FR2969605A1 - New tetrahydro-1H-benzo(b)azepine derivatives useful for preparing fluorescent ligand, which is useful in a binding assay on the vasopressin V2 receptor, and for the marking or the visualization of the V2 receptor - Google Patents

Abstract

Tetrahydro-1H-benzo[b]azepine derivatives (I) and their salts are new. Tetrahydro-1H-benzo[b]azepine derivatives of formula (I) and their salts are new. R1 : H or -COR2; R2 : -(CH 2) mNR3R4 group or -(CH 2CH 2O) n(CH 2) pNR3R4 group; m : 2-4; either R3 : H; and R4 : triazole group of formula (a) or -(CH 2CH 2O) r(CH 2) sNR3R4 group; or NR3R4 : piperid-4-yl group or piperazinyl group; n : 3-5; R5 : -(CH 2) qNR3R4; p, q, s : 2 or 3; and r : 3-6. Independent claims are included for: (1) fluorescent ligand obtained by coupling (I) with a fluorescent compound; and (2) intermediates comprising biphenyl-2-carboxylic acid [4-(5-amino-2,3,4,5-tetrahydro-benzo[b]azepine-1-carbonyl)-phenyl]-amide (II) and N-(1-{4-[(biphenyl-2-carbonyl)-amino]-benzoyl}-2,3,4,5-tetrahydro-1H-benzo[b]azepin-5-yl)-N'-prop-2-ynyl-succinamide (III). [Image] [Image] ACTIVITY : Vasotropic. MECHANISM OF ACTION : Vasopressin V2 receptor modulator. The ability of (I) to modulate vasopressin V2 receptor was tested in Chinese Hamster ovary cell line. The result showed that tetrahydro-1H-benzo[b]azepine derivatives of formula (Id) exhibited an inhibition constant (K i) value of 4+- 0.7 nM.

Description

The present invention relates to 2,3,4,5-tetrahydro-1H-benzo [b] azepine derivatives and their use as a tool in a binding test on the vasopressin V2 receptor. BACKGROUND OF THE INVENTION The V2 vasopressin receptor is a G protein-coupled receptor responsible for the antidiuretic effect of vasopressin. This receptor is of interest to the pharmaceutical industry, particularly for the development of treatments against hyponatremia. The identification of new ligands of this receptor can be done using screening tests based on radioactivity. However, these methods have several disadvantages, such as the disposal of radioactive waste. The use of screening tests based on fluorescence techniques makes it possible to circumvent these limitations. Their development, however, requires the preparation of fluorescent ligands, affine and selective of the receptor studied. The phenomenon of FRET (Fluorescent Resonance Energy Transfer) is widely used in biology, particularly to study molecular interactions. It is based on the use of a fluorescent donor compound (for example a rare earth complex) and an optionally fluorescent acceptor compound, each of these compounds being coupled to a biological molecule. When a biological phenomenon causes the approximation of these molecules, and the donor compound is excited, energy transfer takes place between the donor and the acceptor and will result in a variation of the luminescence emitted by the reaction medium. Studies of biological phenomena based on the FRET technique require the excitation of fluorophores by a light source, which can be provided by a flash lamp or more generally a laser, preferably emitting in the near ultraviolet: nitrogen lasers and YAG lasers, both employed in commercially available fluorometers, emit light at 337 nm and 355 nm, respectively. The use of such lasers nevertheless requires fluorophores capable of absorbing light at these wavelengths. Homogeneous Time-Resolved Fluorescence (HTRFO) is a technology that uses the principles of FRET and Time-Resolved Fluorescence (TRF). This technology requires the availability of a couple of donor / acceptor partners that fulfill certain requirements (see the explanations available by logging on to the following URL: http://www.htrf.com/techn000gy/htrftheory/ The labeled ligands described in the literature are generally peptide and non-selective compounds of the V2 receptor (see for example Min / Rev Mec / Chem 2008, 8 (10), 996-1005) A non-peptidic fluorescent ligand derived from the motif 2,3,4,5-Tetrahydro-1H-benzo [b] azepine has recently been described (Chem et / r 2008, 14, 6247-6254, compound 17a, b) This non-peptidic ligand has a medium affinity ( > 50nM for the V2 receptor, which makes it difficult to carry out a fluorescence-based screening assay (of the Tag-lite or FP type) Object of the Invention There is therefore a need to be able to have compounds that are not peptides which have good affinity for the vasopressin V2 receptor, and which are selective for this receptor vis-à-vis the vasopressin Vi receptor (subtypes (Via, Vip) and oxytocin receptor (OT). There is also a need for fluorescent ligands useful for the development of a binding assay on the V2 receptor, especially using HTRF0 technology. Thus, according to a first aspect, the invention relates to a compound of formula (1): RIHN in which: R1 is hydrogen or a -COR2 group; R2 represents a group - (CFI2) mNR3R4 in which m = 2, 3 or 4 and R3 and R4 each represent hydrogen or R3 and R4 form a piperid-4-ye or piperazinyl group with the nitrogen atom to which They are linked ; or - (CH 2 CH 2 O), (C) pNR 3 R 4 wherein n =, 4 or 5, p = 2 or 3 and R 3 and R 4 are as defined above; or a group wherein R5 is - (CH2) pNR3R4 wherein q = 2 or 3 and R3 and R4 are as defined above; or a group - (CH2CH2O), (CH2) NR3R4 wherein r = 3, 4, 5 or 6, s = 2 or 3 and R3 and R4 are as defined above.

According to one embodiment, R1 represents a -COR2 group in which R2 preferably represents:

a group - (CH 2) n, NR 3 R 4 in which m = 2, 3 or 4, preferably m = 2 or 4, and R 3 and R 4 each represent hydrogen or R 3 and R 4 form a piperazinyl group with the atom of nitrogen to which they are linked; or

a group - (CH2CH2O) n (CH2) pNR3R4 wherein n = 3 and p = 2, and R3 and R4 are each preferably hydrogen; or

a group (CH 2 C C H H N = N wherein R 5 is - (CH 2) 2 NR 3 R 4 and R 3 and R 4 are each preferably hydrogen, or

a group (CHz) wherein R5 is - (CH2CH2O), (CH2) SNR3R4 wherein r = 3

or 6, s = 2 and R3 and R4 preferably each represent hydrogen.

According to another aspect, the invention relates to a fluorescent ligand capable of being used in a binding test on the V2 receptor. This ligand is obtained by coupling a compound of formula (I) with a fluorescent compound. Coupling is conventionally carried out between an -NH or -NH2 residue carried by the compound of formula (I) and a fluorescent compound via a reactive group, for example a group

NHS.

The fluorescent compounds that may be used in the context of the invention are advantageously chosen from: allophycocyanines, in particular that known under the trade name XL665; organic luminescent molecules, such as rhodamines (in particular lissamine rhodamine B), cyanines,

30 squaraines, coumarines, proflavines, acridines, fluoresceins; fluorophores known under the name Bodipy; fluorophores known under the name Atto; fluorophores known under the name Dy, in particular the compound known under the name Dy467; the compounds known under the name AlexaFluor; nitrobenzoxadiazole; fluorescent metal complexes, such as rare earth cryptates, rare earth chelates (in particular the chelates and cryptates of europium, terbium, samarium, dysprosium, neodymium, and more particularly the compound known under the name Lumi4-Tb); inorganic luminescent particles such as nanocrystals ("quantum dots" in English). Preferably, the fluorescent compound is chosen from rhodamines (in particular lissamine rhodamine B), fluoresceins, fluorophores known under the name Dy (in particular the compound known under the name Dy467), and rare earth cryptates ( in particular the compound known under the name Lumi4-Tb). The fluorescent ligand according to the invention corresponds to the general formula (1 '): Rl'HHN

Wherein R'1 represents a residue of a fluorescent compound or a group -COR'2; R'2 represents a group - (CH2) mNR'3R4 in which m = 2, 3 or 4 and R'3 represents hydrogen and R4 represents a residue of fluorescent compound, either R'3 and R4 form a group, or a group - (CH2CH2O) n (CH2) pNR'3R4 in which n = 3, 4 or 5, p = 2 or 3 and R'3 and R'4 are as defined above, or a group (Cl-1), - C Il 0 N = N / NR'6 or -N NR '6 in which R 5 represents a group - (CH2) gNR'3R4 in which q = 2 or 3 and R'3 and R'4 are as defined above, or a group - (CH2CH2O), (CH2), NR'3R4 wherein r = 3, 4, 5 or 6, s = 2 or 3 and R'3 and R4 are as defined above; R'6 represents a fluorescent compound residue; and the fluorescent compound (mentioned in the definition R'4 and R'6) is as defined above According to one embodiment, R'1 represents a group -COR'2 in which R'2 represents a group - (CH2) mNR'3R4 d in which m = 2, 3 or 4, preferably m = 2 or 4, and either R '3 represents hydrogen and R 4 represents a residue of fluorescent compound, or R' 3 and R '4 form a group I -N According to another embodiment, R'1 represents a group -COR'2 in which R'2 represents a group - (CH2CH2O) n (CH2) pNR'3R'4 in which n = 3 and p = 2, R'3 represents hydrogen and R4 represents a fluorescent compound residue. According to another embodiment, R'1 represents a group -COR'2 in which R'2 represents a group (CH1), -C0 N = N in which R'5 represents a group - (CH2) 2NR ' R'4, R'3 is hydrogen and R4 is a fluorescent compound residue. According to another embodiment, R1 represents a group -COR'2 in which R'2 represents a group (CH2) in which R'5 represents a group - (CH2CH2O), (CH2) sNR'3R'4 in which r = 3 or 6, 25 s = 2, R '3 represents hydrogen and R 4 represents a residue of fluorescent compound In another aspect, the invention relates to a ligand capable of being used for the purification of the V2 receptor. is obtained by coupling a compound of formula (I) with biotin, The coupling is conventionally carried out between a N = N residue -NH or -NH 2 carried by e compound of formula (1) and biotin via a Reactive group, for example an NHS group The compounds of formula (1) and (II) as well as the compounds (1) "biotinylated" can be prepared according to the procedure described in diagrams 1 to 6. Scheme 1 describes an example synthesis on solid support Benzazepine 2 is condensed on the bifunctional resin (acetylenic-a cide) 1 (prepared as described in Cher Eur J2008, 14, 6247-6254) using the PyBop coupling agent. It is necessary to use TMSOTf to selectively deprotect the Bacon 3 protecting group. The secondary amine 4 is then capable of reacting with p-nitrobenzoyl chloride thereby resulting in compound 5 which is reduced in the presence of sodium salts. 'tin. The aromatic amine 6 is then condensed on o-phenylbenzoyl chloride 8 to give benzazepine 9. The PEG linker is introduced through a 1,3-type dipole cycloaddition "click" in the presence of salts of copper whose reactivity has been described by B. Sharpless (Angew Chem I Ed, 2002, 41, 2596). The PEG benzazepine amine is then cleaved from the solid support in the presence of TFA to give the expected compound. Compound 13 can be similarly prepared from benzazepine 9 using derivative 11 (azido-PEG-lissamine) and then cleaving compound 12 from the solid support in the presence of TFA. Scheme 2 describes an example of solution synthesis. The condensation of synthons 7 and 14 leads to the ester 15 which is hydrolyzed in an acidic medium to give the acid 16 which is then condensed with benzazepin-5-one 16a to yield derivative 17. A reductive amination makes it possible to obtain compound 18. From this compound, compound 19 is obtained by simple coupling with a yield of 90% and compound 24 is obtained in two stages with an overall yield of 22%. The synthesis of grafted lissamine with different linkers is described in Scheme 3. For lissamine having a piperazinyl linker arm 30, the synthesis begins with a 1.4-Michael addition between monobenzylpiperazine 25 and teryl acrylate, butyl 26 to yield compound 27 in 94% yield. The secondary amine is degraded from its benzyl group by hydrogenolysis and then condensed on the lissamine chlorosulfonyl to give compound 29, the carboxylic acid function of which is deprotected by a solution of TFA. As regards the preparation of compounds 32 and 34, the synthesis is more direct since it is carried out in two stages from commercial products: a first coupling step between, on the one hand, amine-tert-butyl ester (31); or 33 and secondly the lissamine chlorosulfonyl; a second step of deprotection of the carboxyl in the presence of TFA to lead respectively to the compounds 32 and 34. The obtention of benzazepines coupled to lissamine and having different binding arms is described in the scheme 4. The couplings were carried out in a conventional manner by condensing the benzazepine amine 18 on the various derivatives of lissamine in the presence of PyBop as coupling agent, in this way the compounds 35, 36, 37 and 38 are obtained respectively. The synthesis of amines 43 and 45 is described in Scheme 5. From acetylenic benzazepine 21 a 1.3 dipole cycloaddition reaction is carried out in meadows. Figure 6 depicts obtaining fluorescent ligands from compounds 10, 43 and 45. Fluorescent probe conjugated benzazepines were prepared by reacting benzazepine amine derivatives on NHS esters of fluorescent probes. according to methods well known to those skilled in the art. The various reagents used in the syntheses described above are either commercially available or can be obtained according to procedures well known to those skilled in the art. The fluorescent probes DY647-NHS and Lumi4-Tb-NHS are available from Cisbio Bioassays. The other fluorescent probes (fluorescein isothiocyanate, lissamine) and biotin are available from Sigma-Aldrich. Scheme 1 H2N N 2 Boe PyBOP, DIEA, DMF Cl, pyridine anh, 4-DMAP, CH 2 Cl 2 anh 13 Scheme 2 NH 2 1) SOCl 2 NMP, CH 2 Cl 2 anh McO 2 Cl 2 HCl 6N / AcOH 50/50,. CO2H 2) DIEA, CH2Cl2 anh 93% 88% 7 CO2Me 14 15 NCS 1) SOCl2, NMP, CF12Cl2 anh 2) py, 4-DMAP, CH2Cl2 anh 74% 16HN 77% 4-DMAP CH2C12 0 f HO ~~ It Name / O 20 PyBOP, DIEA, DMF anh BpC NH 18 H 1) PyBOP, DIEA, DMF 2) TFA / CH2Cl2 90% CuSO4 Sodium Ascorbate, 5H20 TBTA DMF / H2O (90/10) 26% OH 0 NH2 HN EXAMPLE 3 ## STR2 ## ## STR2 ## ## STR2 ## ## STR2 ## % 94 ° the 27 28

TFA, H2O 9515 CH2O2 31 N / - 96% DIEA, DMAP CH2C121DMF 0 TFA / CH2Cl21H2O 82% HCI .H2N O Figure 4 34 PyBOP, DIEA, DMF 32, PyBOP, DIEA, DMF Lissamine FIN H N-Lissamine NH 38 NH 3 NH 3 NH-N-Lissamine Lissamine Scheme 5 NaN 3 DMF H DCM / TFA O 1 Boc 2 NN 3 Tos 83% 41 98% TFA. H2NN3 42H Boc 'NV 44H c'NOH 39 TsCi Et3N DCM 83% fNI-12 0' 8HN Sodium ascorbate CuSO4, 5H2O DMF / H2O (90110) Sodium ascorbate CuSO4, 5H2O DMF / H2O (90/10) NI-12 29% NH 43 42 21 NH2 Scheme 6 10, R1 = '[(CH2) 2O] 3- (CH2) 2 -NH2 N i N R3 43, R1 = - (CH2) 2 -NH2 45, R1 = [(CH 2) 2 O] 6- (CH 2) 2 -NH 2 46, R 3 = - [(CH 2) 2 O] 3- (CH 2) 2 -NHCO-DY 6 (74%) 47, R 3 = - [(CH 2) 2 O ] 3- (CH2) 2-NHCO-Lumi4-Tb (45%) 48, R3 = - [(CH2) 2O] 3- (CH2) 2 -NH-Biot (77%) 49, R3 = - (CH2) 2-NHCO-DY647 (40%) 50, R3 = - (CH2) 2-NHCO-Lumi4-Tb (16%) 51, R3 = - [(CH2) 2O] 6- (CH2) 2 -NHCO-DY647 ( 89 ° / a) 52, R3 = - [(CH2) 2O] 6- (CH2) 2 -NHCO-Lumi4-Tb (38%) DY647, Lumi4-Tb, Biot OHN NH1. 1H Biot = The intermediates described in Figures 6 form an integral part of the invention. Thus, according to another aspect, the invention relates, as intermediates, to the compounds of formula (II) and (III): HN The fluorescent ligands in accordance with the invention have a non-peptidic structure and are selective for the V2 receptor. to the Via, Vu, and oxytocin (OT) receptors, while maintaining a nanomolar affinity for the V2 receptor. In the context of the present invention, the term "selective" refers to a ratio of affinity constants Ki (calculated for each receiver according to the Cheng-Prusoff equation). 10, preferably 50, more preferably 100. The affinity, the selectivity and the chemical and metabolic stability of these ligands make it possible to envisage in particular: their direct use as part of a V2 ligand-receptor binding is based on in particular on the FRET-HTRF () technique; their use in vivo for labeling the visualization of the V2 receptor, in particular: in studies of homo- or dimerodimerization of the V2 receptor on native tissues; or in medical imaging for monitoring and locating V2 receptors.

The invention is illustrated by the following preparations and examples, given for information only.

Reagents The ert-butyl 5-amino-2,3,4,5-tetrahydro-1H-1-benzazepine-carboxylate was obtained according to the protocol described in 1 Comb. Chem. 2007f 9, 487-500; the azido-PEG3-lissamine rhodamine B chain as well as the "Solid Phase Organic Taging" resin (SPOrT) were obtained according to the protocol described in Chem. Eur. 1 2008, 14, 6247-54. Tert-Butyl 5-aminopentanoate was prepared according to the protocol described in Ore Chem, 2007, 72, 6162-6170. The sulfonyl chloride of lissamine rhodamine B is a commercial compound, available as a mixture of two isomers for substitution of the sulfonyl chloride group at the 2-position (ortho) or 4 (para) -ississamine. Solid Phase Syntheses The solid support reactions were carried out in polypropylene tubes equipped with polyethylene sintered and polypropylene plugs, with orbital shaking at room temperature or in a Chemflex rotating oven (Rabbins Scientific) for 60 ° C. The resins are washed in general with the following solvents: DMF (3 times), CH 2 O 2 (3 times) and MeOH (3 times). Each reaction is monitored using colorimetric tests adapted to the functions to be identified (Biotechnology and bloengineering (Combinatorial Chemistry) 2000/2001, 71 (2), 110-118, 1 Comb Chem., 2004, 6, 165-170 , 1 Comb Chem 2004, 6, 805-810).

Microwave Reactions (MW) Microwave irradiations were performed using an Initiator 2.0 host.

Chromatography Thin-layer chromatography was performed on Merck60 F254 silica gel plates on aluminum foil. The analytical, semi-preparative and preparative high pressure liquid chromatography (HPLC) were respectively carried out on the following apparatus: P580 Dionex chain: ^ Analytical column Speer chroose: 1WD RP18, 4.6 x 50 mm ^ Semi-preparative column: Waters SymrrtetryShr`eld RP18, 0.7pm, 19 x 300 mm Chain Agitate Technologies 1200 Series: ^ Analytical column 2: Agitate a./Ose XDB-C18, 5 μm, 4.6 x 150 mm Shimadzu LC-8A Chain ^ Preparative Column: Waters X-Bridge Column, C18, 5 pm, 19 x 100 o Preparative Column: Vydac C18, 5 pm, 250 x 10

Eluents: A: H 2 O / TFA 0.1% B: MeOH / H 2 O 95/5 + 0.1% TFA C: H 2 O / TFA 0.2% D: MeCN E: H 2 O / 25 mM TEAAc, pH 7

Gradients: gradient 1 Analytical column 2 Flow 1 mL.min-1, 2 min at 10% B then 5 in 10 to 100% B

gradient 2 Analytical column 1 Flow rate 7 mLmin -1, 1 min at 0% C, then 5 min from 0 to 100% C

gradient 3 Semi-preparative column Flow rate 10 mL.min-1, 5 min at 5% of B, 25 min of 5 at 100% of B then 5 min at 100% of B gradient 4 Semi-preparative column Flow rate 10 mLmin-1 , 5 min at 5% of B, 8 min of 5 at 40% of B, 15 min of 40 at 70% of B, 8 min of 70 at 100% of B then 5 min at B

gradient 5 Waters preparative column Flow 20 mL.min-1, 2 min 85% C, 15% D; 17 min from 15% to 70% of D, min from 70% to 100% of D

gradient 6 Vydac preparative column Flow rate 5 mL.min-1, 2 min 90 ° L E, 100 / o D, 17 min from 10% to 70% D, 1 min from 70% to 100% D

The detection is carried out at 220, 254, 320 and 530 nm. The retention times (Tr) are expressed in minutes (min).

Silica column chromatography was performed with Merck 60 silica gel (230-400 Mesh ASTM).

Purifications by flash chromatography were carried out using the Armen Spot IL apparatus.

Eluents: F: 0 G: MeCN Gradient: gradient 7 Column RP18 Flow Rate 15 mL.min-, 0 in 5 to 100% G

Mass Spectrometry (MS) The mass spectra (LC-MS) were performed using an ESI / AFCI multimode source single-quadrupole MSDS 1200 SL spectrometer equipped with a Thermo Hypersft GOLO 1.9 column. The high resolution mass spectra (HR-MS) were obtained using the Agitate Q.Tof apparatus equipped with an electrospray ionisation source and a time-of-use analyzer. ESI-TOF flight).

Nuclear Magnetic Resonance (NMR) NMR spectra were recorded at 200, 300, 400 and 500 MHz on Brucker Advance spectrometers. The chemical shifts (δ) were expressed in parts per million (ppm) and the coupling constants in hertz (Hz) compared to standard deuterated solvents. The following abbreviations are used: s: singulet sl: singlet wide d: doublet dd: doublet of doublets dt: doublet of triplets t: triplet triplet of doublets triplet wide quadruplet multiplet app: apparent td: tl: q: Melting point (PO Melting points were measured on a Sikh / B-540 apparatus.

List of abbreviations 4-DMAP N, N-dimethyl-4-aminopyridine AcOEt ethyl acetate AcOH acetic acid Ala alanine Boc tert-butylcarbonate degree Celsius DIEA N, N-diisopmpylethylamine DMF dimethylformamide DMSO dimethylsulfoxide DY647 fluorophore based on pentamethine marketed by Dyomics DY647-NHS N-hydroxysuccinimide derivative of DY647 eq. Equivalent Et2O h Hept Lumi4Tb Lumi4Tb-NFiS MeCN MeOH min NEt3 NH40Ac NHS NMP PEG Pf PY PyBOP Rf NMR SPOrT TA TBTA TEAAc TFA TMSOTf Ethyl ether hour heptane fluorophore marketed by Cisbio Bioaasay derivative N-hydroxysuccinimide of Lumi4Tb acetonitrile methanol minute triethylamine acetate ammonium N-hydroxysuccinimide N-methyl-2-pyrrolidone polyethylene glycol melting point pyridine hexafluorophosphate (benzotriazol-1-yloxy) tripyrrolidinophosphonium frontal report nuclear magnetic resonance "solid phase organic labeling" (solid phase organic tagging) room temperature Triethylammonium acid trifluoroacetic acid trifluoroacetic acid trimethylsilyl trifluoromethanesulphonate trifluoroacetate retention time Preparation 1 PyBOP, DIEA, DMF OH To the resin SPOrT-OO 2 H (0, 1, 2, 3, 4-yl) methyl] amine 55 mmol) swollen in DMF, was added a solution containing 5-amino-2,3,4,5-tetrahydro-1H-1-benzazepine-1-carb tert-butyl oxylate (193 mg, 0.74 mmol) in the presence of PyBOP (574 mg, 1.10 mmol) and DIEA (257 μL, 1.47 mmol) in DMF (4 mL). The reaction medium was stirred overnight at RT, protected from light. After filtration and washing, the coupling was confirmed by the colorimetric malachite test (negative).

Preparation 2 To a solution of the compound of Preparation 1 (0.55 mmol) in anhydrous CH 2 O 2 was added a solution of TMSOTf (200 μL, 1.10 mmol) in the presence of NEt 3 (78 μL, 0.55 mmol). ) in anhydrous CH2Cl2 (3 mL). The mixture was stirred at RT for 10 min. After filtration and washing, the presence of the deprotected amine was confirmed by a chloranil (positive) colorimetric test. Preparation 3 ooc NO 2, NO 2 4 pyridine anh, 4-DMAP, CH 2 Cl 2 anh 5 To a solution of the compound of Preparation 2 (0.55 mmol) in anhydrous CH 2 O 2 was added a solution of 4-nitrobenzoyl chloride. (341 mg, 1.84 mmol) in the presence of anhydrous pyridine (296 μL, 3.68 mmol) in anhydrous CH 2 Cl 2 (4 mL). After addition of 4-DMAP (5 mg, 0.04 mmol), the reaction medium was stirred at RT overnight under the exclusion of light. After filtration and washing, the coupling was

Confirmed by a chloranil colorimetric (negative) test. Preparation 4 SnC12.2H2O NO2 DMF NH2 In a microwave reactor, the compound of Preparation 3 (0.12 mmol) was swollen in DMF. A solution of SnCl 2, 2H 2 O (543 mg, 2.4 mmol) in DMF (2 mL) was added and the mixture was stirred at RT overnight. After filtration and washing with DMF / AcOH (75/25) ( 3 times), DMF / NEt3 (90/10) (3 times), DMF (3 times), CH2Cl2 (3 times), MeOH (3 times), the reduction was confirmed by a kaiser (positive) colorimetric test.

Preparation 5 NH 2 CH 2 Cl 2 anh To a solution of the compound of Preparation 4 (0.12 mmol) in anhydrous CH 2 O 2, a solution of o-phenylbenzoyl chloride (132 mg, 0.61 mmol) was added in the presence of DIEA (214 μL, 1.22 mmol) in anhydrous CH 2 Cl 2 (1 mL). After addition of 4-DMAP (14 mg, 0.12 mmol), the reaction medium was stirred at RT overnight under the exclusion of light. After filtration and washing, the coupling was confirmed by a chloranil (negative) calorimetric test. Example 1 H2N N3 1) 2) TFA / H2O (9515) 9 HNA a solution of the compound of Preparation 5 (0.05 mmol) in DMF / Piperidine 20%, Cul (44 mg, 0.23) was added mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (27 μL, 0.14 mmol) in DMF / piperidine 20% (1.3 mL). The reaction medium was stirred for 6 h at RT, protected from light. After filtration and washing with DMF / piperidine 20% (3 times), DMF (3 times), CH 2 O 2 (3 times), CH 2 Cl 2 / MeOH (3 times) and MeOH (3 times), the compound was dried at room temperature. 'ether. After cleavage of the resin in TFA / H20 (95/5) for 3 h, semi-preparative column purification (gradient 3) and lyophilization, a colorless oil was obtained (20 mg, 54%). Tr (gradient 1): 13.03. HR-MS (EI) calcd: 817.4032, found: 817.4022. 1H NMR (400 MHz, CDCl3): δ 10.26 (s, 1He), 8.55 (d, J = 7.6Hz, 1H), 8.37 (t, J = 5.6Hz, 1H), 7.75 (st, 2H); ), 7.57-7.52 (m, 2H), 7.48-7.43 (m, 2H), 7.38-7.21 (m, 9H), 7.16 (t, J = 7.6Hz, 1H), 6.98 (t, J); = 6.4 Hz, 1H), 6.63 (d, J = 7.4 Hz, 1H), 5.14 (si, 1H), 4.43 (t, 1 = 5.1 Hz, 2H), 4.30 (t, 1 = 6.0 Hz , 1H), 3.76 (t, .1 = 5.3 Hz, 2H), 3.5 (sl, 1H), 2.96 (q, 1 = 10.9, 5.4 Hz, 2H), 2.44 (q, _7 = 13.8, 7.2 Hz, 2H), 1.99 (si, 1H), 1.88 (si, 1Fi), 1.57 (si, 2H).

Preparation 6 To a solution of the compound of Preparation 5 (0.07 mmol) in DMF / piperidine 20% was added successively Cul (67 mg, 0.35 mmol) in a DMF / piperidine mixture 200/0. (1 mL), then the azido- (PEG) 3-lissamine fluorophore [obtained as described in Chem Eur 2008, 14, 6247-6254] (161 mg, 0.21 mmol) in a DMF / piperidine 20% mixture ( 0.9 mL). The reaction medium was stirred for 6 h at RT, protected from light. After filtration and washing with DMF / piperidine 200/0 (3 times), DMF (3 times), CH 2 Cl 2 (3 times), CH 2 Cl 2 / MeOH (3 times), MeOFI (3 times) and then with ether, it was obtained the lissamine-coupled compound which is used in the subsequent synthesis without further purification.

Example 2 TFAIH20 (95/5) H N 13 A solution of TFA / H20 (95/5) was added to the compound of Preparation 6 (0.07 mmol) and the mixture was stirred at RT for 5 minutes. 3 hrs. The thus obtained compound in solution was recovered after filtration and rinsing with CH2Cl2 and IvIeOH. After purification by semi-preparative column chromatography (gradient 4) and lyophilization, two products were obtained in the form of ortho and para-lissamine isomers (65 mg, 68%).

25 mg (26%) ortho isomer were obtained: 1 H NMR (400 MHz, CD30D): 8.73 (s, 1H), 8.36 8.26 d, 1 = 7.8 Hz, 1H), 8.11 (si, 1H). ), 7.70 (d, 1 = 7.6 Hz, 1H), 7.46 (d, = 7.6 Hz, 1H), 7.38-7.22 (m, 11H), 7.12 (si, 1H), 7.10 (si, 1 H), 7.09 (if, 1H), 7.01 (d, 1 = 8.8 Hz, 2H), 6.90-6.86 (m, 3H), 6.81 (d, _1 = 8.8 Hz, 2H), 6.69 (s). , 2H), 6.52 (d, J = 7.6Hz, 1H), 5.26 (if, 1H), 4.58 (if, 1H), 4.51 (t app, l = 8.2 Hz, 1H), 4.40 ( if, 1H), 3.80 (if, H), 3.57-3.47 (m, 16H), 3.07 (if, 1H), 3.02 (if, 1H), 2.84 (if, 2H), 2.73 ( if, 1H), 2.07 (if, 1H), 1.94 (st, 1H), 1.75 (if, 1H), 1.59 (if, 1H), 1.29 (t, 1 = 6.5Hz, 12H) . Tr (gradient 2): 4.04. HRMS (EI): calcd. For C72H82NI0013S2 679.2747, found: 679.2746.

40 mg (42%) para isomer were obtained: 1 H NMR (400 MHz, CD30D): 8.77 (s, 1H), 8.19 (s, 1H), 8.06 (si, 1H), 8.03 (d, 1 = 8.0 Hz, 1H), 7.90 (if, 1H), 7.71 (d, .1 = 7.7 Hz, 1H), 7.46 (d, 1 = 7.7 Hz, 1H), 7.39-7.20 (m, 11H), 7.16 (d, J = 7.7Hz, 1H), 7.12 (s, 1H), 7.10 (s, 1H), 7.04 (t, 1 = 7.7 Hz, 1H), 7.00 (d, 1 = 8.7 Hz, 2H), 6.85 (t, = 7.3 Hz, 1H), 6.73 (t, = 10.0 Hz, 20H), 6.64 (s, 2H), 6.48 (d, J = 7.7 Hz, 1H), 5.14 (s, 1H), 4.50 (s, 2H), 4.42 (s, 1H), 3.86 (tl, = 4.8 Hz, 1H), 3.64 (tl, .1 = 4.8 Hz, 1H), 3.60-3.48 (m, 14H), 3.27 (s, 2H), 2.96-2.91 (m, 1H), 2.65 (d app, 1 = 7.2 Hz, 2H), 2.59-2.54 ( m, 1H), 1.83 (if, 2H), 1.48 (if, 2H), 1.25 (t, J = 6.9 Hz, 12H). Tr (gradient 2): 4.13. HRMS (EI): calcd for C72H82N10013S2 679.2747, found: 679.2740.

Preparation 7 1) SOCl2, NMP, CH2Cl2 anh 2) DEA, CH2Cl2 anh 93% 14 CO2H +

To a solution of 2-biphenylcarboxylic acid (1 g, 5.0 mmol) in 2.5 ml of anhydrous CH 2 O 2 to 0 ° C under argon was added a few drops of NMP and 9 ml of thionyl chloride. mmol). The mixture was stirred for 30 minutes at 00 ° C. and then for 3 hours at RT. CH 2 Cl 2 was then evaporated and the residue dissolved in 5 mL of anhydrous CH 2 O 2. This solution was added dropwise at 0 ° C under argon to a mixture of methyl 4-aminobenzoate (378 mg, 2.5 mmol) and MEA (860 μL, 5.0 mmol) in 4 mL of anhydrous CH 2 Cl 2. Stirring was continued for 15 min at () oc and then 2 h at RT. The mixture was then diluted in CH 2 Cl 2 and washed with water, 10% citric acid and 1M NaOH. The organic phase, dried over Na 2 SO 4, was then evaporated and the residue precipitated in AcOEt. The product was obtained as a white powder (774 mg, 93%). Rf (Hept / AcOEt 50/50): 0.6. Tr (gradient 4.74): 150-1520 ° C. 1H NMR (300 MHz, CDCl3): δ 7.85-7.93 (m, 3H), 7.35-7.60 (8H), 7.17 (d, = 8.4 Hz, 2H) , 7.05 (if, 1 H), 3.87 (s, 3 H) 13C NMR (75 MHz, CDCl3): δ 167.1, 166.5, 141.7, 139.7, 139.6, 134.7, 131.0, 130.6, 130.4, 129.7, 129.1, 128.8 128.3, 128.0, 125.6, 118.7, 52.0 Preparation 8 HO 6N AcOH 1/1 88% 15 A solution of the compound of Preparation 7 (500 mg, 1.51 mmol) in 50 mL of a 6N HCl mixture 1: 1 was refluxed for 4 h The mixture was then poured into water / ice A white powder (419 mg, 88%) was obtained after filtration and drying under vacuum. Hept / AcOEt 50/50): 0.5 Tr (gradient 1): 13.59, mp: 236-238 ° C. 1H NMR (200 MHz, DMSO-d6: δ 7.20-7.75 (m, 11), 7.84 (d, J = 8.6 Hz, 2, 10.54 (s, 1H), 12.46 (s, 1H), 13C NMR (75 MHz, DMSO-c / 6): 168.2, 166.9, 143. 40.9, 139.9, 139.3 6.7, 130.2, 130.0, 128.3, 128.1, 127.8, 127.3, 27.2, 125.4, 118.8 Preparation 9 1) SOCl2, NMP, CH2O2 anh 2) py, 4-D1MP, CH 2 Cl 2 anh 74% NH 17 A mixture of the compound of Preparation 8 (200 mg, 0.63 mmol), thionyl chloride (142 μL, 1.94 mmol) and NMP (few drops) in anhydrous CH 2 Cl 2 (5 mL) was stirred overnight under argon at RT. The medium was evaporated to dryness and taken up in anhydrous CH2Cl2 (5 mL). This solution was slowly added to a mixture at 0 ° C under argon containing 1,2,3,4-tetrahydro-5H-1-benzazepin-5-one (78 mg, 0.48 mmol), anhydrous pyridine ( 117 μL, 1.45 mmol) and 4-DMAP (12 mg, 0.10 mmol) in anhydrous CH 2 O 2 (2 mL). The medium was stirred at RT for 4h then

Diluted in 5 mL of CH2Cl2, washed with 1N HCl and saturated NaHCO3. The organic phase was dried over Na2SO4 and evaporated. After purification on a silica column (Hept / AcOEt 50/50), a beige powder (165 mg, 74%) was obtained.

Rf Hept / AcOEt 40/60): 0.4. Mp 128-130 ° C. Tr gradient 1): 14.39.

1H NMR (200 MHz, CDCl3): δ 7.58-7.51 (m, 2H 7.28-7.08 (m, 2H), 6.98-6.91 (m, 2H), 6.78 (t app, 1 = 8.6 Hz, 3). H), 6.65 (dd, J = 8.6 Hz, 2H), 6.40 (dd app, J = 7.0, 1.7 Hz, 1H), 3.18 (q, J = 7.1 Hz, 4H), 2.56 (t, = 6.3 Hz, 2H) NMR (75 MHz, CDCl3): δ 202.2, 170.0, 167.4, 143.2, 139.8, 139.7, 135.0, 134.4, 133.1, 130.9, 130.7, 129.8, 129.5, 129.1, 129.0, 128.8, 128.2, 127.9, 118.8, 40.3, 29.8, 22.8 MS (ES) m / z 461.0 ([M + H]) Example 3 A mixture of the compound of Preparation 9 (53 mg, 0.12 mmol) , 532 mg NH40Ac, 6.91 mmol) and NaBF13CN (36 mg, 0.58 mmol) in MeOH (2.5 mL) was irradiated microwaves for 5 min at 1000C under 1 bar. Once the medium evaporated and taken up in CH 2 Cl 2, it was washed with saturated NaHCO 3 and then with saturated NaCl. After purification on a semi-preparative column (gradient 3) and lyophilization, a white powder (31 mg, 57%) was obtained. Rf (AcOEt / MeOH 90/10 + E NEW: 0.1 Tr (gradient 1): 1251. Mp: 177-1780 C. NMR (400 MHz, DMSO-c / 6): δ 10.30 (s, 1H), 8.74 (if, 2H), 7.58-7.53 (m, 2H), 7.49-7.44 (m, 2H), 7.39-7.29 (m, 9H), 7.14-7.09 (m, 3H), 6.76 (d, m.p. , J = 7.1 Hz, 1H), 4.73 (st, 1-1), 4.58 (si, 1H), 2.82 (t, J = 11.3 Hz, 1H), 2.19 (st, 1H), 1.92 (si, 1H), 1.65 (si, 2H) HR-MS (EI) calculated: 462.2176, found: 462.2171.

Example 4 A mixture of the compound of Example 3 (41 mg, 0.09 mmol), Boc-B-Ala-OH 2 mg, 0.13 mmol) in the presence of PyBOP (70 mg, 0.13 mmol) and DIEA (70 μL, 0.40 mmol) in DMF (1.5 mL) was stirred under argon for 15 min. Once the DMF evaporated and the residue taken up in TFA / CH2Cl2. (50/50) (2 mL), the mixture was stirred for 1 min before being evaporated. The residue was purified on a semi-preparative column (gradient 3) to obtain after lyophilization a gray powder (42 g, 90 ° 10) mp: 184 ° C, Tr (gradient 2): 3.79 H NMR (400 MHz, DMSO-c / 6): δ 10.30 (s, 1H, NH), 8.81 (d, J = 7.2 Hz, 1H, NH), 7.75 (s, 2H, NH 2), 7.58-7.52 (m, 2 H), 7.49-7.44 (m, 2H), 7.38-7.28 (m, 8H), 7.24-25 7.20 (m, 3f), 7.01 (t, J = 7.2Hz, 1H,), 6.66 ( d, 1 = 7.2 Hz, 1H), 5.20 (si, 1H), 4.47, 3.08 (s, 2H), 2.92 (t, J = 11.2 Hz, 2.67 (s, 2H), 2.01 (s. 1.60 (si, 2 H 0.) YNH 2 HN 19 HR-MS (EI) calculated: 533.2547, found: 533.2567 Preparation 4-DMAP 0 NH 2 77% 24 A solution containing succinic anhydride (150 mg, 1, 49 mmol) and propargylamine (85 μL, 1.24 mmol) was stirred at RT for 1 h in the presence of 4-DMAP (152 mg, 1.24 mmol) in CH 2 Cl 2 (4 mL). It was extracted with 5% Na2CO3 and then the aqueous phase was acidified with 1N HCl.The product was extracted with AcOEt, dried over Na2SO4 and then under vi. A white powder (149 mg, 77%) was obtained. Rf (CH2Cl2 / MeOH + AcOH): 0.5. Mp: 98-1000C. 1 H NMR 300 MHz, CDCl3):? 7.17 (st, 1H), 3.91 (s, 2H), 2.58-2.39 (m, 4), 2.18 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 175.6, 172.6, 79.5, 71.4, 30.5, 29.3, 29.1.

Preparation 11 HAN 0

) LY-1HNO NHPyBOP, DIEA, DMF 87% 18 NH 21 A solution containing the compound of Example 3 (15 g, 0.10 mmol in the presence of PyBOP (61 mg, 0.12 mmol) and DIEA (51 μL, 0.29 mmol) in anhydrous DMF (0.3 mL) was stirred 5 min at RT then the solution containing the compound of Preparation 10 (54 mg, 0.117 mmol) in anhydrous DMF ( 0.6 mL) was added slowly, the mixture was stirred at RT for 16 h, the residue was evaporated and taken up in AcOEt, washed with saturated NaHCO3 and then with saturated NaCl. organic on Na2SO4 and evaporation, a colorless oil (47 mg, 87%) was obtained.

Tr (gradient 1): 14.18. 1H NMR 400 MHz, CD30D: δ 7.50 (t, J = 7.6 Hz, 2H), 7.36-7.4, 4H), 7.15-7.28

(m, 10 F), 6.95 (t, J = 7.8 Fiz, 1-1), 5.24-5.27 (m, 3.93 = 2.2 Hz, 2H), 3.27

4), 2.60-2.67 (m, 2H), 2.52-2.55 (m, 3H), 97-206 (m, 2H 1.60-1.67 (m, 2F), 1.25 ppm (s, 1H). .

13C NMR (75 MHz, CD30D): δ 141.9, 141.9, 141.4, 141.4, 141.4, 141.4, 140.4, 137.8, 132.5, 131.4, 131.3, 130.3, 129.7, 129.6, 129.5, 129.5, 128.9, 128.6, 128.5, 128.4, 125.6, 120.1, 80.7, 72.3, 51.8, 48.0, 32.7, 31.9, 30.8, 29.6, 26.6.

MS (ES) tn / z 599.2 ([M + H] +.) Preparation 12 S H2N N3 HNN - "---- 0 ------- 0 -" 0 '-' N3 H 22 23 added at 0 ° C and under argon 11-azido-3,6,9-trioxaundecan-1-amine (43 μL, 0.21 mmol) to a solution of fluorescein isothiocyanate (100 mg, 0.26 mmol) in the presence of NEt3 (30 μL, 0.21 mmol) in DMF (3 mL) The reaction mixture was stirred at RT for 2 h and the DMF was evaporated After purification by flash chromatography (gradient 5) and lyophilization, an orange powder was obtained (140 mg, 87%), Tr (gradient 1): 12.85, mp: 116-118 ° C. 1H NMR (400 MHz, CD30D): 8.16 (s,, 7.78 (d , = 8.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 6.69-6.67 (m, 4H), 6.55 (s, 1H), 6.53 (s, 1H), 3.81 (if, 2H 3.72-3.61 (m, 13H), 3.34-3.31 (m, 3H) .3c 75 MHz NMR, CD30D): δ 171.6, 161.7, 154.3, 130.3, 125.8, 113.7, 111.6, 103.7, 71.8, 71.7, 71.6, 71.5, 71.2, 51.9, 45.7 HRMS EI) calcd for C29H3ON5O8S 608.1810, found: 608.1823 HO, HO NEt3, DMF 87% NCS 2 Exemp To a solution containing the compound of Preparation 11 (2 g, 0.04 mmol) and the compound of Preparation 12 (26 mg, 0.04 mmol) in a DMF / H 2 O (90/10) mixture.

(0.8 mL) was added a solution of sodium ascorbate (1 mg, 0.005 mmol) in a minimum of H 2 O and then TBTA (2 mg, 0.005 mmol) in a minimum of DMF and finally CuSO 4. 5H 2 O (0.6 mg, 0.004 mmol) in H 2 O. After stirring for 5 h at RT, the DMF was evaporated and the residue purified by semi-preparative column chromatography (gradient 4). An orange powder (12 mg, 26%) was obtained after

0 lyophilization.

Rf (CH 2 Cl 2 / MeOH 95/5 + 1% AcOH, 0.7, Tr (gradient 1): 14.48, mp 154-155 ° C. HRMS (EI) calcd 1205.4317, found: 1205.4412 Preparation CHO311VIeOH Bn-N NH + N-Bn-N N / 94% A solution of tert-butyl acrylate (12.8 g, 100 mmol) in 50/50 CHCl3 / MeOH (10 mL) ) was added N-benzylpiperazine (588 mg, 3.33 mmol). The reaction medium was stirred at reflux (80 ° C.) for 8 h. It was evaporated and then diluted in CH 2 O 2 to be filtered through silica (eluent CH 2 O 2). A colorless oil (950 mg, 94% was obtained after evaporation of the filtrate Rf (CH2Cl2 / MeOH 92/8): 0.7.1H NMR (300 MHz, CDCl3): δ 7.32-7.26 (m, 5H s, 2 H, 2.67 (t, = 7.0 Hz, 2H), 2.50 (s, 2.41 7.0 Hz, 2 s, 9H), Preparation 4 To a solution of the compound of the preparation 13 940 mg, 3.1 mmol ) in MeOH (20 mL) was added 10% Pd / C (200 mg) The mixture was stirred 15 h under H2 (2 bar)

then the medium was filtered through celite® to give after evaporation a colorless oil (650 g, 98%). The product is used in the rest of the synthesis without additional purification. Preparation Lissamine-SOCl 2 DIEA, CH 2 Cl 2 HN N 47 ° 10 28 0

To a solution of the sulfonyl chloride of lissamine rhodamine B (4.53 g, 7.88 mmol) in CH 2 O 2 (50 mL) was added the compound of the Preparation 14 (1.94 g, 9 mmol), followed by DIEA (6 mL, 34.45 mmol). The mixture was stirred overnight at RT and was evaporated. After purification by chromatography on a silica column (CH 2 Cl 2 / MeOH 97/3), two products were obtained, namely the ortho and para isomers of lissamine. After freeze-drying, para isomer was obtained as a pink powder (2.81 g, 47 °). MS (ES) m / z 756.2 (M + H) Preparation 16 TFAM20 9515 CH 2 Cl 2 -O 2 O 2 O 2 S-NN 96% N + 29 N + A A solution containing the compound of Preparation 15 (2.1 g, 2, 78 mmol) in CH 2 Cl 2 (100 L) was added a 95/5 TFA / H 2 O mixture (100 mL) The mixture was stirred at RT for 24 h before being evaporated The oily residue was solubilized in CH 2 Cl 2 to be precipitated in Et 2 O. A dark pink solid (2.18 g, 96 was obtained after filtration Tr (gradient 1): 11.97 1 H NMR (400 MHz, DMSO d 6): δ 8.09 d, 1.9 Hz , 1H, 7.75 (dd, 1 = 8.1, 1.9 Hz, 1H), 7.37 (d, 1 = 8.1 Hz, 1H), 6.81-6.73 (m, 6H 3.45-3.39 (8H), 3.24-2.77 (m, 8H), 2.50 (t, 1 = 7.4 Hz, 2H), 2.28-2.26 (m, 12H), 13c NMR (75 MHz, DMSO-c / 6): δ 171.3, 157.1, 156.8, 155.0, 148.3, 135.4, 134.5, 132.6, 131.3, 127.8, 126.5, 114.8, 113.5, 113.4, 95.5, 51.4, 50.5, 45.3, 43.2, 28.5, 12.4 HR-MS (EI) calculated for C34H43N4O8S2 699.2517, found 699.2543.

Preparation 17 Lissamine-SOCl2 DIEA NH2. CH 2 Cl 2 59% tert-butyl 3- [2- (2-aminomethoxyethoxy) ethoxy] propionate (1.83 g, 6.6 mmol) was added followed by DIEA (6 mL, 34.45 mmol). ) at 0 ° C to a solution of sulfonyl chloride of lissamine rhodamine B (3.46 g, 6 mmol) in anhydrous CH2Cl2 (90 mL). The mixture was stirred 2 h at RT and then diluted with CH 2 M (8 mL) to extract with 0.1 N HCl followed by saturated NaCl. After drying the organic phase over Na 2 SO 4, evaporation and purification by silica column chromatography (CH 2 Cl 2 / MeOH 95/5), a dark pink powder 2.9 g, 59% was obtained. MS (ES) m / z818.2 ([M + H] +). Preparation 18 To a solution of the compound of Preparation 7 2.9 g, 3.55 mmol) in CH 2 Cl 2 (50 mL) was added a 95/5 TFA / H 2 O mixture (50 mL). The mixture was stirred at RT for 3h before being evaporated. The oily residue was solubilized in CH2Cl2 to be precipitated in Et2O. A dark pink solid (2.45 g, 91%) was obtained after filtration. Tr (gradient 1): 13.43. 1H NMR (400 MHz, CDCl3): δ 8.65 (s, 1H), 7.91 d, = 8.0 Hz, 1H), 7.16 (d, 1 = 8.0 0 Hz, 1H), 7.06 (d, 1 = 9.5 Hz, 2H), 6.71 (dd, = 9.5, 2.5Hz, 2H), 6.60 (d, 1 = 2.3 Hz, 2H), 3.63 (t, 1 = 6.5Hz, 2H), 3.52-3.41 ( m, 18H), 3.10 (t, 1 = 5.3 Hz, 2 1-1), 2.45 (t, = 6.4 Hz, 2H), 1.17 (t, = 7.2 Hz, 12H). 13C NMR (75 MHz, CDCl3): δ 173.7, 157.8, 157.6, 155.5, 146.9, 142.2, 133.7, 132.9, 130.2, 127.5, 126.9, 114.1, 113.6, 95.7, 70.4, 70.3, 70.2, 70.1, 69.7, 66.6, 45.8, 42.9, 34.8, 12.4. MS (ES) m / z 762.1 ([M + H] +).

Preparation 19 HCl .H2N DIEA 9 mL, 5.20 mmol) was added at 0 ° C. to a solution of tert-butyl 5-aminopentanoate (200 g, 0.9 mol) and sodium sulphonyl chloride. lissamine rhodamine B (501 mg, 0.87 mmol) in the presence of 4-DMAP (11 mg, 0.09 mmol) in the mixture CH2Cl2 / DMF 80/20 (9 mL). The mixture was stirred for 30 minutes at 00 ° C. and then overnight at RT. The DMF was then evaporated and the residue taken up in CH 2 O 2 to be washed with HCl (0.1N). After drying the organic phase over Na 2 SO 4 and evaporation, a residue was obtained which is used in the synthesis without further purification.

Preparation The compound of Preparation 19 was taken up in TFA / H 2 O (95/5) in CH 2 Cl 2. The mixture was stirred at RT for 3h before being evaporated. The oily residue was solubilized in CH2Cl2 to precipitate in Et2O. A dark pink solid (470 mg, 82%) was obtained after filtration. Tr (gradient 1): 15.42. 1H NMR (400 MHz, DMSO d6): 8.41d, .7 = 1.9Hz, 1H), 7.93 (dd, J = 8.1, 1.9Hz, 1H), 7.47 (d, 1 = 8.1Hz, 1H) , 7.05 (d, 1 = 2.2Hz, 1H), 7.03 (d, 1 = 2.2Hz, 1H), 6.99-6.96 (m, 2H), 6.94 (d, 1 = 2.2Hz, 2H), 3.67-3.61 (m, 8H), 2.89-2.84 (m, 2H), 2.19 (t, 1 = 7.1 Hz, 1H), 1.53-1.42 (m, 4H), 1.23-1.19 (m, 12H). 13C NMR (75 MHz, CDCl 3): δ 157.9, 157.3, 155.7, 142.6, 133.9, 132.9, 130.3, 127.9, 126.8, 1 14.2, 113.8, 95.8, 49.2, 46.2, 45.9, 42.7, 33.5, 28.9, 21.8, 2.5 . HR-MS (EI) calcd for C32H40N3O8S2 658.2251, found: 658.2258.

Example 6 PyBop, 67% DIEA DMF To a solution of the compound of Preparation 16 (1 eq) in the presence of PyBOP (1 eq) and DIEA (5 eq) in anhydrous DMF was added the compound of the Example 3 (15 g 0.03 mmol, eq). The reaction mixture was stirred for 2 h at RT then the mixture was evaporated to be purified by flash chromatography (gradient 7). There was thus obtained a pink powder (24 mg, 67 ° 10), Tr (gradient 1): 14.54, mp: 233-2340 C. NMR (400 MHz, CD30D): δ 8.61 (s, 1H), 8.02 (d , J = 8.1 Hz, 1H), 7.53-7.21 (m, 16H), 7.10-6.94 (m, 10H), 6.83-6.67 (m, 4H), 6.59 (d, 1 = 7.5Hz, 1H). H), 5.30 (d, J = 10.0 Hz, 1H), 4.54 (if, 1H), 3.65-3.54 (m, 8H), 3.27 (if, 3H), 3.04 (si, 1H), 2.87-2.28 (m, 7H), 2.05 (s, 2H), 1.66 (s, 2H), 1.30-1.26 (m, 12H) .13C NMR (75 MHz, CD30D): δ 173.5, 171.2, 170.9, 159.1, 157.4, 156.9, 147.6, 141.7, 141.2, 139.9, 139.0, 137.4, 135.9, 133.6, 133.5, 132.3, 132.0, 131.2, 130.3, 129.8, 129.5, 129.3, 128.8, 128.6, 128.6, 128.4, 128.3. , 125.6, 119.9, 115.1, 114.9, 96.9, 54.9, 53.0, 51.6, 47.2, 46.8, 34.0, 32.4, 26.3, 13.0 HR-MS (EI) calculated for C64H68N7O9S2 1142.4514, found: 1142.4495.

EXAMPLE 7 This compound was obtained in the form of a pink powder (15 mg, 40%) according to the protocol described in Example 6 using the compound of the present invention. Preparation 18 5 mg, 0.03 mmol) instead of the compound of Preparation 16. Tr (gradient 1): 15.54. Mp 182-183 ° C. 1H NMR (400 MHz, CDCl3): δ 8.83 (s, 1H), 8.0 d, = 7.9 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 7.6 Hz , 1H), 7.41-7.27 (m, 0H), 7.21-7.18 (m, 3H), 7.06 (t, J = 7.7 Hz, 2H), 6.85-6.73 (m, 4H), 6.64 ( if, 2), 6.47 (d, J = 7.6 Hz, 1H), 5.18 (si, 1H), 4.50 (sl 1H), 3.85 (t, J = 6.2 Hz, 2H), 3.71-3.65 ( m, 10H), 3.56-3.46 (m, 10H), 3.25 (si, 2.92 (si, 1H), 1.90 (si, 2H, 1.63, 1H, 1.54 (si, 1H)). , 1.27-1.24 (m, 12H).

NMR (75 MHz, CDCl3):? 158.4, 158.0, 155.8, 142.7, 140.9, 139.9, 139.1, 133.8, 133.4, 130.8, 130.5, 130.2, 129.8, 129.5, 129.1, 128.8, 128.3, 128.2, 127, 9, 127.9, 127.6, 127.4, 127.3, 125.1, 118.8, 114.5, 113.9, 95.9, 77.4, 70.5, 70.4, 70.4, 70.3, 69.9, 67.9, 50.9, 46.9, 46.1, 43.3, 36.8, 31.2, 25.6, 12.8. HR-MS (EI) calcd for C66H73N6O12S2 1205.4722, found: 1205.4703.

Example 8 PyBOP, Dl DMF, 50% 18 / Lissamine NH This compound was obtained as a pink powder (5 mg, 50%) according to the protocol described in Example 6 using the compound of Preparation (4 mg, 0.01 mmol) in place of the compound of Preparation 16. Tr (gradient 1): 15.54. M.p .: 202-203 ° C. 1H NMR (400 MHz, CDCl3): δ 8.65 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 6.8 Hz, 4H), 7.23-7.16 (m, 7H), 7.11-7.04 (m, 5H), 6.84 (t, J = 7.5 Hz, 1 H), 6.76 (td, J = 9.5, 2.3 Hz, 2Fi), 6.63 (s, 2H), 6.46 (s, J = 7.5Hz, 1H), 5.19 (d, J = 10.4Hz, 1H). Fi), 4.46 (si, 1H), 3.53-3.46 (m, 8H), 3.30-3.29 (m, 1H), 2.95 (si, 1H), 2.27 (t, J = 7.6 Hz, 2H). ), 1.99 (if, 1H), 1.88 (if, 1H), 1.75-1.66 (m, 2H), 1.59-1.51 (m, 4H), 1.35-1.30 (m, 4F), 1.24- 1.20 (m, 12H). 13C NMR (75 MHz, CDCl3): 169.9, 158.9, 157.9, 155.7, 146.8, 145.8, 142.9, 140.6, 139.9, 138.7, 135.8, 133.6, 132.9, 130.5, 130.3, 129.5, 128.7, 128.6, 128.4, 127.8, 127.6, 127. 5, 126.8, 124.3, 118.7, 114.2, 113.8, 95.8, 54.4, 46.8, 45.9, 42.6, 42.6, 35.6, 31.2, 29.1, 22.9, 12.6. HR-MS (EI) calculated for C62H65N609S2. 1101.4249, found: 1101.4245.

EXAMPLE 9 H 2 N Hissamine HN Lissamine-SO 2 Cl MEA, OMAR CH 2 Cl 2 AMF 62% 18 NHA a solution of the compound of Example 12 g 0.03 mmol) and of sulfonyl chloride of lissamine rhodamine B (23 mg, 0.04 mmol) in a mixture of CH2Cl2 / DMF 50/50 (0.4 mL) was successively added 4-DMAP (1 mg) and DIEA (23 SIL, 0.13 mmol). The reaction mixture was stirred at RT for 1 h and CH 2 Cl 2 JDMF was evaporated. After purification by flash chromatography (gradient 7) and lyophilization, a pink powder (16 mg, 62%) was obtained. Tr (gradient 1): 15.09. Mp: 234 ° C.

NMR (400 MHz, CDCl3): 8.92 (s, 1H), 8.04 (d, 1 = 8.0 Hz, 1H), 7.72 (s, 1H), 7.55 (d, 1 = 7.9 Hz, 1H), 7.38 (d, 1 = 7.3 Hz, 2H), 7.27-7.25 (m, 5H), 7.19 (d, 1 = 8.2 Hz, 2 1-1), 7.12 (d, 1 = 8.2 Hz, 2H), 7.02 (d, 1 = 8.2 Hz, 2H), 6.96 (t, 1 = 10.5 Hz, 2H), 6.81 (t, 1 = 7.5 Hz, 1H), 6.64-6.57 (m, 5H), 6.46 ( d, 1 = 7.4 Hz, 1H), 4.86 (d, = 9.5 Hz, 1H), 4.34 (t, = 9.4 Hz, 1H), 3.54-3.43 (8H), 3.00 (t, 1 = 9.4 Hz, 1H), 2.09 (s, 2H), 1.83 (st, 1H), 1.23-1.18 (m, 12H). nC NMR (75 MHz, CDCl3): δ 167.5, 158.1, 158.0, 155.7, 140.3, 140.1, 137.6, 135.3, 13 3.8, 13 3.6, 133.5, 131.2, 13.6, 130.5, 13.0.2, 129.2, 12.1.1, 12 8.8, 128.8, 12 8.4, 127.9, 127.6, 127.5, 127.4, 126.5, 118.9, 114.5, 114.5, 113.8, 95.7, 54.6, 46.5, 46.0, 33.2, 25.1, 12.8.

HR-MS (EI) calculated for C57H56N5O8S2. 1002.3565, found: 1002.3568.

Preparation 21 To a solution of tert-butyl 2-hydroxyethylcarbamate (220 mg, 1.42 mmol) in CH 2 O 2, tosyde chloride (406 mg, 2%) was added. 13 mmol) then at 00C NEt3 0.4 mL, 2.84 mmol). The reaction medium was then stirred for 10 min at 00.degree. C. and then 2 h at 83.degree. After evaporation, the residue was purified by flash chromatography (AcOEt 80/20) to obtain a white powder (370 g, 8 Rf (Hept / AcOEt 50/50): 0.6 (off: 63-640 ° C. 1H NMR (400 MHz , CDCl3): 7.69 (d, J = 7.6 Hz, 2Fi), 7.26 (d, J = 7.6Hz, 2.97 (si, 1H), 3.98 (t, 1 = 4.9Hz, 2H), 3.28 (if, 2H), 2.53 (s, 3H), 1.31 (s, 9Fi) 13C 75 MHz NMR, CDCl3): 8155.7, 145.0, 132.7, 130.0, 127.9, 79.7, 69.4, 39.7, 28.3, 21.6 Preparation NaN3 H Boc, N, N3 Tos DMF 40 83% To a solution of the compound of Preparation 21 (370 mg, 1.17 mmol) in anhydrous DMF (3 mL) was added azide Sodium solution (153 mg, 2.35 mmol) The solution was stirred at 50 ° C. for 3 h After evaporation of the DMF, the residue was taken up in AcOEt, washed with water and dried over Na 2 SO 4. After evaporation and purification by flash chromatography (Hept / AcOEt 90/10), a colorless oil (180 mg, 83.9k) was obtained Rf (Hept / AcOEt 50/50): 0.7.1H NMR (400 MHz, CDCl 3 ): 3.34 (t, J = 5) .1 Hz, 2H), .23 (t, = 5.1 Hz, 2H), 1.38 (s, 9H). 13C NMR (75 MHz, CDCl3): 155.9, 79.9, 51.4, 40.3, 28.5. Preparation 23 H DCMITFA Boc'N '' '' - N 50150 TFA. 98% A solution of the compound of Preparation 22 in CH2Cl2 / TFA (50/50) was stirred at RT for 2h. After evaporation and lyophilization, a white powder (190 mg, 98%) was obtained. 1H NMR (400 MHz, CD30D): δ 3.68 (, 5.6 Hz 2H), 3.90 (t, J = 5.6 Hz, 2H). nC NMR 7 MHz, CD30D 8 49.6, 40.0. EXAMPLE 10 To a solution of the compound of Preparation 11 (30 mg, 0.05 mmol, 1 eq.) And the compound of Preparation 23 (1.2 eq.) In a DMF / H 2 O mixture (9 / 1) was added a solution of sodium ascorbate (0.1 eq) in a minimum of H 2 O and then CuSO 4 .5H 2 O (0.1 eq) in H 2 O. After stirring for 2 h at RT, the DMF was evaporated and the residue purified by semi-preparative column chromatography (gradient 3). After lyophilization, a white powder (10 mg, 29 ° C.) was obtained. Tr (gradient 1): 8.08. NMR (400 MHz, CD 3 OD): δ 7.90 (s, 1H), 7.55 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.33-7.23 (m, 8H), 7.19 (t, J = 7.3 Hz, 1H), 7.01 (t, J = 7.3 Hz, 1H), 6.66 (d, 1 = 7.3 Hz, 2H), 5.30 (dd, J = 11.3, 3.5Hz, 1H), 5.10-4.94 (m, 2H), 4.60-4.33 (m, 6H), 2.74-2.71 (m, 2H). , 2.62-2.58 (m, 2H), 2.14-2.00 (m, 2H), 1.72-1.67 (m, 2H). HR-MS EI) calcd for C39H40N8O4. 684.3173, found: 685.3254 [M + H] ÷.

Example II NH2 0 Name / N3 ~ ° 1 H2 6 44 Sodium ascorbate CuSO4, 5H2 O4F / H2O (90110) 33% o The title compound was obtained from the compound of Preparation 11 (23 mg, 0.04 mmol) of 20-azido-3,6,9,12,15,18-hexaoxoicosan-1-amine (available from Iris Biotech G bH) following the protocol described in Example 10 After purification by semi-preparative column chromatography (gradient 3) and lyophilization, a white powder (12 g 33%) is obtained. Tr (gradient 1): 8.34. ¹H NMR (400 MHz, CD30DD): δ 7.89 (s, 1H), 7.55 (t, J = 7.8 Hz, 2He), 7.46 d, = 7.8 Hz, 2H), 7.41 (d, 1 = 7.8 Hz). , 2H), 7.33-7.24 (m, 8H), 7.20 (t, J = 7.3 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.66 (d, J = 7.4 Hz, 2H), 5.31 (dd, J = 11.4, 3.5Hz, 1Fi), 4.59-4.38 (m, 6H), 3.77 (t, J = 4.8Hz, 2H), 3.71-3.54 (m, 22H). ), 3.09-3.07 (m, 2H), 2.74-2.70 (m, 2H), 2.61-2.57 (m, 2f), 2.15-2.00 (m, 2H), 1.74-1.65 (m, 2H); ).

HR-MS (EI): Calculated for C51HEAN8010. 949.4745, found: 948.4826 [M + H] +.

EXAMPLE 12 A solution of the compound of Example 1 (2 pmol, 87 μL, 23 nM) in anhydrous DMSO was charged to an Eppendorf tube. To this solution was added a solution of DY647-NHS (1.52 mg, 2 pmol) dissolved in anhydrous DMSO (152 μL), followed by DIEA (5 μL, 14.7 μmol). The mixture was stirred at room temperature under an inert atmosphere for 1 h. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The solution was diluted in MeCN solution containing 0.2% trifluoroacetic acid (8 mL) and then purified by preparative HPLC (gradient 5). The fractions were collected and concentrated under reduced pressure to give a blue solid (1.48 pmol, 74%). MS (ESI) m / z: 1443 (M + H + 721.52 [(M + 2H 2) EXAMPLE 13 A solution of the compound of Example 1 (400 nmol, 17.5 μL, 22.7) was charged to an Eppendorf tube. nM) in anhydrous DMSO To this solution was added a solution of Lumi4-Tb-NHS (400 nmol) dissolved in anhydrous DMSO (56 μL), then DIEA (1 μL, 2.94 pmol). The mixture was stirred at room temperature under an inert atmosphere for 1 h, followed by HPLC, after which the reaction was complete and the solution was diluted in a solution of triethylamine acetate. 25 mM, pH 7 (8 mL) and then purified by preparative HPLC (gradient 6) The fractions were collected and concentrated under reduced pressure to give a white solid (180 nmol, 450/0) MS (ESI) m / z: 1117.02 [(M + 2H) 2 +] / 2.

Example 14 A solution of the compound of Example 1 (400 nmol, 17.6 μL, 22.7 nM) in dry DMSO was charged to an Eppendorf tube. To this solution was added a Biot = 4015 Biotin-NHS solution (0.16 mg, 480 nmol) dissolved in anhydrous DMSO (100 μl followed by DIEA (0.1 μl, 290 nmol). at room temperature under an inert atmosphere for 1 h The reaction progress was monitored by HPLC After this period, the reaction was complete The solution was diluted in a solution of MeCN containing 0.2% of acid The fractions were collected and concentrated under reduced pressure to give a white solid (310 nmol, 77%) MS (ESI) m / z 1043.85 (m.p. M + H) +.

Example 15 DY 647 \ N = N H 49 This compound (405 nmol, 40%) was obtained using the protocol described in Example 12 from the compound of Example 10 (1 pmol). MS (ESI) m / z: 1310.98 (M + H) +. This compound (165 nmol, 16 ° 10) was obtained using the protocol described in Example 13 from the compound of Example 10 (1 μl SM (ESI) mp: 1061.29 (M + H +) This compound (890 nmol, 89%) was obtained using the protocol described in Example 12 from the compound of Example 11 (pmol) MS (ESI) m / z : 1574.43 (M +) + Example 18 This compound (380 nmol, 38%) was obtained using the protocol described in Example 13 from the compound of Example 11 (1 pmol). m / z: 1193.82 (M + H + Na) 2 + / 2.

Biological Evaluation of Compounds A. Initially, affinity (determined by binding order using a radioligand) and activity (agonist / antagonist character of V2 receptor) of representative compounds of the invention were evaluated. functionalized by lissamine. Argininevasopressin (AVP) and 2-phenyl-4 '- [1,45,6-tetrahydroimidazo [4,5-d] [1] benzazepin-6-yl) carbonyl] -benzanilide (Chem Pharm Bull 1997, 45 (11), 1870-1874) were used as reference compounds. The results are shown in Table 1. Table 1 Compound Affinity Activity (nM) b (nM) a Kart nad AVP 1.36 t 0.45 μ 0.29 ± 0.05 - N 0.57 f 0.18 - 0.096 t 0.030 N 1 Example 2 * 4.0 ± 0.7 - 1.96 f 0.34 Example 6 12.7 ± 0.7 - 2.03 ± 0.04 Example 7 16.8 ± 1.0 - 8.91 ± 0.49 Example 8 7.6 ± 0.7 - 0.66 t 0.12 Example 9 25.3 ± 2.5 - 3.80 f 0.36 * isomer para a The constant of Ki inhibition of compounds tested for the human vasopressin receptor (V2R) was determined on Chinese Hamster Ovary (CHO) cell membranes by competitive binding assay ([3H] AVP displacement) b Kas and K constants; The compounds tested were determined using stably expressing V2R-expressing CHO cells and measuring cAMP accumulation The results are expressed as the mean standard deviation of the mean from three separate experiments repeated three times each Kact reflects the antagonistic nature of a compound and K; nact reflects the agonist nature of a compound. The affinity of AVP was determined directly by saturation binding assay using [3H] -AVP as indicated above to calculate the Kd of [3H] -AVP.

These results show the V2 affinity and agonist character of the compounds of the invention.

B. Affinity (determined by a radioligand binding assay), activity (V2 receptor agonist / antagonist character), and HTRF application of representative functionalized compounds of the invention were also evaluated. fluorescein, DY647 or Lumi4Tb. The results are shown in Table 2. 2969605 Table 2 Affinity Compound (nM) a Activity (nM) b HTRF-FRET® ± 5.2 9.2 ± 0.3 17.2 ± 3.8 7.3 Example 5 Example 12 Example 13 Example 15 Example 16 Example 17 14.6 Example 18 40.6 0.78 ± 0.21 11 0.052 f 0.005 2.33 ± 1.23 0.095 ± 0.038 3.17 ± 0.78 0.069 ± 0.014 1.38 t 0.56 0.42 t 0.12 2.1 ± 0.87 0.38 0.1 2.53 ± 1.19 3.64 ± 0.86 6.2 ± 2.13 a, These experiments were carried out with the V2 receptor labeled with either Lumi4Tb or DY647 depending on the nature of the ligand used (donor or acceptor). This receptor is obtained after transfection of Cos-7 cells expressing the labeled V2 receptor. After incubation for 20 h at 4 ° C, the ligand-receptor pair is excited at 337 nm and the fluorescence emission was measured at 620 nm and 665 nm. The specific signal was calculated according to the equation: deltaF = (R-Rneg) / (Rneg) where R is the ratio of fluorescence at 665 nm and fluorescence at 620 nm, calculated for each test and Rneg is the same ratio for control measurements without ligand.

These results show the V2 affinity and agonist character of the compounds of the invention, and their excellent ability to be used in an HTRF binding assay.

C. Selectivity of the compounds of the invention vis-à-vis the V1 receptors (Via, Vi subtypes) and V2 vasopressin, and the oxytocin receptor (OT) was also evaluated. Arginine vasopressin (AVP), 2-phenyl-4 '- [1,4,5,6-tetrahydimidazo [4,5-d] [1] benzazepin-6-yl) -carbonyl] -benzanilide (Chem Pharm Bull 1997, 45 (111870-1874) as well as compound 17a, b described in Chem Eur J 2008, 14, 6247-6254 (ortho isomer = "compound A", para isomer "compound B"), were The results are shown in Table 3.

Table 3 Affinity (K, n V2 Via Life OT 1.48 ± 0.08b 0.7 0.17 0.49 ± 0.06 1.65 ± 0.49 0.57 ± 0.18 2.74 ± 0 , 19 nd 109 ± 34 187.7 ± 24.7> 1000> 1000 307 ± 18 54.3 ± 6.6> 1000> 1000 495 ± 101 4.0 ± 0.7> 1000> 1000> 1000 26.1 ± 5.2> 1000> 1000 665 ± 47 9.1 ± 0.3 1753 ± 338> 1000 808 ± 61 17.2 ± 3.8 750 ± 89> 1000 304 ± 70 Ki of the compounds tested for the different receptors a was determined on CHO cell membranes by competitive binding assay (displacement of [3H] AVP) A K,> 1000 indicates that displacement of [3H] AVP with the test compound at a concentration of 10 μM did not lead to no competition or inhibition less than 50% b cf table 1

nd not determined These results show the agonist character, selective (vis-à-vis the receivers Via, Vib and OT) and affin compounds of the invention. Compound AVP * isomer para a The constant of inhibition compound A compound B example 2 * example 5 example 12 example 13

Claims (8)

REVENDICATIONS1. wherein: R1 is hydrogen or a group -COR2; REVENDICATIONS1. wherein: R1 is hydrogen or a group -COR2; R 2 is - (CH 2) n, NR 3 R 4 wherein m = 2, 3 or 4 and R 3 and R 4 are each hydrogen or R 3 and R 4 are piperid-4-yl or piperazinyl with the atom nitrogen to which they are linked; or a - (CH 2 CH 2 O) n (CH 2) pNR 3 R 4 group in which n = 3, 4 or 5, p = 2 or 3 and R 3 and R 4 are as defined above; or a group wherein R5 is - (CH2) gNR3R4 wherein q = 2 or 3 and R3 and R4 are as defined above; or a group - (CH2CH2O) r (CH2) SNR3R4 wherein r = 3, 4, 5 or 6, s = 2 or 3 and R3 and R4 are as defined above. 2. A compound according to claim 1 wherein R1 is a -COR2 group. 3. A compound according to claim 2 wherein R2 is - (CH 2) 2 -NR 3 R 4 wherein m = 2 or 4 and R 3 and R 4 are each hydrogen or R 3 and R 4 are piperazinyl with the atom of nitrogen to which they feel bound. 4. A compound according to claim 2 wherein R2 is - (CH2CH2OXCH2) pNR3R4 wherein n = 3 and p = 2. Compound of formula R1HN (I) The compound of claim 2 wherein R2 is -R5 wherein R5 is - (CH2) 2NR3R4. The compound of claim 2 wherein R2 is N-R5 0 N = N wherein R5 is - (CH2CH2O) r (CH2) 2NR3R4 wherein r = 3 or 6 and s = 2. 7. Fluorescent ligand obtained by coupling a compound of formula (1) as defined in any one of claims 1 to 6 with a fluorescent compound. The fluorescent ligand of claim 7, wherein the fluorescent compound is selected from: allophycocyanins, particularly that known under the trade name XL665; organic luminescent molecules, such as rhodamines (in particular lissamine rhodamine B), cyanines, squaraines, coumarins, proflavines, acridines, fluoresceins; fluorophores known under the name Bodipy; fluorophores known under the name Atto; fluorophores known under the name Dy, in particular the compound known under the name Dy467; the compounds known under the name AlexaFluor; nitrobenzoxadiazole; fluorescent metal complexes, such as rare earth cryptates, rare earth chelates (in particular europium, terbium, samarium, dysprosium, neodymium, and especially the compound known as Lumi4-Tb denomination); inorganic luminescent particles such as nanocrystals. Fluorescent ligand according to claim 8, wherein the fluorescent compound is selected from rhodamines (in particular aminesamine rhodamine B), fluoresceins, fluorophores known under the name Dy (in particular H 2 - C 2 H 0 N). = - N 0 (CH 2 II) 2 -Compound known under the name Dy467), and the rare earth esters, in particular the compound known under the name Lumi4-Tb). 10. Compound of formula (II) or (III): HN (m) 11. Use of a fluorescent ligand as defined in any one of claims 7 to 9 in a binding test on the V2 receptor of vasopressin . 12. Use of a fluorescent ligand as defined in any one of claims 7 to 9 for labeling or visualization of the V2 receptor.