USE OF FLUOROPHORE COMPOUNDS OF THE AZA- BODIPY TYPE AS CONTRAST AGENTS IN THE SHORT WAVE INFRARED REGION

Abstract

The present invention relates to the use of an aza-BODIPY fluorophore compound as a contrast agent in the optical window ranging from 1000 to 1700 nm. The invention also relates to the use, as a contrast agent, of a composition comprising said fluorophore compound and a pharmaceutically acceptable excipient and/or a solvent, in a kit comprising an injection system and said fluorophore or said composition, and also to a method for identifying a biological target (such as a healthy or tumour cell, a protein, DNA, RNA, for example).

Claims

1. A method for imaging comprising measuring a fluorophore compound of formula I ##STR00024## R.sup.1, R.sup.2, R.sup.3 and R.sup.4, identical or different, represent a C5-C7 aryl or heteroaryl group, optionally substituted with at least one group chosen from halogen, —NR.sup.cR.sup.d, —OR.sup.d, hydrazine, —CF.sub.3 and —CN, at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is a C5-C7 aryl group substituted with a group —NR.sup.cR.sup.d, and optionally a group chosen from halogen, —OR.sup.d, hydrazine, —CF.sub.3 and —CN, R.sup.5 and R.sup.6, identical or different, represent a hydrogen, a halogen, a C1-C15 group comprising an aldehyde, ketone, carboxylic acid or ester function, a nitrile, —SO.sub.3Na, 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 chalcogen (SePh, TePh), an amide, a C5-C7 aryl or heteroaryl group, optionally substituted by at least one group chosen from a halogen, —NR.sup.cR.sup.d, —OR.sup.d, hydrazine, —CF.sub.3 and —CN, optionally R.sup.3 and R.sup.5 and/or R.sup.4 and R.sup.6 are covalently bonded and together form a C5-C7 aryl or heteroaryl group, optionally substituted by at least one group chosen from halogen, —NR.sup.cR.sup.d, —OR.sup.d, hydrazine, —CF.sub.3 and —CN, R.sup.c and R.sup.d, identical or different, represent hydrogen or a linear or branched C1-C3 alkyl chain, R.sup.a and R.sup.b, identical or different, represent: a halogen, a C1-C50, aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally comprising one or more aromatic or heteroaromatic groups, optionally comprising one or more heteroatoms chosen from O, N, P and/or S, a C1-C50, aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally comprising one or more aromatic or heteroaromatic groups, optionally comprising one or more heteroatoms chosen from O, N, P and/or S, a biological vector covalently coupled via a C1-C50, aliphatic or heteroaliphatic, linear or branched, saturated or unsaturated group, optionally comprising one or more aromatic or heteroaromatic groups, optionally comprising one or more heteroatoms chosen from O, N, P and/or S, a metal complex for therapeutic purposes, formed by a chelating agent and a metal, a radiometallic complex, formed by a chelating agent and a radiometal, or a molecule with a hydrodynamic diameter of less than 10 nm, a cyclic or linear peptide, an antibody, an antibody fragment, a nanobody, an affibody, an aptamer, a short DNA or RNA sequence, a sugar, a polysaccharide, an amino acid, a vitamin, a AMD3100 type molecule, a PSMA ligand, a steroid, a fatty acid, a polyamine, a polyphenol, a DNA base or a caffeine derivative in the optical window ranging from 1000 to 1700 nm.

2. The method according to claim 1, wherein R.sup.5 and R.sup.6 are a hydrogen.

3. The method according to claim 1, wherein R.sup.1 and R.sup.2, identical or different, represent a C5-C7 aryl group substituted by a —NR.sup.cR.sup.d group and optionally a group chosen from halogen, —OR.sup.d, hydrazine, —CF.sub.3 and —CN.

4. The method according to claim 1, wherein R.sup.1 and/or R.sup.2 represent a phenyl group substituted by a —NR.sup.cR.sup.d group.

5. The method according to claim 1, wherein R.sup.a and R.sup.b, identical or different, are chosen from a halogen, from hydrophilic groups of the following formulae: ##STR00025## ##STR00026## from groups comprising a bioconjugable function of the following formulae: ##STR00027## ##STR00028## —NH.sub.2 and —Si(OMe).sub.3, from the groups comprising a biological vector, from PPh.sub.2-Au(I), PPh.sub.2-Pt(II), PPh.sub.2-Pt(IV), phneylpyridine-Au(III), from DOTA-In(III), DOTAGA-In(III), NODAGA-Cu(II), NODAGA-Ga(III), or from α.sub.vβ.sub.3 integrin-targeting c(RGDfK), neuropilin-targeting ATWLPPR, anti-CD44, a thioglucose, a peracetylated thioglucose, folic acid, a AMD3100-type ligand, a PSMA ligand, spermine, spermidine, cadaverine, putrescine, resveratrol, a DNA base, caffeine or progesterone.

6. The method according to claim 1, wherein the fluorophore compound is chosen from fluorophore compounds of formula II: ##STR00029## wherein, Ra, Rb, Rc, Rd, have the definitions given above, Re and Rf, identical or different, represent at least one group from a hydrogen, a halogen, —NR.sup.cR.sup.d, —OR.sup.d, hydrazine, —CF.sub.3 and —CN.

7. The method according to claim 1, Rc and Rd are —CH.sub.3.

8. A composition comprising a fluorophore compound of formula I according to claim 1, and a pharmaceutically acceptable excipient and/or a solvent.

9. The composition according to claim 8, wherein the composition has a pH in the range from 4 to 10.

10. The method according to claim 1, wherein the fluorophore compound is encapsulated.

11. 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.

12. A method for in vitro identification of a biological target comprising: labelling cells of a sample taken or cultured with a composition comprising a fluorophore compound of formula I or II as defined above, measuring fluorescence in the optical window from 1000 to 1700 nm, and identifying target cells.

13. The method according to claim 4, wherein the —NR.sup.cR.sup.d group is in the para position.

14. The method according to claim 6, wherein the fluorophore compound is chosen from compounds of formula II wherein Rc and Rd are —CH.sub.3.

15. The composition according to claim 8, wherein the composition has a pH in the range from 6 to 8.

16. The method according to claim 12, wherein fluorescence is measured via microscopy, flow cytometry, optical imaging by fluorescence reflection (2D or 3D), or optical probe.

17. A composition comprising the fluorophore compound according to claim 6, and a pharmaceutically acceptable excipient and/or a solvent.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0112] FIG. 1 shows a mouse with a U87MG tumour in the right hind leg before and 24 hours after injection of aza-BODIPY (AG22). The upper planes are recorded in NIR imaging: Fluo800 corresponds to excitation at 780nm and collection between 830-900 nm. The lower planes are excited at 830 nm (SWIR800) and the emission is collected using a 1064 nm long pass filter, that means between 1064 and 1700 nm.

[0113] FIG. 2 shows the emission spectra of compounds AG22 and AG04.

[0114] FIG. 3 shows a mouse with a U87MG tumour in the right hind leg, from left to right: before, at 5 h and at 48 h after injection of the AG66 compound.

EXAMPLES

Example 1: Synthesis of Contrast Agents According to the Invention

Materials & Methods

[0115] The reactions were carried out in technical grade Carlo Erba solvents under normal atmosphere, unless otherwise stated. Experiments requiring anhydrous conditions were carried out under argon. Dry solvents were purchased from Carlo Erba, unstabilised and were dried using MB-SPS-800 (MBraun) or PureSolv-MD-5 (Inert®). All commercial reagents were purchased from Sigma-Aldrich® or ACROS Organics® and were used as received without any purification. TOTA-Boc (boc-1-amino-4,7,10-trioxa-13-tridecanamine) was purchased from Iris Biotech GmbH® and .sup.10B-BSH from Katchem®. Reaction monitoring was performed by HPLC-MS and thin layer chromatography on 0.2 mm thick Merck® 60 F254 silica gel plates, revealed by UV (254 nm). Chromatography column purifications were performed on Sigma-Aldrich® technical silica gel, 40-63 μm, 230-400 mesh, 60 Å.

[0116] NMR spectra (.sup.1H, .sup.13C) were recorded on a Bruker 500 Avance III or Bruker 600 Avance III HD (equipped with dual resonance broadband probes). Chemical shifts are expressed in ppm and are given relative to TMS (.sup.1H, .sup.13C)) 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).

[0117] HPLC-MS analyses were 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 simple MSQ Plus quadrupole mass spectrometer equipped with a Phenomenex Kinetex® column (2.6 μm, C18, 100 Å, LC 50×2.1 mm column). The gradient used for the characterisation was as follows (Gradient A):

TABLE-US-00001 Time % H2O (+0.1% % ACN (+0.1% Flow rate (min) formic acid) formic acid) (mL/min) 0 95 5 0.5 5 0 100 0.5 6.5 0 100 0.5 6.6 95 5 0.5 8.5 95 5 0.5 8.51 95 5 0.05

[0118] Semi-preparative HPLC purifications were performed on a Shimadzu HPLC instrument equipped with 2 LC-20AT pumps, a SPD-20A UVNis detector, a FRC-10A fraction collector, a SIL-10AP sampler and a CBM-20A control unit. The column used is a Shim-Pack GIST 5 μm C18 10×250 mm column and the gradients used are as follows:

TABLE-US-00002 Gradient A Gradient B Time % H2O + % ACN + Flow rate Time % H2O + % ACN + Flow rate (min) 0.1% TFA 0.1% TFA (mL/min) (min) 0.1% TFA 0.1% TFA (mL/min) 0 75 25 5 0 60 40 5 5 75 25 5 5 80 40 5 25 0 100 5 25 0 100 5 28 0 100 5 28 0 100 5 30 75 100 5 30 80 40 5 Gradient C Gradient D Time % H2O + % ACN + Flow rate Time % H2O + % ACN + Flow rate (min) 0.1% TFA 0.1% TFA (mL/min) (min) 0.1% TFA 0.1% TFA (mL/min) 0 80 20 5 0 80 20 5 5 80 20 5 5 80 20 5 28 0 100 5 25 0 100 5 33 0 100 5 27 0 100 5 35 80 20 5 28 80 20 5

##STR00014##

18 μL of N,N-dimethylpropargylamine (156 μmol, 2 eq) is dissolved in 2 mL of THF (tetrahydrofuran) under argon (shlenk glassware). Magnesium ethyl bromide (0.17 mL, 170 μmol, 2.2 eq) is then added and the mixture is refluxed for 45 min, allowed to return to room temperature, then transferred via cannula to a second schlenk containing the aza-BODIPY precursor (50 mg, 78 μmol, 1 eq). The mixture is then stirred at reflux for 45 min under argon, then the reaction is stopped by adding 2 mL of EtOH. The solvents are removed by evaporation under reduced pressure. The crude formed is solubilized in 10 mL of AcOEt, then 10 mL of distilled water is added. After stirring and settling in a separating funnel, the organic phase is set aside and the aqueous phase is extracted twice with 10 mL AcOEt. The organic phases are combined, washed twice with 10 mL twice-diluted aqueous NaHCO.sub.3 solution and dried over anhydrous MgSO.sub.4. The resulting solution is filtered and the solvent is removed by evaporation under reduced pressure. The residue is purified by silica gel chromatography column (eluent: 98/2 DCM/MeOH.fwdarw.100% MeOH) to isolate AG22 as a purple glittery powder (51.1 mg, 66.3 μmole, 85%).
.sup.1H NMR (CDCl.sub.3, 500 MHz) δ (ppm): 8.18 (d; .sup.3J=8.9 Hz; 4H); 8.05 (d; .sup.3J=9.0 Hz; 4H); 6.97 (d; .sup.3J=9.0 Hz; 4H); 6.77 (d; .sup.3J=8.9 Hz; 4H); 6.77 (s; 2H); 3.86 (s; 6H); 3.27 (s; 4H); 3.08 (s; 12H); 2.26 (s; 12H).
.sup.13C NMR {1H} (CDCl.sub.3, 150 MHz) 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
HR-MS (ESI) (Da): m/z calculated for C.sub.48H.sub.52BN.sub.7O.sub.2[M+H].sup.+770.42755; found 770.43559.

Analytical HPLC (Gradient A): Tr=4.46 min.

[0119] ##STR00015##

AG22 (30 mg, 39 μmol, 1 eq) is dissolved in 3 mL DCM (dichloromethane). Iodomethane (1.5 mL, large excess) is then added and the reaction medium is stirred for 1 h at room temperature. The solvents are then removed under reduced pressure and the crude obtained is solubilised in 10 mL of a H.sub.2O/DCM mixture (1/1). An extraction of the aqueous phase with DCM (3×5 mL) is carried out, then in a second step the organic phase obtained is extracted with water (8×10 mL). The recovered aqueous phase is evaporated, and the precipitate formed is purified by HPLC (Gradient A). The solid obtained is freeze-dried to provide pure AG24 as a blue-green solid (17.8 mg, 17.2 μmol, 44%).
.sup.1H NMR (DMSO, 500 MHz) δ (ppm): 8.24 (d; .sup.3J.sub.He-Hf=8.9 Hz; 4H); 8.10 (d; .sup.3J.sub.Hb-Hc=9.0 Hz; 4H); 7.22 (s; 2H); 7.11 (d; .sup.3J.sub.Hb-Hc=9.0 Hz; 4H); 6.86 (d; .sup.3J.sub.He-Hf=8.9 Hz; 4H); 4.00 (s; 4H); 3.87 (s; 6H); 3.07 (s; 12H); 2.78 (s; 18H).
HR-MS (ESI) (Da): m/z=calculated for C.sub.50H.sub.58BN.sub.7O.sub.2.sup.2+ [M].sup.2+ 399.73670; found 399.73869.

Analytical HPLC (Gradient A): Tr=4.49 min.

[0120] ##STR00016##

AG22 (250 mg, 0.32 mmol, 1 eq) is dissolved in 50 mL THF and 8 mL H.sub.2O. NaHCO.sub.3 (137 mg, 1.63 mmol, 5.1 eq) is added followed by 4-bromoethylbenzoic acid (144 mg, 0.67 mmol, 2.1 eq). The reaction mixture is left to stir at room temperature overnight. 90 mL Et.sub.2O and 90 mL H.sub.2O are then added and the organic and aqueous phases are separated. The aqueous phase is washed with Et.sub.2O (6×60 mL) to remove the remaining traces of 4-bromoethylbenzoic acid. The aqueous phase is then reduced to ⅓ of its initial volume by evaporation of the water in a rotary evaporator (bath at 35° C.). 10 mL of hydrochloric acid (3M) is then added. The contents of the flask are then centrifuged. The supernatant is removed and the pellet is suspended in 15 mL of Et.sub.2O and centrifuged again. The operation is repeated 3 times and the pellets obtained are then solubilized in MeOH and evaporated to dryness on a rotary evaporator (35° C. bath) to obtain pure AG38 as a black flaky powder (336 mg, 0.28 mmol, 85%).
.sup.1H NMR (DMSO, 500 MHz) δ (ppm): 8.30 (d; .sup.3J=8.9 Hz; 4H); 8.12 (d; .sup.3J=9.0 Hz; 4H); 7.95 (d; .sup.3J=8.3 Hz; 4H); 7.43 (d; .sup.3J=8.3 Hz; 4H); 7.28 (s; 2H); 7.08 (d; .sup.3J=9.0 Hz; 4H); 6.88 (d; .sup.3J=8.9 Hz; 4H); 4.24 (s; 4H); 3.96 (s; 4H); 3.74 (s; 6H); 3.08 (s; 12H); 2.76 (s; 12H).

Analytical HPLC (Gradient A): Tr=4.50 min.

[0121] ##STR00017##

[0122] AG22 (75 mg; 0.097 mmol, 1 eq) is dissolved in 60 mL dry THF in a 250 mL flask under argon. 4-bromoethylbenzoic acid (23 mg; 0.106 mmol, 1.1 eq) is then added and the mixture is left to stir under reflux overnight. After cooling, the supernatant is removed and the precipitate formed is washed with THF (3×15 mL), diethyl ether (2×15 mL) and pentane (2×15 mL). All the supernatants are combined and the solvents are removed under reduced pressure. The residue obtained is purified by silica gel chromatography column (8:2 Toluene/MeOH.fwdarw.100% MeOH) to isolate AG57 as a black flaky powder (20.7 mg, 0.021 mmol, 22%).

.sup.1H NMR (DMSO, 500 MHz) δ (ppm): 8.36 (d; .sup.3J=8.9 Hz; 4H); 8.07 (d; .sup.3J=9.0 Hz; 4H); 7.98 (d; .sup.3J=8.3 Hz; 2H); 7.24 (d; .sup.3J=8.3 Hz; 2H); 7.00 (d; .sup.3=9.0 Hz; 4H); 6.96 (s 2H); 6.82 (d; .sup.3J=8.9 Hz; 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)

Analytical HPLC (Gradient A): 4.48 min.

[0123] ##STR00018##

[0124] AG38 (250 mg, 0.209 mmol, 1 eq) is dissolved in 10 mL anhydrous DMF (dimethylformamide) in a 100 mL flask. HBTU (208 mg, 0.548 mmol, 2.6 eq) is dissolved in 10 mL anhydrous DMF before being added to the reaction mixture. 341 μL (1.959 mmol, 9.3 eq) of DIPEA (diisopropylethylamine) is then added and the mixture is left to stir at room temperature for 1 h. 109.2 mg (0.618 mmol, 2.9 eq) of 2-aminoethylmaleimide hydrochloride is dissolved in 10 mL of anhydrous DMF before being added to the reaction medium which is then stirred at room temperature overnight, evaporated to dryness and then purified by semi-preparative HPLC (25% ACN gradient.fwdarw.100% program 30 min) to isolate pure AG46 as a green solid (151 mg, 0.133 mmol, 63%).

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

.SUP.13.C NMR (DMSO, 150 MHz): 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.

[0125] HR-MS (ESI) (Da): m/z calculated for C.sub.76H.sub.78BN.sub.11O.sub.8.sup.2+ [M] .sup.2+641.80585, found 641.80752.

HPLC-analytical (Gradient A): Tr=4.56 min.

[0126] ##STR00019##

[0127] AG46 (120 mg; 79 μmol; 1 eq) is dissolved in 2 mL ACN (acetonitrile) in a 10 mL flask. 42 mg (166 μmol; 2.5 eq) of BSH is then added and the reaction is left to stir at 40° C. for 48 h. The reaction mixture is transferred to Falcons tubes and centrifuged. The supernatant is removed and the pellet is washed again with ACN (3×15 mL), DCM (1×15 mL), Et.sub.2O (2×15 mL) and pentane (2×15 mL), isolating AG49 as a blue precipitate (62 mg, 41.9 μmol, 53%).

[0128] .sup.1H NMR (DMSO, 500 MHz) 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); 7.82-7.75 (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);

[0129] 4.17 (t; .sup.3J=17.9 Hz; 4H); 3.97-3.90 (m; 4H); 3.74-3.73 (m; 6H); 3.62 (dd; .sup.3J=8.0 Hz; .sup.4J=2H); 3.59-3.55 (m; 2H); 3.54-3.48 (m 4H); 3.07 (s; 12H); 3.03 (d; .sup.3J=8.5 Hz; 1 H); 2.99 (d; .sup.3J=8.5 Hz; 1H); 2.78-2.73 (m; 13H); 1.03 (bs; 11 H).

HR-MS (ESI) (Da): m/z calculated for C.sub.76H.sub.90B.sup.10B.sub.12N.sub.11Na.sup.2+O.sub.8S.sup.2+ [M+ 2Na].sup.2+ 746.90622, found 746.90811.

HPLC-analytical (Gradient A): Tr=5.32 min.

[0130] ##STR00020##

AG35-3 (35 mg; 0.029 mmol; 1 eq) is dissolved in 3 mL DMF in a 50 mL flask. 25 mg HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium) (0.067 mmol; 2.3 eq) dissolved in 3 mL DMF is then introduced, followed by 40 μL DIPEA (0.232 mmol, 8 eq). The reaction was stirred at room temperature for 30 min under argon. A 0.5M solution of H.sub.2N—CH.sub.2—CH—(SO.sub.3.sup.−).sub.2 (TBA or tetrabutylammonium salt) (61 μL, 0.030 mmol, 1.1 eq) in 3 mL DMF is added to the reaction medium, which is then stirred at room temperature for 1 h. 14 mg (0.030 mmol, 1.1 eq) of TOTA-Boc in 3 mL DMF is then added to the solution, which is further stirred for 1 h at room temperature. The reaction mixture is evaporated to dryness, solubilised in 30 mL ACN, then 15 mL HCl (3M) is added. The reaction is stirred at 40° C. for 2 h. The reaction crude is evaporated to dryness and purified by semi-preparative HPLC (gradient 25%.fwdarw.100%, program 40 min), freeze-dried, giving pure AG58 as a green powder (7 mg, 5 μmol, 18%).
.sup.1H NMR (DMSO, 500 MHz) δ (ppm): 8.69 (t; .sup.3J=4.4 Hz; 1H); 8.50 (t; .sup.3J=5.5 Hz; 1H); 8.23 (d; .sup.3J=8.9 Hz; 4H); 8.12 (d; .sup.3J=9.0 Hz; 4H); 7.94 (d; .sup.3J=8.3 Hz; 2H); 7.90 (d; .sup.3J=8.3 Hz; 2H); 7.63 (d; .sup.3J=8.3 Hz; 2H); 7.60 (bs; 3H); 7.56 (d; .sup.3J=8.3 Hz; 2H); 7.18 (s; 2H); 6.98 (d; .sup.3J=8.9 Hz; 4H); 6.87 (d; .sup.3J=8.9 Hz; 4H); 4.60 (s; 2H); 4.28 (s; 2H); 3.95 (t; .sup.3J=4.4 Hz; 2H); 3.80 (s; 2H); 3.72 (t; .sup.3J=4.9 Hz; 1H); 3.63 (s; 6H); 3.51-3.41 (m; 12H); 3.33 (dd; .sup.3J=12.4 Hz; .sup.3J=6.6 Hz; 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).
HR-MS (ESI) (Da): m/z calculated for C.sub.76H.sub.93BN.sub.10O.sub.13S.sub.2.sup.2+ [M].sup.2+ 714.32235, found 714.32560.

HPLC-analytical (Gradient A): Tr=4.32 min.

[0131] ##STR00021##

AG57-3 (21 mg; 0.021 mmol; 1 eq) is dissolved in 2 mL DMF. 20 mg (0.052 mmol; 2.5 eq) of HBTU is dissolved in 2 mL before being 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 min, 10 mg (0.024 mmol; 1.1 eq) of TOTA-Boc were added and the solution was stirred for 1.5 hours at 30° C. The solvents are removed under reduced pressure and the resulting residue is purified by silica gel chromatography column (eluent: 100% DCM.fwdarw.50/50 DCM/MeOH), allowing the TOTA intermediate to be isolated as a black solid.

[0132] Then, in a 25 mL flask under argon, the previous intermediate is dissolved in 4 mL of dry DCM. Iodomethane (1 mL, large excess) is added and the reaction is stirred at room temperature for 1 h. The solvents are removed under reduced pressure and the resulting residue is solubilised in 20 mL ACN. 10 mL of an aqueous HCl (3M) solution is added and the reaction is left to stir at 40° C. for 2 h. The reaction medium is evaporated to dryness and purified by semi-preparative HPLC (20% ACN gradient.fwdarw.100% in 30 min), then lyophilized overnight to isolate pure AG60 as a blue solid (4 mg, 2.4 μmol, 14%).

.sup.1H NMR (ACN, 500 MHz) δ (ppm): 8.21-8.19 (m; 2H); 8.18-8.15 (m; 2H); 8.11-8.08 (m; 4H); 7.89 (t; .sup.3J=8.6 Hz; 2H); 7.86 (s; 1H); 7.52 (bs; 3H); 7.36 (d; .sup.3J=8.0 Hz; 1H); 7.32 (d; .sup.3J=8.1 Hz; 1H); 7.04-7.01 (m; 4H); 7.00 (d; .sup.3J=8.7 Hz; 2H); 6.89-6.86 (m; 4H); 4.01 (s; 1H); 3.86 (s; 1H); 3.85 (s; 1H); 3.81 (5 1 H); 3.80 (s; 3H); 3.76 (s; 3H); 3.66 (q; J=5.5 Hz; 2H); 3.60-3.58 (m; 4H); 3.57-3.55 (m; 6H); 3.53 (t; .sup.3J=5.8 Hz; 2H); 3.44 (quin .sup.3J=6.4 Hz; 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).
HR-MS (ESI) (Da): m/z calculated for C.sub.67H.sub.85BN.sub.9O.sub.6.sup.3+ [M].sup.3+ 374.22331, found 374.22292.

HPLC-analytical (Gradient A): Tr=4.18 min.

[0133] ##STR00022##

In a 10 mL flask, 2-aminoethylmaleimide hydrochloride (10.3 mg; 58 μmol; 1 eq) is dissolved in 2 mL of ACN. .sup.10B-BSH (12.3 mg; 58 μmol; 1 eq) is then added and the reaction is left to stir at room temperature for 1 h. The reaction crude is evaporated to dryness to isolate AG47 as a white solid (23 mg; 58 μmol; 1 eq).

##STR00023##

[0134] In a 100 mL flask, AG38 (200 mg; 178 μmol; 1 eq) is solubilized in 16 mL DMF. HBTU (158 mg; 406 μmol; 2.3 eq) is dissolved in 16 mL before being added to the reaction followed by DIPEA (248 μL; 800 μmol; 8 eq). The reaction was left to stir at room temperature for 30 min under argon. AG47 (72 mg; 186 μmol; 1.05 eq) is dissolved in 16 mL DMF and added to the reaction medium. The reaction is left to stir at room temperature for 1 h. TOTA-Boc (78 mg; 186 μmol; 1.05 eq) is dissolved in 16 mL DMF before being added. After 1 h the contents of the flask are transferred to a separating funnel and 100 mL DCM and 50 mL H.sub.2O are added. The two phases are separated, the aqueous phase is extracted with DCM (3×50 mL). The organic phases are combined, washed with brine (1×100 mL) and evaporated to dryness. The crude obtained is solubilized in 40 mL ACN and 15 mL HCl (3M) is added. The mixture is left to stir for 2 h at 40 ° C. and the crude is evaporated to dryness before being purified by semi-preparative HPLC (gradient A). The resulting product was run on CI.sup.− ion exchange resin (IRA 410) to isolate AG66 as a green precipitate (33 mg; 21 μmol; 12%).

.sup.1H NMR (ACN-d.sub.3/D.sub.2O, 600 MHz, 343 K) δ (ppm): 8.19 (d, .sup.3J=8.2 Hz; 4H); 8.07 (d .sup.3J=8.3 Hz, 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, .sup.3J=5.9 Hz, 2H); 1.80-1.76 (m, 2H); 1.21 (bs; 11H)
.sup.13C NMR (ACN-d.sub.3/D.sub.2O, 125 MHz, 343 K) 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; 115.9; 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.
.sup.11B NMR (ACN-d.sub.3/D.sub.2O, 193 MHz, 343 K): −9.39 (bs, aza-BODIPY); −14.59 (bs, BSH); −16.20 (s, BSH); −19.10 (bs, BSH)
.sup.10B NMR (ACN-d.sub.3/D.sub.2O, 64 MHz, 343 K): −9.08 (bs, aza-BODIPY); −16.27 (s, BSH); −17.28 (s, BSH).
HR-MS (ESI) (Da): m/z calculated for C.sub.80H.sub.107.sup.11B.sup.10B.sub.12N.sub.11O.sub.9S.sup.+ [M].sup.+ 1528.96140, found 1528.96362.

HPLC-analytical (Gradient A): Tr=4.53 min.

Example 2: Small Animal Fluorescence Imaging Procedure

[0135] Five-week-old female NMRI Nu/Nu mice are caged in groups of 5, with food and water at libitum, and day/night lighting from 12:00 to 12:00, according to current ethical recommendations.

[0136] At the age of 6 weeks, U87MG tumor cells (3 million/100 μL) were injected subcutaneously into the right lower limb: the animals were sedated by gas anaesthesia during this injection. The animals are placed back in the cage for the duration of the tumor development, that means approximately 3 weeks.

[0137] When the tumor reaches approximately 100 mm.sup.3 or more, the solution containing the AG22 compound is injected intravenously (25 to 50 μg/mouse), in the tail vein of the animal placed under gas anaesthesia. The animal is then awakened during the external imaging phases. For each imaging session, the animal is placed under gas anaesthesia, the imaging is performed and then the animal is returned to its cage.

[0138] Imaging is performed with an excitation at 830 nm and then the fluorescence signal is collected using a long pass filter between 1064 and 1700 nm.

[0139] We observe (FIG. 1) the mouse with a U87MG tumour in the right hind leg before and 24 hours after injection of the AG22 compound. The upper planes are recorded with NIR imaging. Fluo800 corresponds to excitation at 780 nm and collection between 830 and 900 nm. The lower planes are excited at 830 nm (SWIR800) and the emission is collected using a 1064 nm long pass filter.

[0140] The experiment is repeated with the compound AG66.

[0141] We observe the mouse with a U87MG tumour on the right hind leg, respectively from left to right: before, at 5 h and at 48 h after injection of the AG66 compound.

[0142] The compounds according to the invention can thus be observed in the wavelength range from 1000 to 1700 nm: these wavelengths facilitate in-depth detection with better resolution.

[0143] The compounds according to the invention also allow the delivery of small compounds, such as boron complexes.

LIST OF REFERENCES

[0144] [1] Bruns et al, Nat Biomed Eng. 2017; doi:10.1038/s41551-017-0056/Carr et al, Proc Natl Acad Sci USA. 2018 115 (17):4465-70/Thimsen et al, Nanophotonics 2017; 6 (5): 1043-1054).

[0145] [2] PAC, 1996, 68, 2193 (Basic terminology of stereochemistry (IUPAC Recommendations 1996)) page 2205.

[0146] [3] Gravier et al, Mol Pharm. 2014; doi: 10.1021/mp500329z., page 3134.

[0147] [4] Hirsjärvi et al, Nanomedicine. 2013; doi: 10.1016/j.nano.2012.08.005, page 376.

[0148] [5] Garcia et al, Biomater Sci. 2018; doi: 10.1039/c8bm00396c, page 1755.

[0149] [6] Reagan-Shaw et al, FASEB J, 2007, Vol 22 page 660, doi: 10.1096/fj.07-9574LSF