NOVEL COMPOUNDS AND USES OF SAME FOR NEAR-INFRARED CHERENKOV LUMINESCENCE IMAGING AND/OR FOR DEEP TISSUE TREATMENT BY CHERENKOV DYNAMIC PHOTOTHERAPY

Abstract

Compounds of the general structure (I), which includes: a radioactive entity, which is a beta-energy emitter that produces Cherenkov radiation, a fluorophore that absorbs electromagnetic radiation of a wavelength λ ranging from 300 nm to 500 nm; a fluorophore which emits electromagnetic radiation of a wavelength λ ranging from 650 nm to 950 nm and/or is a photosensitizer which produces reactive oxygen species ROSs; and a vector entity, which may be present or absent. Also, the use of these compounds for an application for near-infrared Cherenkov luminescence imaging and/or for the treatment of deep biological tissues by Cherenkov dynamic phototherapy.

Claims

1-10. (canceled)

11. A compound having the following general structure (I): ##STR00048## wherein: A is a radioactive entity which is a beta-energy emitter which produces Cherenkov radiation, said radioactive entity preferably being a radiochelate, namely a radiometal surrounded by a chelate or a radioelement which is non-metallic; B is a fluorophore which absorbs electromagnetic radiation of a wavelength λ ranging from 300 nm to 500 nm; C is a fluorophore which emits electromagnetic radiation of a wavelength λ ranging from 650 nm to 950 nm, and/or C is a photosensitizer which produces reactive oxygen species ROSs; D may be present or absent, and represents, when it is present, a vector entity, said vector entity preferably being a biomolecule or a nanoparticle vector, L1, L2, L3 are each, independently of one another, an at least divalent linking group or a covalent bond, with the condition that ##STR00049## are not simultaneously present, which means that, if ##STR00050## is present, then ##STR00051## is absent and vice versa, L4 is present if D is present and represents, when it is present, an at least divalent linking group or a covalent bond; n is an integer equal to 1, 2, 3, 4 or 5; m is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8; and wherein: A activates B by CRET-type energy transfer, B transfers the energy received from A to C, by intramolecular FRET-type energy transfer or by TBET-type energy transfer.

12. The compound as claimed in claim 11, having the following structure (I-1): ##STR00052## wherein A, B, C, D, L1, L2, L4, n and m are as defined above.

13. The compound as claimed in claim 11, having the following structure (I-2): ##STR00053## wherein A is a non-metallic radioelement, and B, C, D, L2, L3, L4, n and m are as defined above.

14. The compound as claimed in claim 11, wherein A is: a radiochelate, the radiometal of which is chosen from the group comprising .sup.90Y .sup.177Lu, .sup.69Ga, .sup.89Zr, .sup.64Cu, .sup.89Sr, .sup.212Bi, .sup.213Bi, .sup.44Sc, .sup.225Ac and .sup.44Sc and the chelating agent of which is chosen from the group comprising DOTAGA, DOTA, NOTA, NODAGA and DFO, a non-metallic radioelement chosen from the group comprising .sup.18F, .sup.131L .sup.124I and .sup.32P.

15. The compound as claimed in claim 11, wherein B is chosen from the group comprising a nucleus of the type coumarin; substituted coumarin, in particular substituted with one or more hydroxyls and/or with a pyridinium, itself optionally substituted; pyranine; pyrene; BODIPY; substituted BODIPY, in particular phenyl-BODIPY, hydroxyphenyl-BODIPY, aza-BODIPY; fluorescein; rhodamine, in particular rhodamine 6G, rhodamine 101, rhodamine B, rhodamine 123; eosin, in particular eosin B, eosin Y; tryptophan, and mixtures thereof.

16. The compound as claimed in claim 11, wherein C is chosen from the group comprising a nucleus of the cyanine type, in particular cyanine-7, cyanine-5, cyanine-3; phthalocyanine, in particular silicon, zinc, magnesium, phosphorus, aluminum, indium phthalocyanine, naphthalocyanine, in particular zinc, magnesium, phosphorus, aluminum, silicon, indium naphthalocyanine; chlorin, in particular zinc, magnesium, phosphorus, aluminum, silicon, indium chlorin; bacteriochlorin, in particular zinc, magnesium, phosphorus, aluminum, silicon, indium bacteriochlorin.

17. The compound as claimed in claim 15, wherein B and/or C comprise(s) at least one solubilizing group chosen from the group comprising a sulfonate (SO.sub.3.sup.−); a carboxylate (COO.sup.−); an ammonium (NR.sub.4.sup.+) with R═H, alkyl or aryl; a phosphonate (PO.sub.3.sup.2−); a pyridinium, which is preferably substituted; an imidazolium, and mixtures thereof.

18. The compound as claimed in claim 11, wherein D is a biomolecule, in particular a peptide; a protein; a protein of antibody type; a protein of antibody fragment type, such as Fab, Fab′2, Fab′, ScFv, nanobody, affibody, diabody; an aptamer.

19. A method for near-infrared Cherenkov luminescence imaging, comprising administering to a subject a compound as claimed in claim 11.

20. A method of treating deep biological tissues by Cherenkov photodynamic therapy, comprising administering to a subject in need thereof a compound as claimed in claim 11.

21. The compound as claimed in claim 16, wherein B and/or C comprise(s) at least one solubilizing group chosen from the group comprising a sulfonate (SO.sub.3.sup.−); a carboxylate (COO.sup.−); an ammonium (NR.sub.4.sup.+) with R═H, alkyl or aryl; a phosphonate (PO.sub.3.sup.2−); a pyridinium, which is preferably substituted; an imidazolium, and mixtures thereof.

Description

[0226] FIG. 1 is a scheme of the synthesis of asymmetric cyanines (35) and (36) which will be used to prepare compounds (I) of the invention wherein C is a nucleus of asymmetric cyanine type.

[0227] FIG. 2 is a scheme of the synthesis of the compound 1 of the invention.

[0228] FIG. 3 is a scheme of the synthesis of the compounds 5 (FIG. 3a) and 6 (FIG. 3b) of the invention.

[0229] FIG. 4 is a scheme of the synthesis of the compounds 21 (FIG. 4a) and 11 (FIG. 4b) of the invention.

[0230] FIG. 5 is a scheme of the synthesis of the compounds 4 and 19 of the invention.

[0231] The following examples illustrate the invention; they do not limit it in any way.

EXAMPLE 1

[0232] Preparation of Compounds (I) of the Invention

[0233] The methods for preparing several compounds which are subjects of the invention are described in detail in this example.

[0234] Separations and Analyses by HPLC

[0235] System A: HPLC-MS (Hypersil C18 column, 2.6 μm, 2.1×50 mm) with H.sub.2O 0.1% formic acid (FA) as eluent A and CH.sub.3CN 0.1% FA as eluent B [linear gradient from 5 to 100% of B (5 min) and 100% of B (1.5 min)] at a flow rate of 0.5 ml/min. The UV detection is carried out at 650 and 750 nm.

[0236] System B: HPLC (Hypersil C18 column, 5 μm, 10×250 mm) with H.sub.2O 0.1% FA as eluent A and CH.sub.3CN 0.1% FA as eluent B [linear gradient from 20 to 60% of B over the course of 40 min] at a flow rate of 3.5 ml/min. The UV detection is carried out at 700 and 780 nm.

1/ Synthesis of Asymmetric Cyanines of Formulae (35) and (36) (see FIG. 1)

[0237] In the compounds No. 1, 3, 5-7 and 12-17 of the invention, C is a nucleus of asymmetric cyanine type.

[0238] The method of synthesis of the asymmetric cyanines (35) and (36) used for preparing the compounds of the invention has no precedent and was developed by the inventors. It is therefore described in detail below (see also FIG. 1).

Synthesis of the Compound (30)

[0239] Phosphorus oxychloride (5.6 ml, 60 mmol) is added dropwise at 0° C. to anhydrous DMF (6.5 ml, 84 mmol). After 30 min, cyclohexanone (2.75 ml, 27 mmol) is then added, resulting in a change in color of the reaction mixture which becomes orange and which is brought to reflux for 1 h in a water bath. After having cooled the mixture to ambient temperature, an aniline/ethanol mixture [1:1 (v/v), 90 ml] is added dropwise. An exothermic reaction, generation of HCl and solidification follow on from this. After addition of aniline, the reaction mixture which is deep purple in color is poured into an ice-cold water/concentrated HCl mixture [10:1 (v/v) 80 ml]. Crystals form in the solution stored at 4° C. for 12 h. After filtration, the crystals are washed with cold water and then diethyl ether and dried to give 7.19 g (75%) of the product (30).

Synthesis of (31)

[0240] Sodium azide (650 mg, 10 mmol) is added to a solution of 1-bromo-3-chloropropane (1.57 g, 10 mmol) in 15 ml of DMF (N,N-dimethylformamide). After stirring for 5 h at ambient temperature, the reaction mixture is poured into 80 ml of water and extracted with ether (3×50 ml). The organic phases are combined and washed with water (2×50 ml) and brine (100 ml), then dried over MgSO.sub.4 and finally concentrated under reduced pressure. Added to the residue obtained (0.98 g, 8.23 mmol), which is redissolved in acetone (50 ml), is sodium iodide (2.47 g, 16.47 mmol). The resulting mixture is brought to reflux with stirring for 16 h, then is subsequently poured into 50 ml of water and extracted with ethyl acetate (3×50 ml). The organic phases are washed with water (2×50 ml), dried over MgSO.sub.4 and concentrated under reduced pressure to give 1.27 g (60%) of the product (31), which is in the form of a yellow oil. No purification is necessary. .sup.1H NMR (500 MHz, CDCl.sub.3, 300 K): δ (ppm)=2.03 (m, 2H); 3.25 (t, J=6.6 Hz, 2H); 3.43 (t, J=6.6 Hz, 2H). .sup.13C NMR (125 MHz, CDCl.sub.3, 300 K) δ (ppm)=2.42; 32.46; 51.59.

Synthesis of (32)

[0241] A solution of 2,3,3-trimethylindolenine (377 mg, 2.37 mmol) and of azido-3-iodopropane (31) (1 g, 4.74 mmol) in acetonitrile is brought to reflux for 2 days. The color of the solution changes from pale orange to dark green. The solvent is evaporated off under reduced pressure and 5 ml of dichloromethane are then added. This solution was added dropwise to diethyl ether (50 ml) resulting in the precipitation of a dark green compound. The solid is recovered by filtration, then 5 ml of dichloromethane are added and the process is repeated twice. The solid is vacuum-dried to give 596 mg (68%) of the product (32) which is in the form of a dark green to brown solid.

[0242] .sup.1H NMR (500 MHz, CDCl.sub.3, 300 K): δ (ppm)=1.65 (s, 6H); 2.32 (m, 2H); 3.19 (s, 3H), 3.70-3.77 (m, 2H); 4.91 (t, J=7.2 Hz, 2H); 7.52-7.64 (m, 3H); 7.84-7.89 (m, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3, 300 K): δ (ppm)=196.67; 141.62; 141.08; 130.30; 129.80; 123.40; 115.87; 54.85; 49.04; 47.87; 27.44; 23.26; 17.50.

Synthesis of (33)

[0243] A mixture of 2,3,3-trimethylindolenine (331 mg, 2.08 mmol) and of 3-bromopropylamine hydrobromide (456 mg, 2.08 mmol) is heated at 120° C. in a sealed tube for 10 h. The solid residue formed is cooled and washed abundantly with diethyl ether then a mixture of Et.sub.2O:CHCl.sub.3 (1:1) to give 574 mg (74%) of the product (33).

[0244] .sup.13C NMR (125 MHz, MeOD, 300 K): δ (ppm)=199.28; 143.38; 142.32; 131.36; 130.60; 124.80; 116.40; 56.19; 46.46; 37.86; 29.79; 16.81; 22.85.

Synthesis of (34)

[0245] The compound (32) (300 mg, 0.81 mmol) and sodium acetate (70 mg, 0.85 mmol) are dissolved in 30 ml of dry ethanol, resulting in a green solution. The compound (30) (313 mg, 0.97 mmol) is then added with 10 ml of dry ethanol, resulting in a purple/blue solution. The reaction mixture is brought to reflux for 2 h and the progression of the reaction is monitored by LC-MS (liquid chromatography-mass spectrometry). Half the volume of solvent is distilled under reduced pressure and the more concentrated reaction mixture is poured into 70 ml of Et.sub.2O. The solid is washed with Et.sub.2O (3×50 ml) and vacuum-dried. The solid is then purified with a chromatographic column on silica gel (DCM/MeOH 98/2 vol.) to give 175 mg (36%) of the pure product (34). It should be noted that the color of the compound depends on its state of protonation; it appears blue in TLC because of the acidity of the silica.

[0246] .sup.1H NMR (500 MHz, CDCl.sub.3, 300 K): δ (ppm)=1.66 (s, 6H); 1.87 (p, J=6.2 Hz, 2H); 1.93-2.08 (m, 2H); 2.61-2.67 (m, 2H); 2.79 (t, J=6.1 Hz, 2H); 3.42 (t, J=6.2 Hz, 2H); 3.79 (m, 2H); 5.57 (d, J=12.6 Hz, 1H); 6.70 (d, J=7.8 Hz, 1H); 6.92 (t, J=7.4 Hz, 1H); 7.14-7.23 (m, 5H); 7.37 (m, 3H); 7.62 (d, J=12.6 Hz, 1H); 8.84 (s, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3, 300 K): δ (ppm)=159.86; 158.13; 129.50; 129.40; 129.30; 128.17; 125.77; 122.12; 121.36; 121.23; 121.19; 120.82; 106.69; 93.40; 77.51; 77.26; 77.01; 66.10; 49.03; 46.49; 39.67; 36.09; 29.95; 28.61; 26.94; 26.20; 21.60; 15.52.

Synthesis of the Cyanine (35) (Fluorophore)

[0247] The compounds (34) (105 mg, 0.175 mmol), (33) (119 mg, 0.315 mmol) and sodium acetate (26 mg, 0.315 mmol) are dissolved in 10 ml of dry ethanol to give a green solution. The mixture is brought to reflux with stirring for 7 h and its color changes to dark blue. The progression of the reaction is monitored by UV-Visible and LCMS. The reaction mixture is concentrated under reduced pressure and then poured into 30 ml of Et.sub.2O. The resulting solution is filtered to give a brownish solid which is washed with Et.sub.2O and purified on a size exclusion column using chloroform as eluent, to give 300 mg (40%) of a pure product (35) which is in the form of a green solid. .sup.1H NMR (600 MHz, DMSO, 323 K): δ (ppm)=1.70 (s, 6H); 1.70 (s, 6H); 1.86-1.91 (m, 2H); 2.01-2.07 (m, 4H); 2.74-2.77 (m, 4H); 2.99 (m, 2H); 3.53 (t, J=6.5 Hz, 2H); 4.32 (q, J=7.0 Hz, 4H); 6.32 (d, J=13.9 Hz, 1H); 6.41 (d, J=14.3 Hz, 1H); 7.27-7.67 (m, 8H); 7.87 (s, 2H); 8.27 (t, J=14.0 Hz, 1H); 8.32 (d, J=14.2 Hz, 1H). Mass spectrum: m/z=595.3311 [M-2Br].sup.− (calculated for C.sub.36H.sub.45Br.sub.2ClN.sub.6: 754.1751).

Synthesis of the Cyanine (36) (Fluorophore)

[0248] The compound (35) (300 mg, 0.396 mmol), di-tert-butyl dicarbonate (130 mg, 0.594 mmol) and DIPEA (N,N-diisopropylethylamine) (255 mg, 1.98 mmol) are dissolved in 15 ml of chloroform to give a green solution. The mixture is brought to reflux with stirring, and the progression of the reaction is monitored by LCMS. After cooling to ambient temperature, the crude mixture is washed with water (2×40 ml) and with a 0.2 M solution of hydrochloric acid (30 ml). The organic phases are combined, and concentrated under reduced pressure, and then the compound is isolated and purified with a size exclusion column using chloroform as eluent, to give the pure product (36) which is in the form of a green solid. Mass spectrum: m/z=695.5 [M−Br].sup.− (calculated for C.sub.41H.sub.52BrClN.sub.6O.sub.2: 774.30). HPLC, retention time: 5.95 min. UV-Vis: 777 nm.

2/ Synthesis of the Compound 1 (See FIG. 2)

Synthesis of the Coumarin-Cyanine Conjugate (37)

[0249] 7-Hydroxycoumarin (4.1 mg, 0.026 mmol) and sodium hydride (2.0 mg, 0.0515 mmol) are dissolved in 1 ml of DMF and the mixture is stirred at ambient temperature for 10 min. The cyanine (36) (10 mg, 0.0128 mmol) is subsequently added and then the progression of the reaction is monitored by LCMS. After approximately 20 minutes, the DMF is distilled under reduced pressure, and the product is taken up with CHCl.sub.3 then washed with water and purified on a small plug of silica gel (solvent: DCM). The compound (37) is obtained in the form of a green solid (10 mg, 90%). Mass spectrum: m/z=821.4 [M−Br].sup.− (calculated for C.sub.50H.sub.57BrN.sub.6O.sub.5: 900.35). HPLC, retention time: 5.6 min. UV-Vis: 307, 777 nm.

Synthesis of the Compound (38)

[0250] The compound (37) (10 mg, 0.013 mmol) is dissolved in 2 ml of a TFA/DCM mixture (1/9 vol.) and the resulting solution is stirred for 1 h at ambient temperature. 10 ml of DCM are added to this mixture, and the organic phase is subsequently washed with a saturated solution of NaHCO.sub.3 (2×25 ml), then dried with MgSO.sub.4, then concentrated under reduced pressure to obtain 9 mg (90%) of product (38). Mass spectrum: m/z=721.4 [M−CF.sub.3CO.sub.2.sup.−].sup.− (calculated for C.sub.45H.sub.49N.sub.6O.sub.3: 721.92). HPLC, retention time: 4.4 min. UV-Vis: 307, 777 nm.

Synthesis of the Compound (39)

[0251] DOTA-GA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) (9.1 mg, 0.0198 mmol) and triethylamine (11 mg, 0.105 mmol) are added to a solution of the compound (38) (9 mg, 0.0107 mmol) in 1.5 ml of DMF, and then the resulting mixture is stirred for 24 h at 50° C. After the DMF has been evaporated off, the product is taken up in CHCl.sub.3 and washed with water. The product is subsequently purified using a size exclusion column with CHCl.sub.3 to give 7 mg (50%) of pure product (39). Mass spectrum: m/2z=590.2 (calculated for C.sub.64H.sub.79N.sub.10O.sub.12: 1180.39). HPLC, retention time: 4.3 min. UV-Vis: 307, 777 nm.

Synthesis of the Compound 1 of the Invention

[0252] The precursor (39) is placed in an ammonium acetate buffer, pH 4.5, and placed in the presence of a radioactive source (.sup.90YCl.sub.3 source), so as to obtain the desired specific activity. The mixture is heated at 80° C. for 2 hours and the reaction is monitored by radio-ITLC. The compound 1 of the invention is obtained.

3/ Synthesis of the Compounds 7, 12 and 13 of the Invention

[0253] The compounds 7, 12 and 13 are synthesized according to a synthesis method comparable to that described for the compound 1, but with the following differences: the steps corresponding to the introduction of the radiochelate of .sup.90Y-[DOTAGA] type are not carried out.

[0254] Instead, the .sup.18F-radiolabeling steps for the compounds 7 and 13 are carried out starting from the corresponding alcohol which is converted into a triflic ester which is then placed in the presence of a salt of Na.sup.18F type so as to produce substitution of the triflate with the .sup.18F.

[0255] The synthesis of the compound 12 of the invention is carried out according to a method comparable to the compound 1 (introduction of a BODIPY derivative bearing a 4-hydroxyphenyl group in meso position). The BODIPY compound bearing two non-radioactive .sup.19F fluorine atoms will react with DMAP (dimethylaminopyridine) by substitution of one of the two fluorine atoms, thus resulting in a BODIPY-DMAP adduct wherein the DMAP, now in quaternized form, is a good leaving group. In the presence of a salt of Na.sup.18F type, the .sup.18F will substitute the quaternized DMAP, resulting in the .sup.18F-radiolabeled moiety.

4/ Synthesis of the Compounds 5 and 6 of the Invention (See FIG. 3)

[0256] The compounds 5 and 6 can be obtained from an asymmetric cyanine (40) resulting in the compound (42) or (41) which are respectively the precursors of the compounds 5 (FIG. 3a) and 6 (FIG. 3b) of the invention.

Synthesis of the Compound (40)

[0257] The compound (34) (170 mg, 0.28 mmol), the compound (31) (88 mg, 0.28 mmol) and sodium acetate (23 mg, 0.28 mmol) are dissolved in 12 ml of dry ethanol, giving a brown solution. The mixture is stirred and brought to reflux for 2 h, where it becomes dark green. At the end of UV-Visible and LCMS verifications, the reaction mixture is concentrated under reduced pressure and poured into 75 ml of Et.sub.2O. The solution is filtered to give a brownish solid which is washed with Et.sub.2O and purified by silica gel chromatography (using a DCM/MeOH solvent gradient of 98/2 vol. to 90/10 vol.) so as to give 75 mg of pure product (40) in the form of a dark green solid.

[0258] .sup.1H NMR (500 MHz, CDCl.sub.3, 300 K): δ (ppm)=1.9 (m, 12H); 1.90 (m, 2H); 2.07 (t, J=6.5 Hz, 2H); 2.56-3.08 (m, 6H); 3.53 (t, J=6.0 Hz, 2H); 4.14 (t, J=7.1 Hz, 2H); 4.60 (m, 2H); 6.07 (d, J=13.7 Hz, 1H); 6.58 (d, J=14.4 Hz, 1H); 7.11-7.54 (m, 8H); 8.21 (d, J=13.4 Hz, 1H); 8.42 (d, J=14.2 Hz, 1H).

Synthesis of the Compound 5 (FIG. 3a)

[0259] The coumarin group is reacted with 2-benzyloxy-1,3-dichloropropane, and the adduct is deprotected to give the alcohol bis-coumarin (called multi-B amplifier head) which subsequently reacts with the chloro-cyanine (40). The DOTAGA-ethylenediamine group reacts with the acid function of the intermediate (42) to give the compound (43). The radiolabeling step makes it possible to obtain the compound 5.

Synthesis of the Compound 6 (FIG. 3b)

[0260] Hydroxycoumarin (21 mg, 0.13 mmol) and sodium hydride (6.3 mg, 0.26 mmol) are dissolved in 2 ml of DMF. After 10 min, the compound (40) (50 mg, 0.07 mmol) is added and the course of the reaction is monitored by LCMS. As soon as the reaction is no longer progressing, 20 ml of diethyl ether are added. The solid is filtered off, then washed with diethyl ether and acetone (the purity is monitored by HPLC), to give 11 mg of pure product (41) in the form of a green solid.

[0261] One of the six amine functions of the 1,2,3,4,5,6-benzenehexamethanamine is protected while the other five are involved in the reaction with DOTAGA(tBu).sub.4. The adduct formed (also called multi-A amplifier head) is involved in the reaction with the cyanine. The system obtained is saponified and then radiolabeled with yttrium to give the compound 6.

5/ Synthesis of the Compounds 21 and 11 of the Invention (see FIG. 4)

[0262] The compounds 21 and 11 are obtained from a symmetrical cyanine (44) bearing two azide groups, said cyanine resulting in the intermediate (45), which itself results in the compound (46) which is a precursor of the compound 21 (FIG. 4a) of the invention or in the compound (48) which is a precursor of the compound 11 (FIG. 4b) of the invention.

Synthesis of the Precursor (45)

[0263] Pyranine (74 mg, 0.14 mmol) and triethylamine (43 mg, 0.42 mmol) are dissolved in 2 ml of dry DMSO. The mixture is stirred at 50° C. for 4 h and then a solution of cyanine (44) (50 mg, 0.07 mmol) in 1 ml of DMSO is added. After 4 h at 45° C., 20 ml of DCM are added to the reaction mixture. After filtration which removes the excess pyranine, and then concentration under reduced pressure, the reaction crude is diluted in water (20 ml) and extracted with diethyl ether (2×30 ml) to give, after lyophilization, 41 mg (54%) of pure product (45) in the form of a green solid.

[0264] .sup.1H NMR (500 MHz, methanol-d.sub.4, 300 K); δ (ppm)=0.53 (s, 6H); 1.41 (s, 6H); 1.99 (p, J=6.7 Hz, 4H); 2.10-2.25 (m, 2H); 2.82 (m, 2H); 2.95 (m, 2H); 3.46 (td, J=5.8; 4.0 Hz, 4H); 4.11-4.20 (m, 4H); 6.27 (d, J=14.1 Hz, 2H); 7.06 (t, J=7.5 Hz, 2H); 7.12-7.17 (m, 2H); 7.18 (d, J=8.0 Hz, 2H); 7.24-7.32 (m, 2H); 8.09 (d, J=14.0 Hz, 2H); 8.28 (s, 1H); 9.01 (d, J=9.6 Hz, 1H); 9.23 (s, 2H); 9.50-9.56 (m, 2H).

[0265] ESI-HRMS: m/z=1041.2740 [M-2Na.sup.+ H].sup.− (calculated for C.sub.52H.sub.49N.sub.8O.sub.10S.sub.3.sup.−: 1041.2739). UV-Vis (water): 241.9; 287.4; 360.0; 394.8; 770.4 nm.

Synthesis of the Compound 21 (FIG. 4a)

Synthesis of the Compound (46)

[0266] The compound (45) reacts with DOTA-GA-ethylenediamine-BCN according to the following conditions. 6.3 mg of DOTA-GA-ethylenediamine-BCN (0.0129 mmol) are dissolved in 1 ml of phosphate buffer at pH=7.4. Next, 0.48 ml of a solution of cyanine (45) (1.97 mg; 0.00181 mmol) in water is added. The mixture is subsequently stirred at ambient temperature for 3 h and then lyophilized and the crude product obtained is purified by HPLC to give, after lyophilization, 3.55 mg (78%) of target compound (46). UV-Vis (PBS buffer): 242.0; 288.1; 360.5; 395.2; 770.8 nm. HPLC analysis with system A: 3.9 min, 77% MeCN 0.1% FA. HPLC with system B: 25.0 min, 45% MeCN 0.1% FA.

Preparation of the Compound 21 of the Invention

[0267] A conjugation step (verification of pH, temperature, antibody concentration) makes it possible to obtain the “compound 46-antibody” system, that is to say the compound (47). The bioconjugate is radiolabeled with Y90 to give the compound 21 of the invention.

Synthesis of the Compound 11 (FIG. 4b)

Synthesis of the Compound (48)

[0268] The cyanine (45) is dissolved in 2 ml of dry DMSO and then the DOTA-GA-ethylenediamine-BCN is added. The mixture is stirred at 50° C. for 16 h. The compound (48) is obtained.

[0269] .sup.1H NMR (500 MHz, methanol-d.sub.4, 300 K): δ (ppm)=0.53 (s, 6H); 1.41 (s, 6H); 1.99 (p, J=6.7 Hz, 4H); 2.10-2.25 (m, 2H); 2.82 (m, 2H); 2.95 (m, 2H); 3.46 (td, J=5.8; 4.0 Hz, 4H); 4.11-4.20 (m, 4H); 6.27 (d, J=14.1 Hz, 2H); 7.06 (t, J=7.5 Hz, 2H); 7.12-7.17 (m, 2H); 7.18 (d, J=8.0 Hz, 2H); 7.24-7.32 (m, 2H); 8.09 (d, J=14.0 Hz, 2H); 8.28 (s, 1H); 9.01 (d, J=9.6 Hz, 1H); 9.23 (s, 2H); 9.50-9.56 (m, 2H). ESI-HRMS: m/z=1041.2740 [M-2Na.sup.+ H].sup.− (calculated for C.sub.52H.sub.49N.sub.8O.sub.10S.sub.3.sup.−: 1041.2739). UV-Vis (water): 241.9; 287.4; 360.0; 394.8; 770.4 nm.

Synthesis of the Compound 11

[0270] The bismacrocyclic compound (48) in a buffer is incubated for several hours in the presence of a defined amount (MBq) of yttrium-90 trichloride .sup.90YCl.sub.3 in order to achieve the desired specific activity. The radiochemical purity is verified by RI-TLC.

[0271] The compound 11 of the invention is obtained.

6/ Synthesis of the Compounds 4 and 19 of the Invention (See FIG. 5)

Synthesis of the Compound (49)

[0272] The coumarin (646 mg, 4 mmol) and 4,5-dichlorophthalo-nitrile (788 mg, 4 mmol) are dissolved in 10 ml of DMF (dimethylformamide) in the presence of K.sub.2CO.sub.3 (2.21 g, 16 mmol). The resulting mixture is stirred at 45° C. for 16 h and is then recovered by filtration. The filtrate is concentrated under reduced pressure and purified by column chromatography using a DCM/MeOH 9(5/5 vol.) solvent mixture as eluent, to give 920 mg (70%) of the desired compound (49) in the form of a powder.

[0273] .sup.1H NMR (500 MHz, CDCl.sub.3, 300 K): δ (ppm)=6.45 (d, J=9.6 Hz, 1H); 6.97 (dd, J=8.5; 2.4 Hz, 1H); 7.02 (d, J=2.4 Hz, 1H); 7.26 (s, 1H); 7.59 (d, J=8.4 Hz, 1H); 7.73 (d, J=9.6 Hz, 1H); 7.94 (s, 1H).

[0274] .sup.13C NMR (125 MHz, CDCl.sub.3, 300 K): δ (ppm)=108.17; 111.80; 114.06; 114.16; 115.77; 115.97; 116.68; 117.02; 122.79; 130.16; 131.19; 136.13; 142.55; 155.74; 156.37; 156.60; 159.82.

[0275] MALDI-TOF (calculated for C.sub.17H.sub.7ClN.sub.2O.sub.3: 322.0145, found 323).

Synthesis of the Phthalonitrile-Coumarin-Pyridine System (50)

[0276] The phthalonitrile-coumarin conjugate (40) (400 mg, 1.24 mmol), 4-hydroxypyridine (176 mg, 1.85 mmol) and K.sub.2CO.sub.3 (512 mg, 71 mmol) are dissolved in 20 ml of DMF. The resulting mixture is stirred at 45° C. for 16 h. Next, K.sub.2CO.sub.3 is separated by filtration on a frit and then the filtrate is concentrated under reduced pressure and purified by chromatography using the (DCM/MeOH 90/10) solvent mixture as eluent, to give 200 mg (%) of the desired product (50).

Synthesis of the Diiminoisoindoline-Coumarin-Pyridine System (51)

[0277] A solution of dicyanobenzene (50) in methanol is brought to reflux with ammonia bubbling for several hours. After distillation of the solvent, the compound obtained (51) is immediately used in the next synthesis step.

Steps for Converting (51) into the Compounds 4 and then 19 of the Invention

[0278] The diiminoisoindoline (51) is reacted with a silicon salt. The reaction mixture is brought to reflux and then the solvent is distilled under reduced pressure. The residue obtained is washed with a series of solvents, then dried and immediately used in the following step. The intermediate (52) reacts with 3-azidoethanol in the presence of a base and brought to reflux. After purification, a suspension of phthalocyanine (53) in methyl iodide is brought to reflux. At the outcome, the excess methyl iodide is distilled and the phthalocyanine (54) is purified by semipreparative HPLC. The intermediate (54) and DOTAGA-ethylenediamine-BCN are reacted; after distillation of the solvents, the target conjugate (55) is separated from the compound (54) and the by-products by HPLC.

EXAMPLE 2

[0279] Use of the Compounds of the Invention for Cherenkov-PDT

[0280] An in-tube in vitro study and in vitro study on cells makes it possible to show the properties of transfer by CRET/TBET or CRET/FRET, which is intramolecular, of the compounds of the invention.

[0281] The reactive oxygen species (ROSs) are measured by UV/Visible spectrometry by following the disappearance of the DPBF (diphenylbenzofuran) absorbance band following the reaction with the ROSs generated by the Cherenkov photodynamic process.

[0282] In Vitro Study on Cells

[0283] The cells are plated onto 96-well microplates, and incubated with the solution of a compound of the invention in the total absence of parasitic exogenous light source capable of exciting the photosensitizing compound.

[0284] A control plate is prepared in the presence of the non-radiolabeled compound in order to prove that the toxicity measured does not come: [0285] from a resulting parasitic cytotoxic effect, [0286] from the intrinsic cytotoxicity of the compound, [0287] from a photocytotoxicity resulting from excitation by an exogenous light source.

[0288] Moreover, a control study using the non-radiolabeled parent compound—and in the absence of exogenous light source—makes it possible to confirm the origin of the cytotoxicity.

[0289] In Vivo Cherenkov-PDT Protocol

[0290] It begins with the intravenous injection of a compound of the invention into xenografted mice carrying a deep cancer model of cancer cells.

[0291] A control batch of mice injected with a radiolabeled bioconjugate, of AD structure, that is to say not comprising BC, is prepared. The tumor volume is monitored by carrying out PET imaging of the tumor. This imaging is carried out in the following way: the mice are anesthetized then injected with .sup.18F-fluorodeoxyglucose (.sup.18F-FDG) and are subsequently imaged on a μPET imager. Throughout the experiment, all precautions are taken so that no parasitic light, that is to say light other than the Cherenkov radiation, can reach the tumor labeled with the compound of the invention.

[0292] The following verifications are carried out: [0293] the tumor is deep (>1 cm deep), [0294] the mice have hair, and optionally a screen can be affixed on the study zone.

[0295] The control mouse batch studied makes it possible to show the change in the tumor volume in the case of prolonged exposure to an exogenous, and potentially parasitic, light source. At a sufficient depth, the change in tumor volume for control mice versus treated mice makes it possible to demonstrate the efficacy of the compounds of the invention.

EXAMPLE 3

[0296] Use of the Compounds of the Invention for Near-IR CLI

[0297] CLI Protocol

[0298] A study on an optical imager makes it possible to measure the properties of transfer by CRET/TBET or CRET/FRET, which is intramolecular, of the compound 1 of the invention.

[0299] Moreover, an in vitro study showed that the non-radiolabeled parent compound does not exhibit any cytotoxicity.

[0300] The CLI protocol begins with the intravenous injection of the compound 1 of the invention into xenografted mice carrying a tumor.

[0301] A control batch of mice injected with a radiolabeled bioconjugate of AD type, that is to say not comprising BC, is also prepared.

[0302] When the AD bioconjugate has reached the tumor, after several hours (the number of hours will depend on the nature of the biomolecule), the mice are anesthetized and are then placed in the optical imager.

[0303] The mice are imaged in Cherenkov mode and in bioluminescence mode, first by examining the radiance over the entire spectral window of the optical imager (500-850 nm) and then by using filters in order to examine the radiance on the near-infrared zone exclusively.

[0304] At the end of the experiments, the mice are sacrificed.

[0305] The radiance measurement is the step which makes it possible to demonstrate the transfer to the near-infrared and the efficacy of the compounds of the invention. This involves a direct comparison of the radiance between the batch of control mice injected with AD (the radiolabeled biomolecule, that is to say the Cherenkov radiation alone, not amplified by the BC antenna), and the batch of mice injected with the compound 1 of the invention.

[0306] The comparison of the result obtained with the compound 1 of the invention and the result obtained by the authors Bernhard et al..sup.(7) makes it possible to demonstrate the relevance of a single-molecule probe rather than a multimolecular probe, and the advantage of the compounds of the invention.

LITERATURE REFERENCES

[0307] (1) Grootendorst, M. R., Cariati, M., Kothari, A., Tuch, D. S., Purushotam, A. Cerenkov luminescence imaging (CLI) for image-guided cancer surgery, Clin. Transl. Imaging (2016). 4, 353-366. [0308] (2) Demian van Straten, Vida Mashayekhi, Henriette S. de Bruijn, Sabrina Oliveira and Dominic J. Robinson, Oncologic Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions, Cancers, 2017, 9. 19, 1-54, doi:10.3390/cancers 9020019. [0309] (3) Robertson, R. et al. Optical imaging of Cerenkov light generation from positron-emitting radiotracers. Physics Medicine Biology 54, N355-N365 (2009). [0310] (4) Dothager, R. S., Goiffon, R. J., Jackson, E., Harpstrite, S. & Piwnica-Worms, D. Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems. PLoSone 5, c13300 (2010). [0311] (5) Boschi, F. & Spinelli. A. E. Quantum clots excitation using pure beta minus radioisotopes emitting Cerenkov radiation. RSC Advance 2, 11049-1052 (2012). [0312] (6) Bernhard et al., Chemical Communications, 2014, 50, pp. 6711-6713. [0313] (7) Bernhard et al., Scientific Reports, 2017, 7, 45063. [0314] (8) Bizet et al., Bioorganic & medicinal Chemistry 26 (2018), pp. 413-420. [0315] (9) Anyanee KamKaew et al., Applied Materials & Interfaces, 2016, 8 (40), pp 26630-26637. [0316] (10) C. Göl, M. Malkoç, S. Yeşilot, M. Durmuş, Dyes Pigm, 111 (2014), pp. 81-90. [0317] (11) S. Osati, H. Ali, J. E. van Lier, Tetrahedron Lett, 56 (2015), pp. 2049-2053, [0318] (12) H. Yanik, M. Göksel, S. Yesilot, M. Durmus, Tetrahedron Lett, 57 (2016), pp. 2922-2926. [0319] (13) A. Loudet, C. Thivierge, K. Burgess, Dojin News (2011), p. 137, http://www.dojindo.co.jp/letterj/137, review/01.html. [0320] (14) Wen-Hai Zhan, Jian-Li Hua, Ying-Hua Jin, Xin Teng, and He Tian, Res. Chem. Intermed., Vol. 34, No. 2-3, pp. 229-239 (2008).