MXPA06000843A - Lanthanide complexes preparation and uses thereof - Google Patents

Abstract

The invention relates to compounds and the complexes formed thereby with a lanthanide and use of the complexes for fluorescence or NMR-imagery marking. The complex comprises an Ln ion and a R2 C(X R1) (R3) NR4R5 ligand, where R1 is a functional group, X is a single bond or a hydrocarbon chain, comprising at least one alkylene or alkenylene group with optionally at least one heteroatom or an arylene, R2 is an anionic group A2, or a C1-C4 alkylene or alkenylene with at least one such group A2 and optionally comprising at least one heteroatom, R3 is H or a C1-C5 alkylene or alkenylene, optionally comprising at least one heteroatom and optionally having at least one anionic group A3, R4 is a light-absorbing substituent forming cyclic chelates with Ln and R5 is a substituent forming cyclic chelates with Ln.

Description

i i COMPLEXES OF LANTANIDES, ITS PREPARATION AND ITS USES FIELD OF THE INVENTION The present invention concerns compounds capable of forming complexes with the lanthanides, the complexes obtained and their uses.

BACKGROUND OF THE INVENTION Radioactive labels have been widely used the field of medical imaging and immunology. Because of these drawbacks, they have been largely replaced by fluorescent labels. However, the use of markers fluorescents presents some drawbacks, particularly due to the autofluorescence of the biological media studied and the diffusion of light in the equipment. The complexes of lanthanide ions have been proposed to allow an acquisition in resolution temporary that suppresses these disadvantages. To be used as a luminescent marker in temporal resolution, a complex of a lanthanide ion must have many characteristics, among which the most important ones are hydrophilicity, stability in water, the presence of chromophores capable of generating the antenna effect (Sabbatini, N. et al. Coord. Chem. Rev. 1990, 123, 201) good photo-physical properties (high absorption, excitation in an easily accessible energy range, life time of the excited excited state and quantum efficiency of high luminescence) and a reactive function that allows a covalent graft. The currently proposed compounds rarely possess all of these criteria. For example, the first complexes developed by the firm Wallac Oy under the name Delfia Chelate (Hemilá, I. and collaborators, Anal. Bioche, 1984, 137, 335) do not have good photo-physical properties and it is necessary to proceed to a stage of extraction of the lanthanide to measure its luminescence. The compounds developed by CIS Bio International are cryptats that require the use of fluoride anions to increase luminescence (Hemilá, I. et al., Drug Dicovery Today, 1997, 2, 373). The stability of the compounds also presents serious problems. Accordingly, compounds developed by CyberFluor under the name of BCPDA only form stable luminescent complexes at high concentrations (Marriott, G. et al., Biophysical Journal, 1994, 67, 957).

Complexes of lanthanides, particularly gadolinium, have been used as relaxation or contrast agents for medical images by NMR (Caravan, P. et al., Chem. Rev. 1999, 99, 2293). This use is allowed by the fact that the first coordination sphere of the lanthanide is not completely saturated by the ligand in aqueous solution, water molecules that can then complete the waiting for coordination.

SUMMARY OF THE INVENTION The objective of the present invention is to propose lanthanide complexes that have improved properties in relation to the lanthanide complexes of the prior art. In so the invention has as its object new compounds, their use for the preparation of complexes with lanthanide ions, as well as the use of the complexes obtained as fluorescent markers, as relaxation agents for the NMR, or for the NMR of images. BRIEF DESCRIPTION OF THE FIGURES Figure 1 represents droplets of approximately 750 microns in diameter.

DETAILED DESCRIPTION OF THE INVENTION A compound according to the present invention responds to Formula (I) I 1 - X - C - R 3 i N * 'in which - R 1 is a functional group capable of reacting with the functions present on proteins, antibodies or on mineral or organic materials; X represents a single bond or a hydrocarbon chain consisting of at least one group selected from the alkylene groups and the alkenylene groups optionally comprising at least one heteroatom, and between the arylene groups; - R2 is an anionic group of neutral pH A2 or an alkylene or alkenylene group having from 1 to 4 carbon atoms and containing at least one such A2 group, said alkylene or alkenylene group optionally comprising at least one heteroatom in the chain; - R3 represents H or an alkylene or alkenylene group having from 1 to 5 carbon atoms and optionally containing at least one heteroatom in the chain, said group optionally containing at least one anionic group at neutral pH A3; - R4 represents a grouping that responds to the formula- (C) nC-Z1-CC-Z2-C-A4 in which n is equal to 2, Z1 and Z2, independently represent a heteroatom selected from 0 and N, being at least one a nitrogen atom that is part of an aromatic heterocycle with the two carbon atoms surrounding it, and A4 is a group that is anionic at neutral pH in which the atom containing the anionic charge is in position? in relation to Z2; R5 is a grouping selected from the groupings defined for R4 or between the hydrocarbon chains -CC-E1-CC-E2-C-A5 in which E1 and E2 independently represent a heteroatom selected from O and N, and A5 is a group that is anionic at neutral pH in which the atom containing the anionic charge is in position? in relation to E2. The heteroatom of the substituents X, R2 and R3 can be, in particular, O or N. The substituent R1 can be selected, for example, from amino, thio, cyano, isocyano, acridinyl, hydrazino, halogen acetate, anhydride, triazo, carbonyl, Nitrobenzoyl, sulfonyl, thionyl, halide, epoxide, aldehyde, imidazole. Hydroxyphenyl, mercapto, N-succinic ester, N-sulfosuccinic ester, maleimido, hydroxyl, carboxyl, thiocyano, and isothiocyan. Amino, thio, carbonyl, maleimido, N-succinimide ester, N-sulfosuccinimide ester and isothiocyan are preferred. When the group X is an alkylene or alkenylene group, there are preferably 1 to 10 carbon atoms. When X is an arylene group, there are preferably from 5 to 10 carbon atoms. By arylene group, it is understood, in the present text, a group comprising a single aromatic nucleus or several condensed aromatic nuclei or not, said nucleus possibly consisting of one or more aliphatic hydrocarbon groupings. By way of example of arylene groups, the groups -CdH.-, CH2-C6H4-CH2-, can be mentioned, -C5H4-CH2-, -C6H4-CH2-C6H4-, -C6H3 (CH3) -. X is advantageously selected from a single bond or an alkylene or alkenylene group having 2 or 3 carbon atoms. The substituent R2 is preferably a group A2. The substituent R3 is preferably H or an alkyl in Ci to C3. In the compounds of the present invention each of the substituents R4 and R5 is a monovalent substituent. The substituents R4 and R5 do not together form a divalent group. The R4 substituent is a substituent which has light absorption properties and which makes it possible to form three chelate rings with a lanthanide. Substituents R4 in which n is equal to 1 are preferred. As an example of substituent R4 in which only one of Z1 and Z2 is a nitrogen atom forming part of an aromatic heterocycle, substituents may be mentioned in which one of the segments -CZC- is part of a heterocyclic group selected from the pyridyl, pyrimidyl, quinolyl and isoquinolyl groups. Substituents R4 in which Z1 and Z2 are part of an aromatic heterocyclic group are particularly interesting. As an example of such a substituent, substituents may be mentioned in which each of the segments-C-Z1-C- and -C-Z2-C- forms part of a heterocyclic group selected from the pyridyl, pyrimidyl, quinolyl groups , and iso-quinolyl, the two heterocyclic groups are linked at least by the two carbon atoms that separate Z1 and Z2. As examples of said segments -C-Z2-CC-Z2-C-, mention may be made of the groups 2, 2'-bipyridinyl, 1, 10-phenanthrolinyl, 2, 2-bisquinolyl, 2, 2-bisisoquinolyl and 2, 2 '- bipiri idinilo, said groups may contain alkyl or alkoxy substituents on at least one carbon atom of a heterocycle, preferably an alkyl or alkoxy group having 1 to 5 carbon atoms. By way of example, the following formulas represent respectively a 2,2'-bipyridyl group containing a carboxyl, a monoalkylphosphonate, a monoarylphosphonate, and a phosphonyl, or a phenanthrolinyl group containing a carboxyl group.

R = Alkyl, Aryl The substituent R5 is a substituent that allows to form three chelate cycles with a lanthanide. Among the substituents R5 constituted by a hydrocarbon chain-C-C-E1-C-E2-C-A5, the following groupings can be mentioned: wherein R6 and R7 represent alkyl chains. which have preferably 1 to 5 carbon atoms and optionally contain one or more heteroatoms. The compounds in which R4 and R5 are identical are particularly preferred. As used herein, the term "group that is anionic at neutral pH" means a functional group which, at neutral pH, is in the anionic form, ie carrying a negative charge. In a compound of the invention, the neutral pH anionic groups A2, A3, A4 or A5 can be selected independently from each other between the -C02H groups, S03H, -P (O) (OR) OH, -P (0) E (OH) and -P (O) (OH) 2 in which R is an alkyl group (preferably from Ci to C3) or a group aryl (preferably from C5 to Cg). Depending on the pH of the reaction medium, the compounds (I) are obtained in the cationic, amphoteric or anionic form. In an acid medium, the nitrogen carrying the substituents R4 and R5, as well as optionally the heteroatoms Z1, Z2, E1 and E2 are in the protonated form and the compound is in the cationic form. In basic medium, the different A1 groups are presented in the form of salts and the compound is in the anionic form. At intermediate pHs, on the order of 6 to 8, the compound is presented in the amphoteric form. A complex according to the present invention is constituted by a lanthanide ion Ln complexed by a ligand that responds to Formula (I) above. The lanthanide ion is selected from the europium, terbium, samarium, dysprosium, erbium, ytterbium, neodymium and gadolinium ions. Europium, terbium, samarium or dysprosium will preferably be used if the complex is intended to be used for fluorescence labeling, and europium, dysprosium or gadolinium when the complex is intended to be used as a contrast agent for the IRMN. In a complex according to the invention in which R4 is -CC-Z1-CC-Z2-C-A4, the 3 chelate cycles are formed between the lanthanide cation and respectively: - the N atom, which contains R4 and R5, Z1 and the carbon atoms that separate them; - Z1, Z2 and the two carbon atoms that separate them; - the terminal segment Z2-C-A4. When R5 is of the same type as R4, it forms with the lanthanide ion chelates of the same type as those formed by R4 When R5 is of the type -CC-E1-CC-E2-C-A5, 3 chelate cycles of 5 elements are formed between the lanthanide cation and respectively: - the N atom containing R4 and R5, E1 and the two carbon atoms that separate them; - E1, E2 and the two carbon atoms that separate them; - the terminal segment E2-C-A5. A compound (I) can be obtained by methods well known to those skilled in the art from commercial products or described in the literature by the following scheme: 'X IA IB IC I in which X, R1, R2, R3, R4 and R5 have the meanings given above, and R1', R2 ', R3', R4 ', and R5' represent precursor groups of R1, R2 , R3, R4 and R5 respectively.

In the course of the first two steps, the groupings R4 'and R5' are successively introduced on an IA molecule containing X and the groups R1 ', R2', and R3 'to obtain the compound IC. In the course of the subsequent steps, the groups R1 ', R2', R3 ', R4', and R5 'of the compound IC are respectively transformed into groups R1, R2, R3, R4 and R5. When the groupings R4 'and R5' are identical in order to obtain identical groupings R4 and R5, they are introduced simultaneously in the course of the first stage. When they are different, they are introduced in an indifferent order by reaction of the IAS molecule successively with two different reagents. When the compound (I) is a compound in which the groupings R1 and R2 are carboxyl functions, the group R3 is a hydrogen atom and the group X is a simple bond, a methylene group or an ethylene group, it will be advantageously selected as starting material IA respectively the ethylenic diester of inomalonic acid, the methyl diester of aspartic acid and the methyl diester of glutamic acid, which are commercially available products. When the compound (I) is a compound in which: the groupings X1 and X2 are carboxyl functions, - the group R is a hydrogen atom and - the group X is a propylene or a para-substituted benzene. The 2-amino-adipic acid methyl diester (the preparation of which is described by Larch, E. et al, Helv. Chi. Acta, 1974, 57, 1584) and the methyl ester of acid can be used as starting material IA respectively. (α-amino-4-methoxycarbonyl) benzene acetic acid (the preparation of which is described by Cahuvel, E. et al., J. Med. Chem. 1994, 37, 1339). When the groupings R4 and R5 are identical and their segments -CC-Z1-CC-Z2-C- are derivatives of the 2, 2'-bipyridine, the starting product is reacted during the first stage, with the 6-bromomethyl-6'-bromo-2, '-bipyridine to obtain a dibrominated compound IC. The β-bromomethyl-β '-bromo-2, 2-bipyridine can be obtained by means of a radical-bromination reaction of 6-methyl-6'-bromo-2,2'-bipyridine by means of the N-bromine -succinimide in benzene, 6-methyl-6'-bromo-2, 2'-bipyridine which is obtained according to the method described by Houghton M. et al., J. Chem. Soc., Dalton Trans. 1997, 2725. The reactive scheme of the first stage of this particular case is given below.

When the dibrominated compound IC is subjected to a carboalkylation, followed by a saponification with NaOH and an acidification with HCl, a compound is obtained (I) in which the groupings A4 and A5 are carboxyl groups. The carboalkylation can be carried out according to the procedure described by El-Ghayoury et al., J. Org. Chem., 2000, 65, 7757. When the dibromo compound IC is reacted with dialkyl phosphite (according to the method described by Penicaud et al., Tetrahedron Lett, 1998, 39, 3689), the dialkyl ester of phosphonic acid is obtained, each bromine atom is replaced by a group P (O) (OR) 2. The dialkyl ester of phosphonic acid gives, by means of a saponification with NaOH in the water, followed by an acidification with HCl, a compound (I) in which the groupings A4 and A5 are groups P (0) (OH) OR. By reaction of the phosphonic acid dialkyl ester P (O) (OR) 2 with the trimethylsilyl bromide followed by hydrolysis (according to the method described by McKenna C. et al., Tetrahedron Lett, 1977, 18, 155), a compound (I) in which the two anionic groups A4 and A5 are groups P (0) (0H) 2. The same result can be obtained by means of an acid hydrolysis by means of HCl of the dialkyl ester of the phosphonic acid P (0) (0R) 2. The reaction scheme of the three transformation modes mentioned above is given below. or HCl, H2? When the groupings R4 and R5 are identical and their segments -CC-Z1-CC-Z2-C- are derivatives of 1, 10-phenanthroline, the starting product is reacted during the first stage with the 2- bro-ometyl-9-ethoxycarbonyl-1, 10-phenanthroline. The preparation of 2-bromomethyl-9-ethoxycarboni-1, 10-phenanthroline is described by Ulrich G. et al., (Tetrahedron Lett, 2001, 42, 6113). By subjecting the obtained diester compound to a saponification with NaOH, followed by acidification with dilute HCl, a compound (I) is obtained in which the groupings A4 and A5 are carboxyl. The reaction scheme is given below.

A desired R1 substituent can be obtained by selecting either a starting compound containing it, or a starting compound containing a precursor R1 'of the desired substituent. When a substituent R1 is obtained from a precursor Rl ', the formation of the desired substituent can be made on a compound of Formula (IC) containing the precursor or on a complex formed with a lanthanide cation and a compound of Formula (I) that contains the precursor.

A substituent R1 of the carboxyl type can be obtained by means of a saponification reaction from a precursor group R1 'containing a carboxylic ester function. A substituent R1 of the amino type can be obtained from the reduction of a precursor group R1 'containing a nitro function. An R substituent of the isothiocyan type can be obtained by reaction of a precursor R1 'containing an amino function with the thiophosgene. A substituent R1 of the maleimido type can be obtained by reaction of a precursor Rl 'containing an amino function with the N-succinimide ester of 4-maleimidobutyric acid. An R 1 substituent of the N-succinimide ester type can be obtained from a complex by activation of a carboxyl precursor with N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide followed by a reaction with the N-hydroxy-succinyl ida. A complex according to the invention can be obtained by reaction of a donor compound of the lanthanide cation with a compound of Formula (I). Examples of lanthanide cation donor compounds are hydrated lanthanide halides, hydrated lanthanide nitrates, lanthanide carbonates and lanthanide triflates. The reaction is carried out in solution in a solvent. The solvent is preferably selected from water, methanol, ethanol or acetonitrile. In a preferred embodiment, the compound (I) is reacted with the precursor of the lanthanide ion in a mixture of methanol and water at a pH ranging from 3 to 5, during a period comprised between 10 minutes and 24 hours, a temperature between 25 ° C to 80 ° C. Next, the pH of the solution is brought to 7.0 and the methanol is evaporated before isolating the complex formed. The complexes of the present invention can be used particularly for fluorescence labeling or for nuclear magnetic resonance imaging. For these applications, the preferred R1 groups are the amino, thio and carboxyl groups (which must be activated before covalent coupling with the marker molecule), and the maleimido, N-succinimide and isothiocyan group (which can be directly related). with the molecule to mark). The complexes of the present invention are used for analysis or dosing of compounds by labeling the compounds. The method consists of covalently binding the compound to be dosed, a marker constituted by a complex according to the invention, and detecting or quantifying the presence of the marked compound thanks to the luminescence properties of the label. Europium, terbium, samarium or dysprosium complexes are particularly preferred for this application. When the lanthanide ion complexes according to the invention are intended to be used as relaxation agents for nuclear magnetic resonance, gadolinium, europium or dysprosium complexes are preferably used. The present invention will be described in more detail by the examples given below by way of illustration, to which, however, it is not limited. Example 1 Preparation of Compound 1 Compound 1 has been obtained according to the following synthetic scheme. The (S) -isomer of the selected glutamic ester could be replaced by the (R) isomer or by a mixture of the two isomers.

Preparation of Compound 2 Compound 2 has been prepared according to the procedure described by S. Mameri, et al., In Synthesis, 2003, 17, 2713. In a 250 ml balloon, 1.5 g (6.0 mmol) of 6-methyl is introduced. 6'-bromo-2, 2'-bipyridine, 66 mg (0.4 mmoles) of azo-bis-isobutyronitrile (AIBN), and 1.3 g (7.3 mmoles) of N-bromosuccini ida in 90 ml of benzene. The solution is heated to reflux for 2 hours 30 minutes when irradiated with a standard 100-watt halogen lamp. The solvent is evaporated under reduced pressure and the solid residue is chromatographed on silica using a gradient of CH2Cl2 / hexane from 50/50 to 100/0. 940 mg (2.9 mmol) of Compound 2 (corresponding to a yield of 48%) is obtained, which has the following characteristics: Rf = 0.42, Si02, CH2C12. XH-NMR (CDC13, 200 MHz): d 4.61 (s, 2H), 7.48 (d, 1H, 3J = 7.5 Hz), 7.50 (d, 1H, 3J = 7.5 Hz), 7.68 ( t, 2H, 3J = 8.0 Hz), 7.83 (t, 1H, 3J = 8.Hz), 8.33 (d, 1H, 3J = 8.0 Hz), 8.44 (d, 1H, 3J = 8.0 Hz). 13 C-NMR (CDC13, 50 MHz): d 34.0, 120.1, 120.7, 124.0, 128.2, 138.1, 139.3, 141.6, 154.3, 156.4, 156.9. Preparation of Compound 3 In a Schlenk tube under an argon atmosphere, 470 mg (2.22 mmol) of L-glutamic acid dimethyl ester hydrochloride and 1.23 g of K2CO3 (8.90 mmol) in 100 ml of freshly distilled acetonitrile are introduced over P205. . The solution is heated at 80 ° C for 30 minutes. 1.60 g (4.88 mmol) of Compound 2 are added and heated for 23 hours at 80 ° C. The solution is evaporated to dryness, the residue is redissolved with 100 ml of CH2C12 and 20 ml of water. The aqueous phase is washed with two 20 ml portions of CH2C12 and the sum of the organic phases is dried over Mg2SO4, filtered, then evaporated to dryness. The solid residue is subjected to flash chromatography on silica (f = 5 cm, h = 12 cm) with a mixture of CH 2 Cl 2 / MeOH (100/0 to 97/3) as eluent. 995 mg (1.49 mmol) of Compound 3 (corresponding to a yield of 67%) is obtained, which has the following characteristics: Rf = 0.34, SiO2, CH2Cl2 / MeOH (98/2). XH-NMR (CDC13, 200 MHz): d 2.06-2.20 (, 2H), 2.39- 2.68 (, 2H), 3.50 (s, 3H), 3.54-3.62 (m, 1H), 3.76 (s, 3H) , 3.99- 4.16 (m, 4H), 7.43-7.48 (m, 4H), 7.63 (t, 2H, 3J = 8.0 Hz), 7.71 (t, 2H, 3J = 8.0 Hz), 8.23 (d, 2H, 3J = 8.0 Hz), 8.39 (d, 2H, 3J = 8.0 Hz). 13 C-NMR (CDCl 3, 50 MHz): d 24.8, 30.3, 51.5, 57.2, 62.1, 119.6, 119.7, 123.5, 127.8, 137.3, 139.1, 141.5, 153.8, 157.4, 159.1, 173.1, 173.4. Analysis calculated for C29H2N5? Br2: C, 52.04; H, 4.07; N, 10.46. Found: C, 51.81; H, 3.85; N, 10.19. FAB + / MS: 670.2 ([3 + H] +, 100%). Preparation of Compound 4 In a 250 ml two-neck balloon, 995 mg (1.49 mmol) of Compound 3 and 150 mg (0.21 mmol) of [Pd (PPh3) 2C12] are introduced into 50 ml of ethanol and 50 ml of triethylamine. . The solution is heated at 70 ° C for 15 hours by bubbling a flow of CO. The solution is evaporated to dryness, the solid obtained is redissolved in 100 ml of CH2C12, filtered over celite, then the organic phase is extracted with 20 ml of water. The aqueous phase is washed with two 20 ml portions of CH2CL2 and the combined organic phases are dried over MgSO4, filtered and then evaporated to dryness. The residue is subjected to flash chromatography on silica (f = 5 cm, h = 10 cm) with a mixture of CH 2 Cl 2 / MeOH (99/1 to 90/10) as eluent. 588 mg (0.90 mmol) of 4 are obtained in the form of a slightly orange oil (corresponding to a yield of 60%), which has the following characteristics: Rf = 0.30, SiO2, CH2Cl2 / MeOH (95/5). ^ -RN (CDC13, 200 MHz): d 1.46 (t, 6H, 3J = 7.0 Hz), 2.06-2.19 (m, 2H), 2.38-2.65 (m, 2H), 3.49 (s, 3H), 3.55- 3.63 (m, 1H), 3.76 (s, 3H), 4.02-4.19 (m, 4H), 4.48 (q, 4H, 3J = 7.0 Hz), 7.47 (d, 2H, 3J = 8.0 Hz), 7.75 (t , 2H, 3J = 8.0 Hz), 7.92 (t, 2H, 3J = 8.0 Hz), 8.10 (d, 2H, 3J = 8.0 Hz), 8.40 (d, 2H, 3J = 8.0 Hz), 8.62 (d, 2H , 3J = 8.0 Hz). 13 C-NMR (CDCl 3, 50 MHz): d 14.3, 24.8, 30.4, 51.5, 57.2, 61.8, 62.0, 119.9, 123.5, 124.2, 124.8,137.3,137.7, 147.8, 154.6, 156.5, 159.0, 165.4, 173.2 , 173.5. Analysis calculated for C35H37N508: C 64.11, H 5.69, N 10.68. Found: C 64.07, H 5.55, N 10.53. FAB + / MS: 656.2 ([4 + H] +, 100%).

Preparation of Compound 1 In a balloon equipped with a refrigerant, 588 mg (0.90 mmol) of 4 and 144 mg (3.60 mmol) of NaOH are dissolved in a mixture of 50 ml of MeOH and 15 ml of water. The mixture is heated to 70 before 5 hours. The solution is evaporated to dryness and the solid is dissolved in 10 ml of water on which a 2N HCl solution is slowly added until pH = 2-3. The precipitate that forms is isolated by centrifugation and dried under vacuum. 411 mg (0.60 mmol) of Compound 1 are obtained in the form of pale yellow 1.3 HCl hydrochloride (corresponding to a yield of 67%) whose characteristics are the following: ^ -RMN (CD3OD, 300 MHz): d 2.26-2.48 (m, 2H), 2.80-2.84 (m, 2H), 3.95-3.99 (m, 1H), 4.53-4.81 (, 4H), 7.47 (d, 2H, 3J = 7.5 Hz), 7.63 (t, 2H, 3J = 8.0 Hz), 7.90 (t, 2H, 3J = 8.0 Hz), 8.02 (d, 2H, 3J = 7.5 Hz), 8.42 (d, 2H, 3J = 7.5 Hz), 8.58 (d, 2H, 3J = 7.5 Hz). 13 C-NMR (CD3OD, 75 MHz): d 23.1, 32.1, 57.0, 67.0, 122.3, 125.1, 125.9, 126.1, 139.7, 140.1, 149.0, 154.1, 155.5, 156.1, 168.0, 173.7, 176.4. Analysis calculated for C29H25 5? 8.3HC1: C, 51.15; H, 4.14; N, 10.28. Found: C, 51.01; H, 4.43; N, 9.95. FAB + / MS: 572.5 [(1 + H] +, 100%). Example 2 Preparation of Complex 5 of Formula | Eu. (1- 60 mg of 1.3 HCl (88 pinoles) are dissolved in a mixture of 30 ml of MeOH and 30 ml of water. To this solution was added a mixture of 36 mg (98 μmol) of EuCl3.6H20 dissolved in 3 ml of MeOH and 3 ml of water. The solution is heated at 70 ° C for 1 hour. After cooling, the pH of the solution is increased to 7.4 with a 5% solution of NaOH in water. The solution is concentrated in the rotary evaporator until the appearance of a slight turbidity. THF is then added until the formation of a large precipitate. The precipitate is isolated by centrifugation, then vacuum dried to give 62 mg (74 μmoles) of Compound 5 (corresponding to a yield of 85%) in the form of a beige solid whose characteristics are as follows: 1 H-NMR (D20 / t-BuOH, 200 MHz, all signals are presented in the form of broad singlet): d -9.40 (1H), -8.95 (1H), -4.23 (2H), -3.17 (1H), - 2.21 (1H), 1.88 (1H), 2.73 (1H), 4.17 (1H), 6.06 (1H), 7.12 (1H), 7.80 (1-H), 7.88 (1H), 8.90 (1H), 9.60 (1H), 9.89 (1H) , 11.08 (1H), 11.38 (1H), 12.01 (1H). Analysis calculated for C29H2? aN508Eu.5H20: C, 41.84; H, 3.75; N, 8.41. Found: C, 41.93; H, 3.62; N, 8.44.

FAB + / MS: 720.2 (80%), 722.2 (100.%) -, [5-H20-Na + 2H] +. IR (KBr, cm-1): 3420, 1619, 1574, 1460, 1384, 1274. Photo-physical properties in water: Absorption,? Max [nm] (emax [M-1 .cm-1]): 320 ( point contact zone), 308 (19700), 276 (8700), 267 (9700), 253 (14400). Emission: characteristic of europium compounds with fine bands at 581, 594, 615, 650 and 701 nm. Life time of the excited state: 0.62 ms. Quantum yield (reference [Ru (bipi) 3] 2+ in water: 8% Life time of the excited state in heavy water: 2.48 ms Quantum yield in heavy water: 35% Example 3 Preparation of Complex 6 of Formula: 40 mg (48 μmol) of complex 5 and 12 mg (63 μmol) of ethyl-N, N-dimethyl-3-aminopropylcarbodiimide hydrochloride (EDCI.HC1) salts are suspended in 6 ml of DMSO. To this solution, 7.0 mg (61 μmol) of N-hydroxysuccinimide is added. The solution is stirred at room temperature for 66 hours, during which Complex 5 dissolves, then a white precipitate forms. The solid is isolated by centrifugation and is vacuum dried at 50 ° C for 2 hours. 31 mg (34 μmoles) of 6 (corresponding to a yield of 71%) are obtained whose characteristics are the following: Analysis calculated for C33H25EuN60_o.5H20: C, 43.67; H, 3.89; N, 9.26. Found: C, 43.60; H, 3.80; N, 9.16.

FAB + / MS: 720.1, 722.1 ([6-H20-C4H4N02 + 2H] +, 100%), 817.1, 819.1 ([6-H20 + H] +, 30%). IR (KBr pill, cm "1): 3420, 1739, 1629, 1573, 1459, 1384. Photo-physical properties in water: Absorption,? max [nm] (emax [M ^ .cm "1]): 320 (punctual contact zone), 309 (2000), 276 (10000), 267 (10500), 253 (16000). Europium compounds with fine bands at 581, 593, 615, 649 and 701 nm Life time of the excited state: 0.63 ms Quantum yield (reference [Ru (bipi) 3] 2+ in water): 8%. of life of the excited state in heavy water: 2.47 ms Quantum yield in heavy water: 34% Example 4 Marking of an amine by means of Complex 5 10 mg of Complex 5 (13.1 μmoles) in 5 ml of water are suspended Add 3.5 mg (18.3 μmol) of EDCI.HC1, then 1.7 μl (13.2 μmol) of (+) - a-methylbenzylamine, after 15 minutes, then 1 hour, 1.7 μl of (+) - a-methylbenzylamine at room temperature, stirring is continued for hours. The aqueous phase is washed with 2 times 10 ml of CH2C12, then evaporated to dryness, and 14 mg of pale yellow solid are obtained. After recrystallization with a mixture of Me0H / Et20, centrifugation and drying under vacuum, Complex 7 (8.0 mg, 9.5 μmol) is recovered in the form of a cream powder (73%). ISI-TOP / MS: 847.0513 ([7-H20 + Na] +, 60%), 825.0912 ([7-H20 + H] +, 28%). The formula of complex 7 is represented below.

Example 5 Marking of serum albumin of ASB-res by Complex 6 Complex 6 (2.0 mg) is added to a solution of ASB (5.4 mg) in 1 ml of borate buffer (50 M in water, pH = 7.0) in order to obtain a 6: ASB molar ratio of 30: 1. The solution is stirred at room temperature, leading to a complete dissolution of 6 after 2 hours. After 24 hours of stirring, the solution is deposited on a centrifugal filter (Centricon, Millipore, filter at 30 Kda) and the volume of the solution is reduced to 200-300 μl by filtration. The solution is diluted with 3 ml of water, then the volume is reduced again to 200-300 ml by filtration. This last operation is repeated 3 to 4 times, until the filtration waters are no longer luminescent under UV irradiation (absence of europium). The 200 - 300 μl of residual solution containing the labeled protein and remaining on the filter are recovered and stored in the refrigerator at 4 ° C. Characterization of the labeled ASB The UV-Vis absorption spectrum of the aqueous solution of marked ABS shows an intense absorption due to the europium complexes, which partially coat the absorption due to the protein (max = 278 nm, emax = 38000 M ^ .c "1) By excitation of the solution in the absorption band of the bipyridines (308 nm), a typical emission spectrum of the europium compounds is observed, with a lifetime of the average excited state of 1.1 ms (the decrease is not purely monoexponential) and a luminance quantum yield of 13% The characterization by mass spectrometry in MALDI-TOF mode ("Matrice Assisted Laser Desorption Ionisation-Time Of Fly" = Desorption ionization on matrix assisted by Laser, analysis by time of flight) is carried out as follows: An aqueous solution of labeled ASB is treated with 1% trifluoroacetic acid to decomplement the europium, then the protein is adsorbed on a chromatography column whose hydrophobic solid phase is made up of a C4 chain. After washing with water, the protein is removed with acetonitrile, then analyzed by MALDI-TOF (matrix of -cyano-4-hydroxycinnamic acid). The average mass obtained for the labeled protein without europium is 71700 Da (ASB, M = 66610 Da), which leads to a marker / BSA molar ratio of 9/1 in the labeled protein. Example 6 Preparation of Complex 8 of Formula [Tb. (1- 4H) .H20] Na In a 250 ml balloon equipped with a refrigerant, 40 mg (59 μmol) of Compound 1.3HC1 are dissolved in a mixture of 30 ml of MeOH and 30 ml of water. To this solution is added 25 mg (67 μmol) of TbCl3.6H20 dissolved in 5 ml of MeOH and 5 ml of water. The solution is heated at 70 ° C for one hour. After cooling, the pH of the solution is brought to 7.2 with a 1% solution of NaOH in water. The solution is concentrated in the rotary evaporator until a slight turbidity appears, then THF is added until an important precipitate is formed. A pale yellow solid is isolated by centrifugation, then vacuum dried. 46 mg (56 μmoles) of Complex 6 (corresponding to a yield of 95%) are obtained whose characteristics are the following: Analysis calculated for C. gH ^ Na sOsTb.4H20: C, 42.40; H, 3.56; N, 8.53. Found: C, 42.28; H, 3.31; N, 8.38.

FAB "/ MS: 668.2 ([8-H20-CH2C00Na] -. 100%), 726.2 ([8-H20-Na]", 30%). IR (KBr pellet, cm "1): 3428, 1592, 1574, 1466, 1416, 1387. Photo-physical properties in water: Absorption,? _. Ax [nm] (emax [M" 1 .cm-1] ): 320 (punctual contact zone), 308 (20800), 277 (8900), 267 (10400), 253 (15000). Emission: characteristic of terbium compounds with fine bands at 487, 543, and 621 nm. Life time of the excited state: 1.48 ms. Quantum yield (reference quinine sulfate in H2SO4 IN): 31%. Life time of the excited state in heavy water: 2.53 ms. Quantum yield in heavy water: 53%. Example 7 Preparation of Complex 9 of Formula: In a 10 ml balloon, 50 mg (61 μmol) of Complex 8 was suspended in 5 ml of DMSO. To this solution is added 9 mg (78 μmoles of N-hydroxysuccinimide and 13 mg (68 μmol) of ethyl- N, N-dimethyl-3-aminopropyl-carbodiimide hydrochloride (EDCI.HC1) salts. ambient temperature for 138 hours during which Complex 8 dissolves, then a white precipitate forms.The solid is isolated by centrifugation, washed with THF and dried in vacuo.The addition of THF to the mother liquor causes the formation of a supplementary precipitate, which is recovered by centrifugation.Completely 49 mg (55 μmoles) of Complex 9 (corresponding to a yield of 90%) are obtained whose characteristics are the following: Analysis calculated for C33H25N6O10Tb.4H20: C, 44.21; H, 3.71; N, 9.29. Found: C, 44.01; H, 3.42; N, 9.29.

FAB + / MS: 726. 2 ([9-H20-C-_H4N02] +, 15%), 825. 5 ([9-H20 + H] +, 100%). IR (KBr pill, c "1): 3433, 1741, 1624, 1594, 1574, 1464, 1419, 1375. Photo-physical properties in water Absorption,? Max [nm] (emax [M "1 cm" 1]): 308 (18700), 276, 267, 253. Characteristic emission of the compounds of terbium with fine bands at 487, 543, 583 and 621 nm. Life time of the excited state: 1.50 ms. Quantum yield (reference quinine sulfate in H 2 S0 1 N): 34%. Life time of the excited state in heavy water: 2.42 ms. Quantum yield in heavy water: 62%. Example 8 Labeling of serum albumin of ASB-res by Complex 9 and evidenced by luminescence microscopy in temporal resolution The labeling of serum albumin was performed according to the method described in Example 5, replacing Complex 6 with the Complex 9.

Determination of the molar ratio arcador / BSA The molar ratio marker / BSA (number of complexes 9 covalently bound to the ASB) is determined by differential absorption at 308 nm. The molar absorption coefficients of the native ASB and the labeled ASB were measured at 308 nm. The difference of these two values is divided by the molar absorption coefficient of Complex 9 at 308 nm, to give a marker / BSA molar ratio of 6/1 in the labeled protein. Figure 1 represents droplets of approximately 750 microns in diameter containing marked ABS by means of Compound 9 (left and right columns on each image) and an antibody marked by fluorescein (middle column on each image) serving as reference (Rabbit immunoglobulin labeled with fluorescein produced by Dako-Immunoglobuline under product code F-123). The image obtained by conventional fluorescence microscopy (left) reveals the fluorescence of the two compounds. The image obtained by luminescence microscopy in temporal resolution (term = 0.5 ms, integration time = 5.0 ms) shows the disappearance of the fluorescence of the reference compound at the moment in which the luminescence of the marked ASB lasts. Example 9 Preparation of Complex 10 of Formula [Gd. (1-4H), H2Q] Na In a 100 ml balloon equipped with a refrigerant, 30 mg (44 μmol) of compound 1.3HC1 are dissolved in a mixture of 25 ml of MeOH and 25 ml of water. To this solution is added 19 mg (51 μmol) of GdCl3.6H20 dissolved in 5 ml of MeOH. and 5 mi of water. The solution is heated at 70 ° C for one hour. After cooling the pH of the solution is raised to 7.5 with a 0.5% solution of NaOH in water. The solution is concentrated in the rotary evaporator until a slight turbidity appears, then THF is added until an important precipitate is formed. The pale yellow solid is isolated by centrifugation then vacuum dried to give 30 mg (37 μmol) of Complex 10 (corresponding to a yield of 85%) whose characteristics are the following: Analysis calculated for C29H22Gd at 508.3H20: C, 43.44; H, 3.39; N, 8.73. Found: C, 43.35; H, 3.17; N, 8.55.

FAB "/ MS: 667.2 ([10-H2O-CH2COONa] -, 100%), 725.2 ([10-H2O-Na] -, 45%). 'IR (KBr pill, c "1): 3422, 1637, 1592, 1459, 1419, 1385. EXAMPLE 10 Preparation of Complex 11 of Formula In a 10 ml balloon, 50 mg (62 μmol) of Compound 10 was suspended in 5 ml of DMSO. To this solution are added 9 mg (78 μmol) of N-hydroxysuccinimide and 15 mg (78 μmol) of ethyl-N, N-dimethyl-3-aminopropyl-carbodiimide hydrochloride (EDCI.HC1) salts. The solution is stirred at room temperature for 48 hours during which Complex 10 dissolves, then a white precipitate forms. The solid is isolated by centrifugation, washed with THF and dried in vacuum. The addition of THF to the mother liquors causes the formation of additional precipitate, which is recovered by centrifugation. In total, 45 mg (51 μmoles) of Complex 11 are obtained (corresponding to a yield of 82% whose characteristics are the following: Analysis calculated for C33H25GdN60 _ (.. 3H20; C, 45.20; H, 3.56; N, 9.37. : C, 45.02; H, 3.18; N, 9.21, FAB + / MS: 726.5 ([11-H20-C4H4N02 + 2H] +, 20%), 824.2 ([11-H20 + H] +, 100%). (KBr pellet, c "1): 3435, 1741, 1623, 1573, 1455, 1420, 1376. Example 11 Preparation of Compound 12 This compound is obtained in two stages from Compound 3, according to the following synthetic scheme: Preparation of Compound 13 In a Schlenk tube under an argon atmosphere, 200 mg (0.30 mmol) of Compound 3, 90 μl (0.70 mmol) of diethyl phosphite, 78 mg (0.30 mmol) of PPh3 and 300 μl of diisopropylethylamine are introduced. freshly distilled in 10 ml of toluene. The solution is degassed with argon for 20 minutes. 34 mg (0.03 mmol) of Pd (PPh3) 4 are added and the solution is heated at 100 ° C for 16 hours. 40 μl (0.31 mmoles) of diethyl phosphite and 34 mg (0.03 mmoles) of (PdPh3) are added and the solution is again heated at 100 ° C for 16 hours. The solution is evaporated to dryness. The solid residue is purified by flash chromatography on silica (f = 3 cm, h = 15 cm) with a mixture of CH 2 Cl 2 / MeOH (99/1 to 95/5) as eluent. The pure fractions are evaporated, solubilized in 30 ml of CHC12 and washed with 10 ml of water. The organic phase is dried over MgSO4, filtered and evaporated. 72 mg (0.09 mmol) of Compound 13 (corresponding to a yield of 31%) are obtained in the form of an oil having the following characteristics: Rf = 0.56, SiO2, CH2Cl2 / MeOH (90/10). 2 H-NMR (CDC13, 200 MHz): d 1.35 (t, 12H, 3J = 7.0 Hz), 2.02-2.22 (m, 2H), 2.37-2.71 (, 2H), 3.47 (s, 3H), 3.54-3.61 (m, 1H), 3.75 (s, 3H), 4.01-4.17 (, 4H), 4.18-4.36 (m, 8H), 7.47 (d, 2H, 3J = 7.5 Hz), 7.73 (t, 2H, 3J = 8, 0 Hz), 7.81-7.97 (m, 4H), 8.32 (d, 2H, 3J = 7.5 Hz), 8.59 (dt, 2H, 3J HH = 7, 0 Hz, 3JH-P = 4JH-H = 2.0 Hz). 13 C-NMR (CDC13, 50 MHz): d 16.3, 16.4, 24.7, 30.3, 51.4, 57.1, 61.9, 63.0, 63.1, 119.6, 123.2 (2), 123.4, 127. 4, 127.9, 136.7, 137.0, 137.2, 149.0, 153.5, 154.5, 156. 5, 156.9, 159.0, 173.1, 173.4. 31 P-NMR (CDCl 3, 162 MHz): d 11.73.

Preparation of Compound 12 In a 50 ml balloon equipped with a refrigerant, 51 mg (65 μmoles) of Compound 13 are dissolved in 6 ml of 0.05 N NaOH solution in water. The mixture is heated at 100 ° C for 19 hours. After cooling the aqueous phase is extracted with 4 portions of 5 ml of CH2C12, then it is evaporated to dryness. The product precipitates in a mixture of H20 / THF. 45 mg (51 μmol) of Compound 12 (corresponding to a yield of 79%) are obtained in the form of a cream-colored powder whose characteristics are as follows. XH-NMR (D20 / tBu0H, 300 MHz d 1.18 (t, 6H, 3J = 7.0 Hz), 2.06-2.27 (, 2H), 2.37-2.58 (m, 2H), 3.50 (t, 3H, 3J = 7.5 Hz ), 3.86-3.99 (m, 4H), 4.02-4.24 (m, 4H), 7.48 (d, 2H, 3J = 7.0 Hz), 7.59-7.81 (m, 10H). 13C-NMR (D20 / tBuOH, 75 MHz): d 16.4, 16.5, 27.8, 35.6, 59.8, 62.4, 62.5, 71.6, 121.2, 124.0, 124.1, 125.7, 127.1, 127.4, 138.0, 138.2, 138.5, 154.6, 155.6, 156.3, 156.6, 157.8, 160.6, 181.1, 183.6.31P-NMR (D20, 162 MHz): d 10.17.Analysis calculated for C31.H31 5 a_1O10P2.5H2O: C, 42.43; H, 4.71; N, 7.98. Found: C, 42.35; H, 4.55, N, 7.78, FAB + / MS: 764.2 ([12-Na] +, 10%) Example 12 Preparation of Complex 14 of Formula In a 50 ml balloon equipped with a refrigerant, 19 mg (22 μmol) of Compound 12 are dissolved in 35 ml of water. The pH is adjusted to 3.1 with a diluted HCl solution. To this solution is added 9 mg (25 μmol) of EuCl3.6H0 dissolved in 5 ml of water. The solution is heated at 80 ° C for one hour. After cooling the solution is filtered over celite and the pH is raised to 7.1 with a 0.5% solution of NaOH in water. The solution is evaporated to dryness, the product precipitates in a mixture of H20 / THF. The pale yellow solid is isolated by centrifugation then vacuum dried, and 9 mg (10 μmoles) of complex 14 are obtained. (corresponding to a yield of 47%) whose characteristics are the following: FAB + / MS: 848.2 ([14-H20-Na] +, 35%).

Example 13 Preparation of Compound 15 This compound is obtained in three stages according to the following synthetic scheme: Preparation of Compound 16 In a Schlenk balloon of 500 ml under an argon atmosphere, 450 mg (2.13 mmol) of diethyl aminoalonate hydrochloride salts and 1.18 g (8.54 mmol) of K2C03 in 150 ml of freshly distilled acetonitrile are introduced. . The solution is heated at 80 ° C for one hour. 1.46 g (4.45 min.) Of Compound 2 are added and heated for 21 hours at 80 ° C. The solution is evaporated to dryness and the residue is redissolved with 10 ml of CH2C12 and 20 ml of water. The aqueous phase is washed with two 20 ml portions of CH2C12 and the sum of the organic phases is dried over MgSO4, filtered, then evaporated to dryness. The solid residue is purified by flash chromatography on silica (^ = 4 cm, h = 14 cm) with a mixture of CH2Cl2 / MeOH (100/0 to 99/1) as eluent. 794 mg (1.19 mmoles) of Compound 16 (corresponding to a yield of 56%) are obtained in the form of a pale yellow powder having the following characteristics: Rf = 0.57, SiO2, CH2Cl2 / MeOH (97/3). XH-NMR (CDC13, 200 MHz): d 1.26 (t, 6H, 3J = 7.0 Hz), 4.22 (s, 4H), 4.23 (q, 4H, 3J = 7.0 Hz), 4.47 (s, 1H), 7.43 (dd, 2H, 3J = 7.5 Hz, J = 0.5 Hz), 7.60 (t, 2H, 3J = 7.5 Hz), 7.62 (d, 2H, 3J = 7.5 Hz), 7.75 (t, 2H, 3J = 8.0 Hz ), 8.22 (dd, 2H, 3J = 7.5 Hz, 4J = 1. O Hz), 8.37 (dd, 2H, 3J = 7.5 Hz, 4J = 1.0 Hz). 13 C-NMR (CDC13, 50 MHz): d 14.1, 58.0, 61.4, 67.1, 119.7, 123.4, 127.7, 137.4, 139.0, 141.4, 153.5, 157.4, 158.9, 168.1. Analysis calculated for C29H2Br2N504: C, 52.04; H, 4.07; N, 10.46. Found: C, 51.93; H, 3.93; N, 10.31. FAB + / MS: 670.2 (100%), 672.2 (50%), [16-H] +. Preparation of Compound 17 In a 250 ml two-neck balloon, 778 mg (1.16 mmol) of Compound 16 and 82 mg (0.12 mmol) of [Pd (PPh3) 2C12] are introduced into 75 ml of ethanol and 75 ml of triethylamine. The solution is heated at 70 ° C for 16 hours by bubbling a flow of CO. The solution is evaporated to dryness, the solid obtained is redissolved in 75 ml of CH2C12, filtered on celite, then the organic phase is washed with 15 ml of water. The aqueous phase is extracted with two 20 ml portions of CH2C12 and the whole of the organic phases is dried over MgSO4, filtered and then evaporated to dryness. The residue is purified by flash chromatography on silica (f = 3 cm, h = 16 cm) with a mixture of CH 2 Cl 2 / MeOH (99.5 / 0.5 to 90/10) as eluent. The fractions containing Compound 17 with the triphenylphosphine oxide are dissolved in 40 ml of CH2C12 and extracted with four portions of 3N HCl. The combined aqueous phases are neutralized with NaOH then extracted with three 30 ml portions of CH2C12. The whole of the organic phases is dried over MgSO, is then filtered, evaporated to dryness. 522 mg (0.80 mmol) of Compound 17 are obtained in the form of a colorless oil (corresponding to a yield of 68%), which has the following characteristics: Rf = 0.55, SiO2, CH2Cl2 / MeOH (90/10). XH-NMR (CDC13, 200 MHz): d 1.26 (t, 6H, 3J = 7.0 Hz), 1. 45 (t, 6H, 3J = 7.0 Hz), 4.23 (q, 4H, 3J = 7.0 Hz), 4.24(s, 4H), 4.47 (q, 4H, 3J = 7.0 Hz), 4.48 (s, 1H), 7.64 (dd, 2H, 3J = 7.5 Hz, 4J = 0.5 Hz), 7.80 (t, 2H, 3J = 8.0 Hz), 7.91 (t, 2H, 3J = 7.5 Hz), 8.09 (dd, 2H, 3J = 7.5 Hz, 4J = 1 .0 Hz), 8.40 (dd, 2H, 3J = 7.5 Hz, 4J = 05 Hz), 8 ,. 62 (dd, 2H, 3J = 8.0 Hz, 4J = 1.5 Hz). 13 C-NMR (CDCl 3, 50 MHz): d 14.1, 14.3, 58.0, 61.4, 61.8, 67.1, 120.0, 123.4, 124.2, 124.7, 137.5, 137.7, 147.7, 154.4, 156.5, 158.8, 165.3, 168.2. Analysis calculated for C35H37N508: C, 64.11; H, 5.69; N, 10.68. Found: C, 63.81; H, 5.43; N, 10.43. FAB + / MS: 496.2 (35%), 656.1 ([17 + H] +, 100%). Preparation of Compound 15 In a 50 ml balloon equipped with a refrigerant, 103 mg (0.16 mmol) of Compound 17 and 50 mg (1.25 mmol) of NaOH are dissolved in a mixture of 10 ml of MeOH and 5 ml of water. The mixture is heated at 70 ° C for 5 hours. The solution is evaporated to dryness and the solid is dissolved in 8 ml of water on which a solution of IN HCl is slowly added at 0 ° C until the product precipitates massively (pH = 4-5). The precipitate is isolated by centrifugation and is dried under vacuum. 59 mg (0.08 mmol) of hydrated hydrochloride of 15.3HC1 (corresponding to a yield of 53%) are obtained in the form of a white powder whose characteristics are the following: XH-NMR (NaOD / ^ uOH, 300 MHz): d 3.75 (s, 4H), 4.04 (s, 1H), 6.84 (d, 2H, 3J = 7.5 Hz), 7.15-7.26 (m, 4H), 7.32 (d, 2H, 3J = 7.5 Hz), 7.42 (t, 2H, 3J = 7.5 Hz), 7.56 (d, 2H, 3J = 7.5 Hz). 13 C-NMR (NaOD / '? UOH, 75 MHz): d 60.3, 79.4, 119.9, 122.9, 124.1, 124.4, 138.2, 138.6, 152.8, 153.7, 154.0, 158.7, 168.6, 172.3, 177.3. Analysis calculated for C27H21 5O8.3HC1.3H0: C, 45.87; H, 4.28; N, 9.91. Found: C, 45.75; H, 4.09; N, 9.78. FAB + / MS: 544.2 ([15 + H] +, 20%). Example 14 Preparation of Formula 18 Complex In a 10 ml balloon, 15 mg of 15-3HC1.3H20 (21 μmol) are dispersed in a mixture of 10 ml of MeOH and 10 ml of water. To this solution is added 10 mg (27 μmol) of EuCl3.6H20 dissolved in 5 ml of MeOH and 5 ml of water. The solution is heated at 70 ° C for one hour. After cooling the pH of the solution is raised to 7.3 with a 0.5% solution of NaOH in water. The solution is concentrated in the rotary evaporator until a precipitate appears. The white solid is isolated by centrifugation then vacuum-dried to give 14 mg (19 μmoles) of Compound 18 (corresponding to a yield of 90%) whose characteristics are as follows: FAB + / MS: 692.3 ([18-H20] - , 100%). IR (KBr pill, cm "1): 3442, 1626, 1588, 1460, 1411, 1373.

Example 15 Preparation of Complex 19 of Formula: In a 10 ml balloon, 18 mg (2 μmoles) of Complex 5 are suspended in 5 ml of DMSO. To this solution is added 6 mg (26 μmoles) of monosodium salt of N-hydroxy-succinimide-3-sulphonic acid hydrate and 5 mg (26 μmol) of ethyl-N, N-dimethyl-3-aminopropyl-carbodiimide hydrochloride ( EDCI.HC1). The solution is stirred at room temperature for 46 hours during which Complex 5 dissolves. The addition of THF to the solution causes the formation of a precipitate which is recovered by centrifugation. 15 μmoles) of Complex 19 (corresponding to a yield of 68%) are obtained in the form of a white powder. Example 16 Preparation of Complex 20 of Formula: In a 50 ml balloon, 45 mg (55 μmol) of Complex 8 are suspended in 10 ml of DMSO. To this solution are added 14 mg (60 μmoles) of monosodium salt of N-hydroxy-succinimide-3-sulphonic acid hydrate and 12 mg (63 μmol) of ethyl-N, N-dimethyl-3-aminopropyl-carbodiimide hydrochloride ( EDCI.HC1). The solution is stirred at room temperature for 92 hours during which Complex 8 dissolves. The addition of THF to the solution causes the formation of a precipitate which is recovered by centrifugation. 45 mg (44 μmol) of Complex 20 (corresponding to a yield of 81%) are obtained in the form of a yellow powder whose characteristics are as follows: Analysis calculated for C33H2N6Na013STb.5H20: C, 38.99; H, 3.37; N, 8.27. Found: C, 39.20; H, 3.56; N, 8.39.

FAB + / MS: 682. 2 ([20-H2O-C5H3NNaO7S] +, 95%), 727. 2 ([20-H2O-C6H3NnaO5S + H] +, 55%). Example 17 Preparation of Complex 21 of Formula: In a 10 ml balloon, 19 mg (24 μmol) of Complex 10 are suspended in 5 ml of DMSO. To this solution is added 7 mg (30 μmoles) of monosodium salt of N-hydroxy-succinimide-3-sulphonic acid hydrate and 5 mg (26 μmol) ethyl-N, N-dimethyl-3-aminopropyl-carbodiimide hydrochloride (EDCI.HC1). The solution is stirred at room temperature for 24 hours during which Complex 10 dissolves. The addition of THF to the solution causes the formation of a precipitate which is recovered by centrifugation. 19 mg (19 μmoles) of Complex 21 (corresponding to a t yield of 80%) are obtained under the yellow powder form whose characteristics are as follows: FAB + / MS: 681. 2 ([21-H20-C5H3NNa07S] +, 100%), 726. 3 ([21-H20-C4H3NNa05S + H] +, 40%). Example 18 Preparation of Compound 25 Compound 25 is obtained in four stages according to the following Reaction Scheme: Preparation of Compound 22 In a Schlenk tube under argon atmosphere, 2.04 g (7.6 mmoles) of 7-hydroxy-9-carbomethoxy-2-methyl-phenanthroline (obtained according to Heindel, N et al., J. Heterocycl. Chem. 1968, 5, 869), 2.11 g (15.2 mmol) of K2C03 and 950 μl (15.3 mmol) of methyl iodide in 60 ml of acetonitrile freshly distilled over P2O5. The solution is heated at 80 ° C for 19 hours. The solution is evaporated to dryness, the residue is dissolved in 100 ml of CH2C12 and 15 ml of water. The aqueous phase is extracted with 4 portions of 15 ml of CH2C1 and the sum of the organic phases is dried over MgSO4, filtered, then evaporated to dryness. The residue is purified by chromatography on alumina (f = 5 cm, h = 12 cm) with a mixture of CH 2 Cl 2 / MeOH (99/1) as eluent. 2.05 g (7.3 mmoles) of Compound 22 (corresponding to a yield of 95%) is obtained in the form of a yellow powder having the following characteristics: Rf = 0.54, AI2O3, CH2Cl2 / MeOH (98/2). ^? - NMR (CDCl 3, 200 MHz): d 2.91 (s, 3H), 4.06 (s, 3H), 4.12 (s, 3H), 7.47 (d, 1H, 3J = 8.5 Hz), 7.77 (d, 1H , 3J = 9.0 Hz), 7.83 (s, 1H), 8.08 (d, 1H, 3J = 7.5 Hz), 8.12 (d, 1H, 3J = 9.0 Hz). 13 C-NMR (CDCl 3, 50 MHz): d 25.8, 52.8, 56.2, 102.9, 118.7, 122.2, 123.9, 126.9, 127.4, 136.0, 145.2, 146.1, 148.6, 160.1, 163.2, 166.5. Analysis calculated for C? 6H? N203: C, 68.07; H, 5.00; N, 9.92. Found: C, 67.92; H, 4.93; N, 9.78. Preparation of Compound 23: In a 250 ml balloon, 1 g (3.5 mmol) of Compound 22, 630 mg (3.5 mmol) of N-bromosuccini ida and 30 mg (0.2 mmol) of azo-bis-isobutyronitrile (ATBN) in 10 ml are introduced. of benzene. The solution is irradiated for 30 minutes with a standard halogen lamp of 100 W. The solvent is evaporated under pressure and the residue is purified by chromatography on alumina containing 10% water with a mixture of CH2Cl2 / hexane (50/50) as eluent 468 mg (1.3 mmol) of Compound 23 (corresponding to a yield of 37%) are obtained in the form of a gray powder having the following characteristics: ^? - NMR (CDC13, 200 MHz): d 4.06 (s, 3H) , 4.12 (s, 3H), 4.93 (s, 2H), 7.77 (d, 1H, 3J = 9.0 Hz), 7.83 (s, 1H), 7.7 (d, 1H, 3J = 8.5 Hz), 8, 17 (d, 1H, 3J = 9, 0 Hz), 8, 21 (d, 1H, 3J = 8, 5 Hz). 13 C-NMR (CDCl 3, 50 MHz): d 34.6, 53.0, 56.3, 1033, li20.2, 122.4, 123.7, 127.0, 128.1, 137, 1, 144.5, 145, 9 148.9, 157.6, 163.3, 166.2. Analysis calculated for C? 6H13BrN203: C, 53.21; H, 3. 63; N, 7.76. Found: C, 15.94; H, 3.26; N, 7.51. FAB + / MS: 281.2 ([23-Br] +, 30%), 361.2 (100%), 363.2 (100%), [23 + H] +. Preparation of Compound 24 In a Schlenk tube under argon atmosphere, 96 mg (0.45 mmol) of DL-glutamic acid dimethyl ester hydrochloride and 250 mg (1.81 mmol) of K2C03 in 15 ml of acetonitrile freshly distilled over P205. The solution is heated at 80 ° C for 10 minutes. 360 g (1 mmol) of Compound 23 are added and heated for 18 hours at 80 ° C. A new portion of Compound 23 (52 mg, 0.14 mmol) is added and heated for 24 hours at 80 ° C. The solution is evaporated to dryness, the residue is dissolved in 30 ml of CH2C1 and 10 ml of water. The aqueous phase is extracted with 4 portions of 30 ml of CH 2 Cl 2 and the sum of the organic phases is dried over MgSO 4, filtered, then evaporated to dryness. The residue is purified by chromatography on alumina containing 10% water with a mixture of CH2Cl2 / MeOH (100/0 to 99.3 / 0.7) as eluent. 46 mg (0.06 mmol) of Compound 24 are obtained (corresponding to a yield of 37%) that has the following characteristics: Rf = 0.16, AI2O3, CH2Cl2 / MeOH (98/2). ^ -RM (CDC13, 200 MHz): d 2, 17-2.28 (m, 2H), 2.61 (t, 2H, 3J = 7.5 Hz), 3.44 (s, 3H), 3.71 (t, 1H, 3J = 7.5 Hz), 3.83 (s, 3H), 4.06-4.19 (m, 12H), 4.45-4.70 (m, 4H), 7.82 (d, 2H, 3J = 9.0 Hz), 7.86 (s, 2H), 8.13 (d , 2H, 3J = 8.0 Hz), 8.18 (d, 2H, 3J = 9.0 Hz), 8.24 (d, 2H, 3J = 8.5 Hz). Preparation of Compound 25.3HC1 In a 50 ml balloon, 46 mg (0.06 mmol) of 24 and 10 mg (0.25 mmol) of NaOH are dissolved in a mixture of 9 ml of MeOH and 3 ml of water. The mixture is heated at 75 ° C for 21 hours. The solvents are evaporated under reduced pressure, the solid is dissolved in 5 ml of water and the solution obtained is filtered on celite. The medium is acidified with a dilute solution of hydrochloric acid and the solution is evaporated to dryness. The residue is washed with 2 portions of 2 ml of water. 19 mg (0.02 mmol) of 25.3HC1 hydrochloride are obtained (corresponding to a yield of 39%) in the form of yellow-orange powder whose characteristics are the following: ^? - NMR (CD30D, 200 MHz): d 2.38-2.52 (m, 2H), 2.84 (t, 2H, 3J = 7.0 Hz), 4.17 (t, 1H, 3J = 7.5 Hz), 4.35 (s, 6H), 4.81-4.85 (m, 4H), 7.87 (d, 2H, 3J = 9.5 Hz), 7.89 (s, 2H), 8.16 (d, 2H, 3J = 9.0 Hz), 8.18 (d, 2H) , 3J = 8, 5 Hz), 8, 65 (d, 2H, 3J = 9, 0 Hz). Example 19 Preparation of Compound 26 of Formula [Eu. (25- 4H) .H20] Na In a 50 ml balloon, 19 mg of 25.3HC1 (24 μmol) are dissolved in a mixture of 15 ml of MeOH and 15 ml of water. To this solution is added 10 mg (27 μmol) of BuCl3.6H20 dissolved in 2.5 ml of MeOH and 2.5 ml of water. The solution is heated at 70 ° C for 1 hour. After cooling the pH of the solution is raised to 7.0 with a 0.5% solution of NaOH in water. The solution is concentrated in the rotary evaporator until a slight turbidity appears, then THF is added until a precipitate is formed. The yellow solid is isolated by centrifugation then vacuum drying to give 7 mg (8 μmol) of Compound 26 (corresponding to a yield of 33%) whose characteristics are as follows: FAB- / MS: 791.2 (30%), 828.2 ( [26-H20-Na)] -, 50%).

Example 20 Labeling of an Anti-digoxigenin Antibody by Complex 9 and Characterization by Mass Spectrometry 0.5 mg of Complex 9 is added to a solution of anti-digoxigenin antibodies containing 1.0 mg of antibody dissolved in 500 μl of buffer solution (50 mM borate buffer, pH = 7.0), corresponding to a ratio of 9/30 antibodies. :1. The solution is stirred at room temperature for 24 hours, then the labeled antibody is purified according to the procedure described in Example 5 and stored at 4 ° C. The MALDI-TOF mass spectrometry characterization is carried out according to the procedure described in Example 5, leading to a mass of 49220 Da for the labeled antibody (47880 Da for the free antibody), ie a graft index of 2.5.

Claims (32)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty, and therefore the content of the following is claimed as property: CLAIMS

1. A compound that responds to Formula (I) R2 I R1 - X - C - R3 i N wherein - R1 is a functional group capable of reacting with the functions present on proteins, antibodies or on mineral or organic materials; X represents a single bond or a hydrocarbon chain consisting of at least one group selected from the alkylene groups and the alkenylene groups optionally comprising at least one heteroatom, and between the arylene groups; - R2 is an anionic group of neutral pH A2 or an alkylene or alkenylene group having 1 to 4 carbon atoms and containing at least one such A2 group, said alkylene or alkenylene group optionally comprising at least one heteroatom in the chain; - R3 represents H or an alkylene or alkenylene group having from 1 to 5 carbon atoms and optionally containing at least one heteroatom in the chain, said group optionally containing at least one anionic group at neutral pH A3; - R4 is selected from the groupings corresponding to the formula- (C) nC-Z1-CC-Z2-C-A4 in which n is equal to 1 or 2, Z1 and Z2, each represents a heteroatom selected from O and N, with at least one being a nitrogen atom forming part of an aromatic heterocycle with the two carbon atoms surrounding it, and A4 being a group that is anionic at neutral pH in which the atom containing the anionic charge is in position? in relation to Z2; - R5 is selected from the groupings defined for R4 or between the groups corresponding to the formula -CC-E1-CC-E2-C-A5 in which E1 and E2 independently represent a heteroatom selected from O and N, and A5 is an anionic group at neutral pH in which the atom containing the anionic charge is in position? in relation to E

2. Compound according to claim 1, characterized in that the substituent R1 is selected from amino, thio, cyano, isocyano, acridinyl, hydrazino, halogen acetate, anhydride, triazo, carbonyl, nitrobenzoyl, sulfonyl, thionyl, halide, epoxide, aldehyde, i-idazole, hydroxyphenyl, ercapto, N-succinic ester, N-sulfosuccinic ester, maleimido, hydroxyl, carboxyl, thiocyano, and isothiocyano.

3. Compound according to claim 1, characterized in that the substituent R2 is an anionic A2 group of neutral pH.

4. Compound according to claim 1, characterized in that the substituent R3 is H or a C1 to C3 alkyl.

Compound according to claim 1, characterized in that the groups Z1 and Z2 of R4 are part of an aromatic heterocyclic group.

Compound according to claim 1, characterized in that n is equal to 1.

7. Compound according to claim 1, characterized in that any of the segments -C-Z1-C- or -C-Z2-C- forms part of a heterocyclic group selected from the pyridyl, pyrimidinyl, quinolyl and isoquinolyl groups.

Compound according to claim 1, characterized in that the segment -C-Z1-CC-Z2-C- is selected from the groups 2, 2'-bipyridinyl, 1,10-phenanthrolinyl, 2,2'-bisquinolyl, 2, 2'-bisisoquinolinyl and 2,2'-bipyrimidinyl, said groups may contain alkyl or alkoxy substituents at least one carbon atom of a heterocycle.

9. Compound according to claim 1, characterized in that R5 is selected from the following groupings: wherein R6 and R7 represent alkyl chains having from 1 to 5 carbon atoms and optionally containing one or more heteroatoms.

Compound according to claim 1, characterized in that R4 and R5 are identical.

11. Compound in accordance with the claim 1, characterized in that the neutral pH anionic groups A2, A3, A4 or A5 are independently selected from each other between the groups -C02H, -S03H, -P (0) (OR) OH, -P (0) R (OH) and -P (0) (0H) 2 wherein R is an alkyl group or an aryl group.

12. Compound according to claim 1, characterized in that it is under the cationic form, the nitrogen carrying the substituents R4 and R5, as well as optionally the heteroatoms Z1, Z2, E1 and E2 are in the protonated form.

13. Compound according to claim 1, characterized in that it is in the anionic form, the different groups A1 are presented in the form of salts.

14. Compound according to claim 1, characterized in that it is in the amphoteric form, the nitrogen bearing the substituents Z4 and Z5, as well as optionally the heteroatoms Z1, Z2, E1 and E2 are in the protonated form, and the different groups A1 they come in the form of salts.

Compound according to claim 1, characterized in that X is an amylene group comprising a single aromatic nucleus or several condensed aromatic nuclei or not, said nuclei possibly containing one or more aliphatic hydrocarbon groupings.

16. Compound according to claim 1, characterized in that the group X is an alkylene or alkenylene group, having 1 to 10 carbon atoms.

17. Compound according to claim 1, characterized in that the grouping X is amylene grouping having from 5 to 10 carbon atoms.

Method for preparing a lanthanide complex, characterized in that it consists of reacting a compound (I) according to any one of claims 1 to 17 with a donor compound of the lanthanide cation.

19. Process according to claim 18, characterized in that the lanthanide cation donor compound is selected from the hydrated thiastide halides, the lanthanide hydrated nitrates, the lanthanide carbonates and the lanthanide triflates.

20. Method according to claim 18, characterized in that the reaction is carried out in solution in a solvent selected from water, methanol, ethanol or acetonitrile.

21. Method according to claim 18, characterized in that the compound (I) is reacted with the precursor of the lanthanide ion in a mixture of methanol and water at a pH ranging from 3 to 5, for a period of 10 minutes. and 24 hours, at a temperature between 25 ° C to 80 ° C, then the pH of the solution is adjusted to 7.0 and the methanol is evaporated.

22. The complex obtained by means of a process according to claim 18, constituted by a lanthanide ion Ln, complexed by a ligand that responds to Formula (I).

23. Complex according to claim 22, characterized in that the lanthanide ion is selected from the europium, terbium, samarium, dysprosium, erbium, ytterbium, neodymium and gadolinium ions.

24. Complex according to claim 22, characterized in that the substituent R4 of Compound (I) is -CC-Z1-CC-Z2-C-A4, the three chelate cycles are formed between the lanthanide cation and respectively: - the atom N containing R4 and R5, Z1 and the carbon atoms that separate them; Z1, Z2 and the two carbon atoms that separate them; - Terminal segment Z2-C-A4.

Complex according to claim 24, characterized in that the substituent R5 is of the same type as the substituent R4.

26. Complex according to claim 24, characterized in that the substituent R5 is of the type -CC-E1-CC-E2-C-A5, three chelate cycles of 5 elements that are formed between the lanthanide cation and respectively: - the N atom that it contains R4 and R5, E1 and the two carbon atoms that separate them; E1, E2 and the two carbon atoms that separate them; - Terminal segment E2-C-A5.

27. Procedure for the quantitative or qualitative analysis of a compound, characterized in that it consists in covalently binding said compound, a marker constituted by a complex according to any of claims 25 to 29, and in detecting or quantifying the presence of the marked compound thanks to the luminescence properties of the marker.

28. Method according to claim 27, characterized in that the complex is a complex of europium, terbium, samarium or dysprosium.

29. Process according to claim 27, characterized in that the substituent R1 of the complex is selected from the amino, thio, and carboxyl groups or between the maleimido, N-succinimide and isothiocyano groups.

30. Relaxation agent for nuclear magnetic resonance, characterized in that it is constituted 5 for a complex according to any of claims 22 to 26.

31. Relaxation agent according to claim 30, characterized in that it is constituted by a complex of gadolinyl, europium or dysprosium.

32. Relaxation agent according to claim 30, characterized in that it is constituted by a complex in which the substituent R1 is selected from the amino, thio and carboxyl groups or between the maleimido, N-succinimide ester and 15 isothiocyan twenty 25