MODIFIED CYANINE DYES AND CONJUGATES THEREOF

Abstract

The present invention relates to the field of optical imaging More particularly, it relates to compounds of the cyanine family characterized by improved physico-chemical and biological properties and to their conjugates with biological ligands thereof. The invention also relates to the use of these compounds as optical diagnostic agents in imaging or therapy of solid tumors, to the methods for their preparation and to the compositions comprising them.

Claims

1. A compound of formula (I) ##STR00032## wherein R1 and R2 are independently a —CO—NH—Y group, wherein Y is selected from a linear or branched alkyl, cycloalkyl and heterocyclyl, substituted by at least two hydroxyl groups; R3 is linear or branched alkyl substituted by a group selected from —NH.sub.2, —SO.sub.3H, —COOH and —CONH.sub.2; R4 is linear or branched bivalent alkyl; R5 is selected from —COO— and —CONH—; S is a spacer; T is a targeting moiety; n is an integer equal to 1, 2 or 3; and m is an integer equal to 0 or 1. or a pharmaceutically acceptable salt thereof.

2. The compound of formula (I) according to claim 1, wherein n is 3, R3 is alkyl substituted by —SO.sub.3H, and R1 and R2 are independently selected from the group consisting of ##STR00033##

3. The compound of formula (I) according to claim 1, which is represented by formula (Ia) ##STR00034## wherein R4, R5, S, m and T are as defined in claim 1.

4. The compound of formula (I) according to claim 1, wherein S is selected from —NH(CH.sub.2).sub.pCOO—, —NH(CH.sub.2CH.sub.2O).sub.pCH.sub.2CH.sub.2COO— and —NH(CH.sub.2CH.sub.2O).sub.pCH.sub.2CH.sub.2NH—, wherein p is an integer comprised between 0 and 20.

5. The compound of formula (I) according to claim 1, wherein T is targeting moiety selected from the group consisting of a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or fragment thereof and an aptamer.

6. The compound of formula (I) according to claim 1, wherein T is a moiety interacting with an integrin receptor.

7. A method of using the compound of formula (I) according to claim 1 comprising administering an effective amount of the compound to a patient, wherein the compound is used as fluorescence contrast agent for the detection and demarcation of a tumor margin in guided surgery of the individual patient.

8. The method according to claim 7 wherein the detection and demarcation of the tumor margin is carried out under NIR radiation.

9. The method according to claim 8, wherein said tumor is a tumor selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, esophageal cancer, skin cancer, gastric cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, renal cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer.

10. A pharmaceutical diagnostic composition comprising a compound of formula (I) as defined in claim 1 and at least one pharmaceutically acceptable carrier or excipient.

11. Diagnostic kit comprising a compound of formula (I) as defined in claim 1 together with additional adjuvants thereof for implementing the optical imaging.

12. A compound of formula (I) according to claim 1 selected from ##STR00035##

13. An intermediate compound of formula (II) ##STR00036## wherein R1, R2, R3, R4 and n are as defined in claim 1 and R5′ is selected from —COOH, —CONH.sub.2, —COO-Pg and —CONH-Pg, wherein Pg is a protecting group, or a pharmaceutically acceptable salt thereof.

14. An intermediate of formula (II) according to claim 13, wherein n is 3, R3 is alkyl substituted by —SO.sub.3H, and R1 and R2 are independently selected from the group consisting of ##STR00037##

15. An intermediate of formula (II) according to claim 13, which is represented by formula (IIa) ##STR00038## wherein R4 and R5′ are as defined in claim 13.

Description

DESCRIPTION OF THE DRAWINGS

[0104] FIG. 1 represents the fluorescence signal decay in the healthy muscle tissue after intravenous administration of three doses (0.3 nmol/mouse, circles; 1 nmol/mouse, squares; 3 nmol/mouse, triangles) of compound 1 in Balb/c nu/nu mice (n=5/group).

[0105] FIG. 2 represents the fluorescence decay (left y-axis) in the tumor (black circles) and muscle tissue (white circles) after iv administration of 1 nmol of compound 1 in Balb/c nu/nu mice implanted with 10 million of U87MG cells, and tumor-to-background ratio (squares, right y-axis) over time.

[0106] FIG. 3 shows the tumor-to-background ratio (TBR) of compound 1 administered at the dose of 3 nmol/mouse in Balb/c nu/nu mice implanted with 2.5 million of Detroit-562 cells (n=5).

[0107] FIG. 4 represents the fluorescence decay (left y-axis) in the tumor (black circles) and muscle tissue (white circles) after administration of 1 nmol of compound 1 in Athymic nude mice implanted with 5 million of HT-29 cells (n=5), and tumor-to-background ratio (squares, right y-axis) over time.

[0108] All data are expressed as mean±standard deviation.

EXPERIMENTAL PART

[0109] The invention and its particular embodiments described in the following part are only exemplary and not to be regarded as a limitation of the present invention: they show how the present invention can be carried out and are meant to be illustrative without limiting the scope of the invention.

Materials and Equipment

[0110] All chemicals and solvents used for the reactions were reagent grade. Analytical grade solvents were used for chromatographic purifications. Most of the reagents, unless reported otherwise, are commercial products, including the targeting moieties (e.g. Panitumumab (Vectibix, Amgen; CAS Registry Number of the active substance: 339177-26-3); Cetuximab (Erbitux, Merck; CAS Registry Number of the active substance: 205923-56-4)).

[0111] All synthesized compounds were purified by reverse phase chromatography (RP-HPLC) and characterized by mass spectroscopy using a LC/MS instrument equipped with a UV-VIS detector and an ESI source.

[0112] Analysis were performed with a Waters Atlantis dC18 5 μm, 4.6×150 mm column using a gradient of phase A CH.sub.3COONH.sub.4 10 mM and phase B acetonitrile. Measured mass/charge ratios are listed for each compound. A dual-beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer) was used to determine the absorbance (Abs) of the compounds of the invention. Emission/excitation (Em/Ex) spectra and absolute fluorescence quantum yield (Φ) measurements were carried out on a spectrofluorometer (FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018 integrating sphere accessory. The measurements were performed using an excitation wavelength at maximum absorbance of different dyes, and the sample was excited with a 450W Xenon Light Source. Detection was performed by photomultiplier tubes (PMT-NIR) cooled detector or by TBX-04 detector. Dye solutions were carefully prepared to have an absorbance lower than 0.1 (optical densities) to minimize re-absorption phenomena.

[0113] In vivo imaging experiments were performed using the IVIS Spectrum In Vivo Imaging System (Perkin Elmer Inc.). The system is equipped with with 10 narrow band excitation filters (30 nm bandwidth) and 18 narrow band emission filters (20 nm bandwidth) spanning 430-850 nm.

LIST OF ABBREVIATIONS

[0114] DCC N,N′-dicyclohexylcarbodiimide [0115] DIPEA N,N-Diisopropylethylamine [0116] DMF Dimethylformamide [0117] DMSO Dimethyl sulfoxide [0118] DSC N,N′-Disuccinimidyl carbonate [0119] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0120] HBTU O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate [0121] HPLC High performance liquid chromatography [0122] PBS Phosphate buffered saline [0123] NHS N-hydroxysuccinimide [0124] NMM N-methylmorpholine [0125] RT Room temperature [0126] PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [0127] TEA Triethylamine [0128] TBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [0129] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate [0130] μL Microliter [0131] μM Micromolar [0132] t.sub.R Retention time (HPLC) [0133] cRGDfK Cyclo-(Arg-Gly-Asp-D-Phe-Lys) [0134] aza-cRGD -NH.sub.2 Aza-cyclo-(Arg-Gly-Asp)

[0135] The abbreviations for individual amino acids residues are conventional: for example, Asp or D is aspartic acid, Gly or G is glycine, Arg or R is arginine. The amino acids herein referred to should be understood to be of the L-isomer configuration unless otherwise noted.

EXAMPLE 1: SYNTHESIS OF COMPOUND 4

[0136] ##STR00021## ##STR00022##

Preparation of Intermediate (VIa), Step a

[0137] In a round bottom flask, 5-carboxy-2,3,3-trimethylindolenine (1.50 g, 7.38 mmol), 2,4-butanesultone (1.11 g, 8.15 mmol) and butyronitrile (2 g) were added. The mixture was heated at 120° C. for 2 days. The solid was then treated with dry acetone, filtered under vacuum and washed twice with acetone. The pink solid was dried under vacuum and used with no further purification (2.40 g, 96%). HPLC purity at 270 nm 94%, MS: [M+H]+339.2.

Preparation of Intermediate (VIIa), Step b

[0138] In a round bottom flask intermediate III (1.5 g, 7.38 mmol), ethyl 6-bromohexanoate (1.97 g, 8.85 mmol) and sulfolane (6.8 g) were added. The suspension was heated at 90° C. for 3 days. Then, ethyl acetate (25 mL) was added, the purple dispersion was stirred at RT for 15 minutes and filtered under vacuum. The solid was washed with ethyl acetate three times, then it was redispersed in fresh ethyl acetate (15 mL), stirred at RT for 15 minutes, filtered under vacuum and washed with ethyl acetate. This operation was repeated for other two times. Finally, the pink/violet solid, containing both the desired product and the starting material, was dried under vacuum. It was then dispersed in acetonitrile (4 mL), the dense dispersion was stirred at RT for 15 minutes and filtered. The solid was washed with a few volume of cold acetonitrile. The mother liquor contained prevalently the desired product, whereas the solid contained the starting material. The acetonitrile was evaporated under vacuum and the solid was dispersed again in acetonitrile (4 mL), stirred for 15 minutes, filtered and the solid was washed with cold acetonitrile. The solution was concentrated under vacuum obtaining a pink/violet solid with a HPLC purity of 92% at 270 nm (1.25 g as bromine salt, 40%). MS: [M+H]+346.5.

Preparation of Intermediate (IXa), Step c

[0139] In a round bottom flask glutaconaldehyde dianyl hydrochloride (0.48 g, 1.4 mmol), acetic anhydride (47 mL) and acetic acid (13 mL) were added. The orange-brown solution was heated at 60° C. for 3 h. The reaction mixture was then cooled down to RT and the solution of intermediate (IVa) (0.60 g, 1.4 mmol) in ethanol (10 mL) was dropped at RT in about 20 minutes. The red solution was heated at 35° C. for 2 h, then the temperature was increased at 50° C. and a solution of intermediate (III) (0.48 g, 1.4 mmol) and sodium acetate (0.11 g, 1.3 mmol) in ethanol (19 mL) was added. Immediately after, pyridine (2.0 mL) was added. The reaction mixture was heated at 60° C. for 1.5 h, then solvents were evaporated under vacuum and the dark oil was precipitated in cold water (100 mL). The dark green solid was filtered and washed with cold water. Then, it was dissolved in the minimum amount of ethanol and this solution was dropped under stirring into ethyl acetate (400 mL). The solid was filtered, washed with ethyl acetate, dissolved in dichloromethane and purified on silica gel with a slow dichloromethane-methanol gradient. Fractions containing the pure product were combined, evaporated under vacuum and dried obtaining a green-blue solid (350 mg, 33% yield). HPLC purity at 756 nm 94%, MS: [M+H]+747.3.

Synthesis of Compound 4, Steps d and e

[0140] In a dried round bottom flask, dry DIPEA (115 μL, 0.56 mmol) was added to a solution of intermediate (IXa) (150 mg, 70% purity, 0.14 mmol) in DMF (2 mL). After 30 minutes of stirring under nitrogen flow, a solution of TBTU (309 mg, 0.67 mmol) in DMF (2 mL) was added. After 1 h, a suspension of D-glucamine (87 mg, 0.336 mmol) in DMF (2 mL) was added. The mixture was stirred for 2 h at RT, then concentrated and purified on a pre-packed C18 silica column with a water-acetonitrile gradient. Fractions containing the pure product were collected, concentrated and freeze-dried, obtaining a green-blue powder (95.8 mg, 64% yield). HPLC purity at 756 nm 97%, MS: [M+Na]+1095.3.

[0141] The intermediate (Xa) thus obtained (95 mg, 0.089 mmol) was solubilized in water (10 mL, pH 6.35), then 2N NaOH was added until pH 11.14. The solution was stirred at room temperature maintaining pH 11 through addition of 0.1N NaOH. After 40 h the reaction was completed, the mixture was neutralized with IN HCl and purified on a pre-packed C18 silica column with a water-acetonitrile gradient. Fractions containing the pure product were collected, concentrated and freeze-dried, obtaining a green-blue powder (81 mg, 86% yield). HPLC purity at 756 nm 98%, MS: [M+H]+1045.3.

EXAMPLE 2: SYNTHESIS OF COMPOUND 5

[0142] ##STR00023##

Step f

[0143] In a dried round bottom flask, N-methylmorpholine (10 μL, 0.091 mmol) was added to a solution of Compound 4 prepared as in example 1 (10 mg, 0.0096 mmol) in dry DMF (1 mL). After 30 minutes of stirring under nitrogen flow, a solution of HBTU (13 mg, 0.03 mmol) in DMF (1 mL) was added. After 1 h, a suspension of NH.sub.4Cl (10 mg, 0.19 mmol) in DMF (1 mL) was added. The mixture was kept under stirring for 2 days, then it was dried under vacuum and purified on a pre-packed C18 silica column with a water-acetonitrile gradient. Fractions containing pure product were combined, concentrated and freeze-dried, obtaining a blue solid (2,56 mg, 20% yield). HPLC purity at 756 nm 97.5%, MS: [M+H].sup.+ 1043.1.

EXAMPLE 3: SYNTHESIS OF COMPOUND 7

[0144] ##STR00024## ##STR00025##

Preparation of Intermediate Xb, Step d

[0145] Intermediate IXb was prepared according to the procedure of Example 1, but using the reagent malonaldehyde dianilide hydrochloride for step c). Intermediate IXb (58 mg, 0.081 mmol) was suspended in dry DMF (10 mL). Trometamol (23.5 mg, 0.194 mmol), DIPEA (61 μL, 0.352 mmol) and HATU (73.77 mg, 0.194 mmol) were added. The reaction was stirred under inert atmosphere at RT for 2 hours. The solvent was distilled under reduced pressure and the crude was purified by flash chromatography on pre-packed C18 silica column with a water/methanol gradient. Fractions containing pure product were combined and distilled under vacuum and freeze-dried, giving a bright blue solid (60 mg, 80% yield). HPLC purity at 655 nm: 99.6%. MS: [M+H].sup.+ 927.3.

Synthesis of Compound 7, Step e

[0146] Intermediate Xb (110 mg. 0.106 mmol) was dissolved in ethanol (2 mL) and water (18 mL) was added. The solution was heated at 40° C. and pH was kept 11 with 0.1N NaOH, until the hydrolysis was completed (ca. 6 hours). PH was brought to 7 with 0.1N HCl and the crude was purified by flash chromatography on pre-packed C18 silica column with a water/methanol gradient. Fractions containing pure product were combined, distilled under vacuum and freeze-dried, giving a bright blue solid (18.7 mg, 19% yield). HPLC purity at 655 nm: 95.7%. MS: [M+H].sup.+899.3.

EXAMPLE 4: SYNTHESIS OF COMPOUND 8

[0147] ##STR00026## ##STR00027##

[0148] Intermediates VIc and VIIc were prepared according to the procedure above described for intermediates VIa and VIIa. Analogously to Example 1, they were treated with the corresponding reagent N,N-diphenyl-formamidine, to obtain the trimethine intermediate IXc.

Preparation of Intermediate Xc, Step d

[0149] Intermediate IXc (62 mg, 0.089 mmol) was suspended in dry DMF (15 mL). D-Glucamine (33.9 mg, 0.187 mmol), DIPEA (62 μL, 0.356 mmol) and HATU (71.10 mg, 0.187 mmol) were added and the reaction was stirred under inert atmosphere at RT for 4 hours. The solvent was distilled under reduced pressure and the crude was purified by flash chromatography on pre-packed C18 silica column with a water/acetonitrile gradient. Fractions containing pure product were combined, distilled under vacuum and freeze-dried, giving a bright pink solid (82.4 mg, 91% yield). HPLC purity at 560 nm: 99.8%. MS: [M+H].sup.+ 1021.3.

Synthesis of Compound 8, Step e

[0150] Intermediate Xc (81.4 mg. 0.08 mmol) was dissolved in ethanol (2 mL) and water (18 mL) was added. The solution was stirred at RT and pH was kept 11 with 0.1N NaOH, until the hydrolysis was completed (ca. 46 hours).Then, pH was adjusted to 7 with 0.1 N HCl and the crude purified by flash chromatography on pre-packed C18 silica column with a water/acetonitrile gradient. Fractions containing pure product were combined, distilled under vacuum and freeze-dried, giving a bright pink solid (79 mg, 99.4% yield). HPLC purity at 560 nm: 100%. MS: [M+H].sup.+ 993.3.

EXAMPLE 5: SYNTHESIS OF COMPOUND 1

[0151] ##STR00028##

[0152] Compound 1 was syntesized by conjugation of the dye corresponding to Compound 4 and the cyclic pentapeptide c-RGDfK, wherein the conjugation has been carried out by two alternative procedures A or B. Compound 4 has been prepared as described in Example 1. The peptide c-RGDfK representing the targeting moiety is a commercial reagent.

Procedure A—Conjugation Step Via Direct Coupling

[0153] In a dried round bottom flask, N-methylmorpholine (10 μL, 0.09 mmol) was added to a solution of Compound 4, prepared as described in Example 1, (10 mg, 0.009 mmol) in dry DMF (1 mL). After 30 minutes of stirring under nitrogen flow, a solution of TBTU (6 mg, 0.02 mmol) in DMF (1 mL) was added. After 1 hour, a solution of c(RGD)fK (6 mg, 0.009 mmol) in DMF (1 mL) is added. The mixture was stirred at RT for 20 hours, then other TBTU (4 mg) and N-methylmorpholine (2 μL) were added, and after 2 hours c(RGD)fK (2 mg) was added. The mixture was stirred for additional 6 h, then it was concentrated and the crude was purified on analytical HPLC on C18 silica column with 0.1% ammonium acetate-acetonitrile gradient. Fractions containing the pure product were collected, concentrated and freeze-dried three times, to give a blue solid (4.4 mg, 29% yield). HPLC purity at 756 nm 99%, MS: [M/2].sup.+ 841.0.

Procedure B—Conjugation Step Via Previous Activation with NHS Ester

[0154] Compound 4, prepared as described in Example 1, (4 mg, 0.0038 mmol) was suspended in dry DMF (3 mL) under nitrogen flow. N-methylmorpholine (0.8 μL 0.0076 mmol) and TSTU (2.3 mg, 0.0076 mmol) were added and the solution was stirred at RT for 20 h. The desired product was precipitated by addition of cold diethyl ether, centrifuged and washed twice with diethyl ether.

[0155] The solid was dissolved in a solution of c(RGD)fK (3.0 mg, 0.0042 mmol) in borate buffer at pH 9 (2 mL). The solution was stirred overnight, then the pH was adjusted to 6 with IN HCl and the crude was purified on analytical HPLC on C18 silica column with 0.1% ammonium acetate/acetonitrile gradient. Fractions containing the pure product were collected, concentrated and freeze-dried three times, to give a blue solid (5.2 mg, 83% yield). HPLC purity at 756 nm 99%, MS: [M/2].sup.+ 841.0.

EXAMPLE 6: SYNTHESIS OF COMPOUND 3

[0156] ##STR00029##

[0157] In a dried round bottom flask, N-methylmorpholine (10 μL 0.09 mmol) was added to a solution of Compound 4 prepared as described in Example 1 (10 mg, 0.009 mmol) in dry DMF (1 mL). After 15 minutes of stirring under nitrogen flow, a solution of HBTU (3.6 mg, 0.01 mmol) in DMF (1 mL) was added. After 30 minutes, a solution of aza-cRGD-NH.sub.2 (5.7 mg, 0.01 mmol) in DMF (1 mL) was added. The mixture was stirred for 2 days, then it was concentrated under vacuum and purified on analytic HPLC on C18 silica column with 0.1% ammonium acetate/acetonitrile gradient. Fractions containing the pure product were collected, concentrated and freeze-dried three times, to give a blue powder (3.57 mg, 21% yield). HPLC purity at 756 nm 99%, MS: [M/2].sup.+ 780.8.

EXAMPLE 7: SYNTHESIS OF COMPOUND 9

[0158] The monoclonal antibody EGFR ligand Panitumumab (6 mg) was diluted up to 5 mg/mL in PBS and pH was adjusted by adding 120 μL of 1.0 M potassium phosphate pH 9. Compound 4-NHS ester (prepared as described in Example 5, Procedure B) was dissolved in DMSO at a concentration of 10 mg/ml; then the dye and the antibody were immediately mixed at a molar ratio of 2.5:1 and kept at room temperature in the dark for 3 h. After 3 h, the conjugation reaction mixture was layered onto phosphate buffered saline (PBS)-equilibrated Zeba Spin columns and centrifuged at 1500g for 2 min to separate the conjugate from the free dye. After filtration through a 0.22-μm polyethersulfone (PES) membrane, the conjugated Panitumumab solution in PBS at pH 7.4 was analyzed by SE-HPLC, RP-HPLC, UV/VIS spectrophotometry to determine concentration and purity. The molar conjugation ratio (dyes molecules coupled per antibody) was about 1.3.

EXAMPLE 8: SYNTHESIS OF COMPOUND 10

[0159] The monoclonal antibody EGFR ligand Cetuximab (5 mg/mL) was dialyzed against PBS in a 10K Da MWCO membrane. 10 mg of dialyzed MAb (4.52 mg/mL, 68.8 nmol) were added to 221 μL of 1 M phosphate buffer pH 9. Compound 4-NHS ester (prepared as described in Example 5, Procedure B) was dissolved in DMSO at a concentration of 6.86 mM. 43.3 μL of dye were added to the Cetuximab solution (molar ratio 4.3:1) and kept at room temperature in the dark for 3 h. After this time, the conjugation reaction mixture was layered onto phosphate buffered saline (PBS)-equilibrated Zeba Spin column and centrifuged at 1000 g for 2 min to separate the conjugate from the free dye. After filtration through a 0.22 μm polyethersulfone (PES) membrane, the conjugated Cetuximab solution in PBS at pH 7.4 was analyzed by SE-HPLC, RP-HPLC, UV/VIS spectrophotometry to determine concentration and purity. The molar conjugation ratio (dyes molecules coupled per antibody) was about 1.1.

EXAMPLE 9: SYNTHESIS OF COMPOUND 11

[0160] ##STR00030##

[0161] The small molecule CAIX ligand 4a, described in Wichert et al., Nat Chem 2015, 7, 241-249, was prepared according to the procedure therein disclosed and conjugated to compound 4. 11 mg of compound 4 (10.0 μmol) were dissolved in 1 mL of DMF, then 5.5 mg of PyBOP (10.0 μmol) and 7 μL of DIPEA (40.0 μmol) were added under continuous stirring. After 20 minutes, 9 mg of small molecule 4a (15.0 μmol) were dissolved in 1 mL of DMF and added to the reaction mixture which was stirred for additional 30 minutes at room temperature. Purification was performed by preparative HPLC with a yield of 60%. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI (+) using a Waters Atlantis dC18 column (μm, 4.6×150 mm). HPLC purity at 758 nm: 98%; MS: [M/2].sup.+ 823.3.

EXAMPLE 10: SYNTHESIS OF COMPOUND 12

[0162] ##STR00031##

[0163] The small molecule CAIX ligand 8a, described in Wichert et al., Nat Chem 2015, 7, 241-249, was prepared according to the procedure therein disclosed and conjugated to Compound 4. 15 mg of Compound 4 (14.0 μmol) were dissolved in 1 mL of DMF, then 7.3 mg of PyBOP (14.0 μmol) and 10 μL of DIPEA (57.0 μmol) were added under continuous stirring. After 20 minutes, 22 mg of the small molecule 8a (21.0 μmol) were dissolved in 1 mL of DMF and added to the reaction mixture which was stirred for additional 30 minutes at RT. The purification was performed by preparative HPLC with a yield of 34%. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI (+) using a Waters Atlantis dC18 column (μm, 4.6×150 mm).

[0164] HPLC purity at 758 nm: 95%; MS: [M/2].sup.+ 1051.8.

EXAMPLE 11: STABILITY OF INTERMEDIATE (Xa)

[0165] The stability of intermediate (Xa), prepared as described in Example 1 step d), has been evaluated in different conditions in order to perform the hydrolysis of the ethyl ester in the optimal conditions. In fact, it is known that such type of cyanines are not considerably stable in acidic and basic media. Contrary to other analogues bearing poly-sulfonyl groups, such as for instance compound DA364 described in WO2018/189136, being more stable in acidic conditions, intermediate (Xa) unexpectedly showed a remarkable stability when performing the hydrolysis in strong conditions, such as at pH 11 and 45° C. for several hours, without degradation of the R1 and R2 groups.

[0166] Surprisingly, good results were also obtained performing the hydrolysis with enzymatic porcine and rabbit liver extracts, working at 1 mg/mL, pH 8 and 38° C. in PBS buffer. Complete conversion was obtained after 20-24 hours only, without any degradation.

EXAMPLE 12: OPTICAL PROPERTIES

[0167] The compounds of the invention have been characterized in terms of their optical properties in vitro in aqueous medium (i.e., water/PBS pH 7.4) and in a clinical chemistry control serum (Seronorm, Sero SA), mimicking the chemical composition and optical properties of human serum. All dye or dye-conjugate solutions were freshly prepared.

[0168] In particular, the excitation and emission maxima and the absolute fluorescence quantum yield (Φ) of representative compounds of formula (I) and corresponding dyes of formula (II) are shown in Table II in comparison with the commercial bi-sulfonated heptamethine dye Indocyanine Green (ICG, Sigma), bi-sulfonated pentamethine dye sulfo-Cy5 (Lumiprobe) and bi-sulfonated trimethine dye sulfo-Cy3 (Lumiprobe).

TABLE-US-00003 TABLE 11 Excitation/Emission maxima and absolute fluorescence quantum yields of compounds of formula (I) (compounds 1 and 3) and (II) (compounds 4, 6, 7, 8) Max Ex/Em Φ Φ (nm, PBS pH 7.4) (PBS pH 7.4) (Seronorm) Cy7 ICG 780/810 1.5% 7.8% (Reference) Compound 1 757/786 9.8% 13.0% Compound 3 757/785 10.6% 11.7% Compound 4 755/785 8.9% 13.4% Cy5 Sulfo-Cy5 645/665 27.0% 35.6% (Reference) Compound 6 656/675 35.6% 35.9% Compound 7 655/675 35.1% 35.1% Cy3 Sulfo-Cy3 548/565 6.8% n/a (Reference) Compound 8 557/573 14.5% 20.0% n/a, not available

[0169] The compounds of the invention are characterized by absorption maxima comprised in the range from about 550 nm to 760 nm. The fluorescence quantum yields obtained for the compounds of the invention was remarkably higher than the reference compound, with values from about 1.5 to 7 times greater than the corresponding reference compounds with the same length of polymethine chain Remarkably, the compounds of the invention displayed greater quantum yield in the acqueous buffer PBS than the reference compounds with similar solubility properties.

EXAMPLE 13: AFFINITY TO HUMAN ALBUMIN

[0170] An analysis of the binding affinity of the compounds of the invention to human albumin was carried out and the results compared with the reference Indocyanine Green (ICG).

[0171] Binding affinity to human serum albumin (HSA; Sigma Aldrich, A9511) was measured using two methods, according to the level of binding affinity of the compounds.

[0172] The first method, optimal for compounds which strongly interact with HSA, is based on the analysis of the absorbance spectrum peak shift after the incubation of the dye in solutions containing HSA. Briefly, the samples were incubated at a fixed concentration (1 μM) with HSA dilutions (1×10.sup.−6-4×10.sup.−4M), in phosphate buffer for 5 min in the spectrophotometer at 25° C. before measurements. The measure was performed at the maximum absorbance wavelength of the shifted peak.

[0173] The second method, optimal for compound with low affinity for HSA, is based on measuring the variation of the absorbance of solutions containing the dye and various concentrations of HSA after ultrafiltration. Briefly, each compound was incubated at a fixed concentration (2 μM) with HSA dilutions (1×10.sup.−6-4×10.sup.−4 M), in phosphate buffer. The samples were centrifuged (10,000 g for 30 min at 25° C.) in a Microcon device (10 kDa MWCO, Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane, Millipore) and the absorbance measurements of the filtrates were obtained with the spectrophotometer at the maximum absorbance wavelength of the fluorophore.

[0174] For both methods, the affinity constant (K.sub.A, M.sup.−1) was calculated by fitting the raw data with the following formula:

[00001]$\frac{\Delta A}{b}=\frac{\mathrm{\Delta .Math.}.Math.K_{RL}\left[L\right].Math.R_t}{K_{RL}\left[L\right]+1}$

wherein
ΔA/b=Absorbance measured (b=1 cm)
K.sub.RL=K.sub.A calculated by regression analysis (curve fitting)
Δε.Math.Rt calculated by regression analysis (curve fitting)
[L]=Albumin concentration

[0175] In the first method, ΔA/b corresponds to the absorbance measured for each sample, whereas in the second method ΔA/b is obtained subtracting the absorbance of the control sample (dye without HSA) to the absorbance of each other sample.

[0176] Both methods have demonstrated to provide comparable results, as shown by parallel experiments conducted on the commercial cyanine dye IRDye 800CW carboxylate (LI-COR Inc., Lincoln, USA) using the first method (HSA K.sub.A=215,000 M.sup.−1) and the second method (HSA K.sub.A=216,000 M.sup.−1). However, the measurement of the affinity constant is more precise when the suitable method is used as a function of the affinity level of the compound.

[0177] The results of the binding affinity measured for representative compounds of the invention with one of the two methods are reported in Table III and compared with the results obtained for the clinically available ICG dye and for ICG-RGD and DA364 as reference compounds.

TABLE-US-00004 TABLE III Binding affinity to human serum albumin (HSA) Compound HSA affinity (K.sub.A, M.sup.−1) ICG (Ref. compound) 347,000 ICG-RGD (Ref. compound) 219,000 DA364 (Ref. compound) 28,670 Compound 1 6,290 Compound 3 4,200 Compound 4 5,110 Compound 6 <1,000 Compound 7 3,460 Compound 8 2,070

[0178] As shown in Table III, the dyes and dyes-conjugates of the invention display a remarkably low binding affinity to human albumin compared to the available ICG and to many other known cyanine compounds, with affinity constants of one or two orders of magnitude lower. This advantageous feature relates both to dyes of formula (II) (e.g. Compound 4) and to the corresponding dye when conjugated with a targeting moiety (e.g. Compounds 1 and 3), suggesting that the conjugation of the dyes with a targeting moiety does not affect the affinity to human albumin.

[0179] Rather, the RGD conjugates of formula (I), such as representative Compounds 1 and 3, have unexpectedly displayed much lower affinity for human albumin than other dyes known in the art when conjugated with the RGD targeting moiety, such as ICG-RGD (HSA K.sub.A=219,000 M.sup.−1, Capozza et al., 2018) or DA364 (HSA K.sub.A=28,670 M.sup.−1, WO2016/097317), as reported in Table III. Moreover, the reference compound DA364, when analysed exactly in the same experimental conditions of the present compounds, displayed a higher HSA affinity, with affinity constant (K.sub.A) of 110,000 M.sup.−1.

EXAMPLE 14: RECEPTOR BINDING AFFINITY

[0180] The binding affinity of the conjugates of formula (I) to a specific receptor was determined to assess whether the targeting efficacy of the molecular vector is preserved after the labeling with the dyes of the invention.

Peptide/Peptidomimetic Molecule Conjugates

[0181] As example of peptide/peptidomimetic conjugates, the receptor affinity of representative integrin-binding conjugates was evaluated through calculation of their IC.sub.so (half maximal inhibitory concentration), using an enzyme-linked immunosorbent assay (ELISA), as previously reported (Kapp et al., Sci. Rep. 2017, 7, 39805). Briefly, 96-well ELISA plates were coated overnight at 4° C. with the extracellular matrix (ECM) protein Vitronectin in carbonate buffer (15 mM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6). Each well was then washed with PBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4, 0.01% Tween20, pH 7.4) and blocked for 1 h at RT with TS-B-buffer (Tris-saline/BSA buffer; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2, pH 7.5, 1% BSA). In the meantime, a dilution series of the compound and internal standard was prepared in an extra plate. After washing the assay plate three times with PBS-T, 50 μL of the dilution series were transferred to each well. 50 μL of a solution of human recombinant integrin α.sub.vβ.sub.3 (R&D Systems, 1 μg/mL) in TS-B-buffer was transferred to the wells and incubated for 1 h. The plate was washed three times with PBS-T buffer, and then primary antibody anti-α.sub.vβ.sub.3 was added to the plate. After incubation and washing three times with PBS-T, the secondary anti-IgG peroxidase-labeled antibody was added to the plate and incubated for 1 h. After washing the plate three times with PBS-T, the plate was developed by quick addition of 3,3′,5,5′-tetrametylbenzidine (TMB) and incubated for 5 min in the dark. The reaction was stopped with 3 M H.sub.2SO.sub.4, and the absorbance was measured at 450 nm with a plate reader (Victor3, Perkin Elmer).

[0182] The IC.sub.50 of the representative compounds 1 and 3 was tested in duplicate, and the resulting inhibition curves were analyzed using GraphPad Prism version 4.0 for Windows (GraphPad Software). The inflection point defines the IC.sub.50 value. All experiments were conducted using c(RGDfK) as internal standard.

[0183] The tested molecular probes, either coupled to c(RGDfK) (i.e. Compound 1) or to c(RGD)aza (i.e. Compound 3), showed comparable affinity to the human α.sub.vβ.sub.3 receptor, and similar affinity to the unconjugated reference peptidomimetic c(RGDfK), as reported in Table IV.

TABLE-US-00005 TABLE IV Binding affinity to the human α.sub.νβ.sub.3 integrin receptor of compounds 1 and 3 compared to the peptidomimetic c(RGDfK). Compound Receptor affinity (IC.sub.50, nM) c(RGDfK) 2.69 ± 0.70 Compound 1 2.96 ± 0.49 Compound 3 2.64 ± 0.35

Small Molecule Conjugates

[0184] As example of small molecules conjugates, the inhibitory activity of the representative conjugates towards the CAIX enzyme was evaluated using the commercially available colorimetric kit K473 (BioVision). The protocol was performed according to the manufacturer's instructions, and the inbibitory potency (half maximal inhibitory concentration, IC.sub.50) of the conjugates was compared to that of the unconjugated acetazolamide. Briefly, the kit utilizes the esterase activity of an active carbonic anhydrase enzyme on an ester substrate which releases a chromogenic product. The released product is quantified using an absorbance microplate reader (absorbance: 405 nm). In the presence of a carbonic anydrase specific inhibitor, as for acetazolamide and the conjugates of formula (I), the enzyme loses its activity which results in decrease of absorbance. The small molecules conjugates of formula (I) showed high inhibitory potency towards the enzyme carbonyc anhydrase, with IC.sub.50 values in the low nanomolar range comparable to that of the unconjugated acetaxolamide (acetazolamide: 3.7 nM; Compound 11: 6.5 nM; Compound 12: 1.2 nM).

Protein Molecule Conjugates

[0185] As example of protein molecule conjugates, the receptor affinity of representative EGFR-binding conjugates was evaluated using the AlphaLISA kit AL366H (Perkin Elmer). In this AlphaLISA assay, a biotinylated EGF binds to the Streptavidin-coated Alpha Donor beads, while EGFR-Fc is captured by Anti-Human IgG Fc-specific AlphaLISA Acceptor beads. When EGF is bound to EGFR, Donor beads and Acceptor beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfers in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm. Experiments were conducted by comparing the affinity of the conjugate to the relative unlabeled molecular vector. The unlabeled antibody Trastuzumab, which does not bind the EGFR receptor, was used as negative control. Diluition series of the samples (Compound 9 and Compound 10, and the relative unlabeled molecular vectors Panitumumab and Cetuximab, respectively) were incubated for 30 minutes with the EGFR Acceptor beads, followed by incubation for 30 minutes with the biotynilated EGF. Thus, the Streptavidin-conjugated Donor beads were added to the plate and incubated for 30 minutes. The absorbance was measured at 615 nm using a plate reader (EnSight, Perkin Elmer). No specific binding was observed for the antibody Trastuzumab. Differently, specific receptor binding was observed for Compound 9 (IC.sub.50, 0.2 nM) and Compound 10 (IC.sub.50, 0.4 nM), comparable to the unlabeled parent antibodies Panitumumab (IC.sub.50, 0.5 nM) and Cetuximab (IC.sub.50, 0.4 nM), respectively. Unexpectedly, it was found that large molecules like Cetuximab and Panitumumab maintained the native antigen binding properties even after conjugation with the dyes of the invention.

[0186] Overall, these results demonstrate that the conjugation of either small molecules, peptide/peptidomimetic or protein moieties to a dye compound of formula (II) does not impair the binding affinity of the final compound (I) to the target.

EXAMPLE 15: CELL UPTAKE

[0187] The human melanoma cell line WM-266-4 (ATCC, CRL-1676) was used as in vitro model to assess the cell uptake of representative integrin-binding Compounds 1 and 3, based on the high expression of the integrin receptors, particularly α.sub.vβ.sub.3, on the membrane of these cells (Capasso et al., PlosOne 2014).

[0188] Adherent cells at about 70% confluence were incubated with the compounds 1 or 3 (1 μM) for 2h at 37° C. (5% CO.sub.2) in presence of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. After two washing steps with PBS, cells were detached using 0.1 mM EDTA in PBS, centrifuged and suspended in buffer (PBS, 0.5% BSA, 0.1% NaN.sub.3) for flow cytometry experiments. Fluorescence Activated Cell Sorting (FACS) was used to detect the fluorescence signal within the cells, as measure of cell uptake. Samples were excited with an Argon laser and the emission detected using a 670 nm longpass filter. Values of fluorescence intensity were obtained from the histogram statistic produced by the instrument software.

[0189] To assess the specificity of receptor-mediated cell uptake, experiments were performed by incubating the cells with the molecular probes in presence of high concentration (100 μM) of the unlabeled molecular vector c(RGDfK) as competitor. The residual internalization was calculated by considering the value of fluorescence intensity in absence of the competitor as 100%.

[0190] Furthermore, to assess the effect of biological fluids on the cell uptake, parallel experiments were performed incubating the cells with a compounds of the invention in presence of human serum from male AB plasma (Sigma Aldrich, H4522) or human albumin (Sigma Aldrich, A9511). The residual internalization was calculated by considering the value of fluorescence intensity in absence of the serum as 100%. Such uptake assessment also represents an indication of the percentage of compound which is sequestered by the plasma proteins, in particular albumin, when it diffuses through the vascular compartment before reaching the tissue of interest and the particular targeted receptor.

[0191] In Table V the cell uptake performance of representative Compounds 1 and 3 of the invention is shown.

[0192] The present compounds displayed high cell uptake either in presence of human serum or in the presence of 4% HSA (Compound 1, residual uptake 70%), while they displayed a low residual uptake in the presence of the vector c(RGDfK) as competitor (Compound 1, residual uptake 10%; Compound 3, residual uptake 15%). Thus, for the present compounds it is observed that the internalization in the cells is receptor-mediated and is only slightly affected by the binding to human serum proteins, in particular albumin (about 10-20% of residual uptake), confirming the medium-to-low binding affinity to human albumin of the present compounds (K.sub.A=about 1-6×10.sup.3 M.sup.−1, as shown in example 10).

[0193] Moreover, Table V reports a comparison of the residual cell uptake in presence of human serum for Compounds 1 and 3 with the reference compounds DA364, ICG-RGD (Capozza et al., Photoacoustic 2018, 11, 36-45) and ICG-c(RGDfK), prepared with the same method for ICG-RGD. These results show that the compounds of the present invention have been surprisingly found endowed with a higher efficacy in cell internalization with respect to similar compounds known in the art.

TABLE-US-00006 TABLE V Uptake of the integrin-binding fluorescent probes into WM- 266-4 human melanoma cells in presence of human serum. Residual cell uptake in Compound presence of human serum Cy5.5-cRGD (DA364) 15% ICG-cRGD 12% ICG-c(RGDfK)  8% Compound 1 80% Compound 3 75%

[0194] Notably, neither the interaction of the present compounds with the receptor on the cell surface, nor the internalization of the receptor-probe complex within the cell were impaired by the structure of the conjugated dyes, and particularly by the presence in position R1 and R2 of the compounds of moieties strongly hydrophilic and with high steric hindrance. Thus, the presence of the hydrophilic moieties on the conjugated dyes provide highly efficient and specific receptor binding and probe internalization even in presence of plasma proteins and albumin, which would sequester a conjugate lacking the hydrophilic moieties and negatively affect the binding efficiency.

EXAMPLE 16: BIODISTRIBUTION AND ELIMINATION

[0195] Biodistribution and elimination of the fluorescent agents of the invention was evaluated by optical imaging after intravenous administration in male Balb/c nu/nu mice, 6-10 weeks of age (Charles River Laboratories). Briefly, mice housed 4 per cage and fed with VRF1 (P) sterile diet (Special Diets Services Ltd) up to the end of the acclimation period (5 days). Then, AIN-76a rodent diet irradiated (Research Diets), a special diet that reduces auto-fluorescence, was used up to the end of the experiments. Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer).

[0196] In vivo imaging was performed under gas anesthesia (Sevofluorane 6-8% in oxygen). Animals were intravenously injected with the compounds and imaged longitudinally at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h post-administration. Thus, animals were euthanized by anesthesia overdose, and the main tissues and organs of interest were excised for ex vivo optical imaging. Regions of interest (ROIs) were drawn on the tissues of interest for each fluorescence image at every time point to evaluate signal intensity (expressed as Average adiant Efficiency). The ratio between the fluorescence signal in the muscle (background tissue) and excretory organs (kidney, liver) was then calculated to assess the contrast. The tissue half-life was calculated by fitting the fluorescence signal decay in the muscle with a bi-exponential decay equation model using GraphPad Prism Software (version 5 for Windows).

[0197] The tissue kinetics of Compound 1 is shown in FIG. 1, where the fluorescence signal decay in the muscle tissue was plotted vs. time to assess the washout of the product at different doses. Remarkably, Compound 1 showed a very favorable elimination kinetics with fast tissue washout and improved body elimination and, particularly it displayed very short (<1 hour) tissue half-lives at all doses (0.3, 1, 3 nmol/mouse) tested. The compound quickly distributed in the body and accumulated in the bladder, suggesting renal elimination. Moreover, it displayed fast body elimination and low retention, advantageously allowing for early imaging after administration.

[0198] Table VI shows the ratios between the fluorescence signal detected by optical imaging in the excised excretory organs (liver and kidney) vs. the muscle, 24 hours after the intravenous (iv) administration of 1 nmol/mouse of Compound 1 or 3.

[0199] Ex vivo imaging of excised organs and tissues revealed very low retention of Compounds 1 and 3 in the excretory organs kidney and liver, associated with the faster body elimination and reduced non-specific accumulation.

TABLE-US-00007 TABLE VI Ratio between the fluorescence signal decay in excretory organs and in the muscle 24 hours after iv administration. Kidney to Liver to Compound muscle ratio muscle ratio Compound 1 3.18 ± 0.32 2.92 ± 0.21 Compound 3 2.90 ± 0.55 2.50 ± 0.32

EXAMPLE 17: TUMOR UPTAKE

[0200] The tumor uptake of the compounds of the present invention has been assessed by optical imaging after intravenous administration in mice.

[0201] Tumor uptake experiments were carried out in an animal model of human glioblastoma (subcutanous) overexpressing the integrin receptors, particularly α.sub.vβ.sub.3. Briefly, human glioblastoma U87MG cells (ATCC, HTB-14) were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin. Male Balb/c nu/nu mice, 4-6 weeks of age (Charles River Laboratories), underwent subcutaneous implantation (right flank) of about 10 million cells suspended in 0.1 mL of EMEM. Mice were housed 4 per cage with food and water ad libitum. Animals were fed with VRF1 (P) sterile diet (Special Diets Services Ltd) up to the end of the acclimation period (5 days). Then, AIN-76a rodent diet irradiated (Research Diets), a special diet that reduces auto-fluorescence, was used up to the end of the experiments. Tumor growth was monitored by longitudinal assessments using a caliper up to the target size of 300-600 mm.sup.3 (3-4 weeks after cell implantation). Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer).

[0202] In vivo imaging was performed under gas anesthesia (Sevofluorane 6-8% in oxygen). Animals were intravenously injected (lateral tail vein) with the compounds, and imaged longitudinally at 30 mM, 1 h, 2 h, 3 h, 4 h, and 24 h post-administration. Regions of interest (ROIs) were drawn on the tissues of interest for each fluorescence image at every time point to evaluate signal intensity (expressed as Average Radiant Efficiency). The ratio between the fluorescence signal in the tumor and in the muscle (background tissue) was then calculated to assess the contrast.

[0203] The fluorescence decay values of representative Compound 1 in the tumor and muscle tissue and the tumor-to-backgroud ratio are displayed in FIG. 2. These results show a remarkably high tumor uptake for the representative Compound 1 already at early time-points (1-2 hours after administration), providing a very high tumor-to-background contrast (TBR ˜2-2.5) and low retention in the healthy tissue, suggesting tumor-specific accumulation.

[0204] In fact, the present compounds are quickly eliminated from the body, particularly from the healthy tissues (muscle), while a considerable target-mediated accumulation can be observed for the conjugated dyes of formula (I) in the targeted tissues.

[0205] Parallel experiments were performed in an animal model of human head and neck cancer (orthotopic), using Detroit-562 cells, overexpressing in particular integrin receptor α.sub.vβ.sub.6. Briefly, the human pharyngeal carcinoma cells Detroit-562 (ATCC, CCL-138) were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin. Male Balb/c nu/nu mice, 4-6 weeks of age (Charles River Laboratories), underwent orthotopic implantation in the anterior portion of the tongue of about 2.5 million cells suspended in 0.03 mL di EMEM. Mice were housed 4 per cage with food and water ad libitum. Animals were fed with VRF1 (P) sterile diet (Special Diets Services Ltd) up to the end of the acclimation period (5 days). Then, AIN-76a rodent diet irradiated (Research Diets), a special diet that reduces auto-fluorescence, was used up to the end of the experiments. Tumor growth was monitored by longitudinal assessments using a caliper up to the target size of 10-20 mm.sup.3 (7-10 days after cell implantation).

[0206] Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer). Animals were intravenously injected (lateral tail vein) with 3 nmol/mouse, at 24 hours post-administration were euthanized by anesthesia overdose, and the tongues were excised for ex vivo optical imaging. Regions of interest (ROIs) were drawn on the anterior portion of the tongue (site of tumor cell implantation) and on the posterior region (healthy tissue) to derive the tumor-to-background ratio. One healthy animal (no tumor implantation) was administered with 3 nmol/mouse of the Compound 1 and imaged as described above to be used as negative control.

[0207] Ex vivo imaging performed 24 hours after the administration of the compound 1 revealed a bright region in the tongue site of implantation of the tumor cells. Differently, the healthy region in the back of the tongue showed low signal, similar to that detected in healthy mice, suggesting a low retention in healthy tissue. The administration of the compound of the invention reveals the location of the tumor with high tumor-to-background contrast (as shown in FIG. 3).

[0208] Parallel experiments were performed in an animal model of human colorectal cancer (subcutaneous), using HT-29 cells, expressing low levels of integrin receptors. Briefly, the human colorectal adenocarcinoma cells HT-29 (ATCC, HTB-38) were cultured in McCoy's 5A medium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. Male Athymic nude mice, 4-6 weeks of age (Envigo), underwent subcutaneous implantation (right flank) of about 5 million cells suspended in 0.1 mL of serum-free medium. Mice were housed 4 per cage with food and water ad libitum. Animals were fed with VRF1 (P) sterile diet (Special Diets Services Ltd) up to the end of the acclimation period (5 days). Then, AIN-76a rodent diet irradiated (Research Diets), a special diet that reduces auto-fluorescence, was used up to the end of the experiments. Tumor growth was monitored by longitudinal assessments using a caliper up to the target size of 300-600 mm.sup.3 (3-4 weeks after cell implantation). Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer).

[0209] In vivo imaging was performed under gas anesthesia (Sevofluorane 6-8% in oxygen). Animals were intravenously injected (lateral tail vein) with the compounds of interest, and imaged longitudinally at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h post-administration. Regions of interest (ROIs) were drawn on the tissues of interest for each fluorescence image at every time point to evaluate signal intensity (expressed as Average Radiant Efficiency). The ratio between the fluorescence signal in the tumor and in the muscle (background tissue) was then calculated to assess the contrast.

[0210] The fluorescence decay values in the tumor and muscle tissue and the tumor-to-backgroud ratio are displayed in FIG. 4 for Compound 1.

[0211] These results show high tumor uptake for the representative Compound 1 already at early time-points and fast clearance from the healthy tissues, resulting in a moderate tumor-to-background ratio (TBR ˜1.2-1.5) sufficient to clearly delineate the tumor tissue from the healthy background.

[0212] The target specificity of the representative Compound 1 was compared to that of the reference compound DA364 in an animal model of human cancer which overexpress the human integrin receptor αVβ3, as described above. Both compounds were administered at the dose of 1 nmol/mouse together either with (blocking group) or without (control group) an excess of unlabeled cRGD peptidomimetic (200 nmol/mouse). After 24 hours from injection, the mice were euthanized, the tumor tissues excised and imaged using the IVIS Spectrum fluorescence system (Perkin Elmer). The blocking protocol prompted a 10% reduction in fluorescence signal intensity (the imaging surrogate of uptake) in the tumor tissue of animals administered with DA364. Differently, the blocking protocol prompted a reduction of 75% in fluorescence in the tumors of animals that received Compound 1, denoting a higher in vivo receptor specificity for the compound of the invention (see Table VII).

TABLE-US-00008 TABLE VII Determination of the in vivo receptor specificity by blocking experiment. Reduction of tumor fluorescence Compound after receptor blocking DA364 (Reference) 10% (n = 5/group) Compound 1 75% (n = 5/group)

REFERENCES

[0213] 1. Onda N. et al., Int J Cancer, 2016, 139, 673-682 [0214] 2. Stummer W. et al., Neurosurgery, 2015, 77(5), 663-673 [0215] 3. Licha et al., Photochemistry and Photobiology, 2000, 72(3), 392-398 [0216] 4. Tummers Q. et al., PlosOne 2015 [0217] 5. Achilefu S. et al., J Med Chem, 2002, 45, 2003-2015 [0218] 6. Bunschoten A. et al., Bioconjugate Chem., 2016, 27, 1253-1258 [0219] 7. U.S. Pat. No. 6,913,743 B2 [0220] 8. WO2018/189136 [0221] 9. WO2016/097317 [0222] 10. Capozza et al., Photoacoustics, 2018, 11, 36-45 [0223] 11. U.S. Pat. no. 6,329,531 B1 [0224] 12. Ebert et al., J. Biomed. Optics, 2011, 16(6), 066003 [0225] 13. WO2005/123768 [0226] 14. WO2012/088007 [0227] 15. T. W. Green and P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y. 2007, 4.sup.th Ed., Ch. 5 [0228] 16. Kapp et al., Sci Rep, 2017, 7, 3905 [0229] 17. Wichert et al., Nat Chem 2015, 7, 241-249 [0230] 18. Li et al., FASEB J 2005, 19, 1978-85 [0231] 19. Williams et al., Chem Biol Drug Des 2018, 91, 605-619 [0232] 20 . Mujumdar R. B. et al., Bioconjugate Chem. 1993, 4(2), 105-111 [0233] 21. Yamana et al, Chem. Pharm. Bull., 1972, 20(5), 881-891