NOVEL FLUORESCENT COMPOUNDS FOR LABELING TUMOR TISSUE
20230203014 · 2023-06-29
Assignee
Inventors
- Joanne TRAN-GUYON (MONTIGNY LE BRETONNEUX, FR)
- Vincent GUYON (MONTIGNY LE BRETONNEUX, FR)
- François SCHERNINSKI (PARIS, FR)
Cpc classification
C07D403/08
CHEMISTRY; METALLURGY
C07D209/60
CHEMISTRY; METALLURGY
International classification
Abstract
Novel fluorescent compounds that can be used for labelling tumour tissue, and the method for labelling tumour tissue the novel fluorescent compounds. Also, the application of the tumour tissue labelled with the novel fluorescent compounds as a monitoring tool, a diagnostic tool, or a tool for assisting with cancer surgery.
Claims
1-10. (canceled)
11. A compound of formula (I) ##STR00032## in which n.sub.1 and n.sub.2 are each an integer from 0 to 15, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each selected independently from H, OH, SH, NH.sub.2, SO.sub.3R.sub.10 and X—R.sub.11—Y, R.sub.10 and R′.sub.10 being independently H, Na or K, X, X′ and X″ being independently O, S or NH, R.sub.11, R′.sub.11 and R″.sub.11 being selected independently from C.sub.1 to C.sub.15 alkyl, aryl, heteroaryl, (C.sub.1 to C.sub.15 alkyl)aryl, (C.sub.1 to C.sub.15 alkyl)heteroaryl, aryl(C.sub.1 to C.sub.15 alkyl) and heteroaryl(C.sub.1 to C.sub.15 alkyl); Y, Y′ and Y″ being selected independently from H, halogen, COOR′.sub.10 or amide; R.sub.7 and R.sub.8 each being selected independently from H, OH, SH, NH.sub.2, C.sub.1 to C.sub.15 alkyl, and X′—R′.sub.11—Y′; R.sub.9 being selected from H, OH, SH, NH.sub.2 and X″—R″.sub.11—Y″, said compound comprising at least one group X—R.sub.11—Y, X′—R′.sub.11—Y′ or X″—R″.sub.11—Y″ with Y, Y′ and/or Y″ which is COOR′.sub.10.
12. The compound as claimed in claim 11, for which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are not all simultaneously H.
13. The compound as claimed in claim 11, selected from the compounds of the following formulas: ##STR00033## ##STR00034## in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined above.
14. The compound as claimed in claim 11, selected from the compounds of the following formulas: ##STR00035## ##STR00036## ##STR00037## in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined above.
15. The compound as claimed in claim 11, selected from the compounds of the following formulas: ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## .
16. A method of preparation of the compounds as claimed in claim 11, comprising a reaction step between: ##STR00047## and ##STR00048## .
17. A method for labeling tumor tissue with one of the compounds as claimed in claim 11.
18. A method for labeling tumor tissue with one of the compounds prepared as claimed in claim 16.
19. A method for labeling and/or detection of tumor tissue, and/or in the surgical treatment of tumors, comprising using the compound as claimed in claim 11.
21. A method for labeling and/or detection of tumor tissue, and/or in the surgical treatment of tumors, comprising using the compound as prepared in claim 16.
19. A method for labeling and/or detection of tumor tissue, and/or in the surgical treatment of tumors, comprising using a composition comprising the compound as claimed in claim 11.
21. A method for labeling and/or detection of tumor tissue, and/or in the surgical treatment of tumors, comprising using a composition comprising the compound as prepared in claim 16.
22. A method for detecting tumor tissue comprising a step of labeling tumor tissue with one of the compounds as claimed in claim 11, and a step of detection by medical fluorescence imaging or fluorescence spectrometry.
23. A method for detecting tumor tissue comprising a step of labeling tumor tissue with one of the compounds as prepared in claim 16, and a step of detection by medical fluorescence imaging or fluorescence spectrometry.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0021]
[0022]
DETAILED DESCRIPTION
[0023] The present invention relates firstly to a compound of formula (I)
##STR00002##
(I)
[0024] in which [0025] n.sub.1 and n.sub.2 are each an integer from 0 to 15, [0026] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each selected independently from H, OH, SH, NH.sub.2, SO.sub.3R.sub.10 and X—R.sub.11—Y, [0027] R.sub.10, R′.sub.10 being independently H, Na or K, [0028] X, X′, X″ being independently O, S or NH, [0029] R.sub.11, R′.sub.11, R″.sub.11 being selected independently from C.sub.1 to C.sub.15 alkyl, aryl, heteroaryl, (C.sub.1 to C.sub.15 alkyl)aryl, (C.sub.1 to C.sub.15 alkyl)heteroaryl, aryl(C.sub.1 to C.sub.15 alkyl) and heteroaryl(C.sub.1 to C.sub.15 alkyl); [0030] Y, Y′, Y″ being selected independently from H, halogen, COOR′.sub.10 or amide; [0031] R.sub.7 and R.sub.8 each being selected from H, OH, SH, NH.sub.2, C.sub.1 to C.sub.15 alkyl, and X′—R′.sub.11—Y′; R.sub.9 being selected from H, OH, SH, NH.sub.2 and X″—R″.sub.11—Y″, [0032] said compound comprising at least one group X—R.sub.11—Y, X′—R′.sub.11—Y′ or X″—R″.sub.11—Y″ with Y, Y′, and/or Y″ which is COOR′.sub.10.
[0033] In the sense of the present invention, “C.sub.1 to C.sub.15 alkyl” means a cyclic, linear, or branched hydrocarbon chain containing from 1 to 15 carbon atoms, preferably from 2 to 6 carbon atoms and even more preferably from 4 to 6 carbon atoms, in particular 5 carbon atoms and that may in particular be a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 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, or palmityl chain.
[0034] In the sense of the present invention, “aryl” means an aromatic group, containing one or more aromatic rings, optionally substituted.
[0035] In the sense of the present invention, “heteroaryl” means an aromatic group, containing one or more aromatic rings, optionally substituted, and comprising at least one heteroatom different than carbon and hydrogen.
[0036] In the sense of the present invention, “aralkyl” means an aryl group substituted with one or more alkyl groups; said alkyl groups may be C.sub.1 to C.sub.15 alkyl groups, preferably containing from 1 to 15 carbon atoms.
[0037] In the sense of the present invention, “heteroaralkyl” means a heteroaryl group substituted with one or more alkyl groups; said alkyl groups may be C.sub.1 to C.sub.15 alkyl groups, preferably containing from 1 to 15 carbon atoms.
[0038] According to a particular embodiment, in the above formula, n.sub.1 or n.sub.2 are, independently of one another, equal to 1, 2, 3, 4 or 5, even more preferably 3 or 4.
[0039] According to another particular embodiment, in the above formula n.sub.1=n.sub.2 and is preferably equal to 1, 2, 3, 4 or 5, even more preferably 3 or 4.
[0040] According to another particular embodiment, the molecule is symmetric. In this case, it comprises a single group X″—R″.sub.11—Y″ with Y″ which is COOR′.sub.10 that is carried by R.sub.9 and/or 2 groups X—R.sub.11—Y with Y which is COOR′.sub.10, one carried by one of R.sub.1, R.sub.2, R.sub.3 or R.sub.7, preferably one of R.sub.1, R.sub.2 or R.sub.3 and the other carried by one of R.sub.4, R.sub.5, R.sub.6 or R.sub.8, preferably one of R.sub.4, R.sub.5 or R.sub.6.
[0041] According to a particular embodiment, in the above formula, R.sub.10, and/or R′.sub.10, may be identical. Similarly, X, X′, and/or X″ may be identical, Y, Y′, and/or Y″ may be identical, R.sub.11, R′.sub.11, and/or R″.sub.11 may be identical.
[0042] The compounds according to the invention may in particular be selected from the compounds of the following general formula:
##STR00003##
for which X″ may be O, S or NH, corresponding to the following formulas:
##STR00004##
##STR00005##
##STR00006##
[0043] The compounds of formula (I) according to the invention may in particular be selected from the compounds of formula (I) for which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are not all simultaneously H, which excludes in this case the compounds according to the following formula (II):
##STR00007##
[0044] The compound of formula (I) according to the invention may preferably be selected from the compounds of the following general formulas:
##STR00008##
##STR00009##
in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined above.
[0045] According to a particular embodiment, the compounds according to the invention may be selected from the compounds of the following formulas
##STR00010##
##STR00011##
##STR00012##
in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined above.
[0046] According to a preferred embodiment, the compounds according to the invention may be selected from the following compounds:
##STR00013##
##STR00014##
##STR00015##
##STR00016##
##STR00017##
##STR00018##
##STR00019##
##STR00020##
##STR00021##
[0047] The present invention relates secondly to the method of preparation of the compounds of formula (I) according to the invention comprising a reaction step between:
##STR00022##
and
##STR00023##
[0048] This reaction is preferably carried out by heating under reflux in the presence of sodium acetate in a mixture of acetic acid and acetic anhydride.
[0049] The invention relates thirdly to the method for labeling tumor tissue with one of the compounds according to the invention or prepared according to the method of the invention.
[0050] In the sense of the present invention, “tumor tissue” means tissue consisting of tumor cells, which are abnormal proliferating cells, and of a supporting tissue, also called tumoral stroma or interstitial tissue, composed of cells and extracellular substance in which the tumoral vascularization is located.
[0051] The fluorescent compounds according to the invention have the particular feature, after they have diffused in the body, of being trapped in tumor tissue, whereas they are eliminated from healthy tissues. This particular feature makes it possible to use these fluorescent compounds directly, without prior coupling to another labeling molecule, thus making their use simpler, quicker and more effective than that of the compounds of the prior art. It was observed that this elimination from healthy tissues increases over time. In general, between 24 and 72 hours, preferably between 36 and 60 hours and more preferably 48 hours after administration of these compounds, their elimination from healthy tissues is total. However, they remain trapped in the tumor tissues. This property gives a clear differentiation of tumor tissues relative to healthy tissues and thus these compounds can be used in applications of monitoring, diagnosis and/or as an aid to surgery in a context of cancerous diseases. This differentiation lasts for 6 to 48 hours, preferably 12 to 36 hours, allowing targeted programming of diagnosis or surgery.
[0052] The compounds according to the invention may thus be used in particular in the context of cancers, for example hormone-dependent cancers, such as breast cancer or digestive system cancers, such as pancreatic cancer. In fact, in pancreatic cancer, the tumors are particularly difficult to remove completely by surgery, as they are not easily delimited. The use of the compounds according to the invention makes it possible to obtain better visualization of the contours of the tumors owing to the differentiation of labeling between tumor tissue and healthy tissue, and thus more effective tumor resection by surgery.
[0053] 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 for labeling tumor tissue.
[0054] This method of labeling tissues requires administration of the compounds by the intravenous or intraarterial route, or in another vessel, in particular a lymphatic vessel, or by local injection, or by local application, preferably by the intravenous route.
[0055] The invention further relates to a composition comprising one of the compounds according to the invention or prepared according to the method of the invention and at least one pharmaceutically acceptable adjuvant.
[0056] The invention also relates to one of the compounds according to the invention or prepared according to the method of the invention or a composition comprising one of the compounds according to the invention or prepared according to the method of the invention for use thereof in a method of labeling and/or detection of tumor tissue, and/or in the surgical treatment of tumors.
[0057] The invention also relates to a method for 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 fluorescence imaging or fluorescence spectrometry.
EXAMPLES
Example 1
[0058] ##STR00024##
Compound (1)
[0059] A mixture of 4-[(5-carboxypentyl)oxy]-6-sulfo-1-(4-sulfobutyl)-2,3,3-trimethyl-benz(e)indolium (internal salt and disodium salt) (9 g; 15 mmol), 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (1.30 g; 7.50 mmol), and sodium acetate (3 g; 36.6 mmol) in a 60/30 mixture of acetic acid and acetic anhydride is heated under reflux for 10 minutes. The reaction mixture is cooled to room temperature and the precipitate is separated by filtration and washed with diethyl ether to give 4.33 g (yield: 43.9%) of a green solid. The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 2
[0060] ##STR00025##
Compound (2)
[0061] Sodium methylate (440 mg; 7.6 mmol) is added to compound (1) (1 g; 0.76 mmol) in solution in 500 mL of methanol. The reaction mixture is heated under reflux for 16 h and concentrated under vacuum, and then filtered. The residue obtained is washed with cold methanol and with acetone and dried under vacuum to give 450 mg of a green solid (yield: 45%). The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 3
[0062] ##STR00026##
Compound (3)
[0063] MeSNa (106 mg; 1.5 mmol) is added to compound (1) (400 mg; 0.30 mmol) in solution in 20 mL of a 50/50 mixture of methanol/NMP (N-methyl-2-pyrrolidone). The reaction mixture is heated under reflux for 4 h, and then diethyl ether (20 mL) is added to the mixture. The precipitate is filtered and washed with the same solvent to give 254 mg of crude product (yield: 61%; sulfur odor). The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 4
[0064] ##STR00027##
Compound (4)
[0065] A mixture of 6-sulfo-1-(4-sulfobutyl)-2,3,3-trimethylbenz(e)indolium (internal salt and DCHA salt) (2 g; 4.7 mmol), 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (0.40 g; 2.35 mmol), and sodium acetate (0.9 g; 11 mmol) in a 50/20 mixture of acetic acid and acetic anhydride is heated under reflux for 15 minutes. The precipitate is separated by filtration, washed with ethanol and acetone and dried under vacuum to give 1.6 g (yield: 63.8%) of a brick-red powder. The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 5
[0066] ##STR00028##
Compound (5)
[0067] 8 mL of 1 M methanolic KOH, 16 mL of DMSO and compound (4) (500 mg; 0.25 mmol) are added to 3-(4-hydroxyphenyl)propionic acid (660 mg; 4 mmol). The reaction mixture is stirred at room temperature for 8h, and then 150 mL of ethyl acetate is added dropwise. The precipitate is separated by filtration, washed with ethanol and acetone and dried under vacuum to give 260 mg (yield: 45%) of a green powder. The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 6
[0068] ##STR00029##
Compound (6)
[0069] 4-Aminohydrocinnamic acid (816 mg; 4.9 mmol), 25 mL of DMSO and triethylamine (500 mg; 4.9 mmol) are added to compound (4) (520 g; 0.49 mmol). The reaction mixture is stirred at room temperature for 8 h, and then 200 mL of acetone is added dropwise. The precipitate is separated by filtration, washed with acetone and dried under vacuum to give 430 mg (yield: 74%) of a red powder. The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 7
[0070] ##STR00030##
Compound (7)
[0071] 1 mL of 1 M methanolic KOH, 16 mL of DMSO and compound (4) (500 mg; 0.25 mmol) are added to 4-mercaptohydrocinnamic acid (91 mg; 0.5 mmol). The reaction mixture is stirred at room temperature for 30 minutes, and then 50 mL of ethyl acetate is added dropwise. The precipitate is separated by filtration, washed with ethanol and acetone and dried under vacuum to give 310 mg (yield: 54%) of a green powder. The crude product is purified by column flash chromatography (inverse phase silica gel C18, acetonitrile 0-25% / water).
Example 8: Comparison of a Compound According to the Invention and A Fluorescent Agent of the Prior Art (ICG) for in Vivo Imaging of Mammary Tumors
[0072] ICG (or indocyanine green / Infracyanine) is a fluorescent agent of the prior art, already approved for use in humans for evaluation of cardiac and hepatic function, as well as in ophthalmology, for retinal diseases. It is also undergoing evaluation in many clinical trials throughout the world for guidance of surgery during tumoral exereses, or mapping of the ganglia draining the tumors, by near infrared imaging. ICG was compared with compound (2) according to the invention, synthesis of which is described above in example 2; this compound is called CJ215 in this study.
[0073] The study included 30 mice in total, distributed in three groups. The tumoral grafts were all carried out with 50 000 cells 4T1-Dendra2 /20 .Math.l injected in 2 mammary glands contralaterally for each of the mice.
[0074] The injections of the biomarkers (compound 2 called CJ215 in this study and ICG) were carried out on D9 post-tumoral grafting (to limit the appearance of necrosis in the tumors).
[0075] The variation of the intensity of the fluorescence signals recorded for each of the biomarkers over time was evaluated from the microscopy image. The capacity of the two markers for producing a signal specifically localized to the tumor was assessed quantitatively by calculating the ratio of the specific signal associated with the tumor to the nonspecific signal in the surrounding tissues.
[0076] The imaging protocol was carried out at times 2 h, 24 h, 48 h, 4 and 6 days post-injection for all the mice. All the images made at each acquisition time were acquired on the IVIS Spectrum imager (Perkin Elmer) with the following parameters: [0077] For detection of the GFP form of Dendra2 (detection of the tumor): [0078] Excitation at 465 nm [0079] Emission between 520 and 580 nm [0080] For detection of the biomarkers: [0081] Excitation at 745 nm [0082] Emission between 800 and 840 nm
[0083] The quantitative measurements were performed on the raw images, which had not been deconvoluted. For the two fluorophors, the acquisition time was parameterized in automatic mode. In this mode, the system determines the acquisition time taken to reach the stipulated target value (6000 counts) in the time allowed (fixed at 2 min).
[0084]
[0085] Measurement of the ratio of the tumor/abdomen intensities illustrated in this figure makes it possible to show that: [0086] the intensity ratios are significantly higher for compound 2 (CJ215) compared to ICG, regardless of the time post-injection (from 1.5 times at 2 h to more than 3 times at D+6), which translates into a capacity for identifying a specific signal in the tumors earlier and more specifically with compound 2 (CJ215). These results also indicate the possibility of very significantly improving the specificity of the signal to the tumor by increasing the time between injection of compound 2 (CJ215) and imaging; [0087] the signal/noise ratio increases continuously for compound 2 (CJ215) up to 6 days post-injection, the last day of examination considered in this protocol. At this stage, ICG is no longer observable in the tumors (starting from 48 h). The great stability of the intratumoral signal of compound 2 (CJ215), compared to the surrounding tissues, which eliminate the product, thus offers an improved capacity for identifying the tumors and thus contributes to significant improvement in the fine delimitation of the tumoral margins, which is still problematic with ICG.
Example 9: Comparison of Two Compounds According to the Invention And a Fluorescent Agent of the Prior Art (ICG) in ex Vivo Imaging of Pancreatic Tumors
[0088] A model of orthotopic pancreatic adenocarcinoma in the mouse was developed. The tumoral cells were amplified subcutaneously in SCID mice and the resultant fragments were then implanted surgically in the pancreas of irradiated BALB/c nude mice.
[0089] The development of the tumor was monitored in vivo by MRI (4.7T, PharmaScan, Bruker Biospin) at three time points, D14, 28 and 36. The animals were subjected to a 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 an emission filter at 770 nm.
[0090] After sessions of in vivo imaging performed at 2 h, 48 h and 164 h, 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, whereas the average volumes of the tumors were about 70 mm.sup.3. Indocyanine green (ICG), a dye widely used in the per-operative imaging of tumors, was included as a control.
##STR00031##
(CJ319)
[0091] The ex vivo fluorescence imaging described in
[0092] These results show the superiority of the compounds of the invention relative to a fluorescent agent of the prior art at the level of their specific distribution in a tumor tissue.