Ruthenium (II) Complexes and Conjugates Thereof for Use as Photosensitizer Agent in Photodynamic Therapy

Abstract

The present invention relates to a compound of the following formula (I):

##STR00001##

or a pharmaceutically acceptable salt and/or solvate thereof,
for use as photosensitizer agent in photodynamic therapy.

The present invention relates also to a pharmaceutical composition comprising such a compound and at least one pharmaceutically acceptable excipient.

The present invention relates also to a conjugate comprising such a compound linked to a biomolecule such as a peptide, a protein, an aptamer, an affibody, an antibody or an antigen binding fragment thereof.

Claims

1-15. (canceled)

16. A method of treatment by photodynamic therapy comprising administering to an animal or a human in need thereof an effective amount of a compound of the following formula (I): ##STR00076## or a pharmaceutically acceptable salt and/or solvate thereof, as photosensitizer agent, wherein R.sup.1 to R.sup.10 each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, COR.sup.11, OR.sup.12 and NR.sup.13R.sup.14, R.sup.11 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.15 and NR.sup.16R.sup.17, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), P.sup.1 represents one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, P.sup.2 represents one or several substituents selected in the group consisting of halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20.sub.R.sup.21, or P.sup.1 and P.sup.2 together with the pyridyl groups to which they are bonded represent: ##STR00077## R.sup.x, R.sup.y and R.sup.z each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, R.sup.18 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.22 and NR23R.sup.24, R.sup.19, R.sup.20, R.sup.21, R.sup.22, R.sup.23 and R.sup.24 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), X.sup.m− is a pharmaceutically acceptable anion, m is 1, 2 or 3, wherein the optionally substituted C.sub.1-C.sub.6 alkyl is a C.sub.1-C.sub.6 alkyl optionally substituted with one or more substituents selected from halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2, wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl or aryl, and wherein the optionally substituted C.sub.2-C.sub.6 alkenyl, the optionally substituted C.sub.2-C.sub.6 alkynyl, the optionally substituted carbocycle, the optionally substituted aryl, the optionally substituted heteroaryl, the optionally substituted heterocycle, and the optionally substituted CO-(C.sub.1-C.sub.6 alkyl) are respectively a C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocycle, aryl, heteroaryl, heterocycle, or CO-(C.sub.1-C.sub.6 alkyl) optionally substituted with one or more substituents selected from C.sub.1-C.sub.6 alkyl, halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2, wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl or aryl.

17. The method according to claim 16, wherein X′ is selected in the group consisting of PF.sub.6.sup.−, Cl.sup.−, Br.sup.−, I.sup.−, BF.sub.4.sup.−, (C.sub.1-C.sub.6 alkyl)-C(O)O.sup.−, (C.sub.1-C.sub.6 haloalkyl)-C(O)O.sup.−, (C.sub.1-C.sub.6 alkyl)-CO.sub.3.sup.−, (C.sub.1-C.sub.6-haloalkyl)-SO.sub.3.sup.−, SO.sub.4.sup.2− and PO.sub.4.sup.3−.

18. The method according to claim 16, wherein R.sup.1 to R.sup.10 each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted aryl, OR.sup.12 and NR.sup.13R.sup.14.

19. The method according to claim 16, wherein R.sup.1 R.sup.10 each represent H.

20. The method according to claim 16, wherein P.sup.1 represents one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3 COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, and P.sup.2 represents one or several substituents selected in the group consisting of halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3 COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21.

21. The method according to claim 16, wherein P.sup.1 represents one or several substituents selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl and COR.sup.18, and P.sup.2 represents one or several substituents selected in the group consisting of optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl and COR.sup.18.

22. The method according to claim 16, wherein P.sup.1 and P.sup.2 each independently represent one or several substituents selected in the group consisting of: a C.sub.1-C.sub.6 alkyl optionally substituted with one or more substituents selected among halogen, COR′, COOR′, CONR′R″, OR′, NR′R″ and heterocycle, wherein R′ and R″ are independently of each other H or C.sub.1-C.sub.6 alkyl, the heterocycle being optionally substituted by one or more substituents selected among halogen, C.sub.1-C.sub.6 alkyl and oxo group, a C.sub.2-C.sub.6 alkenyl optionally substituted with at least one substituent selected among halogen, COR′, COOR′, CONR′R″, OR′, NR′R″ and heterocycle, wherein R′ and R″ are independently of each other H or C.sub.1-C.sub.6 alkyl, the heterocycle being optionally substituted by one or more substituents selected among halogen, C.sub.1-C.sub.6 alkyl and oxo group, and COR.sup.18.

23. The method according to claim 16, wherein the compound of formula (I) is a compound of the following formula (I-A): ##STR00078##

24. The method according to claim 16, wherein the compound of formula (I) is selected from the group consisting of: ##STR00079## ##STR00080## ##STR00081## and the pharmaceutically acceptable salts and/or solvates thereof.

25. The method according to claim 16, wherein the photodynamic therapy is intended to treat a disease selected from a cancer; a bacterial infection; a fungal infection; a viral infection; and a skin disorder.

26. The method according to claim 25, wherein the cancer is a lung cancer, a bladder cancer, an oesophageal cancer, a colon cancer, a stomach cancer, a liver cancer, a skin cancer, an ovarian cancer, a pancreatic cancer, a head and neck cancer, or a brain cancer; the bacterial infection is a sinusitis, a diabetic foot, or a burned wound; the fungal infection is a mycosis; the viral infection is herpes; and the skin disorder is acne, or a port wine stain.

27. A compound of the following formula (I): ##STR00082## or a pharmaceutically acceptable salt and/or solvate thereof, wherein R.sup.1 to R.sup.10 each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, COR.sup.11, OR.sup.12 and NR.sup.13R.sup.14, R.sup.11 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.15 and NR.sup.16R.sup.17, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), P.sup.1 represents one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, P.sup.2 represents one or several substituents selected in the group consisting of halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, or P.sup.1 and P.sup.2 together with the pyridyl groups to which they are bonded represent: ##STR00083## R.sup.x, R.sup.y and R.sup.z each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, R.sup.18 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.22 and NR23R.sup.24, R.sup.19, R.sup.20, R.sup.21, R.sup.22, R.sup.23 and R.sup.24 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), X.sup.m− is a pharmaceutically acceptable anion, m is 1, 2 or 3, wherein the optionally substituted C.sub.1-C.sub.6 alkyl is a C.sub.1-C.sub.6 alkyl optionally substituted with one or more substituents selected from halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2 wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl or aryl, and wherein the optionally substituted C.sub.2-C.sub.6 alkenyl, the optionally substituted C.sub.2-C.sub.6 alkynyl, the optionally substituted carbocycle, the optionally substituted aryl, the optionally substituted heteroaryl, the optionally substituted heterocycle, and the optionally substituted CO-(C.sub.1-C.sub.6 alkyl) are respectively a C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocycle, aryl, heteroaryl, heterocycle, or CO-(C.sub.1-C.sub.6 alkyl) optionally substituted with one or more substituents selected from C.sub.1-C.sub.6 alkyl, halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2 wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.l-C.sub.6 alkyl, C.sub.l-C.sub.6 haloalkyl or aryl, with the proviso that said compound is not: ##STR00084## ##STR00085## ##STR00086## ##STR00087## in which of R.sup.α, R.sup.β, R*, R**, R*** and R**** are each independently H, CH.sub.3, COOH or NH.sub.2, provided that at least one or two of R.sup.α and R.sup.β is COOH or NH.sub.2, and ##STR00088##

28. The compound according to claim 27, wherein: R.sup.1 to R.sup.10 each represent H; and P.sup.1 and P.sup.2 each independently represent one or several substituents selected in the group consisting of: a C.sub.1-C.sub.6 alkyl optionally substituted with one or more substituents selected among halogen, COR′, COOR′, CONR′R″, OR′, NR′R″ and heterocycle, wherein R′ and R″ are independently of each other H or C.sub.1-C.sub.6 alkyl, the heterocycle being optionally substituted by one or more substituents selected among halogen, C.sub.1-C.sub.6 alkyl and oxo group, a C.sub.2-C.sub.6 alkenyl optionally substituted with at least one substituent selected among halogen, COR′, COOR′, CONR′R″, OR , NR′R″ and heterocycle, wherein R′ and R″ are independently of each other H or C.sub.1-C.sub.6 alkyl, the heterocycle being optionally substituted by one or more substituents selected among halogen, C.sub.1-C.sub.6 alkyl and oxo group, and COR.sup.18.

29. The compound according to claim 27, being selected from the group consisting of: ##STR00089## ##STR00090## and the pharmaceutically acceptable salts and/or solvates thereof.

30. A pharmaceutical composition comprising at least one compound according to claim 27 and at least one pharmaceutically acceptable excipient.

31. A method for the preparation of a compound according to claim 27 comprising the following steps: (i) reacting a compound of the following formula (II) ##STR00091## in which R.sup.1 to R.sup.10 are as defined in claim 27, R.sup.30 and R.sup.31 each independently represent halogen, OR.sup.32 or S(O)(C.sub.1-C.sub.6 alkyl).sub.2, R.sup.32 is H or C.sub.1-C.sub.6 alkyl, with a compound of the following formula (III) ##STR00092## in which P.sup.1 and P.sup.2 are as defined in claim 27, and (ii) reacting the product resulting from step (i) with a salt A.sup.m+X.sup.m−, wherein X.sup.m− is as defined in claim 27 and Am.sup.m+ is a counter cation.

32. A conjugate comprising a compound of the following formula (I): ##STR00093## or a pharmaceutically acceptable salt and/or solvate thereof, wherein R.sup.1 to R.sup.10 each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, COR.sup.11, OR.sup.12 and NR.sup.13R.sup.14, R.sup.11 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.15 and NR.sup.16R.sup.17, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), P.sup.1 represents one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, P.sup.2 represents one or several substituents selected in the group consisting of halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, or P.sup.1 and P.sup.2 together with the pyridyl groups to which they are bonded represent: ##STR00094## R.sup.x, R.sup.y and R.sup.z each independently represent one or several substituents selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, N.sub.3, COR.sup.18, OR.sup.19 and NR.sup.20R.sup.21, R.sup.18 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.22 and NR.sup.23R.sup.24, R.sup.19, R.sup.20, R.sup.21, R.sup.22, R.sup.23 and R.sup.24 are each independently selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl and optionally substituted CO-(C.sub.1-C.sub.6 alkyl), X.sup.m− is a pharmaceutically acceptable anion, m is 1, 2 or 3, wherein the optionally substituted C.sub.1-C.sub.6 alkyl is a C.sub.1-C.sub.6 alkyl optionally substituted with one or more substituents selected from halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2m wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl or aryl, and wherein the optionally substituted C.sub.2-C.sub.6 alkenyl, the optionally substituted C.sub.2-C.sub.6 alkynyl, the optionally substituted carbocycle, the optionally substituted aryl, the optionally substituted heteroaryl, the optionally substituted heterocycle, and the optionally substituted CO-(C.sub.1-C.sub.6 alkyl) are respectively a C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, carbocycle, aryl, heteroaryl, heterocycle, or CO-(C.sub.1-C.sub.6 alkyl) optionally substituted with one or more substituents selected from C.sub.1-C.sub.6 alkyl, halogen, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkene, C.sub.2-C.sub.6 alkyne, aryl, N.sub.3, oxo, NR.sup.aR.sup.b, COR.sup.c, CO.sub.2R.sup.d, CONR.sup.eR.sup.f, OR.sup.g, CN and NO.sub.2, wherein R.sup.a to R.sup.g are, independently of one another, H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl or aryl; linked to a biomolecule.

33. The conjugate according to claim 32, wherein the biomolecule is a peptide, a protein, an aptamer, an affibody, an antibody or an antigen binding fragment thereof.

34. A method of treatment by photodynamic therapy comprising administering to an animal or a human in need thereof an effective amount of a conjugate according to claim 32, as photosensitizer agent.

35. The method according to claim 34, wherein the photodynamic therapy is intended to treat a disease selected from a cancer, a bacterial infection, a fungal infection, a viral infection, or a skin disorder.

Description

DESCRIPTION OF THE FIGURES

[0177] FIG. 1. Measured UV/Vis spectra of the complexes 1-7 in CH.sub.3CN.

[0178] FIG. 2. Time-dependent biodistribution of complex 6 in organs of healthy BALB/c mice.

[0179] FIG. 3. Free Cys-34 SH content of HSA at various complex 12—HSA ratios (closed triangle, open triangle, closed square) and various conditions: 3 h incubation (open triangle), 0.5 h incubation (closed triangle) and 0.5 h incubation in the presence of 0.5% SDS (closed square). The effect of a ruthenium complex without a maleimide function (complex 13) was also tested as negative control (open circle).

EXAMPLES

1) Synthesis

Materials

[0180] All chemicals were obtained from commercial sources and were used without further purification. Solvents were dried over molecular sieves if necessary. The Ru(II) complexes Dichlorobis(1,10-phenanthroline)ruthenium(II) [RuCl.sub.2(phen).sub.2] and Dichlorobis(4,7-Diphenyl-1,10-phenanthroline)ruthenium(II) [RuCl.sub.2(bphen).sub.2] were synthesised as previously published using the respective ligands (Sullivan, B. et al., 1978). The substituted bipyridine ligands 2,2′-Bipyridine-4,4′-dicarbonitrile, (E,E′)-4,4′-Bis(N,N-dimethylaminovinyl)-2,2′-bipyridine and 2,2′-Bipyridine-4,4′-dicarboxaldehyde were synthesised as reported (Wuest, J. D. 2011 and Le Bozec, H., 2001). The Ru(II) complexes [Ru(phen).sub.2(dppz-7-aminomethyl)](PF.sub.6).sub.2 was synthesized as previously reported (Gasser, G. et al., 2015).

Instrumentation and Methods

[0181] .sup.1H and .sup.13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific, Courtaboeuf, France) and operated in positive ionization mode, with a spray voltage at 3.6 kV. No Sheath and auxiliary gas was used. Applied voltages were 40 and 100 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 275° C. Detection was achieved in the Orbitrap with a resolution set to 100,000 (at m/z 400) and a m/z range between 150-2000 in profile mode. Spectrum was analyzed using the acquisition software XCalibur 2.1 (Thermo Fisher Scientific, Courtaboeuf, France). The automatic gain control (AGC) allowed accumulation of up to 2*10.sup.5 ions for FTMS scans, Maximum injection time was set to 300 ms and 1 μscan was acquired. 10 pi was injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific, Courtaboeuf, France) with a continuous infusion of methanol at 100 μL.min.sup.−1. For analytic and preparative HPLC the following system has been used: 2× Agilent G1361 1260 Prep Pump system with Agilent G7115A 1260 DAD WR Detector equipped with an Agilent Pursuit XRs 5C.sub.18 (Analytic: 100 Å, C.sub.18 5 μ.m 250×4.6 mm, Preparative: 100 Å, C18 5 μm 250×300 mm) Column and an Agilent G1364B 1260-FC fraction collector. The solvents (HPLC grade) were Millipore water (0.1% TFA, solvent A) and acetonitrile (0.1% TFA, solvent B). The sample was dissolved in 1:1 (v/v) CH.sub.3CN/ H.sub.2O 0.1% TFA solution and filtered through a 0.2 μm membrane filter. Gradient: 0-3 minutes: isocratic 95% A (5% B); 3-17 minutes: linear gradient from 95% A (5% B) to 0% A (100% B); 17-25 minutes: isocratic 0% A (100% B). The flow rate was 1 mL/min (for preparative purposes: 20 mL/min) and the chromatogram was detected at 250 nm, 350 nm, 450 nm.

Synthesis of Ruthenium Complexes

(Bipyridine)bis(1,10-phenanthroline)ruthenium(II)hexafluorophosphate [Ru(bpy)(phen).SUB.2.](PF.SUB.6.).SUB.2 .(1) (Comparative)

[0182] ##STR00063##

[0183] The synthesis of [Ru(bpy)(phen).sub.2](PF.sub.6).sub.2 is already published in Crosby, G. et al., 1976.

(4,4′-Dimethyl-2,2′-bipyridine)bis(1,10-phenanthroline)ruthenium(II)hexafluorophosphate [Ru(Me-bpy)(phen).SUB.2.](PF.SUB.6.).SUB.2 .(2) (Comparative)

[0184] ##STR00064##

[0185] The synthesis of [Ru(Me-bpy)(phen).sub.2](PF.sub.6).sub.2 is already published in Jones Jr, W. E. et al., 1989.

(4,4′-Dibromo-2,2′-bipyridine)bis(1,10-phenanthroline)ruthenium(II) hexafluorophosphate[Ru(Br-bpy)(phen).SUB.2.](PF.SUB.6.).SUB.2 .(3) (Comparative)

[0186] ##STR00065##

[0187] RuCl.sub.2(phen).sub.2 (150 mg, 0.28 mmol, 1.0 equiv.) and 4,4′-Dibromo-2,2′-bipyridine (105 mg, 0.34 mmol, 1.2 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (40 mL) and were refluxed for 18 h under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 5 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was isolated by column chromatography on silica gel with an CH.sub.3CN /aq. KNO.sub.3 (0.4 M) solution (10:1). The fractions containing the product were united and the solvent was removed. The residue was dissolved in CH.sub.3CN and undissolved KNO.sub.3 was removed by filtration. The solvent was removed again and the product was dissolved in H.sub.2O (50 mL). Upon addition of NH.sub.4PF.sub.6 the product precipitated as a PF.sub.6 salt. The solid was obtained by filtration and was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 78%. .sup.1H NMR (500 MHz, CD.sub.3CN) δ=8.76 (2H, d, .sup.4J=2.0 Hz), 8.68 (2H, dd, .sup.3J=8.3 Hz, .sup.4J=1.3 Hz), 8.55 (2H, dd, .sup.3J=8.3 Hz, .sup.4J=1.3 Hz), 8.27 (2H, d, .sup.3J=8.9 Hz), 8.25 (2H, dd, .sup.3J=5.3 Hz, .sup.4J=1.3 Hz), 8.22 (2H, d, .sup.3J=8.9 Hz), 7.84 (2H, dd, .sup.3J=5.3 Hz, .sup.4J=1.3 Hz), 7.81 (2H, dd, .sup.3J=8.3 Hz, .sup.3J=5.2 Hz), 7.55 (2H, dd, .sup.3J=8.3 Hz, .sup.3J=5.3 Hz), 7.50 (2H, d, .sup.3J=6.1 Hz), 7.47 (2H, dd, .sup.3J=6.1 Hz, .sup.4J=2.0 Hz). .sup.13C NMR (125 MHz, CD.sub.3CN) δ=158.3, 154.0, 153.9, 153.6, 148.7, 148.4, 138.0, 137.9, 134.7, 132.0, 132.0, 131.7, 129.1, 129.0, 129.0, 127.0, 126.9. HR-MS (ESI+m/z): Calcd. [M-2PF 6] 2+: 386. 96526; found: 386. 96576. EA (%): Calcd. for (C.sub.34H.sub.22Br.sub.2F.sub.12N.sub.6P.sub.2Ru): C 38.33, H 2.08, N 7.89; found. C 38.62, H 2.01, N 7.78.

(2,2′-bipyridine-4,4′-carboxamide)bis(1,10-phenanthroline)ruthenium(II) hexafluorophosphate [Ru(CONH2-bpy)(phen).SUB.2.](PF.SUB.6.).SUB.2 .(4) (Comparative)

[0188] ##STR00066##

[0189] RuCl.sub.2(phen).sub.2 (150 mg, 0.28 mmol, 1.0 equiv.) and 2,2′-Bipyridine-4,4′-dicarbonitrile (64 mg, 0.31 mmol, 1.1 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (30 mL) and were refluxed for 18 h under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 5 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was purified by column chromatography on silica gel with an CH.sub.3CN /aq. KNO.sub.3 (0.4 M) solution (10:1). The fractions containing the product were united and the solvent was removed. The residue was dissolved in CH.sub.3CN and undissolved KNO.sub.3 was removed by filtration. The solvent was removed again and the product was dissolved in H.sub.2O (50 mL). Upon addition of NH.sub.4PF.sub.6 the product precipitated as a PF.sub.6 salt. The solid was obtained by filtration and was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 16%. .sup.1H NMR (400 MHz, CD.sub.3CN) δ=8.97 (2H, s), 8.67 (2H, d, .sup.3J=8.3 Hz), 8.58 (2H, d, .sup.3J=8.3 Hz), 8.30-8.22 (4H, m), 8.18 (2H, d, .sup.3J=5.2 Hz), 7.87-7.84 (4H, m), 7.79 (2H, dd, .sup.3J=8.3 Hz, .sup.3J=5.2 Hz), 7.61-7.57 (4H, m), 7.25 (2H, s), 6.48 (2H, s).

[0190] .sup.13C NMR (100 MHz, CD.sub.3CN) δ=165.7, 158.8, 154.0, 153.9, 153.5, 148.6, 148.3, 143.0, 138.2, 138.0, 132.1, 132.0, 129.1, 129.0, 127.0, 127.0, 126.0, 123.1. HR-MS (ESI+m/z): Calcd. [M-2PF 6] 2+: 352.06056; found: 352.06063. EA (%): Calcd. for (C.sub.36H.sub.26F.sub.12N.sub.8O.sub.2P.sub.2Ru): C 43.52, H 2.64, N 11.28; found. C 43.33, H 2.47, N 11.15.

((E,E′)-4,4′-Bis(N,N′-dimethylaminovinyl)-2,2′-bipyridine)bis(1,10-phenanthroline)ruthenium(II) hexafluorophosphate [Ru(Me2Nvin-bpy)(phen).SUB.2.](PF.SUB.6.).SUB.2 .(5) (Comparative)

[0191] ##STR00067##

[0192] [Ru(Me-bpy)(phen).sub.2](PF.sub.6).sub.2 (2) (100 mg, 0.11 mmol, 1.0 equiv.) was dissolved in dry DMF (1.5 mL) and Cert-Butoxy bis(dimethylamino)methane (0.2 mL, 0.97 mmol, 8.8 equiv.) was added. The mixture was heated at 140 ° C. for 16 h under N.sub.2 atmosphere. The solution was cooled down and an aq. solution of NH.sub.4PF.sub.6 was added. The resulting precipitate was collected by vacuum filtration and the solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was isolated via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O and afterwards dried in high vacuum. Yield: 41%. .sup.1H NMR (400 MHz, CD.sub.3CN) δ=8.61 (2H, dd, .sup.3J=8.3 Hz, .sup.4J=1.3 Hz), 8.48 (2H, dd, .sup.3J=8.3 Hz, .sup.4J=1.3 Hz), 8.38 (2H, dd, .sup.3J=5.3 Hz, .sup.4J=1.3 Hz), 8.25-8.18 (4H, m), 8.07 (2H, d, .sup.4J=2.2 Hz), 7.87 (2H, dd, .sup.3J=5.3 Hz, .sup.4J=1.3 Hz), 7.82 (2H, dd, .sup.3J=8.2 Hz, .sup.3J=5.3 Hz), 7.52-7.48 (4H, m), 6.99 (2H, d, .sup.3J=6.2 Hz), 6.77 (2H, dd, .sup.3J=6.2 Hz, .sup.4J=2.1 Hz), 5.08 (2H, d, .sup.3J=13.4 Hz), 2.94 (12H, s). .sup.13C NMR (100 MHz, CD.sub.3CN) δ=157.6, 153.5, 153.5, 151.6, 150.6, 149.2, 149.1, 147.8, 137.0, 137.0, 131.9, 131.9, 129.0, 129.0, 126.9, 126.7, 120.3, 117.1, 92.9, 40.1. HR-MS (ESI+m/z): Calcd. [M-2PF 6] 2+: 378.11260; found: 378.11289. EA (%): Calcd. for (C.sub.42H.sub.38F.sub.12N.sub.8P.sub.2Ru): C 48.24, H 3.66, N 10.71; found: C 47.97, H 3.59, N 10.76.

(4,4′-Dimethyl-2,2′-bipyridine)bis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) hexafluorophosphate [Ru(Me-bpy)(bphen).SUB.2.](PF.SUB.6.).SUB.2 .(6)

[0193] ##STR00068##

[0194] The synthesis of [Ru(Me-bpy)(bphen).sub.2](PF.sub.6).sub.2 is already published (Mazuryk, O. et al., 2014) but in this study another synthetic route was employed. RuCl.sub.2(bphen).sub.2 (200 mg, 0.24 mmol, 1.0 equiv.) and 4,4′-Dimethyl-2,2′-bipyridine (53 mg, 0.29 mmol, 1.2 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (10 mL) and were refluxed for 18 h under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 10 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the suspension was sonicated. 60 mL of H.sub.2O were added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 93%. .sup.1H NMR (400 MHz, CD.sub.3CN) δ=8.44 (2H, s), 8.29 (2H, d, .sup.3J=5.5 Hz), 8.22-8.16 (m, 4H), 8.10 (2H, d, .sup.3J=5.5 Hz), 7.75 (2H, d, .sup.3J=5.5 Hz), 7.72-7.53 (24H, m), 7.21 (2H, d, .sup.3J=5.8, .sup.4J=1.7 Hz), 2.56 (6H, s). .sup.13C NMR (125 MHz, CD.sub.3CN) δ=157.7, 153.1, 152.9, 152.2, 151.4, 149.9, 149.8, 149.5, 149.4, 136.7, 136.7, 130.8, 130.7, 130.7, 130.6, 130.6, 130.1, 130.1, 130.1, 129.9, 129.9, 129.1, 127.1, 127.0, 127.0, 126.9, 125.8, 21.3. HR-MS (ESI+m/z): Calcd. [M-2PF 6] 2+: 475.13300; found: 475.13388. EA (%): Calcd. (C.sub.60H.sub.44F.sub.12N.sub.6P.sub.2Ru)×(H.sub.2O).sub.2: C 56.47, H 3.79, N 6.59; found: C 56.46, H 3.85, N 6.11.

((E,E′)-4,4′-Bis(N,N′-dimethylaminovinyl)-2,2′-bipyridine)bis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) hexafluorophosphate [Ru(Me.SUB.2.Nvin-bpy)(bphen).SUB.2.](PF.SUB.6.).SUB.2 .(7)

[0195] ##STR00069##

[0196] [Ru(Me-bpy)(bphen).sub.2](PF.sub.6).sub.2 (7) (150 mg, 0.12 mmol, 1.0 equiv.) was dissolved in dry DMF (1.5 mL) and tert-Butoxy bis(dimethylamino)methane (0.3 mL, 1.45 mmol, 12.1 equiv.) was added. The mixture was heated at 140 ° C. for 18 h under N.sub.2 atmosphere. After this time, more tert-Butoxy bis(dimethylamino)methane (0.4 mL, 1.94 mmol, 16.2 equiv.) was added the mixture was heated at 145° C. for 72 h under N.sub.2 atmosphere. The solution was cooled down and an aq. solution of NH.sub.4PF.sub.6 was added. The resulting precipitate was collected by vacuum filtration and the solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was isolated via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O and afterwards dried in high vacuum. Yield: 67%. .sup.1H NMR (500 MHz, CD.sub.3CN) δ=8.47 (2H, d, .sup.3J =5.5 Hz), 8.22-8.13 (8H, m), 8.09 (2H, d, .sup.3J=5.5 Hz), 7.80 (2H, d, .sup.3J=5.5 Hz), 7.69-7.52 (22H, m), 7.21 (2H, d, .sup.3J=6.3 Hz), 6.87 (2H, dd, .sup.3J=6.3 Hz, .sup.4J=2.0 Hz), 5.13 (2H, d, .sup.3J=13.3 Hz), 2.96 (12H, s). .sup.13C NMR (125 MHz, CD.sub.3CN) δ=157.4, 152.9, 152.7, 151.5, 150.6, 149.7, 149.6, 149.2, 149.2, 149.2, 149.2, 149.2, 147.7, 136.9, 136.8, 130.8, 130.7, 130.7, 130.5, 130.5, 130.1,130.0, 130.0, 129.7, 129.7, 127.1, 126.9, 126.8, 126.8, 120.2, 117.0, 92.7, 40.7. HR-MS (ESI+m/z): Calcd. [M-2PF 6] 2+: 530.17520; found: 530.17584. EA (%): Calcd. for (C.sub.66H.sub.64F.sub.12N.sub.8P.sub.2Ru)×(H.sub.2O) 0.5: C 58.32, H4.08, N 8.24; found: C 58.17, H 3.83, N 8.66.

(4′-Methyl-2,2′-bipyridinyl-4-aldehyde)bis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) hexafluorophosphate (8):

[0197] ##STR00070##

[0198] Ru(bphen).sub.2Cl.sub.2 (200 mg, 1.0 equiv.) and 440 -Methyl-2,2′-bipyridinyl-4-aldehyde (57 mg, 1.2 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (10 mL) and were refluxed overnight under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 10 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 79%. .sup.1H NMR (400 MHz, CD.sub.3CN) δ=10.18 (s, 1H), 8.93 (s, 1H), 8.64 (s, 1H), 8.29 (1H, d, J=5.5 Hz), 8.27 (1H, d, J=5.5 Hz), 8.20 (4H, d, J=2.2 Hz), 8.15 (1H, d, J=5.8 Hz), 8.11 (1H, d, J=3.2 Hz), 8.10 (1H, d, J=3.2 Hz), 7.78-7.69 (m, 4H), 7.67-7.57 (m, 22H), 7.29-7.27 (m, 1H), 2.60 (s, 3H). .sup.13C NMR (100 MHz, CD.sub.3CN) δ=191.5, 160.3, 157.0, 154.9, 153.2, 153.1, 152.9, 152.3, 151.8, 150.4, 150.3, 150.2, 149.4, 149.3, 149.2, 148.9, 142.8, 136.6, 136.6, 130.8, 130.7, 130.7, 130.6, 130.1, 130.1, 130.0, 129.9, 129.8, 127.2, 127.0, 126.7, 126.2, 122.9, 21.2. ESI-HRMS (pos. detection mode): calcd for C60H42N6O1Ru m/z [M].sup.2+ 482.1236; found: 482.1226. Elemental analysis calcd for C60H42F12N6O1P2Ru (%): C 57.47, H 3.38, N 6.70; found: C 57.56, H 3.32, N 6.64.

(4′-Methyl-2,2′-bipyridinyl-4-carboxylic acid)bis(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) hexafluorophosphate (9):

[0199] ##STR00071##

[0200] Ru(bphen).sub.2Cl.sub.2 (200 mg, 1.0 equiv.) and 4′-Methyl-2,2′-bipyridinyl-4-carboxylic acid (57 mg, 1.2 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (10 mL) and were refluxed overnight under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 10 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 83%. .sup.1H NMR (400 MHz, CD.sub.3CN) δ=9.09 (s, 1H), 8.67 (s, 1H), 8.35 (1H, d, J=5.5 Hz), 8.32 (1H, d, J=5.5 Hz), 8.23 (2H, d, J=1.5 Hz), 8.22 (2H, d, J=2.0 Hz), 8.16 (2H, d, J=5.5 Hz), 8.03 (1H, d, J=5.8 Hz), 7.82-7.74 (m, 4H), 7.67-7.62 (m, 22H), 7.28 (1H, d, J=5.5 Hz), 2.58 (s, 3H). .sup.13C NMR (100 MHz, CD.sub.3CN) δ=166.5, 159.2, 157.3, 153.8, 153.1, 153.0, 152.2, 151.7, 150.1, 150.1, 150.0, 150.0, 149.4, 149.3, 149.3, 149.1, 142.9, 136.7, 136.7, 136.6, 130.8, 130.7, 130.6, 130.6, 130.1, 130.1, 129.9, 129.9, 129.9, 129.5, 127.2, 127.2, 127.1, 127.0, 126.5, 124.1, 21.1. ESI-HRMS (pos. detection mode): calcd for C60H42N6O2Ru m/z [M].sup.2+ 490.1215; found: 490.1201. Elemental analysis calcd for C60H42F12N6O2P2Ru (%): C 56.74, H 3.33, N 6.62; found: C 56.80, H 3.24, N 6.59.

[Ru(bphen).SUB.2.(Me-aminomethyl)](PF.SUB.6.).SUB.2 .(10)

[0201] ##STR00072##

[0202] Ru(bphen).sub.2Cl.sub.2 (200 mg, 1.0 equiv.) and 5-(aminomethyl)-2,2′-bipyridine (57 mg, 1.2 equiv.) were dissolved in a 1:1 mixture of H.sub.2O/EtOH (10 mL) and were refluxed overnight under N.sub.2 atmosphere. The solvent was evaporated and the residue redissolved in 10 mL of H.sub.2O. A saturated, aq. NH.sub.4PF.sub.6 solution was added and the resulting precipitate was collected by vacuum filtration. The solid was washed with H.sub.2O (50 mL) and Et.sub.2O (50 mL). The product was dried in high vacuum. Yield: 88%. .sup.1H-NMR (CD.sub.3CN, 400 MHz): 8.59 (1H, d, J=1.3 Hz), 8.44 (1H, s), 8.29 (1H, d, J=5.5 Hz), 8.26 (1H, d, J=5.5 Hz), 8.15-8.07 (6H, m), 7.93 (1H, d, J=5.9 Hz), 7.74-7.70 (3H, m), 7.62-7.47 (22H, m), 7.39 (1H, dd, J=5.9, 1.7 Hz), 7.24 (1H, d, J=5.7 Hz), 4.38 (2H, s), 2.53 (3H, s). .sup.13C-NMR (CD.sub.3CN, 100 MHz): 158.4, 156.6, 152.9, 152.6, 152.5, 152.3, 151.8, 151.3, 149.5, 149.4, 149.4, 148.9, 148.7, 148.6, 142.9, 136.3, 136.2, 130.3, 130.2, 130.1, 123.0, 129.6, 129.5, 129.4, 129.4, 129.3, 127.4, 126.8, 126.7, 126.7, 126.4, 125.6, 124.5, 42.9, 20.8.

[Ru(bphen).SUB.2.(Me-maleimidemethyl)](PF.SUB.6.).SUB.2 .(11)

[0203] ##STR00073##

[0204] [Ru(bphen)2(Me-aminomethyl)](PF.sub.6).sub.2 (30 mg, 1.0 equiv.) and maleic anhydride (47 mg, 20.0 equiv.) were suspended in acetic acid (10 mL) under a nitrogen atmosphere. The mixture was refluxed for 10 h. The solution was then cooled down and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The crude product, which precipitated as a PF.sub.6 salt, was collected by filtration and washed three times with H.sub.2O and Et.sub.2O. The product was purified by column chromatography on silica gel with a CH.sub.3CN /aq. KNO.sub.3 (0.4 M) solution (10:1). The fractions containing the product were united and the solvent was removed. The residue was dissolved in CH.sub.3CN and undissolved KNO.sub.3 was removed by filtration. The solvent was removed and the product was dissolved in H.sub.2O. Upon addition of NH.sub.4PF.sub.6 the product precipitated as a PF.sub.6 salt. The solid was obtained by centrifugation and was washed with H.sub.2O and Et.sub.2O. Yield: 78%. .sup.1H-NMR (CD.sub.3CN, 400 MHz): 8.65 (1H, s), 8.57 (1H, d, J=1.3 Hz), 8.32 (1H, d, J=5.5 Hz), 8.29 (1H, d, J=5.5 Hz), 8.21-8.15 (6H, m), 8.11 (1H, d, J=5.5 Hz), 7.79-7.75 (3H, m), 7.69-7.56 (22H, m), 7.23 (2H, dd, J=5.8, 1.4 Hz), 6.90 (2H, s), 4.84 (2H, s), 2.57 (3H, s). .sup.13C-NMR (CD.sub.3CN, 100 MHz): 171.6, 158.5, 157.3, 153.0, 153.0, 152.9, 152.2, 151.5, 149.9, 149.9, 149.8, 149.4, 149.3, 149.2, 149.2, 149.1, 136.7, 136.6, 135.7, 130.7, 130.7, 130.5, 130.5, 130.0, 123.0, 129.8, 129.4, 127.1, 127.0, 126.9, 126.5, 126.2, 123.0, 40.6, 21.1. ESI-HRMS (pos. detection mode): calcd for C.sub.64H.sub.45N.sub.7O.sub.2Ru [M-2PF.sub.6].sup.2+ m/z 522.6334; found: 522.6347.

[Ru(bphen).SUB.2.(Me-maleimidemethyl)](Cl).SUB.2 .(12)

[0205] ##STR00074##

[0206] The counter ion PF.sub.6.sup.− of compound 11 was exchanged to Cl.sup.− by elution with MeOH from the ion exchange resin Amberlite IRA-410 to afford compound 12.

[0207] Elemental analysis calcd for C.sub.64H.sub.46Cl.sub.2N.sub.7O.sub.2Ru+H.sub.2O (%): C 67.78, H 4.18, N 8.65; found: C 67.73, H 3.94, N 8.36.

(4,4′-Dimethyl-2,2′-bipyridine)bis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichlorine [Ru(Me-bpy)(bphen).SUB.2.](Cl).SUB.2 .(13)

[0208] ##STR00075##

[0209] The synthesis of compound 13 is described in Mazuryk et al., 2014.

2) Photophysical Properties

[0210] Photophysical measurements were performed to evaluate the potential of the complexes of the invention 6 and 7 and the comparative examples as photosensitizers in PDT therapies.

Spectroscopic Measurements

[0211] The absorption of the samples in cuvettes has been measured with a Lambda 800 UV/VIS Spectrometer (PerkinElmer Instruments) and in 96 well plates with a SpectraMax M2 Spectrometer (Molecular Devices). The emission was measured by irradiation of the sample in fluorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla) at 355 nm. Luminescence was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator equipped with 1200 g/mm grating blazed at 500 nm. As a detector a XPI-Max 4 CCD camera (Princeton Instruments) has been used.

Results

[0212] At first, the absorption of the complexes in CH.sub.3CN was measured since the wavelengths used in PDT has a direct influence on the light penetration depth into the tissue and therefore influence the success of a treatment. All investigated complexes have a transition at about 263 nm for the phenanthroline-based complexes 1-5 and about 279 nm for the 4,7-diphenyl-1,10-phenanthroline-based complexes 6-7. Smaller bands varying from 280-320 nm (FIG. 1) were assigned to ligand centered (LC) transitions. Furthermore, these complexes have as the lowest energy absorption band a metal-to-ligand charge transfer (MLCT) transition. For the prototype complex, [Ru(bipy).sub.3].sup.2+, this band occurs at 450 nm, whereas this transition occurs for the complexes investigated in this study between 441 to 480 nm. Importantly, the compounds 5-7 have a long absorption tail towards the therapeutic spectral window.

[0213] Upon excitation at 355 nm, the emission of the complexes in CH.sub.3CN was determined. The maximum of the emission signal was measured between 600-710 nm (Table 1). Worthy of note, complexes 5 and 7 which showed the highest red shift of the MLCT transition, have also the highest emission maximum at 694-710 nm. This leads for all investigated complexes to a large Stokes shift implying minimal inference between excitation and luminescence.

TABLE-US-00001 TABLE 1 Spectroscopic properties of characterised complexes 1-7 in CH.sub.3CN at room temperature. Emission Compound UV/Vis λ / nm (ε / M.sup.−1 cm.sup.−1 * 10.sup.−3) λ.sub.em / nm 1 200 (73.2), 225 (64.3), 264 (86.5), 600 284 (44.1), 446 (15.0) 2 202 (77.9), 222 (61.5), 264 (81.7), 606 280 (43.9), 421 (12.8), 449 (13.9) 3 201 (72.9), 223 (91.0), 263 (95.2), 645 289 (45.1), 388 (11.5), 441 (14.8) 4 201 (100.1), 223 (91.3), 263 (105.8), 308 654 (28.2), 386 (13.8), 438 (16.7), 441 (16.8) 5 201 (89.3), 224 (81.2), 265 (91.1), 703 379 (25.6), 458 (23.1) 6 192 (183.4), 279 (126.3), 623 441 (23.2), 457 (23.2) 7 192 (168.8), 280 (102.5), 694 371 (35.0), 465 (30.1)

3) Singlet Oxygen Generation

Singlet Oxygen Measurements

Direct Evaluation

[0214] The samples were prepared in an air saturated CH.sub.3CN or D.sub.2O solution with an absorbance of 0.2 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using a mounted M450LP1 LED (Thorlabs) whose irradiation, centered at 450 nm, has been focused with aspheric condenser lenses. The intensity of the irradiation has been varied using a T-Cube LED Driver (Thorlabs) and measured with an optical power and energy meter. The emission signal was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator equipped with 600 g/mm grating blazed at 1200 nm. A longpass glass filter was placed in front of the monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. The slits for detection were fully open. As a detector an EO-817L IR-sensitive liquid nitrogen cooled germanium diode detector (North Coast Scientific Corp.) has been used. The singlet oxygen luminesce at 1270 nm was measured by recording spectra from 1100 to 1400 nm. For the data analysis, the singlet oxygen luminescence peaks at different irradiation intensities were integrated. The resulting areas were plotted against the percentage of the irradiation intensity and the slope of the linear regression calculated. The absorbance of the sample was corrected with an absorbance correction factor. As reference for the measurement in an CH.sub.3CN solution phenalenone (Φ.sub.phenaleone=0.95).sup.33 and for the measurement in a D2O solution [Ru(bipy).sub.3]Cl.sub.2 (Φ.sub.Ru(bipy)3C12=0.22).sup.31 was used and the singlet oxygen quantum yields were calculated using the following formula:

[00001]${\Phi }_{\mathrm{sample}}={\Phi }_{\mathrm{reference}}*\frac{S_{\mathrm{sample}}}{S_{\mathrm{reference}}}*\frac{I_{\mathrm{reference}}}{I_{\mathrm{sample}}}$$I=I_0*\left(1-10^{-A}\right)$$\Phi =\mathrm{singlet}\mathrm{oxygen}\mathrm{quantum}\mathrm{yeild},$$S=\mathrm{slope}\mathrm{of}\mathrm{the}\mathrm{linear}\mathrm{regression}\mathrm{of}\mathrm{the}\mathrm{plot}\mathrm{of}\mathrm{the}\mathrm{areas}\mathrm{of}\mathrm{the}$$\mathrm{sign}\mathrm{let}\mathrm{oxyen}\mathrm{luminescence}\mathrm{peaks}\mathrm{against}\mathrm{the}\mathrm{ir}\mathrm{radiation}\mathrm{intensity},$$I=\mathrm{absorbance}\mathrm{correction}\mathrm{factor},$$I0=\mathrm{light}\mathrm{intensity}\mathrm{of}\mathrm{their}\mathrm{radtion}\mathrm{source},$$A=\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{sample}\mathrm{at}\mathrm{irradiation}\mathrm{wavelength}.$

Indirect Evaluation

[0215] For the measurement in CH.sub.3CN: The samples were prepared in an air-saturated CH.sub.3CN solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 24 μM) and imidazole (12 mM). For the measurement in PBS buffer: The samples were prepared in an air-saturated PBS solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 20 μM) and histidine (10 mM). The samples were irradiated on 96 well plates with an Atlas Photonics LUMOS BIO irradiator for different times. The absorbance of the samples was measured during these time intervals with a SpectraMax M2 Microplate Reader (Molecular Devices). The difference in absorbance (A0-A) at 420 nm for the CH.sub.3CN solution or at 440 nm a PBS buffer solution was calculated and plotted against the irradiation times. From the plot the slope of the linear regression was calculated as well as the absorbance correction factor determined. The singlet oxygen quantum yields were calculated using the same formulas as used for the direct evaluation.

Results

[0216] The investigation of the luminescence lifetimes of the complexes 1-7 in comparison between a degassed and air-saturated CH.sub.3CN solution showed that the excited state was able to interact with .sup.3O.sub.2. Additionally, the DFT calculations were able to characterize the lowest energy absorption band as a MLCT transition with a triplet state. With this in hand, a quantitative evaluation of singlet oxygen (.sup.1O.sub.2) was performed to assess the potential of the PSs in PDT, by two methods: 1) direct by measurement of the luminescence of .sup.1O.sub.2, 2) indirect by measurement of the variation in absorbance of a reporter molecule, as described above. In the first method, the efficiency of the production of .sup.1O.sub.2was assessed by measuring its phosphorescence at 1270 nm. Worthy of note, the possibility of detection in this experiment is affected by its environment as well as the used setup. With the setup used in this study, we could only detect Φ(.sup.1O.sub.2) larger than 0.20 based on a low peak-to-noise ratio. In the second method (indirect method), .sup.1O.sub.2 is reacting with imidazole (in CH.sub.3CN) and histidine (in PBS buffer) to a trans-annular peroxide adduct. This can further quench the absorbance of the reporter molecule p-nitrosodimethyl aniline (RNO), which has been monitored by UV/VIS spectroscopy. In both methods, the .sup.1O.sub.2 production has been compared with a reference molecule, namely a solution of phenalenone in CH.sub.3CN (Φ(.sup.1O.sub.2).sub.phenaleone=0.95).sup.33 and a solution of [Ru(bipy).sub.3]Cl.sub.2 in water Φ(.sup.1O.sup.2).sub.Ru(bipy)3Cl2=0.22).sup.31. The results (Table 2) obtained show that the substitution of the bipyridine has an influence on the ability of the complexes to act as a photocatalyst. The Φ(.sup.1O.sub.2) in CH.sub.3CN using the direct and indirect method were found to be in the same range for complexes 1-4 and 6, namely between 0.53-0.69. In comparison, the values changed drastically in an aqueous solution. As an example, the Φ(.sup.1O.sub.2) for compound 3 and 6 in an aqueous environment was not detectable by the direct method and were determined to be 0.16 and 0.03, respectively by the indirect method. However, compounds 1-2 and 4 still showed a good singlet production with values between 0.23-0.46, as determined by direct and indirect method. These values are comparable with those previously reported for related compounds..sup.31-32 Additionally, the (E,E)-4,4′-bis(N,N′-dimethylaminovinyl)-2,2′-bipyridine substituted complexes 5 and 7 were investigated. As previously described in their excited state behaviour (emission, luminescence, lifetime) and anticipated by DFT calculations, these complexes showed different photophysical properties in comparison to the other complexes investigated in this work. They have untypically low Φ(.sup.1O.sub.2) values in CH.sub.3CN (0.22-0.35) for Ru(II) polypyridyl complexes. Subsequently, the .sup.1O.sub.2 production was also quite low in an aqueous environment.

TABLE-US-00002 TABLE 2 Singlet oxygen quantum yields (Φ(.sup.1O.sub.2)) in CH.sub.3CN and aqueous solution determined by direct and indirect methods by excitation at 450 nm. Average of three independent measurements, +−10%. Com- CH.sub.3CN CH.sub.3CN D.sub.2O PBS pound Direct Indirect Direct indirect 1 0.57 0.54 0.27 0.46 2 0.69 0.53 0.31 0.34 3 0.55 0.56 n.d. 0.16 4 0.62 0.59 0.25 0.26 5 0.24 0.30 n.d. 0.21 6 0.61 0.63 n.d. 0.03 7 0.22 0.35 n.d. 0.07 n.d. = not determinable, Φ(.sup.1O.sub.2) < 0.20

4) Dark Cytotoxicity and (Photo-)Toxicity

Material and Methods

Cell Culture

[0217] HeLa and CT-26 cell lines were cultured in DMEM media (Gibco, Life Technologies, USA) supplemented with 10% of fetal calf serum (Gibco). U87 and U373 cell lines were cultured in MEM media with addition of 1% of MEM NEAA (non-essential aminoacids) (Gibco) and 10% of fetal calf serum. RPE-1 cells were cultured in DMEM/F-12 (Gibco) supplemented with 10% of fetal calf serum. RPE-1 stable cells lines were cultured as RPE-1 cells with addition of geneticin (0.5 mg/ml) (Gibco). All cell lines were complemented with 100 U/ml penicillin-streptomycin mixture (Gibco), and maintained in humidified atmosphere at 37° C. and 5% of CO.sub.2.

Dark Cytotoxicity and (Photo-)Toxicity

[0218] Dark and light cytotoxicity of the Ru(II) complexes was assesed by fluorometric cell viability assay using resazurin (ACROS Organics). For dark and light cytotoxicity, cells were seeded in triplicates in 96 well plates at a density of 4000 cells per well in 100 μl, 24 h prior to treatment. The medium was then replaced with increasing concentration of the tested complexes and cells were incubated for 4 h. Medium was then replaced for fresh complete medium. Cells used for light cytotoxicity experiment were exposed to: 480 nm light for 10 min, 510 nm for 40 min, 540 for 60 min or 595nm for 120 min in a 96-well plate using a LUMOS-BIO photoreactor (Atlas Photonics). Each well was individually illuminated with a LED at constant current. After irradiation cells were kept for another 44 h in the incubator and the medium was replaced by fresh complete medium containing resazurin (0.2 mg ml.sup.−1 final concentration). After 4 h incubation at 37° C., the fluorescence signal of the resorufin product was read by SpectraMax M5 mictroplate reader (ex: 540 nm em: 590 nm). IC.sub.50 values were calculated using GraphPad Prism software.

[0219] Having assessed that complexes 1-7 were producing .sup.1O.sub.2, the inventors then investigated their cytotoxicity in the dark and upon light irradiation. The potential of the complexes to act as PDT PSs was determined on mouse colon carcinoma cells (CT-26), human glioblastoma cells (U87 and U373), human cervical carcinoma cells (HeLa) as well as non-cancerous retina pigmented epithelial cell line (RPE-1) according to the method described above. The obtained results along with the calculated phototoxic index (PI) (IC.sub.50 in the dark/IC.sub.50 upon light irradiation) are gathered in Table 3. Ideally, a PDT PS should be non-toxic in the dark and highly toxic upon light activation. Promisingly, complexes 1-5 and 7 were found to be non-cytotoxic in all chosen cell lines in the dark (IC.sub.50>100 μM). Compound 6 showed a slight cytotoxicity (IC.sub.50 range from 3.09 to 28.77 μM) which is not detrimental for its use as photosensitizer. The toxicity of the compounds upon light irradiation (480 nm, 10 min, 3.21 J cm.sup.−2) was then investigated. No or only poor toxicity was observed for comparative complexes 1-5 (IC.sub.50 range from >100 to 52.54 μM). In contrast, complexes of the invention (6 and 7) showed a notable phototoxicity upon light irradiation (PI values range from 6.5 to 42.5). More importantly, both complexes showed potency in the treatment of the human glioblastomas (U87 and U373 cell lines). It is known that glioblastomas are difficult to treat and current therapies are not significantly improving the survival of patients (Lim, M., 2018).

[0220] To determine if complex 6 was efficiently killing cells when irradiated with longer wavelengths than 480 nm (i.e. closer to the biological window: 600-900 nm), we tested its ability to kill CT-26 mouse colon carcinoma cells at 510, 540 and 595 nm. Light irradiation of the treated cells at 510 nm (40 min) or 540 nm (60 min) caused phototoxic effect (PI values of 20.6 and 9.6, respectively). Even irradiation at 595 nm (2 h) generated toxicity in cells (PI value of 23.47). It has to be noted that the lack of CO.sub.2 atmosphere during the 2 h irradiation also contributed to the obtained results (Table 4). Nevertheless, obtained PI value is reliable, dark control cells were also incubated for 2h at 37.sup.2C in non-CO.sub.2 atmosphere. Overall, these results make compound 6 an impressive candidate as PDT PS.

TABLE-US-00003 TABLE 3 IC.sub.50 values for the complexes 1-7 incubated with cell lines in the dark and upon light irradiation (480 nm, 10 min; 3.21 J cm.sup.−2). Comparative complexes Complexes of the invention IC.sub.50/μM 1 2 3 4 5 6 7 CT-26 Dark >100 >100 >100 >100 >100  3.09 ± 0.30 94.47 ± 7.38 Light >100 91.24 ± 7.54 85.71 ± 9.47 72.59 ± 7.44 52.54 ± 6.04  0.19 ± 0.04  6.62 ± 0.70 PI — >1 >1 >1 >2 16.3 14.3 U87  Dark >100 >100 >100 >100 >100 28.45 ± 1.97 >100 Light 93.68 ± 2.50 71.40 ± 7.54 >100 >100 >100  0.67 ± 0.13  7.90 ± 0.54 PI >1 >1 — — — 42.5 >12.7 U373 Dark >100 >100 >100 >100 >100 23.37 ± 0.53 >100 Light >100 >100 >100 >100 >100  1.89 ± 0.07 14.85 ± 0.81 PI — — — — — 12.37 >6.7 HeLa Dark >100 >100 >100 >100 >100 13.57 ± 1.30 >100 Light >100 >100 >100 >100 >100  0.61 ± 0.06 15.21 ± 1.29 PI — — — — — 22.2 >6.5 RPE-1 Dark >100 >100 >100 >100 >100 28.77 ± 0.94 >100 Light >100 >100 >100 >100 >100 0.825 ± 0.03  8.95 ± 0.50 PI — — — — — 34.9 >11.2

TABLE-US-00004 TABLE 4 IC.sub.50 values on CT-26 mouse colon carcinoma cells for complex 6 in the dark and upon light irradiation with wavelengths longer than 480 nm. CT-26 IC.sub.50 [μM] Dark Light PI 510 nm 40 min 4.18 ± 0.56  0.20 ± c0.005 20.6 540 nm 60 min 3.27 ± 0.64 0.34 ± 0.005 9.6 595 nm 2 h 1.408 ± 0.003 0.06 ± 0.004 23.47

5) In Vivo Biodistribution of Complex 6

[0221] Due to the very encouraging in vitro results obtained for compound 6, we have then tested its behavior in vivo.

Material and Methods

[0222] Twenty four, 8 week old healthy BALB/c female mice were used in this study. 0.015 mg/ml solution of complex 6 was prepared in Milli-Q water and filtrated (0.2 μm cellulose acetate membrane, VWR). For the introduction of solution of complex 6, IV injection was used (300 μl per mouse). Organ samples, including brain, liver, spleen, kidneys and lung, were collected from treated mice after 2 h, 6 h and 24 h post-injection. Each time six mice were sacrificed. Remaining six animals were used as a control.

[0223] For these experiments, we have decided to use the chloride salt of the complex 6 to improve its solubility. The time-dependent biodistribution of this compound in different organs was determined in healthy 8-week-old BALB/c mice according to the above-described method. The amount of ruthenium in the tested samples was assessed using Inductive Coupled Plasma Mass-Spectrometry (ICP-MS). Worthy of note, the animals treated with compound 6 behave normally, without signs of pain, stress or discomfort. Blood analysis after 24 h treatment showed no sign of immune response compared to untreated control. As shown in FIG. 2, from all harvested organs, only liver had clearly increased levels of Ru after 6 h post IV injection. After 24 h, the amount of ruthenium in the liver decreased. This is a very promising result that could indicate that complex 6 is metabolized by the liver in living organisms.

6) Binding of Compounds 12 and 13 to Albumin

[0224] The following experiments have been carried out in order to demonstrate that compound 12, which bears a maleimide unit, is able to covalently bound to albumin and thus to form a conjugate.

Material and Method

[0225] UV-visible (UV-Vis) spectrophotometry was monitored on an Agilent Cary 8454 diode array spectrophotometer in the wavelength range between 190 and 1100 nm.

[0226] Interaction of complex 12 at the Cys-34 residue of HSA was investigated spectrophotometrically via the dithiodipyridine (DTDP) method described previously (Pichler et al., 2013). The available Cys-thiol content in HSA was determined to be 22%. Complex binding was tested in the following setup: 133 μM HSA (29 μM free thiol) and various amounts of complex (0-120 μM) were incubated for 3 h or 30 min at pH=7.00 (PBS). A first series of UV-Vis spectra (a) were recorded before addition of 110 μM DTDP and a second series of UV-Vis spectra (b) were recorded after addition of 110 μM DTDP and after another 40 min waiting. Cys-34 residues of HSA which are not conjugated to complex 12 react with DTDP to form the UV active compound 2-thiopyridone. Blank experiment with compound 13 was carried out as well as a control experiment. The effect of protein unfolding on the thiol binding of complex 12 was studied by the addition of 0.5% (m/m) SDS to the protein prior to its reaction with the complex. This experiment allows to determine the amount of free Cys-34 residue of HSA as a function of the added equivalent of the complexes. The results of this experiment are presented on FIG. 3).

Results

[0227] Complex 13 applied as negative control, affects barely the quantity of free thiol groups. Complex 12, on the other hand, interacts in a significant extent with Cys-34. Incubation of the complex with HSA for 3 h (open triangle) or 30 min (closed triangle) did not result in remarkable differences, at the same time the interaction with the native protein does not show quantitative binding at this site. Measurements implemented with the unfolded protein using 0.5% SDS as denaturing agent revealed nearly quantitative interaction between 12 and the Cys-34 thiol group of HSA. Two scenarios are possible regarding the interaction with native protein: (i) concurrent binding at other sites in HSA reduces the effective concentration of 12, or (ii) structural heterogeneity applies in the HSA stock, i.e. the availability of thiol groups for 12 is different. First interpretation does not fit to thermodynamic considerations (irreversible binding at Cys-34 should be preferred over intermolecular interactions), while the second assumption can explain the elevated saturation phase of the curves, but not their relatively low slope.

[0228] All in all, complex 12 was found to interact with the Cys-34 thiol group of HSA, although the interaction is not quantitative.

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