Biocompatible modular tetrazine platform

Abstract

A modular tetrazine platform, can be used as diagnostic, theragnostic agent and/or medicinal product. An inverse electron demand Diels-Alder reaction can be used for synthesizing bifunctionalized tetrazines, which can be obtained from monofunctionalised tetrazines.

Claims

1. A compound of formula (I) ##STR00072## or one of the tautomers thereof, wherein: is a double bond or single bond; ring A is selected from the group consisting of bicyclo[6.1.0]nonane, cyclooctane, bicyclo[6.1.0]nonene, cyclooctene, difluorocyclooctene, hydroxycyclooctene, methylcyclopropane, norbornene, 5,6-dihydrodibenzo[a,e][8]annulene, and 5,6-dihydrodibenzo[b,f]azocine group; X.sup.1 is S, NH, or O; L.sup.1, L.sup.2 and L.sup.3 are each independently a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized; R.sup.1, R.sup.2 and R.sup.3 are each independently a detectable group, a bioactive group, a cytotoxic agent, an affinity group, or a solubilising group; said detectable group being selected from the group consisting of a fluorophore, chromophore, probe for nuclear imaging, and MRI probe, wherein the fluorophore is selected from among cyanine derivatives; Alexa fluor 647; a coumarin selected from hydroxycoumarin, aminocoumarin, and methoxy coumarin; a rhodamine selected from X-rhodamine, and rhodamine B; a fluorescein or BODIPY, and wherein the chromophore is selected from among phenolphthalein, gentian violet or Congo Red; said bioactive group being selected from the group consisting of an antibody, peptide, peptidomimetic, protein, folic acid, an aptamer, a nanoparticle or liposome; said affinity group being selected from the group consisting of biotin, avidin, streptavidin and hexa-histidine peptide; said solubilising group being selected from the group consisting of linear or branched poly(ethylene glycol) chains, linear or branched poly(glutamic acid) chains and cholesterol; provided that at least one of R.sup.1 and R.sup.2 is a detectable group, and at least one of R.sup.2 and R.sup.3 is a bioactive group.

2. The compound of formula (I) according to claim 1, wherein: the cytotoxic agent is selected from the group consisting of monomethyl auristatin E, maytansinoid DM1, Duocarmycin, Calicheamicin, alpha-amanitin, a group carrying a radiometal, a silica nanoparticle and a gold nanoparticle.

3. A pharmaceutical composition comprising a compound of formula (I) according to claim 1 and a pharmaceutically acceptable vehicle.

4. A method for synthesising a compound of formula (I) according to claim 1, comprising the contacting of a bifunctionalized tetrazine of formula (II) ##STR00073## wherein: X.sup.1 is S, NH or O; L.sup.1 and L.sup.2 are each independently a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized; R.sup.1 and R.sup.2 are each independently a detectable group, a bioactive group, a cytotoxic agent, an affinity group or a solubilising group; provided that at least one of R.sup.1 and R.sup.2 is a detectable group; with an alkyne or alkene of formula (III): ##STR00074## wherein: represents a triple bond or double bond L.sup.3 is a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized; provided that at least one of R.sup.2 and R.sup.3 is a bioactive group.

5. The method according to claim 4 further comprising a forming the bifunctionalized tetrazine of formula (II) by nucleophilic substitution on a tetrazine of formula (IV): ##STR00075## wherein: Y.sup.2 is a halogen or leaving group selected from the group consisting of mesylate, tosylate, triflate and 3,5-dimethyl-1H-pyrazol-1yl groups; X.sup.1 is S, NH or O; L.sup.1 is a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized; in the presence of a thiol of formula (V)
R.sup.2-L.sup.2-SH  (V) wherein: L.sup.2 is a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized provided that at least one of R.sup.1 and R.sup.2 is a detectable group.

6. The method according to claim 5, further comprising a step to form the tetrazine of formula (IV) by nucleophilic monosubstitution of a tetrazine of formula (VI): ##STR00076## where Y.sup.1 and Y.sup.2 are each independently a halogen or leaving group selected from the group consisting of mesylate, tosylate, triflate and 3,5-dimethyl-1H-pyrazol-1yl groups; in the presence of a nucleophile of formula (VII)
R.sup.1-L.sup.1-X.sup.1-H  (VII) where: X.sup.1 is S, NH, or O; and L.sup.1 is a single bond or spacer selected from the group consisting of alkyl, alkoxy, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl groups, wherein the alkyl groups optionally contain in the alkyl chain one or more groups selected from the group consisting of —O—, —NH—, —S—, —C(O)—, —C(O)NH— and —NHC(O)—, wherein the heteroaryl is an aromatic ring having 5 to 15 carbon atoms or cyclic systems containing 1 to 3 rings that are fused together or covalently bound; wherein one or more carbon atoms in one or more of these rings are replaced by oxygen, nitrogen and/or sulfur atoms; the nitrogen and sulfur atoms optionally being oxidized and the nitrogen atoms optionally being quaternized.

7. The compound of claim 1, wherein the detectable group is a fluorophore being a cyanine derivative selected from the group consisting of: Cyanine3, Cyanine5, Cyanine5.5, Cyanine7, and Sulfonated Cyanines.

8. The compound of claim 1, wherein L.sup.1, L.sup.2 and L.sup.3 are each independently selected from the group consisting of —CH.sub.2CH.sub.2NH—, —NHCH.sub.2CH.sub.2NH—, —CH.sub.2OCONH—CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2—, —CH.sub.2OCO—, —COCH.sub.2CH.sub.2NH—, —CH.sub.2CH.sub.2NHCO—CH.sub.2CH.sub.2— and —CH.sub.2CH.sub.2NHCO-p-Ph-.

9. The method according to claim 5, wherein Y.sup.2 is chlorine.

10. The method according to claim 5, wherein L.sup.1 and L.sup.2 are each independently selected from the group consisting of —CH.sub.2CH.sub.2NH—, —NHCH.sub.2CH.sub.2NH—, —CH.sub.2OCONH—CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2—, —CH.sub.2OCO—, —COCH.sub.2CH.sub.2NH—, —CH.sub.2CH.sub.2NHCO—CH.sub.2CH.sub.2— and —CH.sub.2CH.sub.2NHCO-p-Ph-.

11. The method according to claim 6, wherein Y.sup.1 and Y.sup.2 are chlorine.

12. The method according to claim 6, wherein L.sup.1 is selected from the group consisting of —CH.sub.2CH.sub.2NH—, —NHCH.sub.2CH.sub.2NH—, —CH.sub.2OCONH—CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2—, —CH.sub.2OCO—, —COCH.sub.2CH.sub.2NH—, —CH.sub.2CH.sub.2NHCO—CH.sub.2CH.sub.2— and —CH.sub.2CH.sub.2NHCO-p-Ph-.

13. The compound of claim 1, having the following formula (I-a): ##STR00077## or one of the tautomers thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a scheme for synthesis of the trifunctionalised platforms (1) of the invention involving the mono- and bi-functionalised tetrazine intermediates (IV) and (II).

(2) FIG. 2 gives the multimodal SPECT image of a mouse xenografted with BT-474 cells (breast tumour) treated with compound I-1 radiolabelled with indium 111 (FIG. 2A), and with compound I-1 radiolabelled with indium co-injected with non-labelled excess trastuzumab (FIG. 2B).

(3) FIG. 3 gives a histogram showing the mean dose percentage injected per gram of organ, determined by gamma count in different organs of mice treated with compound I-1 radiolabelled with indium (batch A—light grey) or of mice treated with compound I-1 radiolabelled with indium with excess trastuzumab (batch B—dark grey).

(4) FIG. 4 is a photograph with (b) and without (a) fluorescence, of a mouse treated with compound I-1 radiolabelled with indium 111.

(5) FIG. 5 is a photograph with ex vivo fluorescence of the tumour and of various organs of a mouse treated with compound I-1 radiolabelled with indium 111.

(6) FIG. 6 gives a histogram showing the mean biodistribution of compound I-1 radiolabelled with indium 111 in the tumour and in various organs, for the mice in batch A (light grey) or mice in batch B (dark grey).

(7) FIG. 7 is a photograph with in vivo fluorescence of a mouse treated with compound I-3 after 1 h, 4 h or 24 h post-injection.

(8) FIG. 8 is a photograph with ex vivo fluorescence of the tumour and of various organs of a mouse treated with compound I-3 after 24 h post-injection.

(9) FIG. 9 gives a histogram showing the mean biodistribution of compound 1-3 in the tumour and in various organs, for the mice in batch C.

EXAMPLES

(10) The present invention will be better understood on reading the following examples giving a nonlimiting illustration of the invention.

Abbreviations

(11) ACN: acetonitrile

(12) BCN: bicylononyne

(13) BODIPY: boron-dipyromethene

(14) DCM: dichloromethane

(15) DIPEA: diisopropylethylamine

(16) DMF: dimethylformamide

(17) DMSO: dimethylsulfoxide

(18) DODT: 3,6-dioxa-1,8-octanedithiol

(19) DOTAGA: 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid

(20) Fab′: Fab′ fragment of pertuzumab (anti-HER2)

(21) HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide

(22) hexafluorophosphate

(23) HPLC: high performance liquid chromatography

(24) MeOH: methanol

(25) NODAGA: 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid

(26) PBS: phosphate buffered saline

(27) rpm: revolutions per minute

(28) TCO: trans-cyclooctene

(29) TFA: trifluoroacetic acid

(30) TIS: triisopropylsilane

(31) TSTU: N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate

(32) Material

(33) Unless otherwise stated, the chemical reagents and solvents were obtained from usual suppliers of chemical products (Sigma-Aldrich, Alfa-Aesar, Fisher Scientic, BioSolve) and were used without prior purification. The polyazamacrocycles used were obtained from Chematech (Quetigny, France). The optical probes of the Cyanine family were courteously supplied by Prof. Anthony Romieu (Université de Bourgogne) and synthesised following the protocols described in the literature.

(34) Purifications with semi-preparative HPLC were performed on an UltiMate 3000 Dionex system (Thermo Scientific) equipped with UV-visible detector on a reverse phase column (C18).

(35) Purifications with flash chromatography were performed on a puriFlash®430 system (Interchim) using pre-packed Interchim columns in normal phase or reverse phase (size of particles 15 μm or 25 μm).

(36) LC-MS analyses were performed on an UltiMate 3000 liquid chromatography system equipped with DAD detector and coupled to a MSQ Plus mass detector (Thermo Scientific), in ESI mode. Chromatographies were carried out on a Kinetex™ C.sub.18 column, 2.6 μm, 100 Å, 50×2.1 mm (Phenomenex) with HPLC-quality eluents (Eluent A: H.sub.2O 0.1% formic acid (FA); eluent B: acetonitrile 0.1% FA) with 5% to 100% gradient of B in 5 min, 100% plateau for 1.5 min. The purity of the compounds was determined from LC-MS chromatograms at 214 nm wavelength.

(37) High resolution mass spectra (HRMS ESI) of the end compounds were performed on a PACSMUB platform (DIJON, France), with LTQ Orbitrap XL mass spectrometer, equipped with an ESI source (Thermo Scientific).

(38) .sup.1H NMR spectra were obtained on a Bruker Avance III NanoBay NMR spectrometer. The spectra were obtained at ambient temperature. The following abbreviations were used to describe multiplicity of signals: s=singlet, d=doublet, t=triplet, m=multiplet.

Part I. Chemical Synthesis

1. Amine Intermediates of Formula (VII)

1.1. Cyanine 5.0-NH.SUB.2

2-((1E,3E,5E)-5-(1-(6-((2-ammonioethyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium

(39) ##STR00045##

(40) Cyanine 5.0 (20.0 mg, 32.7 μmol), TSTU (11.8 mg, 39.3 μmol, 1.2 eq) and DIPEA (6.9 μL, 39.3 μmol, 1.2 eq) were dissolved in 700 μL of anhydrous DMF. The reaction medium was left under agitation at ambient temperature for 1 h, after which ethylenediamine (21.8 μL, 327 μmol, 10.0 eq) and DIPEA (57.1 μL, 327 μmol, 10.0 eq) were added. After 25 min, the solvent was removed under reduced pressure. Purification of the reaction product was conducted using reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA).

(41) Cyanine 5.0-NH.sub.2 was obtained in the form of a blue powder (11.1 mg, 52% TFA salt). .sup.1H NMR (500 MHz, CDCl.sub.3) ∂: 1.36 (t, 3H; CH.sub.3), 1.41 (m, 2H; CH.sub.2), 1.64 (s, 6H; CH.sub.3), 1.65 (s, 6H; CH.sub.3), 1.67 (m, 2H; CH2), 1.74 (m, 2H; CH.sub.2), 2.24 (t, 2H; CH.sub.2), 3.13 (m, 2H; CH.sub.2), 3.51 (m, 2H; CH.sub.2), 3.94 (t, 2H; CH.sub.2), 4.01 (q, 2H; CH.sub.2), 6.13 (d, 1H; CH), 6.16 (d, .sup.3J=13.5 Hz, 1H; CH), 6.58 (t, 2H; CH), 7.07 (m, 2H; CH.sub.Ar), 7.19 (m, 2H; CH.sub.Ar), 7.32 (m, 4H; CH.sub.Ar), 7.81 (dd, 2H; CH), 8.26 (t, 1H; CONH), 8.38 (m, 3H; NH.sub.3.sup.+); MS: C.sub.35H.sub.47N.sub.4.sup.+ ([M].sup.+) m/z calculated: 539.4; found: 539.4.

2. Thiol Intermediates of Formula (V) and (VII)

2.1. Rhodamine B-SH

N-(6-(diethylamino)-9-(2-((2-mercaptoethyl)carbamoyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium

(42) ##STR00046##

(43) Rhodamine B (200 mg, 418 μmol), HATU (191 mg, 502 μmol, 1.2 eq) and DIPEA (87 μL, 502 μmol, 1.2 eq) were dissolved in 2 mL of anhydrous DMF. The solution was left under agitation at ambient temperature for 2 h, and 2-aminoethanethiol hydrochloride (95 mg, 836 μmol, 2.0 eq) and DIPEA (139 μL, 836 μmol, 2.0 eq) were afterwards added to the reaction medium. After 2 h, the DMF was removed under reduced pressure. Purification of the reaction product was obtained with flash chromatography on silica (eluents: DCM/MeOH). Rhodamine B-SH was obtained in the form of a purple powder (179.3 mg, 85%). .sup.1H NMR (300 MHz, CDCl.sub.3): Equilibrium between the non-cyclic and cyclic forms of rhodamine; MS: C.sub.30H.sub.36N.sub.3O.sub.2S.sup.+ ([M].sup.+) m/z calculated: 502.3; found: 502.2.

2.2. BODIPY-SH

2,8-diethyl-5,5-difluoro-10-(4-((2-mercaptoethyl)carbamoyl)phenyl)-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide

(44) ##STR00047##

(45) BODIPY-COOH (100 mg, 235 μmol) and carbonyldiimidazole (46 mg, 282 μmol, 1.2 eq) were dissolved in 1 mL of anhydrous DMF. The solution was left under agitation at ambient temperature for 1 h, after which 2-aminoethanethiol hydrochloride (54 mg, 470 μmol, 2.0 eq) was added. After 20 min, the DMF was removed under reduced pressure. Purification of the reaction product was performed with flash chromatography on silica (eluents: DCM/MeOH). BODIPY-SH was obtained in the form of a red powder (65.0 mg, 57%). .sup.1H NMR (300 MHz, CDCl.sub.3): ∂=0.95 (t, 6H; CH.sub.3), 1.23 (s, 6H; CH.sub.3), 1.43 (t, 1H; SH), 2.27 (q, 4H; CH.sub.2), 2.51 (s, 6H; CH.sub.3), 2.81 (dt, 2H; CH.sub.2), 3.66 (dt, 2H; CH.sub.2), 6.70 (t, 1H; NH), 7.37 (d, 2H; CH.sub.Ar), 8.18 (d, 2H; CH.sub.Ar) MS: C.sub.26H.sub.32BFN.sub.3OS.sup.+ ([M−F].sup.+) m/z calculated: 464.2; found: 464.1.

2.3. Disulfonated Cyanine 3.0-SH

1-Ethyl-2-((1E,3E)-3-(1-(6-((2-mercaptoethyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate

(46) ##STR00048##

(47) Disulfonated cyanine 3.0 (24.5 mg, 38.8 μmol), TSTU (16.1 mg, 53.5 μmol, 1.4 eq) and DIPEA (10.0 μL, 57.3 μmol, 1.5 eq) were dissolved in 1 mL of anhydrous DMF. The solution was left under agitation at ambient temperature for 15 min, and 2-aminoethanethiol hydrochloride (11.0 mg, 96.8 μmol, 2.5 eq) and DIPEA (13.6 μL, 77.7 μmol, 2.0 eq) were added afterwards. After 25 min at ambient temperature the reaction was complete. Dithiothreitol (15.5 mg, 100 μmol, 2.6 eq) and water (400 μL) were added and the solution left under agitation for 3 h at ambient temperature. The solvents were afterwards removed under reduced pressure. Purification of the reaction product was performed with reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). Disulfonated cyanine 3.0-SH was obtained in the form of a pink powder (18.2 mg, 68%). .sup.1H NMR (300 MHz, D.sub.2O): ∂=1.27 (m, 4H; CH.sub.2), 1.56 (m, 2H; CH.sub.2), 1.62 (s, 12H; CH.sub.3), 1.73 (m, 3H; CH.sub.3), 2.17 (m, 2H; CH.sub.2), 2.43 (t, .sup.3J=7.0 Hz, 2H; CH.sub.2), 3.18 (t, 3J=7.0 Hz, 2H; CH.sub.2), 4.02 (m, 4H, CH.sub.2), 6.32 (t, .sup.3J=11.5 Hz, 2H; CH), 7.27 (m, 2H, CH.sub.Ar), 7.80 (m, 2H, CH.sub.Ar), 7.85 (s, 2H, CH.sub.Ar), 8.37 (t, .sup.3J=11.5 Hz, 1H, CH); MS: C.sub.33H.sub.44N.sub.37S.sub.3.sup.+ ([M].sup.+) m/z calculated: 690.2; found: 690.3.

2.4. Cyanine 5.0-SH

1-Ethyl-2-((1E,3E,5E)-5-(1-(6-((2-mercaptoethyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium

(48) ##STR00049##

(49) Cyanine 5.0 (20.0 mg, 32.7 μmol), TSTU (11.8 mg, 39.3 μmol, 1.2 eq) and DIPEA (6.9 μL, 39.3 μmol, 1.2 eq) were dissolved in 800 μL of anhydrous DMF. The solution was left under agitation at ambient temperature for 10 min, after which 2-aminoethanethiol hydrochloride (7.4 mg, 65.4 μmol, 2.0 eq) and DIPEA (11.4 μL, 65.4 μmol, 2.0 eq) were added. After 1 h at ambient temperature, dithiothreitol (13.1 mg, 85.0 μmol, 2.6 eq) an water (400 μL) were added. The solution was left under agitation for 7 h at ambient temperature, and the DMF afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). Cyanine 5.0-SH was obtained in the form of a blue powder (22.2 mg, purity: 88%, product oxidized to disulfide bridge: 9%)). .sup.1H NMR (500 MHz, CD.sub.3CN): ∂=1.34 (t, .sup.3J=7.0 Hz, 3H; CH.sub.3), 1.42 (m, 2H; CH.sub.2), 1.60 (m, 1H; SH), 1.63 (m, 2H; CH.sub.2), 1.66 (s, 12H; CH.sub.3), 1.78 (m, 2H; CH.sub.2), 2.13 (t, .sup.3J=6.9 Hz, 2H; CH.sub.2), 2.54 (m, 2H, CH.sub.2), 3.27 (m, 2H, CH.sub.2), 4.00 (t, .sup.3J=7.5 Hz, 2H; CH.sub.2), 4.06 (q, .sup.3J=7.0 Hz, 2H, CH.sub.2), 6.22 (d, .sup.3J=13.7 Hz, 2H; CH), 6.55 (t, .sup.3J=12.5 Hz, 1H; CH), 6.74 (s broad, 1H, NH), 7.25 (m, 4H, CH.sub.Ar), 7.40 (m, 2H, CH.sub.Ar), 7.48 (m, 2H, CH.sub.Ar), 8.08 (dd, .sup.3J=13.7, 12.5 Hz, 2H, CH); MS: C.sub.35H.sub.46N.sub.3OS.sup.+ ([M].sup.+) m/z calculated: 556.3; found: 556.5.

2.5. Disulfonated Cyanine 5.0-SH

1-Ethyl-2-((1E,3E,5E)-5-(1-(6-((2-mercaptoethyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate

(50) ##STR00050##

(51) Disulfonated cyanine 5.0 (16.0 mg, 24.4 μmol), TSTU (8.8 mg, 29.2 μmol, 1.2 eq) and DIPEA (15.3 μL, 87.6 μmol, 3.6 eq) were dissolved in 500 μL of DMF. The solution was left under agitation for 5 min, after which cystamine dihydrochloride (8.2 mg, 36.5 μmol, 1.5 eq) dissolved in 500 μL borate buffer (2.5 M, pH 8) and DIPEA (3.2 μL, 18.4 μmol) were added.

(52) After 1 h at ambient temperature, dithiothreitol (6.8 mg, 43.8 μmol, 1.8 eq) was added and the reaction medium left under agitation overnight at ambient temperature. The solvents were removed under reduced pressure and product purification carried out with reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). Disulfonated cyanine 5.0-SH was obtained in the form of a blue powder (7.3 mg, 42%). MS: C.sub.35H.sub.46N.sub.3O.sub.7S.sub.3.sup.+ ([M].sup.+) m/z calculated: 716.2; found: 716.4.

2.6. (R)-NODAGA-SH

(S)-2,2′-(7-(1-carboxy-4-((2-mercaptoethyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid

(53) ##STR00051##

(54) (R)-NODAGA(tBu).sub.3 (159 mg, 292 μmol), TSTU (97.0 mg, 321 μmol, 1.1 eq) and DIPEA (61.0 μL, 350 μmol, 1.2 eq) were dissolved in 2 mL of anhydrous DMF. The solution was left under agitation at ambient temperature for 40 min, and 2-(tritylsulfanyl)ethanamine (102 mg, 321 μmol, 1.1 eq) then added. After 5 h, the DMF was removed under reduced pressure followed by the addition of 10 mL of TFA/DODT/TIS deprotection solution, 90:5:5 (v/v/v). The reaction medium was left under agitation at ambient temperature overnight, the white solid which had formed was removed by filtration and the TFA evaporated under a stream of nitrogen. The product was precipitated in diethyl ether at 4° C. and isolated by centrifugation (4000 rpm, 4° C.). Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). (R)-NODAGA-SH was obtained in the form of a hygroscopic white powder (89.5 mg, 71%, purity: 95%). .sup.1H NMR (300 MHz, D.sub.2O): ∂ (ppm)=2.10 (m, 1H; CH.sub.2), 2.19 (m, 1H; CH.sub.2), 2.51 (t, .sup.3J=7.8 Hz, 2H; CH.sub.2), 2.68 (t, .sup.3J=6.3 Hz, 2H; CH.sub.2), 3.11 to 3.22 (m, 4H; CH.sub.2), 3.22 to 3.31 (m, 2H; CH.sub.2), 3.34 (s, 2H; CH.sub.2), 3.40 (t, .sup.3J=6.3 Hz, 2H; CH.sub.2), 3.70 (dd, 1H; .sup.3J=6.0, 7.8 Hz, 1H; CH), 3.98 (s, 4H; CH.sub.2); MS: C.sub.17H.sub.31N.sub.4O.sub.7S.sup.+ ([M+H].sup.+) m/z calculated: 435.2; found: 435.1.

3. Monofunctionalised Tetrazines of Formula (IV)

3.1. NODAGA-NH-Tz-Cl (IV-1)

(S)-2,2′-(7-(1-carboxy-4-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)amino)ethyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid

(55) ##STR00052##

(56) (R)-NODAGA-NH.sub.2 (95.3 mg, 228 μmol) was dissolved in 1 mL of borate buffer (2.5 M, pH 8) and the pH adjusted to pH 8 through the addition of 2.5 M NaOH. Dichloro-s-tetrazine (34.5 mg, 228 μmol, 1.0 eq) in 300 μL ACN was added. After being left under agitation at ambient temperature for 2 h. the solvents were removed under reduced pressure.

(57) Purification of the reaction product was carried out with reverse phase semi-preparative HLPC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). NODAGA-NH-Tz-Cl was obtained in the form of an orange powder (43.8 mg, 36%). .sup.1H NMR (500 MHz, D.sub.2O): ∂=1.20 (m, 1H; CH.sub.2), 2.05 (m, 1H; CH.sub.2), 2.43 (t, .sup.3J=7.5 Hz, 2H; CH.sub.2), 3.01 to 3.15 (m, 4H; CH.sub.2), 3.22 (m, 2H; CH.sub.2), 3.29 (s, 4H; CH.sub.2), 3.46 to 3.59 (m, 3H; CH, CH.sub.2), 3.71 (m, 2H; 2H; CH.sub.2), 3.58 (s, 4H; CH.sub.2); MS: C.sub.19H.sub.31ClN.sub.9O.sub.7.sup.+ ([M+H].sup.+) m/z calculated: 532.2; found: 532.1.

3.2. DOTAGA-NH-Tz-Cl (IV-2)

2,2′,2″-(10-(1-carboxy-4-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)amino)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

(58) ##STR00053##

(59) DOTAGA-NH.sub.2 (243 mg, 443 μmol) was dissolved in 7 mL of borate buffer (2.5 M, pH 8) and the pH was adjusted to pH 8 through the addition of 2.5 M NaOH. Dichloro-s-tetrazine (68 mg, 443 μmol, 1.0 eq) in 2 mL of ACN was added.

(60) After an agitation time of 2 h at ambient temperature, the solvents were removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). DOTAGA-NH-Tz-Cl was obtained in the form of an orange powder (154 mg, purity: 99%). .sup.1H NMR (500 MHz, D.sub.2O): ∂=1.20 (m, 1H; CH.sub.2), 2.05 (m, 1H; CH.sub.2), 2.43 (t, .sup.3J=7.5 Hz, 2H; CH.sub.2), 3.01 to 3.15 (m, 4H; CH.sub.2), 3.22 (m, 2H; CH.sub.2), 3.29 (s, 4H; CH.sub.2), 3.46 to 3.59 (m, 3H; CH, CH.sub.2), 3.71 (m, 2H; 2H; CH.sub.2), 3.58 (s, 4H; CH.sub.2); MS: C.sub.19H.sub.31ClN.sub.97.sup.+ ([M+H].sup.+) m/z calculated: 532.2; found: 532.1.

3.3. (R)-NODAGA-S-Tz-Cl (IV-3)

(S)-2,2′-(7-(1-carboxy-4-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)thio)ethyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid

(61) ##STR00054##

(62) (R)-NODAGA-SH (40 mg, 92 μmol) and dichloro-s-tetrazine (41.7 mg, 276 μmol, 3 eq.) were dissolved in 3 mL of anhydrous DMF. After agitation for 10 min at ambient temperature, the DMF was removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% trifluoroacetic acid, ACN 0.1% trifluoroacetic acid). (R)-NODAGA-S-Tz-Cl was obtained in the form of an orange powder (46 mg, 91%, purity: 96%). .sup.1H NMR (500 MHz, D.sub.2O): ∂=2.04 (m, 1H; CH.sub.2), 2.11 (m, 1H; CH.sub.2), 2.46 (m, 2H; CH.sub.2), 3.08 to 3.11 (m, 4H; CH.sub.2), 3.23 (m, 4H; CH.sub.2), 3.29 (s, 4H, CH.sub.2), 3.55 (t, .sup.3J=7.1 Hz, 1H; CH), 3.58 (m, 2H; CH.sub.2), 3.67 (m, 2H; CH.sub.2), 3.826 (s, 2H; CH.sub.2), 3.831 (s, 2H; CH.sub.2); MS: C.sub.19H.sub.30ClN.sub.8O.sub.7S.sup.+ ([M+H].sup.+) m/z calculated: 549.2; found: 548.9.

3.4. BODIPY-S-Tz-Cl (IV-4)

10-(4-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)thio)ethyl)carbamoyl)phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2′1′-f][1,3,2]diazaborinin-4-ium-5-uide

(63) ##STR00055##

(64) BODIPY-SH (29.1 mg, 60.2 μmol) and dichloro-s-tetrazine (10.6 mg, 70.2 μmol, 1.2 eq) were dissolved in 1.5 mL of DMF/ACN mixture 0.6:0.4 (v/v) and DIPEA (12.5 μL, 72.2 μmol, 1.2 eq) was added to the reaction medium.

(65) After agitation for for 1 h at ambient temperature, the solvents were removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). BODIPY-S-Tz-Cl was obtained in the form of a red powder (24.1 mg, purity: 90%, product reduced to dihydrotetrazine: 10%). .sup.1H NMR (300 MHz, CDCl.sub.3): ∂=0.96 (t, .sup.3J=7.5 Hz, 6H; CH.sub.3), 1.23 (s, 6H; CH.sub.3), 2.28 (q, .sup.3J=7.5 Hz, 4H; CH.sub.2), 2.51 (s, 6H; CH.sub.3), 3.65 (t, .sup.3J=6.3 Hz, 2H; CH.sub.2), 3.92 (dt, .sup.3J=5.7, 6.3 Hz, 2H; CH.sub.2), 6.71 (t, .sup.3J=5.7 Hz, 1H; NH), 7.39 (d, .sup.3J=8.2 Hz, 2H; CH.sub.Ar), 8.18 (d, .sup.3J=8.2 Hz, 2H; CH.sub.Ar); HRMS: C.sub.28H.sub.31BClFN.sub.7OS.sup.+ ([M−F].sup.+) m/z calculated: 578.20709; found: 578.20828.

3.5. Rhodamine B-S-Tz-Cl (IV-5)

N-(9-(2-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)thio)ethyl)carbamoyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium

(66) ##STR00056##

(67) Rhodamine B-SH (200 mg, 399 μmol) and dichloro-s-tetrazine (60.3 mg, 399 μmol, 1.0 eq.) were dissolved in 2 mL of ACN, and DIPEA (69.5 μL, 399 μmol, 1.0 eq) was added to the reaction medium. The solution was left under agitation for 5 h at ambient temperature and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). Rhodamine B-S-Tz-Cl was obtained in the form of a pink powder (47.8 mg, 19%). .sup.1H NMR (300 MHz, CDCl.sub.3): equilibrium between the cyclic and non-cyclic forms of rhodamine; HRMS: C.sub.32H.sub.35ClN.sub.7O.sub.2S.sup.+ ([M].sup.+) m/z calculated: 616.22560; found: 616.22622; C.sub.32H.sub.34ClN.sub.7NaO.sub.2S.sup.+ (cyclic form) ([M−H+Na].sup.+) m/z calculated: 638.20754; found: 638.20749.

3.6. Disulfonated Cyanine 3.0-S-Tz-Cl (IV-6)

2-((1E,3E)-3-(1-(6-((2-((6-chloro-1,2,4,5-tetrazin-3-yl)thio)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)prop-1-en-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium-5-sulfonate

(68) ##STR00057##

(69) Disulfonated cyanine 3.0-SH (15.8 mg, 22.9 μmol) and dichloro-s-tetrazine (4.3 mg, 28.5 μmol, 1.2 eq) were dissolved in 1 mL of anhydrous DMF, and DIPEA (5.5 μL, 31.8 μmol, 1.4 eq) was added to the reaction medium under agitation at ambient temperature. After 1 h, the reaction was complete and the solvent removed under reduced pressure. The reaction product was purified by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). Disulfonated cyanine 3.0-S-Tz-Cl was obtained in the form of a pink powder (6.0 mg). HRMS (negative mode): C.sub.35H.sub.41ClN.sub.7O.sub.7S.sub.3.sup.− ([M−H].sup.−) m/z calculated: 802.19236; found: 802.18923.

4. Bifunctionalised Tetrazines of Formula (IIa)

4.1. DOTAGA-NH-Tz-S-Rhodamine B (II-1)

N-(9-(2-((2-((6-((2-(4-carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)butanamido)ethyl)amino)-1,2,4,5-tetrazin-3-yl)thio)ethyl)carbamoyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium 2,2,2-trifluoroacetate

(70) ##STR00058##

(71) Rhodamine B-SH (39.7 mg, 79.0 μmol) and DOTAGA-NH-Tz-Cl (50.0 mg, 79.0 μmol, 1.0 eq.) were dissolved in 1 mL of anhydrous DMF, and DIPEA (65.7 μL, 395 μmol, 5.0 eq.) was added to the reaction medium. The solution was left under agitation at 75° C. for 3 h, and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). DOTAGA-NH-Tz-S-Rhodamine B was obtained in the form of a red oil (37.0 mg, 43%).sup.1H NMR (500 MHz, D.sub.2O) ∂: 1.13 (m, 12H; CH.sub.3), 2.01 (m, 2H; CH.sub.2), 2.56 (m, 2H; CH.sub.2), 2.96 (m, 2H; CH.sub.2), 3.00 to 3.48 (m, 12H; CH.sub.2), 3.48 to 3.63 (m, 8H; CH.sub.2), 3.69 (m, 10H; CH.sub.2), 3.74 to 4.37 (m, 6H; CH.sub.2), 3.99 (m, 1H; CH), 7.11 (m, 2H; CH.sub.Ar), 7.24 (m, 3H; CH.sub.Ar), 7.63 (m, 2H; CH.sub.Ar), 7.72 (m, 2H; CH.sub.Ar), 8.02 (m, 1H; CH.sub.Ar); HRMS: C.sub.53H.sub.72N.sub.13O.sub.11S.sup.+ ([M].sup.+) m/z calculated: 1098.51895; found: 1098.52126; C.sub.53H.sub.71N.sub.13NaO.sub.11S.sup.+ ([M−H+Na].sup.+) m/z calculated: 1120.50089; found: 1120.50291; C.sub.53H.sub.70KN.sub.13NaO.sub.11S.sup.+ ([M−2H+Na+K].sup.+) m/z calculated: 1158.45677; found: 1158.454311.

4.2. DOTAGA-NH-Tz-S-BODIPY (II-2)

10-(4-((2-((6-((2-(4-carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)butanamido)éthyl)amino)-1,2,4,5-tetrazin-3-yl)thio)ethyl)carbamoyl)phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2,1′-f][1,3,2]diazaborinin-4-ium-5-uide

(72) ##STR00059##

(73) BODIPY-SH (11.7 mg, 24.2 μmol) and DOTAGA-NH-Tz-Cl (15.3 mg, 24.2 μmol, 1.0 eq. were dissolved in 1 mL of anhydrous DMF, and DIPEA (22.8 μL, 121.0 μmol, 5.0 eq.) was added to the reaction medium. The solution was left under agitation at 75° C. for 1 h 30, and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). DOTAGA-NH-Tz-S-BODIPY was obtained in the form of a red powder (18.0 mg, 69%, purity: 95%). .sup.1H NMR (500 MHz, MeOD) ∂: 0.99 (m, 6H; CH.sub.3), 1.32 (s, 6H; CH.sub.3), 2.35 (m, 4H; CH.sub.2), 2.48 (s, 6H; CH.sub.3), 3.01 to 4.13 (m, 27H; CH.sub.2, macrocycle), 3.52 (m, 2H; CH.sub.2), 3.80 (m, 2H; CH.sub.2), 7.46 (m, 2H; CH.sub.Ar), 8.01 (m, 2H; CH.sub.Ar); HRMS: C.sub.49H.sub.69BF.sub.2N.sub.13O.sub.10S.sup.+ ([M+H].sup.+) m/z calculated: 1080.50667; found: 1080.50639; C.sub.49H.sub.67BF.sub.2N.sub.13NaO.sub.10S.sup.+ ([M+Na].sup.+) m/z calculated: 1102.48861; found: 1102.48834; C.sub.49H.sub.67BF.sub.2N.sub.13Na.sub.2O.sub.10S.sup.+ ([M−H+2Na].sup.+) m/z calculated: 1124.47056; found: 1124.46975.

4.3. DOTAGA-NH-Tz-S-cyanine 5.0 (II-3)

2-((1E,3E,5E)-5-(1-(6-((2-((6-((2-(4-carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)butanamido)ethyl)amino)-1,2,4,5-tetrazin-3-yl)thio)ethyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium

(74) ##STR00060##

(75) Cyanine 5.0-SH (14.9 mg, 26.8 μmol) and DOTAGA-NH-Tz-Cl (19.5 mg, 30.8 μmol, 1.1 eq.) were dissolved in 1 mL of anhydrous DMF, and DIPEA (28.0 μL, 149.5 μmol, 5.6 eq.) was added to the reaction medium. The solution was left under agitation at 75° C. for 1 h, and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). DOTAGA-NH-Tz-S-cyanine 5.0 was obtained in the form of a blue powder (15.1 mg, 49%). .sup.1H NMR (500 MHz, CD.sub.3CN): ∂=1.33 (t, .sup.3J=7.0 Hz, 3H; CH.sub.3), 1.42 (m, 2H; CH.sub.2), 1.61 (m, 2H; CH.sub.2), 1.67 (s, 12H; CH.sub.3), 1.76 (m, 2H; CH.sub.2), 2.12 (t, 2H; CH.sub.2), 2.76 to 3.97 (m, 35H; macrocycle+CH.sub.2), 4.00 (t, 2H; CH.sub.2), 4.06 (q, .sup.3J=7.0 Hz, 2H, CH.sub.2), 6.21 (d, .sup.3J=13.0 Hz, 2H; CH), 6.53 (t, .sup.3J=13.0 Hz, 1H; CH), 7.25 (m, 4H, CH.sub.Ar), 7.40 (m, 2H, CH.sub.Ar), 7.47 (m, 2H, CH.sub.Ar), 8.07 (dd, 2H, CH); HRMS: C.sub.58H.sub.82N.sub.13O.sub.10S.sup.+ ([M].sup.+) m/z calculated: 1152.60228; found: 1152.60445; C.sub.58H.sub.82N.sub.13NaO.sub.10S.sup.2+ ([M+Na].sup.2+) m/z calculated: 587.79575; found: 587.79558.

4.4. DOTAGA-NH-Tz-S-disulfonate Cyanine 5.0 (II-4)

2-((1E,3E,5E)-5-(1-(6-((2-((6-((2-(4-carboxy-4-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)butanamido)ethyl)amino)-1,2,4,5-tetrazin-3-yl)thio)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium-5-sulfonate

(76) ##STR00061##

(77) Disulfonated cyanine 5.0 —SH (5.4 mg, 7.54 μmol) and DOTAGA-NH-Tz-Cl (5.7 mg, 9.05 μmol, 1.2 eq.) were dissolved in 800 μL of anhydrous DMF, and DIPEA (8.47 μL, 45.25 μmol, 6.0 eq.) was added to the reaction medium. The solution was left under agitation at 75° C. for 5 h, and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). DOTAGA-NH-Tz-S-disulfonated cyanine 5.0 was obtained in the form of a blue powder (3.75 mg). .sup.1H NMR (500 MHz, D.sub.2O) ∂: 1.31 (m, 5H; CH.sub.2.sup.+CH.sub.3), 1.59 (m, 2H; CH.sub.2), 1.61 (s, 6H; CH.sub.3), 1.67 (s, 6H; CH.sub.3), 1.81 (m, 2H; CH.sub.2), 1.88 (m, 2H; CH.sub.2), 2.20 (t, 2H; CH.sub.2), 2.44 (m, 2H; CH.sub.2), 2.97 to 3.63 (m, 26H; 2 CH.sub.2+11 CH.sub.2, macrocycle), 3.82 (m, 5H; 2 CH.sub.2+CH), 4.07 (m, 4H; CH.sub.2), 6.15 (d, 1H; CH), 6.23 (d, 1H; CH), 6.48 (t, 1H; CH), 7.33 (d, 2H; CH.sub.Ar), 7.79 à 7.88 (m, 4H; CH.sub.Ar), 7.96 (m 1H; CH), 8.01 (m 1H; CH);

4.5. (R)-NODAGA-NH-Tz-S-BODIPY (II-5)

(S)-10-(4-((2-((6-((2-(4-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)-4-carboxybutanamido)ethyl)amino)-1,2,4,5-tetrazin-3-yl)thio)ethyl)carbamoyl)phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2,1′-f][1,3,2]diazaborinin-4-ium-5-uide

(78) ##STR00062##

(79) BODIPY-SH (17.0 mg, 40.1 μmol) and (R)-NODAGA-NH-Tz-Cl (27.4 mg, 51.5 μmol, 1.3 eq.) were dissolved in 1 mL of anhydrous DMF, and DIPEA (48.3 μL, 275.5 μmol, 5.0 eq.) was added to the reaction medium. The solution was left under agitation at 75° C. for 1 h, and the solvent afterwards removed under reduced pressure. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). (R)-NODAGA-NH-Tz-S-BODIPY was obtained in the form of a red powder (8.1 mg). HRMS: C.sub.45H.sub.62BF.sub.2N.sub.12O.sub.8S.sup.+ ([M+H].sup.+) m/z calculated: 979.45899; found: 979.45948; C.sub.45H.sub.61BF.sub.2N.sub.12NaO.sub.8S.sup.+ ([M+Na].sup.+) m/z calculated: 1001.44094; found: 1001.44055.

5. Bifunctionalised Tetrazines of Formula (IIb)

5.1. (R)-NODAGA-S-Tz-S-peptide (II-6)

2,2′-(7-((S)-4-((2-((6-(((2S,5S,11S,17S,20S,23S,26R)-17-((1H-indol-3-yl)methyl)-26-acetamido-1-amino-23-(4-aminobutyl)-5-(carboxymethyl)-11-(3-guanidinopropyl)-20-(4-hydroxybenzyl)-2-(hydroxymethyl)-1,4,7,10,13,16,19,22,25-nonaoxo-3,6,9,12,15,18,21,24-octaazaheptacosan-27-yl)thio)-1,2,4,5-tetrazin-3-yl)thio)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid

(80) ##STR00063##

(81) (R)-NODAGA-S-Tz-Cl (8.5 mg, 15.5 μmol) and peptide Ac-CKYWGRGDS-NH.sub.2, 2 TFA (25 mg, 18.7 μmol, 1.2 eq.) were dissolved in 600 μL of ultrapure water. The solution was left under agitation at ambient temperature, protected from light, for 30 min. Purification of the reaction product was carried out by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% formic acid, ACN 0.1% formic acid). The product (R)-NODAGA-S-Tz-S-peptide, which can also be written Ac-C(Tz-S-NODAGA)KYWGRGDS-NH.sub.2, was obtained in the form of an orange powder (5.8 mg, 55%, purity: 82%). HRMS: C.sub.67H.sub.98N.sub.23O.sub.21S.sub.2.sup.+ ([M+H].sup.+) m/z calculated: 1624.67; found: 1624.57; C.sub.67H.sub.97N.sub.23NaO.sub.21S.sub.2.sup.+ ([M+Na].sup.+) m/z calculated: 1646.66; found: 1646.49.

5.2. BSA-S-tetrazine-S-NODAGA (II-7)

(82) ##STR00064##

(83) Bovine Serum Albumin (BSA) was incubated with (R)-chlorotetrazine-NODAGA (10 equiv.) for 1 hour at ambient temperature (pH=5.15). After purification by ultracentrifugation, the BSA-S-tetrazine-S—(R)-NODAGA (II-7) compound was obtained. LC-HRMS after deconvolution of the mass spectrum: 67004 (corresponds to compound I-3) and 66430 (corresponds to the protein residue without probe).

5.3. Fab′-S-tetrazine-S-DOTAGA (II-8)

(84) ##STR00065##

(85) The Fab′ fragment was incubated with (R)-chlorotetrazine-NODAGA (50 equiv.) for 30 minutes at 37° C. (pH=7.3). LC-HRMS of the reaction after deconvolution of the mass spectrum: 49687 (corresponds to compound I-3) and 48660 (corresponds to the Fab′ residue without probe). The compound was not purified before being used for formation of the compound (disulfonated cyanine 5.0-BCN-pyridazine, S—(R)-NODAGA).sub.2, S-Fab′ (I-4).

5.4. Reactivity Selectivity of (R)-NODAGA-S-Tz-Cl for Thiols Versus the Amines of a Peptide

(86) The chemoselectivity of the reaction of the chlorotetrazines monosubstituted by a nucleophile of thiol type, with respect to the nucleophiles present on the proteins, was assessed using two model peptides: Ac-CKYWGRGDS-NH.sub.2 and Ac-MKYWGRGDS-NH.sub.2. These peptides contain the most usual amino acids having nucleophilic side chains such as lysine (amine), serine (alcohol) and tyrosine (phenol). They differ through the presence of a cysteine (thiol) in one thereof, which is substituted by a methionine (thioether) in the other.

(87) The peptides (20 μM), in solution in a phosphate buffer (0.01 M; different pHs), were incubated in the presence of 2.5 equivalents of (R)-NODAGA-S-Tz-Cl at 25° C. The conversion rate to product, resulting from substitution of the chlorine of (R)-NODAGA-S-Tz-Cl by the peptides, was determined by LC-MS at 214 nm. The results are given in the table below.

(88) Conversion rate of the peptides in the presence of 2.5 equiv. of (R)-NODAGA-S-Tz-Cl at different pHs. (quant.: quantitative; <1%: detection limit of the method used).

(89) TABLE-US-00006 pH 5.97 6.29 7.13 Ac-CKYWGRGDS-NH.sub.2 3.5 min 96% 96% quant.  30 min 97% quant. quant. Ac-MKYWGRGDS-NH.sub.2 3.5 min — <1% <1%  30 min — <1% <1%

(90) These results show that the chlorotetrazines mono-substituted by a nucleophile of thiol type selectively react with the nucleophiles of thiol type carried by the Ac-CKYWGRGDS-NH.sub.2 peptide, within a few minutes and under mild conditions (buffered aqueous solution, 25° C., neutral pH). In the absence of cysteine, no reaction was observed.

6. Intermediates of Formula (III)

6.1. Cyanine 5.0-BCN

2-((1E,3E,5E)-5-(1-(6-((2-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino)ethyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium 2,2,2-trifluoroacetate

(91) ##STR00066##

(92) Cyanine 5.0-NH.sub.2 (25.7 mg, 33.5 μmol) and bicyclononyne-N-hydroxysuccinnimide (16.3 mg, 55.8 μmol, 1.7 eq.) were dissolved in 1 mL of anhydrous DMF, and DIPEA (8.41 μL, 48.6 μmol, 1.5 eq.) was added to the reaction medium. The solution was left under agitation 40 min at ambient temperature, and the solvent afterwards removed under reduced pressure. The reaction product was purified by reverse phase semi-preparative HPLC (eluents: H.sub.2O 0.1% TFA, ACN 0.1% TFA). Cyanine 5.0-BCN, TFA was obtained in the form of a blue powder (11.5 mg, 41%, TFA salt, purity: 90%). .sup.1H NMR (500 MHz, CDCl.sub.3): ∂=0.86 (m, 2H; CH), 1.31 (m, 1H; CH), 1.39 (t, .sup.3J=7.5 Hz, 3H; CH.sub.3), 1.47 (m, 2H; CH.sub.2), 1.53 (m, 2H; CH.sub.2), 1.66 (s, 6H; CH.sub.3), 1.67 (s, 6H; CH.sub.3), 1.71 (m 2H; CH.sub.2), 1.79 (m 2H; CH.sub.2), 2.08 to 2.27 (m, 6H; CH.sub.2), 2.30 (t, .sup.3J=7.3 Hz, 2H; CH.sub.2), 3.31 (m, 2H; CH.sub.2), 3.54 (m, 2H; CH.sub.2), 3.99 (t, .sup.3J=7.6 Hz, 2H; CH.sub.2), 4.04 (q, .sup.3J=7.2 Hz, 2H; CH.sub.2), 4.07 (d, .sup.3J=8.4 Hz, 2H; CH.sub.2), 6.04 to 6.22 (m, 1H; OCONH), 6.17 (d, .sup.3J=13.5 Hz, 1H; CH), 6.26 (d, .sup.3J=13.5 Hz, 1H; CH), 6.66 (t, .sup.3J=12.5 Hz, 1H; CH), 7.05 (d, .sup.3J=7.9 Hz, 1H; CH.sub.Ar), 7.10 (d, .sup.3J=8.0 Hz, 1H; CH.sub.Ar), 7.22 (m, 2H; CH.sub.Ar), 7.35 (m, 4H; CH.sub.Ar), 7.78 (dd, .sup.3J=12.0, 13.5 Hz, 1H; CH), 7.79 (dd, .sup.3J=12.0, 13.5 Hz, 1H; CH), 8.26 (m, 1H; CONH); HRMS: C.sub.46H.sub.59N.sub.4O.sub.3.sup.+ ([M].sup.+) m/z calculated: 715.45817; found: 715.45752.

6.2. Trastuzumab-BCN

(93) ##STR00067##

(94) 26.7 μL stock solution of 2 mM BCN-NHS in DMSO (53.3 nmol, 4.0 eq.) were added to the trastuzumab antibody (Herceptin; Roche, U.K.) (2 mg, 13.3 nmol) in solution in a bicarbonate buffer (0.2 M, pH 8.5) in the presence of 10% (v/v) DMSO (trastuzumab concentration: 2 mg/mL). The solution was left under agitation in a thermomixer (800 rpm, 25° C.) for 2 h. Excess BCN was removed by ultrafiltration on Amicon Ultra Ultracel-30 kDa (Merck Millipore). At the ultrafiltration step, the product was concentrated and the bicarbonate buffer exchanged for PBS buffer (0.01 M, pH 7.4). Trastuzumab-BCN was isolated in solution in 51 μL PBS at a concentration of 35.5 mg/mL (1.81 mg, 12.1 nmol, labelling yield: 91%). The BCN/antibody ratio was 3.3 (determined by MALDI/TOF mass spectrometry).

7. Trifunctionalised Platforms of Formula (I)

7.1. Trastuzumab-BCN-Pyridazine, NH-DOTAGA, Disulfonated S-Cyanine 5.0 (I-1)

(95) ##STR00068##

(96) 75.3 μL stock solution of DOTAGA-NH-Tz-disulfonated S-cyanine 5.0 at 5 mM in ultrapure water (376.6 nmol, 33.0 eq.) was added to trastuzumab-BCN (1.71 mg, 11.4 nmol) in solution in PBS (pH 7.4)+10% (v/v) DMSO (finale trastuzumab-BCN concentration: 10 mg/mL). The solution was left under agitation in a thermomixer (800 rpm, 37° C.) for 16 h. Excess probe was removed by FPLC purification (Akta Pure Healthcare, GE) on a desalting column (Hitrap desalting, 5 mL, pre-packed with Sephadex G-25 Superfine) with AcONH.sub.4 buffer (0.1 M, pH 5.8, Trace Select) as eluent. Trastuzumab-BCN-pyridazine, NH-DOTAGA, disulfonated S-Cyanine 5.0 (I-1) was obtained in solution in 484 μL of ammonium acetate buffer at a concentration of 2.1 mg/mL (correction factor=0.095) (1.02 mg, 6.8 nmol, 59%). The probe/antibody ratio was 1.7 (determined by spectrophotometry). Analysis by gel electrophoresis under denaturing conditions showed a fluorescent signal for the molecular weight corresponding to compound I-1, indicating a bond of covalent type between the probe and trastuzumab antibody. The stability of compound I-1 was verified by incubation at 37° C., in the dark, in human plasma (analysis by gel electrophoresis of a sample after an incubation time of 1, 2, 4, 8, 24 and 48 h).

7.2. NODAGA-BCN-Pyridazine, S—(R)-NODAGA, S-Peptide (I-2)

2,2′-(7-((1S)-4-((2-((((1-(((2S,5S,11S,17S,20S,23S,26R)-17-((1H-indol-3-yl)methyl)-26-acetamido-1-amino-23-(4-aminobutyl)-5-(carboxymethyl)-11-(3-guanidinopropyl)-20-(4-hydroxybenzyl)-2-(hydroxymethyl)-1,4,7,10,13,16,19,22,25-nonaoxo-3,6,9,12,15,18,21,24-octaazaheptacosan-27-yl)thio)-4-((2-((S)-4-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)-4-carboxybutanamido)ethyl)thio)-6,6a,7,7a,8,9-hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyridazin-7-yl)methoxy)carbonyl)aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid

(97) ##STR00069##

(98) 2 μL stock solution of (R)-NODAGA-S-Tz-S-peptide at 30 mM in DMSO (60 nmol) and 12 μL stock solution of NODAGA-BCN at 50 mM in PBS (600 mmol, 10 eq.) were combined in PBS at ambient temperature (finale (R)-NODAGA-S-Tz-S-peptide concentration: 2 mM and in DMSO: 6.7%). The progress of the reaction was monitored by UV-Vis spectrophotometry. After a reaction time of 50 min, the conversion rate to NODAGA-BCN-pyridazine, S—(R)-NODAGA, S-peptide (I-2) was 98% (determined by LC-MS at 214 nm). MS: C.sub.95H.sub.140N.sub.26O.sub.30S.sub.2 ([M+2H].sup.2+) m/z calculated: 1095.5; found: 1096.0.

7.3. Disulfonated Cyanine 5.0-BCN-Pyridazine, S—(R)-NODAGA, S-BSA (I-3)

(99) ##STR00070##

(100) The compound (R)-NODAGA-S-tetrazine-S-BSA was combined with disulfonated cyanine 5.0-BCN by click chemical reaction, in an aqueous solution (phosphate buffer, pH=7.13) at 37° C. overnight. The end product disulfonated cyanine 5.0-BCN-pyridazine, S—(R)-NODAGA, S-BSA (I-3) was purified by steric exclusion chromatography. The probe/protein ratio was 0.7 (determined by spectrophotometry). LC-HRMS after deconvolution of the mass spectrum: 67851 (corresponds to compound I-3) and 66430 (corresponds to the protein residue without probe). Analysis by gel electrophoresis under denaturing conditions showed a fluorescent signal for the molecular weight corresponding to compound I-3, indicating a bond of covalent type between the probe and albumin. The stability of compound I-3 was verified by incubation at 37° C., in the dark, in human plasma (gel electrophoresis analysis of a sample after an incubation time of 1, 2, 4, 8, 24 and 48 h).

7.3. (Disulfonated Cyanine 5.0-BCN-pyridazine, S—(R)-NODAGA).SUB.2., S-Fab′ (I-4)

(101) ##STR00071##

(102) Disulfonated cyanine 5.0-BCN was added and the reaction mixture incubated at 37° C. overnight (pH=7.3) to obtain the product (disulfonated cyanine 5.0-BCN-pyridazine, S—(R)-NODAGA).sub.2, S-Fab′ (I-4). The non-purified reaction was analysed by LC-HRMS, after deconvolution of the mass spectrum. The masses obtained were: 51503 (corresponds to compound I-4+2 copper cations) and 48659 (corresponds to the protein residue without probe).

Part II. Imaging Results

(103) Compound I-1, after radiolabelling with indium 111, was studied for multimodal imaging via single photon emission computed tomography (SPECT) and for optical imaging (fluorescence). Compound I-3 was studied by optical imaging. In vivo experiments were conducted on a mouse model bearing a breast tumour xenograft (model BT-474).

(104) 8.1. Imaging Using Compound I-1 Radiolabelled with Indium 111

(105) The objective of this experiment was to evidence the specific nature of compound I-1 radiolabelled with indium 111, used as imaging agent.

(106) For this purpose, two batches of mice were studied: batch A: mice treated with compound I-1 radiolabelled with indium 111 (n=4); and batch B: mice treated with compound I-1 radiolabelled with indium 111 and non-labelled excess trastuzumab(n=3).

(107) 24 h after injection, images of the mice of batches A and B were recorded by single photon emission computed tomography (SPECT) and by optical imaging. Injection of a large excess of non-labelled trastuzumab (which saturates the HER2 receptors) allowed evidencing of the specificity of the imaging agent.

(108) If no significant change is observed between the images of batches A and B, then accumulation of the imaging agent in the examined tissue is not specific. On the contrary, if the signal observed with batch A decreases or disappears from the image obtained with batch B, then the accumulation of the imaging agent in the examined tissue is specific.

(109) Single Photo Emission Computed Tomography (SPECT)

(110) Examples of illustrative SPECT images are given for batch A, FIG. 2A and for batch B, FIG. 2B.

(111) These results show that: regarding treatment with compound I-1 radiolabelled with indium 111 (batch A), the tumour can be seen under SPECT imaging (cf. white mark in FIG. 2A); when a mouse is treated with compound I-1 radiolabelled with indium 111 with large excess of non-labelled trastuzumab, the signal is seen to disappear compared with batch A.

(112) This experiment therefore evidences the specific accumulation of the imaging agent in tumour tissue.

(113) FIG. 3 shows the biodistribution of radioactivity (mean dose percentage injected per gram of organ), in different organs of mice (n=4) treated with compound I-1 radiolabelled with indium 111 (batch A—light grey), or of mice (n=3) treated with compound I-1 radiolabelled with indium 111 with excess trastuzumab (batch B—dark grey).

(114) In FIG. 3, a distinct difference is observed between batches A and B regarding the tumour compared with the other organs.

(115) These results show the specificity of compound I-1 radiolabelled with indium 111 used as imaging agent, for the tumour in the studied model.

(116) Optical Imaging (Fluorescence)

(117) In vivo and ex vivo optical images were obtained for mice treated with compound I-1 radiolabelled with indium 111. FIG. 4 gives an in vivo photograph of a mouse treated with compound I-1 radiolabelled with indium 111 (FIG. 4(a)) in which the siting of the tumour is distinctly visible (top left protuberance from the animal's back).

(118) The same image taken by superimposing the fluorescence signal (FIG. 4(b)) shows an intense signal (white) coinciding with the siting of the tumour.

(119) These in vivo results confirm the efficacy of compound I-1 radiolabelled with indium 111 as imaging agent, in particular for the targeting of tumours.

(120) FIGS. 5 and 6 give the results obtained on isolated organs (ex vivo). In FIG. 5, the intensity of fluorescence emanating from the tumour is much more intense than the intensity from the other isolated organs. FIG. 6 confirms that compound I-1 radiolabelled with indium 111 acts as specific imaging agent with respect to the tumour. A marked difference in biodistribution is observed between batches A and B for the tumour compared with the other organs for which the signals are comparable.

(121) To conclude, the results of fluorescence confirm the results obtained with nuclear imaging. The advantage of site-specific double labelling with trastuzumab is therefore demonstrated for applications in multimodal SPECT/optical imaging.

(122) 8.2. Imaging Using Compound I-3

(123) A study was conducted using compound I-3 for optical imaging of a batch of mice previously xenografted with breast tumour BT-474 cells, and treated with compound I-3 (n=3, lot C).

(124) After treatment (t.sub.0), in vivo fluorescence images of the mice in batch C were acquired at t=1 h, 4 h, and 24 h (FIG. 7); and ex vivo on isolated organs after 24 h (FIGS. 8 and 9).

(125) FIG. 7 shows the change in the in vivo fluorescence signal in one mouse of the group in batch C. It shows that after injection of compound I-3, the fluorescence signals are intense (bright areas) in the tumour region even after 24 h, compared with the remainder of the organism.

(126) FIG. 8 illustrates the results obtained on isolated organs and on the tumour (ex vivo) for one mouse in batch C. A more intense fluorescence signal in the tumour is observed compared with the other organs.

(127) Quantification relating to FIG. 8 is given in FIG. 9.

(128) To conclude, as for compound I-1, the experiments show that compound I-3 is an efficient imaging agent.