FR2727415A1 - New bi:functional ruthenium complexes - Google Patents

Abstract

New bifunctional ruthenium (II) or ruthenium (III) complexes comprise: (i) a gp. capable of recognising a peptide sequence contg. at least one histidine residue, consisting of a central Ru atom bonded to a CO3 ligand or to two ligands selected from Cl, Br, I or H2O; and (ii) ligands, at least one of which bears at least one functional gp. with intrinsic reactivity towards other opt. modified amino acids. 5 cpds (I) are specifically claimed e.g.: cis-dichloro-bis-(4,4'-bis4-(4-benzoylbenzoyloxy)butoxycarbonyl- 2,2'-bipyridine) ruthenium (II); (Ia); and cis-carbonato-bis(4,4'-bis-4-(4-benzoylbenzoyloxy)butoxycarbonyl- 2,2'-bipyridine) ruthenium (II).

Description

RUTHENlUN COMPLEXES ESPECIALLY USEFUL FOR
DETERMINING THE PROTEIN TOPOLOGY AND THEIR PROCESS
OF PREPARATION.

 The subject of the present invention is new ruthenium complexes, which can be used in particular for determining the topology of proteins.

 These ruthenium complexes can also find other uses, for example as anticancer agents, as reagents for studying the transfer of fluorescence on proteins and as stabilizers of proteins.

 Numerous ruthenium (II) and (III) complexes are known, carrying various ligands, such as carbonyl, phosphine, alkylamine and / or heterocyclic amine groups.

 So far these complexes have been synthesized for uses such as catalysis, kinetic and mechanistic studies of ligand substitution reactions and photochemical studies, in particular for the chemical storage of light energy.

 More recently, ruthenium complexes have found new applications, in particular in the field of biological macromolecules.

Thus, Clarke et al, in Inorganica
Chimica Acta, vol. 124, 1986, p. 13-28, described ruthenium complexes of the formula! <H2O) (NH3) 5 Ru1112 + as anticancer agents and as inhibitors of DNA synthesis. They have shown that these complexes have an affinity for guanine residues.

Bannwarth et al in Helvetica Chimica
Acta, vol. 71, 1988, p. 2085-2099, described Ru (II) complexes as fluorescent markers usable in biological probes.

 More recently, Casimiro et al in J.

Phys. Chem., Vol. 97, 1993, p. 13073-13077, showed that the neutral complex Ru (bipy) 2 C03 reacted in a specific way with the histidines present on the surface of proteins such as cytochromes c.

 Muheim et al in J. Am. Chem. Soc., Vol.

115, 1993, p. 5312-5313, showed that the RuII rII (bpy) 2CO3 complex could be used to bind two histidines on opposite strands of a cytochrome c ss sheet to stabilize this protein.

 According to the present invention, this affinity property is used for the histidine of certain ruthenium complexes, but they are further modified in terms of structures to make them bifunctional, in order to determine the protein topology. Thus, the subject of the invention is bifunctional ruthenium complexes having, on the one hand, the property of specifically recognizing an amino acid sequence present on the surface of one of the subunits composing the protein studied and, on the other hand on the other hand, the property of exhibiting an interaction with a second subunit of the protein considered, thanks to the presence of a functional group carried by one of the ligands of the complex.

 Indeed, the determination of the topology of proteins using complexes must use proximity / contact relationships between the different protein subunits constituting a given protein.

 Also, the present invention relates to new bifunctional ruthenium complexes having these two properties.

According to the invention, the bifunctional complex of ruthenium (II) or (ICI) carrying several ligands, comprises
a group capable of recognizing a peptide sequence incorporating at least one histidine, said group being formed by the central ruthenium atom of the complex linked to the CO3 ligand or to two identical or different ligands, chosen from Cl, Br,
I and H2O; and
- At least one functional group having intrinsic reactivity vis-à-vis other amino acids possibly modified, this functional group being carried by at least one of the other ligands of the complex.

dans laquelle
a) X1 et X2 qui peuvent être identiques ou différents, représentent Br, I, C1 ou H2O, ou X1 et X2 forment ensemble CO3 ;
b) Y est une simple liaison ou un groupe bivalent choisi parmi -C (O) O-, -CONH-, -O-, -S-, -CH2O-, -CH=CH-,-CH2-CH=CH-, -CH2S- et -'C- ;;
d) R1 est un groupe monovalent choisi parmi les groupes photosensibles, les groupes fluorescents, les groupes luminescents, les groupes complexants, les enzymes, l'avidine, la biotine, les chromophores fluorescents, les chromophores absorbant la lumière, les groupes marqués par un élément radioactif, les anticorps et les fragments d'anticorps, et
e) m est tel que 1 S m s 5 ; et
f) R2 représente ~(CH2)n- avec 0 S n 5 2 ou
R2 est un cycle benzénique condensé avec les deux cycles pyridines pour former le cycle orthophénanthroline.According to one embodiment of the invention, the ruthenium complex corresponds to the formula

in which
a) X1 and X2 which may be the same or different, represent Br, I, C1 or H2O, or X1 and X2 together form CO3;
b) Y is a single bond or a bivalent group chosen from -C (O) O-, -CONH-, -O-, -S-, -CH2O-, -CH = CH -, - CH2-CH = CH- , -CH2S- and -'C- ;;
d) R1 is a monovalent group chosen from photosensitive groups, fluorescent groups, luminescent groups, complexing groups, enzymes, avidin, biotin, fluorescent chromophores, light absorbing chromophores, groups marked with a radioactive element, antibodies and antibody fragments, and
e) m is such that 1 S ms 5; and
f) R2 represents ~ (CH2) n- with 0 S n 5 2 or
R2 is a benzene ring condensed with the two pyridine rings to form the orthophenanthroline ring.

 In these ruthenium complexes the chemical group RuX1X2 gives the complex the specific reactivity / recognition property of given peptides incorporating histidines, and the presence of the bipyridine or phenanthroline ligands functionalized with the R1 group gives the complex the property of being able to interact with the chalne peptide of a second subunit of a protein under consideration.

 This interaction can be of different types.

 Thus, when R1 is a photosensitive or photoactivable group, the irradiation of R1 will allow the formation of a stable covalent bond with the peptide chain of a second subunit of the protein considered.

 When R1 is a fluorescent group, a luminescent group or a chromophore, the interaction may be obtained after an appropriate modification of the second protein subunit, by fluorescence transfer.

 When R1 is a protein, for example an enzyme, an antibody or an antibody fragment, the interaction will be obtained after possible modification of the protein subunit by reactions of the enzyme-enzyme substrate type, and antigen-antibody type. .

 Thus, the bifunctional complexes of the invention are suitable for determining the topology of proteins. However, they also have advantageous properties for use as anticancer agents, as agents for stabilizing the structure of proteins, for example by establishing a bond between histidine groups, or as reagents for studying fluorescence transfers on proteins. , in the case where the functional group is a fluorescent group.

 In the ruthenium complex of the invention, R1 can also represent a group marked by a radioactive element such as iodine, tritium, carbon-14, indium.

 In this case, the radioactive element can be introduced just at the time of use.

 Preferably, according to the invention, a photosensitive group such as the benzophenone, bromobenzophenone and aryl azide groups is used for R1, because these groups can be activated by irradiation at a wavelength greater than 280-300 nanometers, which does not denature proteins.

dans laquelle R3 représente H, 3H ou Br. Thus, R1 preferably corresponds to formula (II)

in which R3 represents H, 3H or Br.

 In the complexes of the invention, Y and Z can be of various types. In particular, satisfactory results are obtained with the complexes in which Y represents -C (O) z and Z represents -OC (O) -.

 Preferably, in these complexes, the bipyridines are bipyridines without an intermediate hydrocarbon chain, that is to say that R2 represents - (CH2) n- with n equal to 0.

 The ruthenium complexes of the invention can be prepared by conventional methods from the appropriate derivatives of bipyridine or phenanthroline.

dans laquelle R11 R2, Z, Y, et m sont tels que définis ci-dessus, avec un sel de ruthénium de formule RuCl3, pH2O dans laquelle p est un nombre entier allant de 1 a 3.Thus, it is possible to prepare the ruthenium complexes of formula (I) in which Y, Z, R1, R2 and m have the meanings given above, and X1 and X2 represent Cl, by reaction of a bipyridine derivative or phenanthroline of formula

in which R11 R2, Z, Y, and m are as defined above, with a ruthenium salt of formula RuCl3, pH2O in which p is an integer ranging from 1 to 3.

To carry out this reaction, the technique described by Sullivan et al in
Inorganic Chemistry, vol. 17, no 12, 1978, p. 3335, which consists in refluxing the ruthenium salt, for example RuC13, 3H2O with bipyridine and Licol in an organic solvent such as dimethylformamide.

When we want to prepare the ruthenium complexes of formula (I) in which X1 and X2 form CO3, we can operate as follows
a) first of all prepare the ruthenium complex of formula (I) in which X1 and X2 represent C1 by the process described above, then
b) bringing the ruthenium complex thus obtained into contact with Na2CO3 to exchange the C1 atoms of the complex for CO3.

 This contacting can be carried out in particular using the technique described by Johson et al in Inorganic Chemistry, vol. 17, no 8, 1978, p.

2212.

 When we want to prepare a ruthenium complex of formula (I) in which at least one of the Xl represents Br, I or H2O, we can operate as follows: first prepare a ruthenium complex of formula (I ) in which X1 and X2 represent C1, by the method described above, then subject this complex to an exchange reaction in one or more stages in order to replace at least one of the C1s with Br, I or H2O.

 Exchange reactions of this type are described in particular in Inorganic Chemistry, vol.

17, no 12, 1978, p. 3336.

The bipyridines or phenanthrolines of formula (III) used as starting material in these processes can be prepared by conventional techniques from the corresponding bipyridines or phenanthrolines of formula

 where R2 is as defined above.

Thus, in the case where Y is a single bond, the dimethylated derivative is reacted with lithium to transform the methyl groups into CH2Li groups which are then reacted with a halogenated derivative of formula X- (CHZ) m-1 -OP where X represents a halogen atom such as Br or I, and
P an alcohol protecting group, to form, after deprotection of the alcohol, the derivative of formula

 By subsequent reaction of the OH groups with a compound of formula R1ZH, the desired bipyridine or phenanthroline derivative is obtained.

 In the case where Y represents -C (O) O-, the dimethylated derivative of formula (IV) can be oxidized by means of chromium oxide to replace the two CH3s with COOH.

 By reaction of these COOH groups with an appropriate dialcohol, one of the functions of which is suitably protected and then deprotected, the -C (O) O- (CH2) m-OH arm can be formed.

 The subsequent reaction of this OH group with a compound of formula R1ZH will lead to the desired bipyridine or phenanthroline derivative.

 In the case where Y represents -CONH-, the dimethylated derivative of formula (IV) can be oxidized as before to replace the two CH3s with COOH, then react the CH groups with an amino alcohol whose alcohol function is protected then deprotected for obtain the -CO-NH- (CH2) s-OH arm.

 The reaction of OH with R1ZH will lead, as before, to the desired derivative.

que lton fait réagir avec l'alcoolate de sodium de formule NaO(CH2)mR4 où R4 est un atome d'halogène ou un groupe hydroxyle protégé, pour obtenir le composé de formule

qui peut etre réduit ensuite, par exemple par PC13, pour donner le dérivé de formule

In the case where Y represents -O-, we can start from dinitro-N, N'-dioxide of formula

which is reacted with the sodium alcoholate of formula NaO (CH2) mR4 where R4 is a halogen atom or a protected hydroxyl group, to obtain the compound of formula

which can then be reduced, for example by PC13, to give the derivative of formula

 By reaction with R1ZH, after deprotection of the OH group when R4 is a protected hydroxyl group, this derivative will lead to the desired bipyridine or phenanthroline derivative.

 The same process can be used in the case where Y represents S using the thiolate of Na instead of the alcoholate.

que l'on réduit par l'hydrure double de lithium et d'aluminium, pour former le diol correspondant de formule

In the case where Y represents -CH2O-, it is possible to start from the diacid of formula

which are reduced by the double hydride of lithium and aluminum, to form the corresponding diol of formula

 By reaction of this diol with an appropriate halide and then with a compound of formula R1ZH, the desired bipyridine or phenanthroline derivative will be obtained as before.

 In the case where Y represents -CH2S-, it is possible to start from 4,4'-dimethyl-2,2'-bipyridine which, radically brominated, is reacted with the corresponding thiolate.

In the case where Y represents -HC = CH-, it is possible to start from the diacid of formula (IX) which is reduced to dialdehyde of formula

By carrying out a Wittig reaction between this dialdehyde and Ph3P = CH-R, the derivative of formula is obtained

pour donner le composé de

In the case where Y represents -CH2-CH = CH-, it is possible to start from the dimethylated derivative (IV) which is transformed into the CH2Li derivative. This compound can then react on a compound of the formula type

to give the compound of

 In the case where Y represents - C t C-, one starts from 4,4 'dibromethyl-bipyridine which is condensed with the appropriate lithium alkynide.

avec une bromopyridine de formule

avec n=l ou 2
D'autres caractéristiques et avantages de l'invention apparaltront mieux a la lecture des exemples suivants, donnés bien entendu à titre illustratif et non limitatif, en référence aux figures annexées.The starting bipyridines of formula (IV) are commercial products or can be prepared by coupling with palladium or copper, a bromopyridine of formula

with a bromopyridine of formula

with n = l or 2
Other characteristics and advantages of the invention will appear better on reading the following examples, given of course by way of illustration and not limitation, with reference to the appended figures.

 The functional properties, in particular of recognition of ruthenium complexes of the type of those described in the present invention are demonstrated by the complexation of the carbonated bifunctional derivatives with an appropriate peptide. This complexation demonstrates the affinity of complex 11 for the histidine residue incorporated in a given peptide sequence.

 FIG. 1 is a diagram corresponding to the synthesis of the complexes described in the examples.

 Figure 2 illustrates the formula of the complexes obtained.

 In the examples which follow, the compounds were identified by spectrometry, chromatography, microanalysis and determination of the melting points, under the following conditions.

The 1H and 13C NMR spectra were obtained on a BRUCKER AC 300 pulsed wave spectrometer, operating at 300 MHz for proton NMR and 75.47 MHz for carbon, and equipped with a computer.
ASPECT 3000. The values of the chemical shifts are expressed in parts per million (ppm) and the coupling constants in Hertz (Hz). The reference used is tetramethylsilane (TMS, 0.00 ppm). In the description of the spectra, the multiplicities of the signals are indicated using the following abbreviations s, d, t, q, m, M meaning respectively singlet, doublet, triplet, quadruplet, multiplet and solid.

The infrared IR spectra were performed on a PERKIN ELMER System 2000 FT device
IR, spectral processing is carried out with the IR Data Manager program on a DIGITAL computer
DECpc 450D2LP (range 4000-450 cm ~ l, the wave numbers of the absorption bands are expressed in cm-i).

UV ultraviolet spectra were performed on a model XONTRON spectrophotometer
UVIKON 860 with Victor VPC IIc acquisition system.

 The melting points were obtained on a Boche 535 device.

Low resolution mass spectra were obtained on a FINNIGAN-MAT 4600 quadruple device coupled and controlled by a computer system
INCOS, while the high resolution spectra were carried out on a VARIAN MAT 311 spectrometer. The mass spectra in FAB are carried out on a NERMAG R3010 spectrometer.

TLC thin layer chromatographies are performed on MERCK Si 60 silica plates
F254 on glass or plastic support. The revelations are carried out either by ultraviolet illumination
(254 nm), either with bisublimated iodine, or by spraying one of the following developers: vanillin, panisaldehyde, ninhydrin, potassium permanganate.

Example 1: Preparation of cis-Dichloro-bis (4,4'-bis [4-
(4-benzoylbenzoyloxy) butoxycarbonyl] -2.2 '
bipyridine) ruthenium (II) 9.

a) preparation of 4,4'-dicarboxy-2,2'-bipyridine
2.

4.4'-dimethyl-2,2 '-bipyridine
(64.0 mmol; 12.0 g) is dissolved in 100 ml of concentrated sulfuric acid. The solution is cooled to OOC and the chromium oxide (39.0 g; 6 eq.) Is added in small portions over 1 hour. The blue-green mixture is heated at 75 ° C. for 4 hours, then stirred overnight at room temperature. The solution is poured into a water-ice mixture (100 ml), then filtered through a sintered glass. After washing with water (SxS0 ml), the light yellow precipitate is dissolved in 50 ml of a 1N KOH solution until the solution becomes basic (formation of potassium dicarboxylate).

After filtration through celite, the filtrate is acidified with a 1N HCl solution (50 ml). The precipitated diacid is washed with water (3x50 ml): thus obtains 14.5 g of 2
(YId: 92.7%).

TLC: Rf 0.08 (CH2Cl2-EtOAc: 9-1); Rf 0.75 (CHCl3
MeOH-AcOH: 14-6-2);
1 H NMR (300 MHz, CF3COOD): # 9.17 (s, 2 H, H3, H3), 9.07 (d, 2 H, J5-6 = J5'-6 '= 5.3 Hz, H6, H62), 8.49 (d, 2 H, J5-6 = J5i-6 '= 5.3 Hz, Hs, H5')
13C NMR (75.47 MHz, CF3COOD + D2O): 145.96 (2C, C7,
C7 '), 125.89 (2C, C2, C2'), 121.65 (2C, C6, C6 '), 119.25
(2C, C4, C4 '), 111, 72 (2C, C5, C5,), 107.92 (2C, C3, C3');
IR (KBr): 3114 # (CH) arom, 2457 u (OH) acid, 1722 #
(C = O) acid arom, 1604-1563 # (C = N) and # (C = C) bipy, 766 # (CH) arom;
W (0.1 N NaOH, 0.0171g / l): 216 (19736), 293 (11782)
SM (70eV / DCI / NH3 / intensity%): m / z: 245 (100;
[M + 1] +), 201 (25.76; [M-CO2] +)
SMHR: theoretical mass: 244.04840 mass found 244.0474;
Microanalysis for C12H8N2O4.1 / 2H2O: Calc. : C, 56.92;
H, 3.58; N, 11.04; Exp. : C, 57.26; H, 3.77; N, 11.20.

b) preparation of 4-t-butyldimethylsilyloxybutanol
3.

60% NaH dispersed in oil
(44.8 mmol; 1.792 g) is suspended in anhydrous THF (88 ml). 1,4-Butanediol (44.8 mmol; 4.0 ml) is added dropwise, then the medium is stirred for 45 minutes at room temperature. A white precipitate then forms. After addition of tbutyldimethylsilyl chloride (44.8 mmol; 6.752 g), the mixture is stirred vigorously for 45 minutes. The solution is poured into 700 ml of ether, washed with 240 ml of 108 K2CO3 and 240 ml of NaCl. The decanted organic phase is dried over Na2SO4, filtered and then concentrated in vacuo.

The resulting oil is purified by chromatography on silica (eluent: EtOAc / Heptane 40/60). 7.8 g are obtained.
(38.2 mmol) of 3 (YId: 85%).

TLC: Rf 0.48 (EtOAc / Heptane: 40/60);
1 H NMR (300 MHz, CDCl3): b 0.05 (s, 6 H, 2 CH3), 0.9
(s, 9 H, 3 CH3), 1.60-1.70 (m, 4 H, CH2-CH2), 2.60 (s, 1
H, OH), 3.55-3.70 (m, 4H, 2 CH2O)
13C NMR (75.47 MHz, CDCl3): 0.00 (2C, Me2Si), 18.00
(1C, Si-C (CH3) 3), 25.80 (3C, Si-C (CH3) 3), 29.40 (1C,
CH2-CH2OH), 29.80 (1C, CH2-CH2OSi), 62.50 (1C, CH2OH), 63.30 (1C, CH2OSi);
IR (KBr): 3350 # (OH), 2931-2859 # (CH2) 1472 # (CH2), 1390 6 (t-Bu-Si), 1256 (CH3-Si), 1102 # (Si-OC), 837 6
(Si-O);
MS (70eV / DCI / NH3 /% intensity): m / z: 222 (77.22;
[M + 18] +), 205 (100; [M + 1] +);
Microanalysis for C10H24O2Si: Calc. : C, 58.76; H, 11.83; Exp. : C, 58.52; H, 11.68.

c) preparation of 4,4'-bis- [4- (t-
butyldimethylsilyloxy) butoxycarbonyl] -2.2 '
bipyridine 4.

 2 (8.19 mmol; 2.0 g), DCC (17.9 mmol; 3.7 g) and 3 (18.8 mmol; 3.84 g) are dissolved in the CH2Cl2 / THF mixture (150 ml / 20 ml). After 10 min of stirring, the DMAP (9.0 mmol; 1.1 g) is added and the medium stirred for 24 hours at room temperature. The reaction mixture is filtered in order to remove the dicyclohexylurea formed. The filtrate is washed successively with 2x50 ml of 1N HCl, 2x50 ml of a saturated NaHCO3 solution and 2x50 ml of a saturated NaCl solution. The organic phase is dried (Na2SO4), filtered and evaporated in vacuo. 4 is chromatographed on silica 60H (eluent: Heptane-EtOAc 60-40). 3.73 g (6.0 mmol) of 4 are obtained in the form of a white solid (yield: 74%).

TLC: Rf 0.8 (EtOAc / Heptane: 40/60); Pf: 54.5-55 C;
1 H NMR (300 MHz, CDCl3): # 8.94 (s, 2 H, H3, H3 '), 8.85 (d, 2 H, J5-6 = J5'-6' = 5 Hz, H6, H6 '), 7.89 (d, 2 H, J56 = J5'-6' = 5 Hz, H5, H5, H5 '), 4.42 (t, 4 H, J8-9 = J8'-9' = 6, 2
Hz, 2 H8, 2 H8 '), 3.68 (t, 4 H, J10-11 = J10'-11' = 6.0 Hz, 2
H11, 2 H11 '), 1.88 (m, 4 H, 2 Hg, 2 H9'), 1.67 (m, 4 H, 2 H10, 2 H10 '), 0.89 (s, 18 H, 9 H13, 9 H13 '), 0.05 (s, 12 H, 6 H12 6 H12');
13C NMR (75.47 MHz, CDCl3) :: 164.98 (2C, C7, (C7 '), 156.31 (2C, C2, C2'), 149.80 (2C, C6, C6), 138, 70 (2C,
C4, C4e), 122.93 (2C, Cs, C5 '), 120.31 (2C, C3, C3'), 65.63 (2C, C8, C8 '), 62.31 (2C, C11, C11 '), 29.05 (2C,
C10, C10 '), 25.69 (6C, 3C13, 3C13'), 25.12 (2C, C9, C9 '), 18.05 (2C, C14, Ci4'), 0.00 (4C, 2C12, 2C12 ');
IR (KBr): 2955 #as (CH) alkyl, 2830 ') s #s (CH) alkyl, 1730 u (C = O) ester arom, 1580-1558 # (C = N) and # (C = C) bipy, 1463 # (CH) of CH2 and CH3, 1380-1362 6 (CH) of tBu, 1256 # (CH3-Si), 1110 # (Si-OC), 1100 6 (CO) ester, 836 # (Si- O), 776 # (CH) arom;
UV (MeOH, 0.0672 mg / ml): 207 (42250), 240 (11879), 298
(12445);
MS (70eV / DCI / NH3 / intensity%): m / z: 617 (100;
[M] +)
HRMS [M-tBu] +: theoretical mass: 559.26594 mass found: 559.2638;
Microanalysis for C32H52N2O6Si2: Calc. : C, 62.30; H, 8.50; N, 4.54; Exp. : C, 62.20; H, 8.46; N, 4.54.

preparation of 4,4'-bis- [4
(hydroxy) butoxycarbonyl] -2,2'-bipyridine 5.

 4 (3.48 mmol; 2.15 g) dissolved in 30 ml of anhydrous THF, is added 3 ml of acetic acid, then 12 ml (12.0 mmol; 3.4 eq.) Of a solution molar of tetrabutylammonium fluoride in anhydrous THF. The medium is stirred at 200C for 24 h, then a saturated NaHCO3 solution is added in order to neutralize the medium. After extraction with CH2C12 (3x50 mi), drying (Na2SO4), and concentration under vacuum, S is purified by chromatography on silica 60H (eluent: EtOAc-MeOH: 80-20). 1.17 g (3.01 mmol) of 5 are obtained in the form of a white solid (yield: 86.5%).

TLC: Rf 0.7 (EtOAc / MeOH: 80/20);
Pf: 91.0-91.50C;
1 H NMR (300 MHz, CDCl3): d 8.90 (s, 2 H, H3, H3 '), 8.83
(d, 2 H, J5-6 = J5'6 '= 4.94 Hz, H6, H6'), 7.87 (d, 2 H,
J6-5 = J6'-5 '= 4.94 Hz, H5, H5'), 4.41 (t, 4 H, J8-9 = J8'-9 '= 6.7 Hz, 2 Hs, 2 H8 '), 3.72 (t, 4 H, J10-11 = J10'-11' = 5.8
Hz, 2 H11, 2 H11 '), 1.90 (m, 4 H, 2 H9, 2 H9'), 1.72 (m, 4 H, 2 H10, 2 H10 '), 1.39 (t, 2H, J11-12 = J11'-12 '= = 5.06
Hz, H12, H12 ')
13C NMR (75.47 MHz, CDCl3): 164.97 (2C, C7, C7 '), 156.33 (2C, C2, C2'), 149.88 (2C, C6, C6 '), 138.63 (2C,
C4, C4 '), 122.97 (2C, C5, C5'), 120.31 (2C, C3, C3 '), 65.45 (2C, C8, Ce'), 62.08 (2C, C11, C11 '), 28.69 (2C,
C10, C10 '), 24.95 (2C, C9, C9');
IR (KBr): 3419 u (OH) alcohol, 2970 #as (CH) alkyl, 2890 #s (CH) alkyl, 1713 u (C = O) ester arom, 1593-1558 # (C = H) and # ( C = C) bipy, 1474 # (CH) of CH2, 1131 # (COC) ester, 1059 # (CO) alcohol, 763 6 (CH) arom, 725 # (CH) alkyl;
W (CH2Cl2, 0.0526 mg / ml): 228 (10692), 241 (11496), 299 (12442);
SM (70eV / DCI / NH3 / intensity%): m / z: 389 (100 [M + 1] +)
Microanalysis for C20H24N2O6: Calc. : C, 61.85; H, 6.23; N, 7.21; Exp. : C, 61.85; H, 6.19; N, 7.06.

e) preparation of 4,4'-bis [4- (4
benzoylbenzoyloxy) butoxycarbonyl] -2.2 '
bipyridine 7a
Diol 5 (5.15 mmol; 2.0 g), DCC (9.8 mmol 1 2.02 g) and 4-benzoylbenzoic acid 6a (8.95 mmol; 2.0 g) are solution in 150 ml of
CH2C12. After 10 min of agitation at 20 ° C., the DMAP
(0.89 mmol; 0.11 g) is added and the medium stirred for 24 hours at room temperature. The cloudy solution is heated slightly (400C) in order to dissolve 7a. Only the dicyclohexylurea (DCU) formed remains insoluble. The reaction mixture is then filtered. The filtrate is washed with 2x50 ml of 1N HCl, 2x50 ml of a saturated NaHCO3 solution and 2x50 ml of a saturated NaCl solution. The organic phase is then dried (Na2SO4), filtered and concentrated in vacuo. 7a is purified by chromatography on silica 60H
(eluent: Heptane-EtOAc 60-40). 2.9 g (3.6 mmol) of 7a are obtained in the form of a white solid (Yield: 708).

To a solution of 4-benzoylbenzoic acid 6a (240 mg, 1.06 mmol) and 4,4'-bis- [4-
(hydroxy) butoxycarbonyl] -2,2'-bipyridine 5 (200 mg; 0.52 mmol) in 10 ml of anhydrous THF, a commercial solution of DEAD (177 l; 1.13 mmol) is slowly added followed by a solution of triphenylphosphine (300 mg; 1.14 mmol) in 5 ml of anhydrous THF. The solution discolors quickly. The reaction medium is then evaporated and directly purified by chromatography on silica 60H (eluent: THF-Heptane 50-50). 375 mg (0.47 mmol) of white solid are obtained (yield: 44%).

TLC: Rf 0.5 (EtOAc / Heptane: 50/50 or CHCl3 / Et2O 1/1);
Pf: 118-119 C;
1 H NMR (300 MHz, CDCl3): d 8.94 (s, 2 H, H3, H3 '), 8.86
(d, 2 H, J5-6 = J5'-6 '= 4.94 Hz, H6, H6'), 8.14 (d, 2 H,
J14-15 = J14'-15 '= 8.2 Hz, H14, H14'), 7.89 (d, 2 H, J56 = J5'-6 '= 4.94 HZ, H5, H5'), 7.83 (d, 4 h, J = 14-15 = J14'-15 '= 8.2 Hz, H15, H15'), 7.81 (d, 4 H, J19-20 = J19'-20 '= 7, 4 Hz,
H19, H19 '), 7.61 (t, 2 H, J20-21 = J20'-21' = 7.4 Hz, H21,
H21 '), 7.49 (t, 4 H, J19-20 = J19'-20' = J20-21 = J20'-21 '= 7.4
Hz, H20, H20 '), 4.44 (m, 8 H, 2 H8, 2 H8', 2 H11, 2 H11 '), 2.03-1.98 (m, 8 H, 2 Hg, 2 H9 '2 Hic, 2 H10');
13C NMR (75.47 MHZ, CDCl3) :: # 195.64 (2C, C17, C17 '), 165.46 and 164.86 (4C, C7, C7', C12, C12 '), 156.29 ( 2C,
C2, C2 '), 149.86 (2C, C6, C6'), 141.11 (2C, C16, C16 '), 138.45 (2C, C4, C4'), 136.64 (2C, Cie, C18 '), 133.03
(2C, C13, C13 '), 132.68 (2C, C21, C21'), 129.85 and 129.49 and 129.21 (12C, 2C14, 2C14 ', 2C15, 2C15', 2C19, 2C19 ') , 128.17 (4C, 2C20, 2C20 '), 122.93 (2C, C5, C5'), 120.27
(2C, C3, C3 '), 65.03 and 64.51 (4C, C8, C8' C11, C11 '), 25.18 and 24.66 (4C, C9, Cgw, Cio, Cio');
IR (KBr): 2928 #as (CH) alkyl, 2852 #s (CH) alkyl, 1727 # (C = O) ester arom, 1646 u (C = O) ketone, 1627-1578 u
(C = N) and # (C = C) bipy, 1448 # (CH) of CH2, 1285 # (CO) ester, 1127 # (COC) ester, 712 # (CH) alkyl, 766 6 (C
H) arom;
W (100 l of a 0.4176 mg / ml solution in CH2Cl2 in 2.5 ml of MeOH): 252 (25895);
SM (FAB / thioglycerol): m / z: 805 ([M + 1] +), 595 ((M- 2PhCO] +), 525 ([M- (CH2) 4Obzp)] +), 507 ((M { O (CH2) 4Obzp}] +), 375 ([M- (COO (CH2) 4Obzp) -PhCO}] +), 242
([M-2 {(CH2) 4Obzp}] +), 209 ([M-2 [CO2 (CH2) 4Obzp}] +);
HRMS:: theoretical mass: 804.26827 mass found 804.2705.

f) Preparation of cis-Dichloro-bis
(4,4'-bis [4- (4-benzoylbenzoyloxy) butoxycarbonyl] -2,2'-bipyridine) ruthenium (II) 9.

RuCl3 @ nH2O (65.0 mg; 0.25 mmol) is dissolved in 8 ml of anhydrous DMF in the presence of 7a (400 mg; 0.5 mmol) and LiCl (73.6 mg; 1.74 mmol). The medium is brought to reflux of DMF for 8 hours under an argon atmosphere. After cooling to 20 ° C., the homogeneous purple medium is added with 30 ml of acetone. The solution is then put in the freezer
(-200C) overnight. After filtration of the black crystals obtained, the solids are washed with ethyl ether (3 × 25 ml). 161 mg of complex 9 are obtained in the form of a pulverulent black solid (yield: 36.4%).

IR (KBr): 3385 # (OH) H2O, 1722 u (C = O) ester arom., 1650-1570 u (C = O) ketone and # (C = N) bipy, 1548 # (C = C) bipy , 1264 # (CO) ester, 1128 # (COC) ester, 787 # (CH) arom; UV (MeOH, 0.05915 mg / ml): 389 (12985), 309 (55323), 243 (62087), 210 (85279), SM
(FAB / thioglycerol): m / z: 1500 ([MR] +), 1465 ([MR-Cl] +), 1429 ([MR-2C1] +), 1222 ([M-2R] +), 1185 ( [M-2R-Cl] +), 1149 ([MR-2C1] +), with R = (CH2) 4O2CBzp.

EXAMPLE 2 Preparation of cis-Dichloro bis (4,4'-bis [4- (4- (4-bromobenzoyl) benzoyloxy) butoxycarbonyl] -2,2'-bipyridine) ruthenium (II) 10.

The same operating mode as in Example 1 is followed to prepare compound S and it is reacted with 4- (4-bromobenzoyl) benzoic acid 6b which is prepared as follows
a) preparation of 4- (4-bromobenzoyl) benzoic acid
6b:
For this preparation, the following reaction scheme is followed

 The 4-bromobenzotque acid (2.5 g; 12.31 mmol) is dissolved in 35 ml of anhydrous THF, then cooled to -100 C. 19 ml (24.89 mmol; 2.02 eq.) Is added slowly. of n-butyllithium (1.31M solution in anhydrous hexane). The temperature of the medium must remain below -95 C. The solution is then stirred for 2 h at -780C. Ethyl 4-bromobenzoate (2.82 g; 12.3 mmol) in 15 ml of THF is added while keeping the temperature below -700C. After stirring for 3 h at -200C, the reaction mixture is poured into 50 ml of a 5% HCl solution. After extraction with ether (3 × 50 ml), the ethereal phase is washed with 50 ml of a 1M NaOH solution. 6b reprecipitates again during a slow addition of a 10% HCl solution (30 ml). After filtration and drying under vacuum, 1.95 g (6.4 mmol) of 6b are obtained in the form of a white solid (yield: 52%).

TLC: Rf 0.4 (Heptane / EtOAc: 1/2);
Pf: 269-270 C;
1 H NMR (300 MHz, DMSO): 8.09 (6.2 H, J7-8 = 8.2 Hz, 2
Hs), 7.82 (d, 2 H, J2-3 = 7.6 Hz, 2 H3), 7.80 (d, 2 H, J23 = 7.6 Hz, 2 H2), 7.70 (d , 2H, 2H7);
13C NMR (75.47 MHz, DMSO): # 195.93 (1C, Cs), 168.06
(1C, C10), 141.50 (1C, C6), 137.00 (1C, C4), 135.829
(1C, Cg), 133.22-133.12 (4C, 2C2, 2C3), 131.05-130.85
(4C, 2C7, 2Ce), 128.59 (1C, C1);
IR (KBr): 3002 # (CH), 1690 u (C = O) acid as a dimer, 1647 # (C = O) ketone, 1548 # (C = C) arom, 732 # (C-
H) arom;
W (MeOH + 1 drop of THF, 0.0074 mg / ml): 263 (17469);
SM (70eV / EI / intensity%): m / z: 304/306 (68/60; [M] +), 259/261 (8.0 / 7.2; [M-COOH] +), 225 ( 12.08, [M-
Br] +), 183/185 (91.8 / 86.6; [M-PhCOOH] +), 155/157 (30.3 / 28.3; [M-COPhCOOH] +), 149 (100, [ M-PhBr] +).

b) preparation of 4,4'-bis [4- (4-
bromobenzoyl) benzoyloxy) butoxycarbonyl] -2.2 '
bipyridine 7b
DEAD (660 Rl; 4.22 mmol) is slowly added to a solution of 6b (1.15 g; 3.77 mmol) and 4 (732 mg; 1.88 mmol) in 20 ml of anhydrous THF. triphenylphosphine (1.10 g; 4.20 mmol; 5 ml anhydrous THF). An immediate discoloration of the solution is observed from yellow to white accompanied by the precipitation of 7b. 1.30 g (1.35 mmol) of 7b are obtained in the form of a white solid (yield: 72%). After filtration, an additional 230 mg are extracted from the filtrate (overall yield: 84.5%).

TLC: Rf 0.75 (Et2O-CHCl3; 50-50);
Pf: 180-180.60C;
1 H NMR (300 MHz, CDCl3): d 8.93 (s, 2 H, H3, H3 '), 8.84 (d, 2 H, J5-6 = J5'-6' = 4.80 Hz, H6 , H6 '), 8.14 (d, 4 H,
J14-15 = J14'-15 '= 8.13 Hz, 2H14, 2H14'), 7.88 (d, 2 H, J56 = J5'-6 '= 4.80 Hz, H5, H5'), 7 , 79 (d, 4 H, J14-15 = J14'-15 '= 8.13 Hz, 2Hîs, 2H15'), 7.70-7.60 (M, 8 H, 2H19, 2H19 '.

2H20, 2H20 '), 4.47 (m, 8H, 2H8, 2H8', 2H11, 2H11 '), 2.00 (m, 8H, 2H9, 2H9', 2H10, 2H10 ');
13C NMR (75.47 MHz, CDCl3): # 195.59 (2C, C17, C17 '),
165.41 and 164.87 (4C, C7, C7 ', C12, C12'), 156.31 (2C,
C2, C2 '), 149.91 (2C, C6, C6'), 140.66 (2C, C16, C16 '),
138.49 (2C, C4, C4e), 135.21 (2C, Cie, C18 '), 133.26
(2C, C13, C13 '), 131.59 and 131.30 (8C, 2C19, 2C19', 2C20, 2C20 '), 129.42 and 129.35 (8C, 2C14, 2C14', 2C15, 2C15 ') , 127.94 (2C, 2C21, 2C21 '), 122.97 (2C, C5, C5'), 120.28
(2C, C3, C3 '), 65.06 and 64.61 (4C, C8, C8', C11, C11 '), 25.26 and 25.207 (4C, Cg, Csl, C10, C10');
IR (KBr): 2968 #as (CH) alkyl, 1719 # (C = O) aromatic ester, 1651 # (C = O) ketone, 1584-1540 u (C = N) and # (C = C) bipy , 1286 # (CO) ester, 1123 # (COC) ester, 760 6 (CH) arom, 733 # (CH) backbone (CH2) 4, 660 # (C-Br);
W (100 l of a 0.1007 mg / ml solution in CH2Cl2 in 2.5 ml of MeOH): 261 (53232);
SM (70eV / IC / NH3 / intensity%): m / z: 960/962/965
(47.45 / 100 / 60.64; (M = 1] +);
Microanalysis for C48H38Br2N2O10: Calc. : C, 59.89; H, 3.98; N, 2.91; Br, 16.60; Exp. : C, 59.96; H, 4.29; N, 2.86; Br, 16.47.

c) Preparation cis-dichloro-bis (4,4'-bis (4- (4- (4
bromobenzoyl) benzoyloxy) butoxycarbonyl] -2.2 '
bipyridine) ruthenium (II) 10.

 The procedure necessary for the synthesis of 10 is identical to that in Example 1.f. 963.0 mg of complex 10 are obtained in the form of a black powdery powder (yield: 44%).

IR (KBr): 3417 u (OH) H20, 1720 # (C = O) aromatic ester, 1651 # (C = O) ketone, 1585-1540 # (C = N) bipy and v (C = C) bipy , 1272 # (CO) ester, 1106 # (COC) ester, 758 # (CH) arom, 733 # (CH) backbone (CH2) 4; UV (MeOH, 0.04865 mg / ml): 393 (11824), 310 (40003), 205 (93254).

EXAMPLE 3 Preparation of cis-Carbonato-bis (4,4'-bis (4- (4-benzoylbenzoyloxy) butoxycarbonyl] -2,2'-bipyridine) ruthenium (II) 11.

 9 (28.0 mol; 50.0 mg) is dissolved in 2 ml of argon degassed water. After adding a large excess of Na2CO3 (46.0 mol; 46.0 l of a 1M solution), the solution is stirred at reflux for 2 hours.

The red-burgundy solution is cooled to 20 ° C. and then lyophilized. 11 are obtained in the form of a purple powder (yield: 95%).

1H NMR (300 MHz, D2O): 89.57-8.86 (M, 16H, 2H1, 2H10, 4H18, 4H18 ', 2H4, 2H7), 8.3-8.13 (M, 8H, 2H2, 2Hg , 4H19), 8.04 (m, 2H, 2H25), 7.8-7.4 (M, 20H, 4H19 ', 4H24' 4H24 ', 2H25', 4H23, 4H23 ', 2H25), 3.56 ( m, 16H, 4H12, 4H12 ', 4H15, 4H15'), 1.28 (m, 16H, 4H13, 4H13w, 4H14, 4H14 '); IR (KBr): 3430 # (OH) H20, 1730 u (C = O) aromatic ester, 1650-1570 u (C = O) ketone and # (C = N) bipy, 1539 u (C = C) bipy , 1265 # (C = O) ester, 1120 # (COC) ester, 787 # (CH) arom; W (MeOH, 0.0486 mg / ml): 361 (5064), 306
(22152), 247 (20731), 203 (37565).

EXAMPLE 4: Preparation of the
cis-Carbonato-bis (4,4'-bis ([4- (4- (4
bromobenzoyl) benzoyloxy) butoxycarbonyl]
2,2'-bipyridine) ruthenium (II) 12.

The procedure necessary for the synthesis of 12 is identical to that described in Example 3. 12 are obtained in the form of a powder of purple color
(YId: 95%) -
1H NMR (300 MHz, D2O): # 9.35-8.78 (M, 16H, 2H1, 2H10, 4H18, 4H18 ', 2H4, 2H7), 8.31-8.13 (M, 8H, 2H2, 2H9, 4H19), 8.04-7.48 (M, 20H, 4H19 ', 4H24, 4H24', 4H23, 4H23 '), 3.67 (m, 16H, 4H12, 4H12, 4:15, 4H15'), 1 , 62 (m, 16H, 4H13, 4H13, 4H141 4H14 '); IR (KBr): 3442 v (OH) H2O, 1721 v (C = O) aromatic ester, 1651 v (C = O) ketone1618-1540 v
(C = N) bipy and v (C = C) bipy, 1282 # (CO) ester, 1105 6
(COC) ester, 786 6 (CH) arom, 735 6 (CH) skeleton
(CH2) 4, 663 6 (C-Br); UV (MeOH, 0.05565 mg / ml): 369
(10319), 306 (38762), 249 (33359), 203 (56736).

EXAMPLE 5 Preparation of cis-Carbonato-bis (4,4'-bis [4- (4- (4-3H-benzoyl) benzoyl oxy) butoxycarbonyl] -2,2'-bipyridine) ruthenium (II) 13.

Complex 12 (4.8 mmol; 10 mg) is dissolved in 1 ml of MeOH. After adding a large excess of Pd / C
(20 mg) and 2.5 ml of TEA (18 mmol; 4 eq.), The solution is installed on a Toepler type pump. The tritium gas bulb is broken and the balloon saturated in 3H. The solution is stirred at room temperature for 8 hours. After filtration of the catalyst and washing with 3 × 10 ml of MeOH, the solvent is evaporated. The product is purified by reprecipitation in EtOH, then stored in MeOH.

3 H NMR (320.135 MHz, D2O): 6 8.01 (s, 1 3H, 1 3H25), 7.61
(s, 2 3 H, 2 3 H 25 '); UV (MeOH): 367, 306, 247, 203;
Specific activity determined by W å 306 nm: AS

11 Ci / mmol.

EXAMPLE 6 Preparation of the complex 14.

After complete dissolution of it (2.8 pinol; 5 mg) in the phosphate buffer (pH 7), 2 mg of the peptide
Ala-His-Ala-Ala-Ala-His-Ala (3.1 Cunol; 1.1 eq.) In solution in the buffer are added. The medium is placed under argon, and thus left for 24 h. After addition of 6 equivalents of NH4PF6 (16.8 pmol; 2.7 mg), the solution is stirred for 2 h, the buffer evaporated and the whole taken up in MeOH. Compound 14 is obtained in the form of a red powder with yields of between 30 and 50%.

1 H NMR (300 MHz, D2O); # 9, 61-8.78 (M, 16H, 2H1, 2H10, 4H18, 4H18 ', 2H4, 2H7), 8.35 (s, 1H, Im), 8.11 (s, 1H, Im) 7, 98-7.48 (M, 32H, 2H2, 2Hg, 4H191 4H19 ', 4H241 4H24', 4H231 4H23 ', 2H25, 2H25'), 7.21 (s, 1H, Im), 7.11 (s, 1H , Im), 4.70-4.05 (M, 7H, 7H has some on the peptides), 3.70 (m, 16H, 4H12, 4H12 ', 4H15.4H15'), 3.30-3.00 (M, 4H, 2CH2 in histidines), 1.60 (m, 16H, 4H13, 4H13 ', 4H14, 4H14w), 1.53-1.35 (M, 15H, 5 Me of alanines); IR (KBr): 3446 and 3378 v (NH) amide, 3132 v (CH) arom., 2925v (CH) alkyl, 2341 v (OH) acid, 1713-1580v (C = O) amide, ketone and ester arom. , v (C = N) and v (C = C) bipy, 1376 # (CH) CH3, 1262 # (CO) ester arom ,. 1123 # (C-O-C) ester, 996-955 # (C-H), 848 6 (P-F), 830 # (C-H) arom. ; UV (MeOH, 0.1188 mg / ml): 499 (2229), 370 (5729), 307 (24789), 243 (21624), 203 (50827).

Claims (10)

 1. Bifunctional complex of ruthenium (II) or (III) carrying several ligands, comprising  a group capable of recognizing a peptide sequence incorporating at least one histidine, said group being formed by the central ruthenium atom of the complex linked to the CO3 ligand or to two ligands, identical or different, chosen from C1, Br, I and H2O; and  - At least one functional group having an intrinsic reactivity towards other amino acids possibly modified, this functional group being carried by at least one of the other ligands of the complex.  2. Ruthenium complex, corresponding to the formula

 in which  a) X1 and x2 which may be identical or different, represent Br, I, C1 or H2O, or X1 and X2 together form CO3  b) Y is a single bond or a bivalent group chosen from -C (O) O-, -CONH-, -O-, -S-, -CH2O-, -CH = CH-, -CH2S, -CH2-CH = CH and -C = C-  c) Z is a single bond or a bivalent group chosen from -OC (O) -, -NHCO-, -O-, -S-; -OCH2 -, - SCH2-, -CH = CH- and -C-C- ;;  d) R1 is a monovalent group chosen from photosensitive groups, fluorescent groups, luminescent groups, complexing groups, enzymes, avidin, biotin, fluorescent chromophores, light absorbing chromophores, groups marked with a radioactive element, antibodies and antibody fragments;  e) m is such that 1 S m 5 5; and  f) R2 represents - (CH2) n- with 0 S n S 2 or R2 is a benzene ring condensed with the two pyridine rings to form the orthophenanthroline ring.  3. Ruthenium complex according to claim 2, characterized in that R2 represents  (CH2) n with n equal to 0.  4. Ruthenium complex according to any one of claims 2 and 3, characterized in that Y represents -C (O) O-.  5. Ruthenium complex according to any one of claims 2 to 4, characterized in that Z represents -OC -OC (O) -.  6. Ruthenium complex according to any one of claims 2 to 5, characterized in that R1 is a photosensitive group.  7. Ruthenium complex according to claim 6, characterized in that R1 corresponds to the formula

 in which R3 represents H, 3H or Br.  8. Ruthenium complex chosen from cis-dichloro-bis (4,4'-bis [4- (4-benzoyl benzOyloxy) butoxycarbonyl] -2,2'-bipyridine) ruthenium  (II), cis-dichloro-bis) 4,4'-bis [4- (4- (4- (4bromobenzoyl) benzoyloxy) butoxycarbonyl] -2,2'bipyridine) ruthenium (II), cis-carbonato-bis (4 , 4 'bis [4- (4-benzoylbenzoyloxy) butoxycarbonyl] -2,2'- bipyridine) ruthenium (II), cis-carbonato-bis (4,4' bis [4- (4 (4-bromobenzoyl) benzoyloxy ) butoxycarbonyl] - 2,2'-bipyridine) ruthenium (II) and cis-carbonato-bis (4,4'-bis [4- (4- (4- (3H) benzoyl) benzoyloxy) butoxycarbonyl) -2, 2'-bipyridine) ruthenium (II).  9. Process for the preparation of a ruthenium complex corresponding to the formula

 in which R1, R2S, Z, Y and m are as defined above, with a ruthenium chloride of formula RuC13, p H2O in which p is an integer ranging from 1 to 3.

B 11991.3 MDT in which Y, Z, R1, R2 and m have the meanings given in claim 2, and X1 and X2 represent C1, characterized in that it consists in reacting a bipyridine of formula  10. Process for the preparation of a ruthenium complex corresponding to the formula

  in which Y, Z, R1, R2 and m have the meanings given in claim 2, and X1 and X2 together form CO3, characterized in that it comprises the following steps  a) preparing a ruthenium complex of formula (I) in which Y, Z, R1, R2 and m are as defined above, and X1 and X2 represent C1, by implementing the process of claim 9, and  b) bringing the ruthenium complex obtained in step a) into contact with sodium carbonate to replace the two C1s by C03.