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  • C07D403/08—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing alicyclic rings
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
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  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
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• • G—PHYSICS
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  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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  • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
  • C09K2211/10—Non-macromolecular compounds
  • C09K2211/1018—Heterocyclic compounds
  • C09K2211/1025—Heterocyclic compounds characterised by ligands
  • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Definitions

• the present invention relates to novel fluorescent compounds which can be used for labeling tumor tissue, their method of preparation, as well as their application as a tool for monitoring, diagnosing or assisting in cancer surgery.
• Fluorescent compounds have been used for over fifty years in medicine as markers in non-invasive imaging techniques for monitoring and / or diagnosis.
• Certain existing fluorescent markers have a limited persistence of fluorescence, which makes it necessary to operate on the patient following the injection of the marker and does not make it possible to obtain a satisfactory delineation of the tissue. tumor. In other cases, the accumulation of the label in the tissues is not high enough, which leads to poor labeling and therefore detection problems. The localization of lesions or tumors then their elimination, for example by surgery, is then not complete.
• ICG indocyanine green
• Another major drawback of existing compounds is that they cannot be used directly. In fact, in order to be used, they need to be coupled with other targeting molecules such as antibodies, proteins, molecules specific to tumor tissue, folic acid, or even steroids.
• the present invention overcomes the problems of the prior art explained above by providing fluorescent molecules having a preferential distribution in tumor tissues over healthy tissues and sufficient persistence for their use in techniques of. imaging for monitoring, diagnosis, and / or surgical assistance.
• These new molecules have the major advantage of being able to be used alone and directly, without prior coupling, due to their specific affinity for tumor tissue. They also have, compared to the molecules of the prior art, a much higher remanence in the tumor tissues, going up to a duration of several days, which allows a more extensive elimination of these fluorescent molecules circulating outside the tumor tissues, and thus improved visualization thanks to a better signal-to-noise ratio.
• the present invention relates to a compound of formula (I) [Chem. 1] where n1 and n2 are each an integer from 0 to 15,
• Ri, R2, R3, R4, R5 and He are each independently selected from H, OH, SH, NH 2 , SO3R10 and X-R11-Y,
• R11, R'11, R ”n being independently selected from C1 to C15 alkyl, aryl, heteroaryl, (C1 to Cis alkyl) aryl, (C1 to Cis alkyl) heteroaryl, aryl (C1 to C15 alkyl) and heteroaryl (C1-C15 alkyl);
• Y, Y ’, Y being independently selected from H, halogen, COOR’10 or amide; R7 and Re being each selected from H, OH, SH, NH2, C1-C15 alkyl, and X'-R'n-Y ';
• R 9 being chosen from H, OH, SH, NH2 and X ”-R” nY ”, said compound comprising at least one group X-R11-Y, X'-R'n-Y 'or X” -R ”n - Y ”with Y, Y ', and / or Y” which is COOR'10.
• the present invention also relates to the process for preparing the compounds of formula (I) according to the invention, as well as the process for labeling tumor tissue with one of the compounds according to the invention or prepared according to the process of the invention. .
• the first subject of the present invention relates to a compound of formula (I)
• ni and n2 are each an integer from 0 to 15, Ri, R2, R3, R4, R6 and R6 are each independently selected from H, OH, SH, NH 2 , SO3R10 and X-R11-Y,
• X, X ’, X being independently O, S or NH,
• R11, R'11, R ”H being independently selected from C1 to C15 alkyl, aryl, heteroaryl, (C1 to C18 alkyl) aryl, (C1 to Cis alkyl) heteroaryl, aryl (C1 to C15 alkyl) and heteroaryl (C1-C15 alkyl);
• Y, Y ’, Y being independently selected from H, halogen, COOR’10 or amide;
• R7 and R8 being each selected from H, OH, SH, NH2, C1-C15 alkyl, and X'-R'n-Y '; R 9 being chosen from H, OH, SH, NH2 and X ”-R” nY ”, said compound comprising at least one X-R11-Y, X'-R'n-Y 'or X” -R ”n group - Y ”with Y, Y ', and / or Y” which is COOR'10.
• C 1 to C 15 alkyl means a hydrocarbon chain, cyclic, linear or branched, containing from 1 to 15 carbon atoms, preferably from 2 to 6 carbon atoms and more preferably still from 4 to 6 carbon atoms, in particular 5 carbon atoms and possibly being in particular a methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl chain, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, isopentyl, neopentyl, 2-pentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, heptyl, octyl, nonyl, decyl, dodecyl, palmityl.
• heptyl octyl
• heteroaryl means an aromatic group, containing one or more aromatic rings, optionally substituted, and comprising at least one heteroatom other than carbon and hydrogen.
• arylalkyl is understood to mean an aryl group substituted by one or more alkyl groups, said alkyl groups possibly being C1 to C15 alkyl groups, preferably containing from 1 to 15 carbon atoms. carbon.
• heteroarylalkyls is understood to mean a heteroaryl group substituted by one or more alkyl groups, said alkyl groups possibly being C 1 to C 15 alkyl groups, preferably containing from 1 to 15 carbon atoms. carbon.
• m or n2 are independently of one another, equal to 1, 2, 3, 4 or 5, more preferably still 3 or 4.
• n-i n2 and is preferably equal to 1, 2, 3, 4 or 5, more preferably still 3 or 4.
• the molecule is symmetrical.
• it comprises a single group X ”-R” nY ”with Y” which is COOR'10 which is carried by R9 and / or 2 groups X-R11-Y with Y which is COOR'10, one carried by one of Ri, R2, R30U Rz, preferably one of Ri, R2 or R3 and the other carried by one of R4, Rs, R6 or Rs, preferably one of R4, Rs or R6.
• R10, and / or R'10 may be identical.
• X, X ', and / or X may be the same, Y, Y', and / or Y” may be the same, Ru, R'11, and / or R ”n may be the same.
• the compounds according to the invention can in particular be chosen from the compounds of the following general formula: for which X ”can be O, S or NH, then corresponding to the formulas below:
• the compounds of formula (I) according to the invention can in particular be chosen from the compounds of formula (I) for which Ri, R2, R3, R4, Rs and Re ne are not all simultaneously H, which in this case excludes the compounds according to formula (II) below:
• the compound of formula (I) according to the invention can preferably be chosen from the compounds of the following general formulas:
• the compounds according to the invention can be chosen from the compounds of the following formulas [Chem. 5] [Chem. 7] in which R1, R2, R3, R4, Rs, Re, R7, Rs and R9 are as defined above. According to a preferred embodiment, the compounds according to the invention can be chosen from the following compounds:
• the second object of the present invention relates to the process for preparing the compounds of formula (I) according to the invention comprising a reaction step between:
• the third object of the invention relates to the method of labeling tumor tissue with one of the compounds according to the invention or prepared according to the method of the invention.
• tumor tissue is understood to mean the tissue consisting of tumor cells which are abnormal proliferative cells, and supporting tissue, also called tumor stroma or interstitial tissue, made up of cells and extra-cellular material in which the tumor vasculature is located.
• the fluorescent compounds according to the invention have the particularity, after their diffusion in the body, of being trapped in the tumor tissue, while they are eliminated from healthy tissue. This feature makes it possible to use these fluorescent compounds directly, without prior coupling to another labeling molecule, thus making their use simpler, faster and more efficient than that of the compounds of the prior art. It has been observed that this elimination from healthy tissue increases over time. In general, between 24 and 72 hours, preferably between 36 and 60 hours and more preferably 48 hours after the administration of these compounds, their elimination from healthy tissues is complete. However, they remain trapped in tumor tissue. This property makes it possible to have a clear differentiation of tumor tissues from healthy tissues and thus the use of these compounds in monitoring, diagnostic and / or surgical assistance applications in the context of cancerous diseases. This differentiation lasts for 6 to 48 hours, preferably 12 to 36 hours, making it possible to program the diagnosis or the surgery in a targeted manner.
• the compounds according to the invention can thus in particular be used in the context of cancers, for example hormone-dependent, such as breast cancer or digestive cancers, such as pancreatic cancer.
• hormone-dependent such as breast cancer
• digestive cancers such as pancreatic cancer.
• pancreatic cancer tumors are particularly difficult to remove in their entirety by surgery because they are not easily delimited.
• the use of the compounds according to the invention makes it possible to obtain a better visualization of the contours of the tumors thanks to the differentiation of labeling between the tumor tissue and the healthy tissue, and thus a more efficient tumor resection by surgery.
• the invention also relates to the use of one of the compounds according to the invention or prepared according to the method of the invention in a method of labeling tumor tissue.
• This method of labeling tissues requires the administration of the compounds intravenously, intraarterially, or into another vessel, in particular a lymphatic vessel, either by local injection or by local application, preferably intravenously.
• Another object of the invention relates to a composition
• a composition comprising one of the compounds according to the invention or prepared according to the process of the invention and at least one pharmaceutically acceptable adjuvant.
• the invention also relates to one of the compounds according to the invention or prepared according to the process of the invention or a composition comprising one of the compounds according to the invention or prepared according to the process of the invention for its use. in a method of labeling and / or detecting tumor tissue, and / or in the surgical treatment of tumors.
• the invention also relates to a method of detecting tumor tissue comprising a step of labeling tumor tissue with one of the compounds according to the invention or prepared according to the method of the invention, and a step of detection by medical imaging in fluorescence or fluorescence spectrometry.
• FIG. 1 shows the median values and standard deviations of the tumor / abdomen intensity ratios as a function of post-injection times of Compound 2 (CJ215) and ICG.
• FIG. 2 shows the results of ex vivo imaging of pancreatic tumors after injection of two compounds according to the invention and a fluorescent agent of the prior art (ICG).
• the reaction medium is cooled to room temperature and the precipitate is separated by filtration and washed with diethyl ether to provide 4.33 g (yield: 43.9%) of a green solid.
• the crude product is purified by flash column chromatography (reverse phase C18 silica gel, 0-25% acetonitrile / water).
• Example 8 Comparison of a compound according to the invention and of a fluorescent agent of the prior art (ICG) for in vivo imaging of mammary tumors
• ICG fluorescent agent of the prior art
• ICG or indocyanine green / Infracyanine
• ICG is a fluorescent agent of the prior art, already approved for use in humans for the evaluation of cardiac and hepatic functions, as well as in ophthalmology, for retinal pathologies. It is also being evaluated in numerous clinical trials around the world for guiding the surgical procedure during tumor resection, or the mapping of lymph nodes draining tumors, by near infrared imaging.
• the ICG was compared with the compound (2) according to the invention, the synthesis of which is described in the preceding example 2, this compound is called CJ215 in this study.
• the study included a total of 30 mice divided into three groups.
• the tumor grafts were all carried out with 50,000 4T1-Dendra2 / 20 ⁇ l cells injected into 2 mammary glands contralateral for each of the mice.
• the injections of biomarkers (compound 2 called CJ215 in this study and ICG) were carried out on D9 post-tumor grafting (in order to limit the appearance of necrosis in tumors).
• the change in the intensity of the fluorescence signals observed for each of the biomarkers over time was evaluated by microscopic image.
• the ability of the two markers to produce a signal specifically localized to the tumor was assessed quantitatively by calculating the ratio of the specific signal bound to the tumor to the non-specific signal in the surrounding tissues.
• the imaging protocol was carried out at times 2h, 24h, 48h, 4 and 6 days post-injection for all the mice. All the images produced at each acquisition time were acquired on an I VIS Spectrum imager (Perkin Elmer) with the following parameters:
• the quantitative measurements were carried out on the non-deconvoluted raw images.
• the acquisition time was set in automatic mode. In this mode, the system determines the acquisition time necessary to reach the imposed target value (6000 counts) within the allotted time (fixed at 2 min).
• Figure 1 reports the median values and standard deviations of the tumor / abdomen intensity ratios as a function of post-injection times of CJ215 and ICG.
• Example 9 Comparison of two compounds according to the invention and of a fluorescent agent of the prior art (ICG) in ex vivo imaging of pancreatic tumors.
• a model of orthotopic pancreatic adenocarcinoma in mice has been developed. Tumor cells were amplified subcutaneously in SCID mice and the resulting fragments were then surgically implanted into the pancreas of irradiated BALB / c nude mice.
• MRI 4.7T, PharmaScan, Bruker Biospin
• Animals were subjected to weak fluorescence in order to minimize autofluorescence. Fluorescent imaging was performed with a charge coupled device (CCD) camera (PhotonRT, BiospaceLab) with excitation at 700 nm and emission filter at 770 nm.
• CCD charge coupled device
• ex vivo fluorescent images were acquired.
• the fluorescent compounds according to the invention 2 (CJ215) and CJ319 (the structure of which is detailed below) were injected intravenously at 2 mg / kg, 39 days after implantation of the tumor fragments, while the volumes average tumors were about 70 mm 3 .
• Indocyanine green (ICG) a dye widely used in intraoperative tumor imaging, was included as a control.

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