine and progesterone.

4. The use according to claim 1, wherein the fluorophore compound is selected from the fluorophore compound of formula II: in, - Ra, Rb, Rc, Rd have the definitions given above, -Re and Rf are the same or different and represent a radical selected from hydrogen, halogen, -NR c R d 、-OR d , hydrazine, -CF3 and -CN.

5. The use according to claim 1, wherein the fluorophore compound is selected from the compounds of formula I or II, wherein Rc and Rd are -CH3.

6. The method according to claim 1, wherein R a or R b wherein the halogen is selected from the group consisting of fluorine and chlorine.

7. The method according to claim 1, wherein R a or R b wherein the C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group contains one or more heteroatoms selected from O, N, P and / or S, and is in the form of one or more hydrophilic functional groups selected from quaternary ammonium, sulfate, sulfonate and phosphonate functional groups.

8. The method according to claim 1, wherein R a or R b wherein the C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group contains one or more bioconjugated functional groups selected from the group consisting of amines, carboxylic acids, pentafluorophenyl groups, tetrafluorophenyl groups, squarates, thiols, isothiocyanates, isocyanates, oxadiazolylmethyl sulfones, azides, substituted or unsubstituted tetrazines, triazoles, trans-cyclooctene, cyclooctyne, dibenzocyclooctyne, bicyclononyne, and PPh2AuCl complexes.

9. The method according to claim 1, wherein R a or R b wherein the C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group contains one or more bioconjugated functional groups selected from the group consisting of N-hydroxysuccinimide type activated esters and maleimides.

10. The method according to claim 1, wherein R a or R b In the invention, the biocarrier is covalently coupled via a C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, wherein the C1-C50 aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group is in the form of one or more bioconjugated functional groups.

11. The method according to claim 1, wherein R a or R b In the embodiment, the metal is a radio metal.

12. The method according to claim 1, wherein R a or R b In the method, the antibody is selected from the group consisting of antibody fragments, nanobodies and affibodies.

13. The method according to claim 1, wherein R a or R b wherein the sugar is a polysaccharide.

14. The method according to claim 1, wherein R 5 or R 6 wherein the aromatic chalcogenide is SPh, SePh or TePh.

15. The use according to claim 3, wherein R a or R b yes 16. The use according to claim 1, wherein the fluorophore compound is contained in a composition further comprising a pharmaceutically acceptable excipient and / or solvent.

17. Use according to claim 16, wherein the pH of the composition is in the range of 4 to 10.

18. The use according to claim 1, wherein the fluorophore compound is encapsulated.

19. A kit for imaging in the optical window ranging from 1000 nm to 1300 nm, said kit comprising an injection system and a composition comprising a fluorophore compound of formula I or II as defined in any one of claims 1 to 15 and a pharmaceutically acceptable excipient and / or solvent.

20. Use of a composition comprising a fluorophore compound of formula I or II as defined in any one of claims 1 to 15 in the preparation of a reagent for in vitro or in vivo recognition of a biological target, said in vitro or in vivo recognition comprising at least the following steps: - labeling ligands or cells of a collected or cultured sample with a composition comprising a fluorophore compound of formula I or II as defined in any one of claims 1 to 15, -Measure fluorescence in the optical window from 1000nm to 1300nm, and - identifying said biological target.

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