Metal Complexes Bearing Bisstyryl-Bipyridine Ligand and Their Use as Photosensitizer Agent in One and Two-Photon Photodynamic Therapy
20220409727 · 2022-12-29
Inventors
Cpc classification
C07D213/38
CHEMISTRY; METALLURGY
A61K41/008
HUMAN NECESSITIES
C07D213/22
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K41/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to metal complexes bearing at least one (E-E′)-4,4′-bisstyryl-2,2′-bipyridine ligand (LIG1) of the following formula (I): or a pharmaceutically acceptable salt and/or solvate thereof. The present invention also relates to pharmaceutical compositions comprising these complexes and at least one pharmaceutically acceptable excipient. The present invention also relates to the use of compounds of formula (I) or pharmaceutical compositions comprising thereof as drug and as photosensitizer agent in photodynamic therapy. The present invention relates to methods of preparation of said complexes.
##STR00001##
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 M is selected among ruthenium, rhenium, osmium, rhodium, iridium and platinum, LIG.sub.1 is a bidentate ligand having the following formula: ##STR00077## wherein the wavy lines indicate the points of attachment to M, R.sup.1 and R.sup.2 each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.9 and NR.sup.10R.sup.11, R.sup.3 to R.sup.6 each independently represent a substituent selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.12 and NR.sup.13R.sup.14, R.sup.7 and R.sup.8 each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.15 and NR.sup.16R.sup.17, R.sup.9 to R.sup.11 are each independently selected in the group consisting of H and C.sub.1-C.sub.6 alkyl, and R.sup.12 to R.sup.17 are each independently selected in the group consisting of H, C.sub.1-C.sub.6 alkyl and CO—(C.sub.1-C.sub.6 alkyl), LIG.sub.2 is a bidentate ligand having the following formula (a) or (b): ##STR00078## wherein the wavy lines indicate the points of attachment to M, LIG.sub.3 is a bidentate ligand having the following formula (c) or (d): ##STR00079## wherein the wavy lines indicate the points of attachment to M, each represents a single or a double bond, provided that each cycle A, B, C and D is a heteroaromatic cycle, T.sub.1 is NR.sub.a1 or CR.sub.a1, T.sub.2 is NR.sub.a2 or CR.sub.a2, T.sub.3 is NR.sub.a3 or CR.sub.a3, T.sub.4 is NR.sub.a4 or CR.sub.a4, T.sub.7 is NR.sub.a7 or CR.sub.a7, T.sub.8 is NR.sub.a8 or CR.sub.a8, T.sub.9 is NR.sub.a9 or CR.sub.a9 and T.sub.10 is NR.sub.a10 or CR.sub.a10, provided that when T.sub.1 is NR.sub.a1, then T.sub.2 is CR.sub.a2, when T.sub.3 is NR.sub.a3, then T.sub.4 is CR.sub.a4, when T.sub.7 is NR.sub.a7, then T.sub.8 is CR.sub.a8 and when T.sub.9 is NR.sub.a9, then T.sub.10 is CR.sub.a10, Z.sub.1 is N or CR.sub.b1, Z.sub.2 is N or CR.sub.b2, Z.sub.3 is N or CR.sub.b3, Z.sub.4 is N or CR.sub.b4, Z.sub.5 is N or CR.sub.b5, Z.sub.6 is N or CR.sub.b6, Z.sub.9 is N or CR.sub.b9, Z.sub.10 is N or CR.sub.b10, Z.sub.11 is N or CR.sub.b11, Z.sub.12 is N or CR.sub.b12, Z.sub.13 is N or CR.sub.b13 and Z.sub.14 is N or CR.sub.b14, provided that at least two of Z.sub.1 to Z.sub.3 and at least two of Z.sub.4 to Z.sub.6 and at least two of Z.sub.9 to Z.sub.11 and at least two of Z.sub.12 to Z.sub.14 are not N, R.sub.a1 to R.sub.a12 and R.sub.b1 to R.sub.b16 each independently represent 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 or NR.sup.20R.sup.21, or Z.sub.3 and Z.sub.4 in formula (b) are linked together so that LIG.sub.2 represents: ##STR00080## and/or Z.sub.11 and Z.sub.12 are linked in formula (d) together so that LIG.sub.3 represents: ##STR00081## wherein R.sup.x and R.sup.y 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 to 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 and n are independently 1, 2, 3 or 4, wherein n is 1 when M is rhenium, n is 2 when M is ruthenium or osmium, n is 3 when M is rhodium or iridium and n is 4 when M is platinum, and y1 is 1, 2 or 3, y2 and y3 are independently 0, 1 or 2, provided that y1+y2+y3 is 3.
17. The method according to claim 16, wherein R.sup.1 and R.sup.2 are one or several substituents each independently selected from the group consisting of H, halogen, OR.sup.9 and NR.sup.10R.sup.11.
18. The method according to claim 17, wherein R.sup.1 and R.sup.2 are both OR.sup.9, with R.sup.9 being a C.sub.1-C.sub.6 alkyl.
19. The method according to claim 16, wherein LIG.sub.1 is of following formula: ##STR00082##
20. The method according to claim 16, wherein LIG.sub.2 and LIG.sub.3 are different from LIG.sub.1.
21. The method according to claim 16, wherein R.sub.a1 to R.sub.a12 and R.sub.b1 to R.sub.b16 each independently represent H, halogen, C.sub.1-C.sub.6 alkyl, aryl, OR.sup.19 or NR.sup.20R.sup.21, and R.sup.x and R.sup.y each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, aryl, OR.sup.19 and NR.sup.20R.sup.21, with R.sup.19 to R.sup.21 being each independently selected in the group consisting of H and C.sub.1-C.sub.6 alkyl.
22. The method according to claim 16, wherein R.sub.a1 to R.sub.a12 and R.sub.b1 to R.sub.b16 are H or aryl, and R.sup.x and R.sup.y represent H.
23. The method according to claim 16, wherein y1 is 1, 2 or 3, y2 is 2, 1 or 0 respectively and y3 is 0.
24. The method according to claim 23, wherein LIG.sub.2 is a bidentate ligand which is selected from the group consisting of: ##STR00083## ##STR00084## with R.sub.b1 to R.sub.b16 and R.sup.x as defined in claim 16.
25. The method according to claim 24, wherein LIG.sub.2 is of formula (b-1).
26. The method according to claim 16, wherein M is ruthenium or osmium.
27. The method according to claim 16, wherein the compound of formula (I) is selected from the group consisting of: ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
28. The method according to claim 16, wherein the photodynamic therapy is intended to treat a disease selected from the group consisting of cancer; bacterial infection; fungal infection; viral infection; and skin disorders.
29. The method according to claim 28, wherein the cancer is selected from the group consisting of lung cancer, bladder cancer, oesophageal cancer, colon cancer, stomach cancer, liver cancer, skin cancer, ovarian cancer, pancreatic cancer, head and neck cancer and brain cancer, the bacterial infection is selected from the group consisting of sinusitis, diabetic feet and burned wounds, the fungal infection is mycoses, the viral infections is herpes and the skin disorders are selected in the group consisting of acne and port wine stains.
30. A pharmaceutical composition comprising at least one compound of formula (I) as defined in claim 16 and at least one pharmaceutically acceptable excipient.
31. A method of treatment by photodynamic therapy comprising administering to an animal or a human in need thereof an effective amount of a pharmaceutical composition according to claim 30.
32. The method according to claim 31, wherein the photodynamic therapy is intended to treat a disease selected from cancer; bacterial infection; fungal infection; viral infection; and skin disorders.
33. A compound of formula (I): ##STR00093## or a pharmaceutically acceptable salt and/or solvate thereof, wherein M is selected among ruthenium, rhenium, osmium, rhodium, iridium and platinum, LIG.sub.1 is a bidentate ligand having the following formula: ##STR00094## wherein the wavy lines indicate the points of attachment to M, R.sup.1 and R.sup.2 each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.9 and NR.sup.10R.sup.11, R.sup.3 to R.sup.6 each independently represent a substituent selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.12 and NR.sup.13R.sup.14, R.sup.7 and R.sup.8 each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, OR.sup.15 and NR.sup.16R.sup.17, R.sup.9 to R.sup.11 are each independently selected in the group consisting of H and C.sub.1-C.sub.6 alkyl, and R.sup.12 to R.sup.17 are each independently selected in the group consisting of H, C.sub.1-C.sub.6 alkyl and CO—(C.sub.1-C.sub.6 alkyl), LIG.sub.2 is a bidentate ligand having the following formula (a) or (b): ##STR00095## wherein the wavy lines indicate the points of attachment to M, LIG.sub.3 is a bidentate ligand having the following formula (c) or (d): ##STR00096## wherein the wavy lines indicate the points of attachment to M, each represents a single or a double bond, provided that each cycle A, B, C and D is a heteroaromatic cycle, T.sub.1 is NR.sub.a1 or CR.sub.a1, T.sub.2 is NR.sub.a2 or CR.sub.a2, T.sub.3 is NR.sub.a3 or CR.sub.a3, T.sub.4 is NR.sub.a4 or CR.sub.a4, T.sub.7 is NR.sub.a7 or CR.sub.a7, T.sub.8 is NR.sub.a8 or CR.sub.a8, T.sub.9 is NR.sub.a9 or CR.sub.a9 and T.sub.10 is NR.sub.a10 or CR.sub.a10, provided that when T.sub.1 is NR.sub.a1, then T.sub.2 is CR.sub.a2, when T.sub.3 is NR.sub.a3, then T.sub.4 is CR.sub.a4, when T.sub.7 is NR.sub.a7, then T.sub.8 is CR.sub.a8 and when T.sub.9 is NR.sub.a9, then T.sub.10 is CR.sub.a10, Z.sub.1 is N or CR.sub.b1, Z.sub.2 is N or CR.sub.b2, Z.sub.3 is N or CR.sub.b3, Z.sub.4 is N or CR.sub.b4, Z.sub.5 is N or CR.sub.b5, Z.sub.6 is N or CR.sub.b6, Z.sub.9 is N or CR.sub.b9, Z.sub.10 is N or CR.sub.b10, Z.sub.1 is N or CR.sub.b11, Z.sub.12 is N or CR.sub.b12, Z.sub.13 is N or CR.sub.b13 and Z.sub.14 is N or CR.sub.b14, provided that at least two of Z.sub.1 to Z.sub.3 and at least two of Z.sub.4 to Z.sub.6 and at least two of Z.sub.9 to Z.sub.11 and at least two of Z.sub.12 to Z.sub.14 are not N, R.sub.a1 to R.sub.a12 and R.sub.b1 to R.sub.b16 each independently represent 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 or NR.sup.20R.sup.21, or Z.sub.3 and Z.sub.4 in formula (b) are linked together so that LIG.sub.2 represents: ##STR00097## and/or Z.sub.11 and Z.sub.12 are linked in formula (d) together so that LIG.sub.3 represents: ##STR00098## wherein R.sup.x and R.sup.y 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 to 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 and n are independently 1, 2, 3 or 4, wherein n is 1 when M is rhenium, n is 2 when M is ruthenium or osmium, n is 3 when M is rhodium or iridium and n is 4 when M is platinum, and y1 is 1, 2 or 3, y2 and y3 are independently 0, 1 or 2, provided that y1+y2+y3 is 3, with the proviso that said compound is not: ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
34. The compound according to claim 33, wherein R.sup.1 and R.sup.2 are one or several substituents each independently selected from the group consisting of H, halogen, OR.sup.9 and NR.sup.10R.sup.11 and/or R.sub.a1 to R.sub.a12 and R.sub.b1 to R.sub.b16 each independently represent H, halogen, C.sub.1-C.sub.6 alkyl, aryl, OR.sup.19 or NR.sup.20R.sup.21, and R.sup.x and R.sup.y each independently represent one or several substituents selected in the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, aryl, OR.sup.19 and NR.sup.20R.sup.21, with R.sup.19 to R.sup.21 being each independently selected in the group consisting of H and C.sub.1-C.sub.6 alkyl and/or y1 is 1, 2 or 3, y2 is 2, 1 or 0 respectively and y3 is 0 and/or M is ruthenium or osmium.
35. The compound according to claim 34, being selected from the group consisting of: ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113##
Description
DESCRIPTION OF THE FIGURES
[0197]
[0198]
[0199]
[0200]
[0201]
[0202]
[0203] The present invention is illustrated by the following examples.
EXAMPLES
[0204] 1. Synthesis
[0205] The synthesis of the complexes is presented.
[0206] Structures
##STR00072## ##STR00073## ##STR00074## ##STR00075##
[0207] Materials
[0208] All chemicals were obtained from commercial sources and used without further purification. Solvents were dried over molecular sieves if necessary. The Ru(II) precursors Ru(DMSO).sub.4Cl.sub.2 and Ru(bipy).sub.2Cl.sub.2, Ru(phen).sub.2Cl.sub.2 and Ru(bphen).sub.2Cl.sub.2 were synthesised as previously reported using the respective ligand (Bratsos, I. and Alessio, E., 2010, Meyer, T. J. et al., 1987 and Wu, Q. et al, 1995).
[0209] Instrumentation and Methods
[0210] .sup.1H and .sup.13C NMR spectra were recorded on a Bruker 400 MHz or 500 MHz NMR spectrometer. Chemical shifts (5) are reported in parts per million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the residual proton solvent peaks as internal standards. Coupling constants (J) are reported in Hertz (Hz) and the multiplicity is abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet). ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific) and operated in positive ionization mode, with a spray voltage at 3.6 kV. No Sheath and auxiliary gas were 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). The automatic gain control (AGC) allowed accumulation of up to 2.105 ions for FTMS scans, Maximum injection time was set to 300 ms and 1 μscan was acquired. 10 μL was injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific) with a continuous infusion of methanol at 100 μL.Math.min.sup.−1. Elemental microanalyses were performed on a Thermo Flash 2000 elemental analyser. The absorption of the samples has been measured with a SpectraMax M2 Spectrometer (Molecular Devices). 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 5C18 (Analytic: 100 Å, C18 5 μm 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). Inductive coupled plasma mass spectrometry (ICP-MS) experiments were carried out on an iCAP RQ ICP-MS instrument (Thermo Fisher).
[0211] Synthesis of the Ligands
(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine
[0212] The synthesis of 4,4′-Bisstyryl-2,2′-bipyridine is already published (Meyer, T. J. et al., 1987) but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (1000 mg, 5.43 mmol, 1.0 equiv.) was dissolved in dry DMF under nitrogen atmosphere and Benzaldehyde (1.2 mL, 11.84 mmol, 2.2 equiv.) was added to the solution. Afterwards potassium tert-butoxide (2436 mg, 21.72 mmol, 4.0 equiv.) was added slowly. The colour of the solution turned to green and the mixture was stirred for 24 h. After that the mixture was poured into H.sub.2O (400 mL) and the suspension cooled down to 5° C. The crude product which precipitated, was filtered and washed with Methanol. The product was purified by recrystallization from boiling acetic acid. The obtained solid was dissolved in Dichloromethane and the mixture was washed with a 5% LiCl aqueous solution, brine and H.sub.2O. The solvent was removed and the product was isolated by recrystallization from boiling acetic acid. 1292 mg of (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine (3.58 mmol, 66%) were yielded as a beige solid.
4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine
[0213] The synthesis of 4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine is already published (Wu, Q. et al) but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (1000 mg, 5.43 mmol, 1.0 equiv.) was dissolved in dry DMF (100 mL) under nitrogen atmosphere and potassium tert-butoxide (2437 mg, 21.72 mmol, 4.0 equiv.) was added slowly. After 1.5 h of stirring, 4-(Dimethylamino)benzaldehyde (1701 mg, 11.40 mmol, 2.1 equiv.) was added to the reaction mixture. The colour of the solution turned to yellow and the mixture was heated at 90° C. for 19 h. After that the mixture was poured into H.sub.2O (400 mL) and the suspension cooled down to 5° C. The crude product which precipitated, was filtered and washed with H.sub.2O and Et.sub.2O. The product was isolated by recrystallization from DCM/Pentane. 1541 mg of (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (3.45 mmol, 64%) were yielded as a yellow solid.
(E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine
[0214] The synthesis of (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine is already published (Wu, Q. et al) but in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (532 mg, 2.89 mmol, 1.0 equiv.) was dissolved in dry DMF (25 mL) under nitrogen atmosphere and 4-Methoxybenzaldehyde (0.88 mL, 7.22 mmol, 2.5 equiv.) was added to the solution. Afterwards potassium tert-butoxide (1360 mg, 12.13 mmol, 4.2 equiv.) was added slowly. The colour of the solution turned to green and the mixture was stirred for 24 h. After that the mixture which turned bright was poured into H.sub.2O (200 mL) and the suspension cooled down to 5° C. The crude product which precipitated, was filtered and washed with Methanol. The product was purified by recrystallization from boiling acetic acid. The obtained solid was dissolved in Dichloromethane and the mixture was washed with a 5% LiCl aqueous solution, brine and H.sub.2O. The solvent was removed and the product was isolated by recrystallization from boiling acetic acid. 925 mg of (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (2.20 mmol, 76%) were yielded as a beige solid.
(E,E′)-4,4′-Bis[m-methoxystyryl]-2,2′-bipyridine
[0215] (E,E′)-4,4′-Bis[m-methoxystyryl]-2,2′-bipyridine was synthesized according to the literature (Aranyos, V. et al., 2001).
(E,E′)-4,4′-Bis[o-methoxystyryl]-2,2′-bipyridine
[0216] (E,E′)-4,4′-Bis[o-methoxystyryl]-2,2′-bipyridine was synthesized according to the literature (Sinha, S., 2015)
(E,E′)-4,4′-Bis[p-fluorostyryl]-2,2′-bipyridine
[0217] 4,4′-Dimethyl-2,2′-bipyridine (1.00 g, 5.43 mmol, 1.0 equiv.) was dissolved in dry DMF (50 mL) under nitrogen atmosphere and 4-fluorobenzaldehyde (1.46 mL, 13.57 mmol, 2.5 equiv.) was added to the solution. Afterwards potassium tert-butoxide (2.44 g, 21.74 mmol, 4.0 equiv.) was added slowly. The colour of the solution turned to green and the mixture was stirred for 24 h. After that the mixture which turned bright was poured into H.sub.2O (500 mL) and the suspension cooled down to 5° C. The crude product which precipitated, was filtered, and washed with methanol. The product was purified by recrystallization from boiling acetic acid. The obtained solid was dissolved in Dichloromethane and the mixture was washed with a 5% LiCl aqueous solution, brine, and H.sub.2O. The solvent was removed to yield (E,E′)-4,4′-Bis[p-fluorostyryl]-2,2′-bipyridine as a beige solid (1.38 g, 3.48 mmol, 64%). .sup.1H-NMR (400 MHz, CD.sub.2Cl.sub.2): δ 8.75 (dd, J=5.0, 0.7 Hz, 2H), 8.68 (dd, J=1.7, 0.7 Hz, 2H), 7.69 (dd, J=8.7, 5.3 Hz, 4H), 7.55 (d, J=16.5 Hz, 2H), 7.52 (dd, J=5.0, 1.7 Hz, 2H), 7.22 (dd, J=8.7, 5.3 Hz, 4H), 7.20 (d, J=16.5 Hz, 2H); ESI-HRMS (pos. detection mode): calcd for C.sub.26H.sub.19N.sub.2F.sub.2, 397.1516; found, 397.1513.
(E,E′)-4,4′-Bis[p-hydroxystyryl]-2,2′-bipyridine
[0218] (E,E′)-4,4′-Bis[p-hydroxystyryl]-2,2′-bipyridine was synthesized according to the literature (n K. K. et al., 1995).
(E,E′)-4,4′-Bis[p-nitrostyryl]-2,2′-bipyridine
[0219] (E,E′)-4,4′-Bis[p-nitrostyryl]-2,2′-bipyridine was synthesized according to the literature (K. K. et al., 1995).
(E,E′)-4,4′-Bis[p-aminostyryl]-2,2′-bipyridine
[0220] (E,E′)-4,4′-Bis[p-aminostyryl]-2,2′-bipyridine was synthesized according to the literature (Gajardo, F. et al., 2011).
Tetraethyl 2,2′-bipyridine-4,4′-bisphosphonate
[0221] Tetraethyl-2,2′-bipyridine-4,4′-bisphosphonate was synthesized according to the literature (Woo, S. J., 2019).
4,4′-bis((E)-2-(4-methoxyphenyl)prop-1-en-1-yl)-2,2′-bipyridine
[0222] Under nitrogen, tetraethyl-2,2′-bipyridine-4,4′-bisphosphonate (200 mg, 0.44 mmol, 1.0 equiv.) was placed in a flame-dried flask. Anhydrous THF (10 mL) was added and the solution was cooled down to 0° C. KHMDS (2.6 mL, 1.30 mmol, 3.0 equiv., 0.5 mol/L solution in toluene) was added and the mixture was allowed to warm up to room temperature. The mixture was heated at 60° C. for 1 h. The mixture was then cooled down and 4-acetanisole (330 mg, 2.20 mmol, 5.0 equiv.) dissolved in anhydrous THF (5 mL) was added. The mixture was heated at 60° C. for 15 h. The mixture was cooled down and distilled water (35 mL) was added. The suspension was centrifuged, the solid was washed with hot ethanol and dried under vacuum to yield a beige powder (65 mg, 0.14 mmol, 32%). .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 8.66 (d, J=5.1 Hz, 2H), 8.39 (s, 2H), 7.49 (d, J=8.8 Hz, 4H), 7.28 (d, J=4.4 Hz, 2H), 6.93 (d, J=8.8 Hz, 4H), 6.79 (s, 2H), 3.85 (s, 6H), 2.35 (d, J=1.32, 6H). .sup.13C-NMR (CDCl.sub.3, 125 MHz): δ=159.6, 156.4, 149.2, 147.3, 141.1, 135.8, 127.4, 124.1, 123.8, 121.5, 114.0, 55.5, 18.0. ESI-HRMS (pos. detection mode): calcd for C.sub.30H.sub.29N.sub.2O.sub.2 m/z [M+H]+ 449.2229; found: 449.2223.
[0223] Synthesis of the Ruthenium Complexes
[Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine).SUB.3.][PF.SUB.6.].SUB.2 .(1)
[0224] (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine (400 mg, 1.11 mmol, 4.0 equiv.) and Ru(DMSO).sub.4Cl.sub.2 (134 mg, 0.28 mmol, 1.0 equiv.) were suspended in dry Ethanol (150 mL) under nitrogen atmosphere and the mixture was refluxed for 24 h. Then the solution was cooled down and undissolved residue was removed via filtration. To the residual solution 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 centrifugation and washed with Ethanol, H.sub.2O and Et.sub.2O. The crude product was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and H.sub.2O. After drying, 323 mg of 1 (0.22 mmol, 79%) were yielded as a red solid. .sup.1H-NMR (CD.sub.3CN, 500 MHz): δ=8.76 (d, .sup.3J=1.8 Hz, 6H), 7.78 (d, 3l=16.4 Hz, 6H), 7.76 (d, .sup.3J=5.9 Hz, 6H), 7.71-7.69 (m, 12H), 7.51 (dd, .sup.3,4J=5.9, 1.8 Hz, 6H), 7.49-7.46 (m, 12H), 7.44-7.40 (m, 6H), 7.33 (d, .sup.3J=16.4 Hz, 6H). .sup.13C-NMR (CD.sub.3CN, 125 MHz): δ=158.2, 152.4, 147.6, 137.3, 136.8, 130.6, 130.1, 128.4, 125.4, 125.1, 121.7. ESI-HRMS (pos. detection mode): calcd for C78H60N6Ru m/z [M].sup.2+ 591.1956; found: 591.1978. Elemental analysis calcd for C78H60F12N6P2Ru+4*H2O (%): C, 60.66, H, 4.44, N, 5.44; found: C, 60.62, H, 4.43, N, 5.86. [Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine).sub.3][Cl].sub.2: The counter ion PF.sub.6 was exchanged to Cl by elution with MeOH from the ion exchange resin Amberlite IRA-410. Elemental analysis calcd for C78H60Cl2N6Ru (%): C, 74.75, H, 4.83, N, 6.71; found: C, 74.36, H, 4.51, N, 6.37.
[Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine).SUB.3.][PF.SUB.6.].SUB.2 .(2)
[0225] (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (338 mg, 0.76 mmol, 4.0 equiv.), Ru(DMSO).sub.4Cl.sub.2 (92 mg, 0.19 mmol, 1.0 equiv.) and LiCl (401 mg, 9.46 mmol, 50.0 equiv.) were dissolved in dry DMF (50 mL) under nitrogen atmosphere. The mixture was refluxed for 48 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 centrifugation and washed with Ethanol, H.sub.2O and Et.sub.2O. The residue was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and H.sub.2O. The solvent was removed under reduced pressure and the crude product recrystallized from DCM/Pentane. The product was isolated via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O. 86 mg of 2 (0.05 mmol, 26%) were yielded as a black solid. .sup.1H-NMR (CD.sub.3CN, 500 MHz): δ=8.62 (d, .sup.3J=1.9 Hz, 6H), 7.66 (d, .sup.3J=16.2 Hz, 6H), 7.65 (d, .sup.3J=6.1 Hz, 6H), 7.55-7.52 (m, 12H), 7.38 (dd, .sup.3,3J=6.1, 1.9 Hz, 6H), 7.03 (d, .sup.3J=16.2 Hz, 6H), 6.81-6.78 (m, 12H), 3.01 (s, 36H). .sup.13C-NMR (CD.sub.3CN, 125 MHz): δ=159.4, 158.1, 152.6, 148.3, 137.7, 130.0, 124.5, 124.4, 120.6, 119.7, 113.2, 40.4. ESI-HRMS (pos. detection mode): calcd for C90H90N12Ru m/z [M].sup.2+ 720.3222; found: 720.3247. Elemental analysis calcd for C90H90F12N12P2Ru (%): C, 62.46, H, 5.24, N, 9.71; found: C, 62.54, H, 5.17, N, 9.79. [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine).sub.3][Cl].sub.2: The counter ion PF.sub.6 was exchanged to Cl by elution with MeOH from the ion exchange resin Amberlite IRA-410. Elemental analysis calcd for C90H90Cl2N12Ru (%): C, 71.51, H, 6.00, N, 11.12; found: C, 71.19, H, 5.93, N, 10.84.
[Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine).SUB.3.][PF.SUB.6.].SUB.2 .(3)
[0226] (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (286 mg, 0.68 mmol, 4.0 equiv.) and Ru(DMSO).sub.4Cl.sub.2 (82 mg, 0.17 mmol, 1.0 equiv.) were suspended dry EtOH (50 mL) under nitrogen atmosphere. The mixture was refluxed for 15 h. The solution was then cooled down and undissolved solid was removed by filtration. A sat. aqueous solution of NH.sub.4PF.sub.6 was added and the crude product, which precipitated as a PF6 salt was collected by filtration. The solid was washed with H.sub.2O and Et.sub.2O. The residue was purified via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O. The collected product was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and H.sub.2O. After drying, 218 mg of 3 (0.13 mmol, 76%) were yielded as a black solid. .sup.1H-NMR (CD.sub.3CN, 500 MHz): δ=8.79 (d, .sup.4J=1.7 Hz, 6H), 7.79 (d, .sup.3J=16.4 Hz, 6H), 7.71 (d, .sup.3J=6.0 Hz, 6H), 7.64 (d, .sup.3J=8.9 Hz, 12H), 7.44 (d, .sup.3,4J=6.0, 1.7 Hz, 6H), 7.17 (d, .sup.3J=16.4 Hz, 6H), 7.00 (d, .sup.3J=8.9 Hz, 12H), 3.83 (s, 18H). .sup.13C-NMR (CD.sub.3CN, 125 MHz): δ=162.0, 158.2, 152.2, 148.0, 137.0, 130.1, 129.5, 125.0, 122.7, 121.3, 115.5, 56.2. ESI-HRMS (pos. detection mode): calcd for C84H72N6O6Ru m/z [M].sup.2+ 681.2290; found: 681.2273. Elemental analysis calcd for C84H72F12N6O6P2Ru (%): C, 61.05, H, 4.39, N, 5.09; found: C, 61.17, H, 4.44, N, 5.21. [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine).sub.3][Cl].sub.2: The counter ion PF.sub.6 was exchanged to Cl by elution with MeOH from the ion exchange resin Amberlite IRA-410. Elemental analysis calcd for C84H72Cl.sub.2N6O6Ru (%): C, 70.38, H, 5.06, N, 5.86; found: C, 70.62, H, 5.28, N, 5.57.
[Ru(bipy)((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine).SUB.2.][PF.SUB.6.].SUB.2 .(4)
[0227] (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (220 mg, 0.49 mmol, 2.0 equiv.), Ru(DMSO).sub.4Cl.sub.2 (119 mg, 0.25 mmol, 1.0 equiv.) and LiCl (1044 mg, 24.63 mmol, 100 equiv.) were suspended in dry DMF (30 mL) under nitrogen atmosphere. The mixture was refluxed for 4 h. The solution was then cooled down and H.sub.2O was added. The crude product, which precipitated was collected by filtration and washed with H.sub.2O and Et.sub.2O. The formation of [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine).sub.2Cl.sub.2] was analysed via HPLC. [Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine).sub.2C.sub.2] and 2,2′-Bipyridine (47 mg, 0.3 mmol, 1.2 equiv.) were suspended in dry Ethanol (50 mL) under nitrogen atmosphere. The mixture was refluxed for 7 h. The solution was then cooled down and undissolved solid was removed by filtration. A sat. aqueous solution of NH.sub.4PF.sub.6 was added and the crude product, which precipitated as a PF.sub.6 salt was collected by filtration. The solid was washed with H.sub.2O and Et.sub.2O. The residue was purified via preparative HPLC as a TFA salt. The solvents were millipore water (0.1% TFA, solvent A) and acetonitrile (solvent B). The following HPLC gradient has been used: 0-3 minutes: isocratic 50% A (50% B); 3-17 minutes: linear gradient from 50% A (50% B) to 0% A (100% B); 17-23 minutes: isocratic 0% A (100% B). The flow rate was 20 mL/min and the chromatogram was detected at 250 nm, 350 nm, 450 nm. The collected product was dissolved in CH.sub.3CN and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The product, which precipitated as a PF6 salt was collected by filtration and washed with H.sub.2O, Et.sub.2O and Pentane. 89 mg of 4 (0.06 mmol, 24%) were yielded as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.61 (s, 4H), 8.50 (d, J=8.2 Hz, 2H), 8.04 (td, J=8.0, 1.5 Hz, 2H), 7.86 (ddd, J=5.7, 1.4, 0.6 Hz, 2H), 7.66 (dd, J=16.2, 1.9 Hz, 4H), 7.64 (d, J=6.3 Hz, 2H), 7.56-7.50 (m, 10H), 7.43-7.34 (m, 6H), 7.02 (dd, J=16.2 Hz, 4H), 6.81-6.77 (m, 8H), 3.02 (s, 12H), 3.01 (s, 12H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=158.1, 158.0, 152.7, 152.6, 151.9, 151.9, 148.5, 138.3, 137.9, 130.0, 128.4, 125.1, 124.4, 120.7, 119.6, 113.2, 40.4. ESI-HRMS (pos. detection mode): calcd for C70H68N10Ru m/z [M].sup.2+ 575.2330; found: 575.2347. Elemental analysis calcd for C70H68F12N10P2Ru (%): C, 58.37, H, 4.76, N, 9.72; found: C, 58.19, H, 4.62, N, 9.72.
[Ru(bipy)((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine).SUB.2.][PF.SUB.6.].SUB.2 .(5)
[0228] (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (490 mg, 1.17 mmol, 2.0 equiv.), Ru(DMSO).sub.4Cl.sub.2 (282 mg, 0.58 mmol, 1.0 equiv.) and LiCl (2470 mg, 58.26 mmol, 100 equiv.) were suspended in dry DMF (75 mL) under nitrogen atmosphere. The mixture was refluxed for 6 h. The solution was then cooled down and purged into H.sub.2O. The crude product, which precipitated was collected by filtration and washed with H.sub.2O and Et.sub.2O. The formation of [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine).sub.2C.sub.2] was analysed via HPLC. [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine).sub.2Cl.sub.2] and 2,2′-Bipyridine (109 mg, 0.70 mmol, 1.2 equiv.) were suspended in dry Ethanol (100 mL) under nitrogen atmosphere. The mixture was refluxed for 6 h. The solution was then cooled down and undissolved solid was removed by filtration. A sat. aqueous solution of NH.sub.4PF.sub.6 was added and the crude product, which precipitated as a PF.sub.6 salt was collected by centrifugation. The solid was washed with H.sub.2O and Et.sub.2O. The residue was purified via preparative HPLC as a TFA salt. The solvents were millipore water (0.1% TFA, solvent A) and acetonitrile (solvent B). The following HPLC gradient has been used: 0-3 minutes: isocratic 50% A (50% B); 3-17 minutes: linear gradient from 50% A (50% B) to 0% A (100% B); 17-23 minutes: isocratic 0% A (100% B). The flow rate was 20 mL/min and the chromatogram was detected at 250 nm, 350 nm, 450 nm. The collected product was dissolved in CH.sub.3CN and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The product, which precipitated as a PF6 salt was collected by filtration and washed with H.sub.2O, Et.sub.2O and Hexane. 248 mg of 5 (0.18 mmol, 31%) were yielded as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.72 (d, J=1.6 Hz, 4H), 8.51 (d, J=8.2 Hz, 2H), 8.06 (td, J=8.0, 1.3 Hz, 2H), 7.85 (dd, J=5.6, 1.1 Hz, 2H), 7.75-7.68 (m, 6H), 7.67-7.61 (m, 8H), 7.60 (d, J=5.9 Hz, 2H), 7.46-7.39 (m, 6H), 7.17 (dd, J=16.4, 2.1 Hz, 4H), 7.05-6.99 (m, 8H), 3.85 (s, 6H), 3.84 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=162.0, 158.1, 152.6, 152.2, 148.1, 138.6, 137.1, 132.8, 130.1, 129.5, 128.5, 125.2, 125.0, 122.7, 121.3, 115.5, 56.2. ESI-HRMS (pos. detection mode): calcd for C66H56N6O4Ru m/z [M].sup.2+ 549.1698; found: 549.1707. Elemental analysis calcd for C66H56F12N6O4P2Ru+1Hexane (%): C, 57.96, H, 4.44, N, 5.87; found: C, 58.43, H, 3.94, N, 5.79.
[Ru(bipy).SUB.2.((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(6)
[0229] Ru(bipy).sub.2Cl.sub.2 (350 mg, 0.72 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (388 mg, 0.87 mmol, 1.2 equiv.) were suspended in dry Ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 6 h. Then the solution was 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 with H.sub.2O and Et.sub.2O. The product was isolated via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O. 449 mg of 6 (0.39 mmol, 54%) were yielded as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 500 MHz): δ=8.62 (d, J=1.7 Hz, 2H), 8.50 (d, J=8.2 Hz, 4H), 8.07-8.02 (m, 4H), 7.87-7.84 (m, 2H), 7.75-7.72 (m, 2H), 7.67 (d, J=16.3 Hz, 2H), 7.57-7.52 (m, 4H), 7.52-7.49 (d, J=6.0 Hz, 2H), 7.44-7.34 (m, 6H), 7.02 (d, J=16.3 Hz, 2H), 6.81-6.76 (m, 4H), 3.01 (s, 12H). .sup.13C-NMR (CD.sub.3CN, 125 MHz): 5=158.1, 158.0, 158.0, 152.7, 152.7, 152.6, 151.9, 148.8, 138.6, 138.0, 130.0, 128.5, 128.5, 125.2, 124.4, 124.3, 120.8, 119.5, 113.1, 40.4. ESI-HRMS (pos. detection mode): calcd for C50H46N8Ru m/z [M].sup.2+ 430.1439; found: 430.1441. Elemental analysis calcd for C50H46F12N8P2Ru (%): C, 52.22, H, 4.03, N, 9.74; found: C, 51.97, H, 4.04, N, 9.71.
[Ru(bipy).SUB.2.((E,E′)-4,4′-Bis[p-(N,N-methoxy)styryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(7)
[0230] Ru(bipy).sub.2Cl.sub.2 (432 mg, 0.89 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-(N,N-methoxy)styryl]-2,2′-bipyridine (450 mg, 1.07 mmol, 1.2 equiv.) were suspended in dry Ethanol (100 mL) under nitrogen atmosphere and the mixture was refluxed for 6 h. Then the solution was 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 with H.sub.2O and Et.sub.2O. The product was isolated via fractionated precipitation from CH.sub.3CN by adding dropwise Et.sub.2O. 358 mg of 7 (0.32 mmol, 36%) were yielded as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 500 MHz): δ=8.71 (d, J=1.4 Hz, 2H), 8.51 (dd, J=8.2, 0.7 Hz, 4H), 8.06 (td, J=8.0, 1.5 Hz, 4H), 7.86-7.84 (m, 2H), 7.76-7.73 (m, 2H), 7.72 (d, J=16.4 Hz, 2H), 7.66-7.62 (m, 4H), 7.59 (d, J=6.0 Hz, 2H), 7.45-7.38 (m, 6H), 7.16 (d, J=16.4 Hz, 2H), 7.03-6.98 (m, 4H), 3.83 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 125 MHz): δ=162.0, 158.1, 158.0, 152.7, 152.6, 152.2, 148.2, 138.7, 138.7, 137.1, 130.1, 129.5, 128.6, 128.5, 125.2, 125.0, 122.6, 121.4, 115.5, 56.2. ESI-HRMS (pos. detection mode): calcd for C48H40N6O2Ru m/z [M].sup.2+ 417.1123; found: 417.1126. Elemental analysis calcd for C48H40F12N6O2P2Ru (%): C, 51.30, H, 3.59, N, 7.48; found: C, 51.23, H, 3.48, N, 7.61. [Ru(bipy).sub.2((E,E′)-4,4′-Bis[p-(N,N-methoxy)styryl]-2,2′-bipyridine)][Cl].sub.2: The counter ion PF.sub.6 was exchanged to Cl by elution with MeOH from the ion exchange resin Amberlite IRA-410. Elemental analysis calcd for C48H40Cl.sub.2N6O2Ru (%): C, 63.70, H, 4.46, N, 9.29; found: C, 63.51, H, 4.30, N, 9.11.
[Ru(phen).SUB.2.((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(8)
[0231] Ru(phen).sub.2Cl.sub.2 (455 mg, 0.86 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (458 mg, 1.03 mmol, 1.2 equiv.) were suspended in dry Ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 19 h. Then the solution was cooled down, undissolved residue was removed via filtration and washed with Ethanol. To the residual solution 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 with water and Et.sub.2O. The solid was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and water. The solvent was removed under reduced pressure and the product was purified via fractionated precipitation from Acetonitrile by adding dropwise Et.sub.2O. The obtained solid was separated by filtration and was washed with H.sub.2O, Et.sub.2O and Pentane. 427 mg of 1 (0.36 mmol, 41%) were yielded as a red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.68-8.63 (m, 4H), 8.54 (dd, J=8.3, 1.4 Hz, 2H), 8.34-8.31 (m, 2H), 8.29-8.14 (m, 6H), 7.89 (dd, J=5.4, 1.4 Hz, 2H), 7.81 (dd, J=8.3, 5.1 Hz, 2H), 7.64 (d, J=16.4 Hz, 2H), 7.57-7.49 (m, 4H), 7.46 (d, J=6.1 Hz, 2H), 7.24 (dd, J=6.0, 1.7 Hz, 2H), 7.00 (d, J=16.4 Hz, 2H), 6.80-6.74 (m, 4H), 3.00 (s, 12H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=158.3, 153.7, 153.6, 152.7, 152.4, 148.9, 148.7, 137.9, 137.6, 137.5, 132.0, 130.0, 129.0, 127.0, 126.8, 124.3, 124.2, 120.7, 119.5, 113.7, 113.1, 40.4. HR-MS (pos. detection mode): calcd for C.sub.54H.sub.46N.sub.8Ru m/z [M].sup.2+ 454.1439; found: 454.1455. Elemental analysis calcd for C54H.sub.46F.sub.12N.sub.8P.sub.2Ru (%): C, 54.21, H, 3.91, N, 9.24; found: C, 54.14, H, 3.87, N, 9.35.
[Ru(phen).SUB.2.((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(9)
[0232] Ru(phen).sub.2Cl.sub.2 (443 mg, 0.83 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (420 mg, 0.99 mmol, 1.2 equiv.) were suspended in dry Ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 24 h. Then the solution was cooled down and undissolved residue was removed via filtration. To the residual solution 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 with water and Et.sub.2O. The product was isolated by column chromatography on silica gel with an Acetonitrile/aq. KNO.sub.3 (0.4 M) solution (10:1). The fractions containing the product were united and the solvent was removed under reduced pressure. The residue was dissolved in Acetonitrile 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 three-times with H.sub.2O and Et.sub.2O. 672 mg of 2 (0.57 mmol, 69%) were yielded as a red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.70 (s, 2H), 8.65 (d, J=8.2 Hz, 2H), 8.55 (d, J=8.2 Hz, 2H), 8.32-8.22 (m, 6H), 7.89 (d, J=4.7 Hz, 2H), 7.81 (dd, J=8.2, 5.2 Hz, 2H), 7.68 (d, J=16.2 Hz, 2H), 7.63-7-53 (m, 8H), 7.30 (d, J=5.5 Hz, 2H), 7.13 (d, J=16.2 Hz, 2H), 7.01 (d, J=8.0 Hz, 4H), 3.83 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=16 2.0, 158.4, 153.7, 153.6, 15 2.7, 148.9, 148.6, 148.1, 137.7, 137.6, 137.0, 13 2.0, 130.0, 129.4, 129.0, 127.0, 126.8, 124.7, 122.6, 121.2, 115.5, 56.1. HR-MS (pos. detection mode): calcd for C.sub.52H.sub.40N.sub.6O.sub.2Ru m/z [M].sup.2+ 441.1123; found: 441.1131. Elemental analysis calcd for C52H.sub.40F.sub.12N.sub.6O.sub.2P.sub.2Ru (%): C, 53.29, H, 3.44, N, 7.17; found: C, 53.18, H, 3.35, N, 7.26.
[Ru(4,7-Diphenyl-1,10-phenanthroline).SUB.2.((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(10)
[0233] Ru(4,7-Diphenyl-1,10-phenanthroline).sub.2Cl.sub.2 (335 mg, 0.40 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (215 mg, 0.48 mmol, 1.2 equiv.) were suspended in dry Ethanol (100 mL) under nitrogen atmosphere and the mixture was refluxed for 24 h. Then the solution was cooled down and undissolved residue was removed via filtration. The residual solution was diluted with a mixture of Ethanol and water 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 centrifugation and washed with water and Et.sub.2O. The solid was dissolved in Acetonitrile and undissolved residue was removed via filtration. The solvent was removed under reduced pressure and the obtained solid was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and water. The solvent was removed under reduced pressure. The product was isolated via fractionated precipitation from Methanol by adding dropwise Et.sub.2O. After drying, 289 mg of 3 (0.19 mmol, 48%) were yielded as a red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.73 (d, J=1.5 Hz, 2H), 8.42 (d, J=5.5 Hz, 2H), 8.26-8.16 (m, 4H), 8.12 (d, J=5.5 Hz, 2H), 7.78 (d, J=5.5 Hz, 2H), 7.70 (d, J=16.3 Hz, 2H), 7.67-7.57 (m, 24H), 7.54 (d, J=8.9 Hz, 4H), 7.34 (dd, J=6.1, 1.7 Hz, 2H), 7.05 (d, J=16.3 Hz, 2H), 6.78 (d, J=9.0 Hz, 4H), 3.01 (s, 12H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=158.3, 153.1, 152.7, 152.4, 149.9, 149.8, 149.5, 149.4, 148.8, 138.0, 136.8, 136.7, 130.8, 130.8, 130.6, 130.1, 130.1, 130.0, 129.9, 127.2, 127.0, 127.0, 127.0, 124.3, 120.8, 119.5, 113.1, 40.4. HR-MS (pos. detection mode): calcd for C.sub.78H.sub.62N.sub.8Ru m/z [M].sup.2+ 606.2065; found: 606.2078. Elemental analysis calcd for C.sub.78H.sub.62N.sub.8RuP.sub.2F.sub.12+1.5 MeOH (%): C, 61.59, H, 4.42, N, 7.23; found: C, 61.73, H, 4.48, N, 6.88.
[Ru(4,7-Diphenyl-1,10-phenanthroline).SUB.2.((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(11)
[0234] Ru(4,7-Diphenyl-1,10-phenanthroline).sub.2Cl.sub.2 (300 mg, 0.36 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (181 mg, 0.43 mmol, 1.2 equiv.) were suspended in dry Ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 24 h. Then the solution was cooled down and undissolved residue was removed via filtration. The residual solution was diluted with a mixture of Ethanol and water 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 with water and Et.sub.2O. The solid was dissolved in Dichloromethane and washed with a 5% LiCl aqueous solution, brine and water. The solvent was removed under reduced pressure. After drying, 395 mg of 4 (0.27 mmol, 74%) were yielded as a red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.78 (s, 2H), 8.41 (d, J=5.5 Hz, 2H), 8.21 (s, 4H), 8.13 (d, J=5.5 Hz, 2H), 7.78 (d, J=5.5 Hz, 2H), 7.75 (d, J=2.3 Hz, 2H), 7.72 (d, J=8.0 Hz, 2H), 7.66-7.58 (m, 26H), 7.41 (d, J=6.1 Hz, 2H), 7.19 (d, J=16.4 Hz, 2H), 7.02 (d, J=8.7 Hz, 4H), 3.84 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 100 MHz): δ=162.0, 158.4, 153.1, 152.8, 150.0, 149.9, 149.5, 149.3, 148.2, 137.1, 136.8, 136.7, 130.8, 130.8, 130.7, 130.1, 130.1, 130.1, 130.0, 129.9, 129.4, 127.2, 127.0, 124.9, 122.7, 121.3, 115.5, 56.2. HR-MS (pos. detection mode): calcd for C.sub.76H.sub.56N.sub.6O.sub.2Ru m/z [M].sup.2+ 593.1749; found: 593.1768. Elemental analysis calcd for C.sub.76H.sub.56F.sub.12N.sub.6O.sub.2P.sub.2Ru+4*H.sub.2O (%): C, 58.95, H, 4.17, N, 5.43; found: C, 58.58, H, 3.94, N, 5.72. [Ru(1,10-phenanthroline).sub.3][PF.sub.6].sub.2([Ru(phen).sub.3][PF.sub.6]2) (Comparative example) [Ru(phen).sub.3][PF.sub.6].sub.2 was synthesized as previously published (Zuloaga, F. and Kasha, M., 1968) using RuCl.sub.2(DMSO).sub.4 precursor. Purity of the sample was assessed by HPLC and elemental analysis. Elemental analysis calcd for C.sub.36H.sub.24F.sub.12N.sub.6P.sub.2Ru (%): C, 46.41, H, 2.60, N, 9.02; found: C, 46.34, H, 2.54, N, 8.83.
[Ru(bpy).SUB.2.((E,E′)-4,4′-Bis[m-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(12)
[0235] Under nitrogen, Ru(bpy).sub.2Cl.sub.2 (200 mg, 0.41 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[m-methoxystyryl]-2,2′-bipyridine (208 mg, 0.50 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) and the mixture was refluxed for 6 h. Then the solution was 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 with water and diethyl ether. The product was purified by silica gel chromatography using acetonitrile/potassium nitrate 0.3 M in water (9:1 v/v) as eluent. The fractions containing the product were collected and the solvent evaporated. The residue was dissolved in acetonitrile and filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in ethanol and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The precipitate was filtered, washed with water and diethyl ether, and dried under vacuum to yield 12 (390 mg, 0.35 mmol, 85%) as a dark red solid. .sup.1H-NMR (400 MHz, CD.sub.3CN): δ 8.72 (d, J=1.9 Hz, 2H), 8.52 (d, J=8.2 Hz, 4H), 8.07 (td, J=7.9, 1.5, 4H), 7.85 (d, J=5.6 Hz, 2H), 7.78-7.61 (m, 6H), 7.54-7.09 (m, 14H), 6.95 (dd, J=7.3, 1.9, 2H), 3.84 (s, 6H). .sup.13C-NMR (CDCl.sub.3, 101 MHz): δ=161.2, 158.2, 158.0, 152.7, 152.4, 147.6, 138.8, 138.3, 137.2, 131.1, 128.6, 125.4, 125.3, 125.2, 121.7, 121.3, 116.4, 113.2, 56.0. ESI-HRMS (pos. detection mode): calcd for C.sub.48H.sub.40N.sub.6O.sub.2Ru m/z [M].sup.2+ 417.1128; found: 417.1126. Elemental analysis calcd for C.sub.48H.sub.40F.sub.12N.sub.6O.sub.2P.sub.2Ru (%): C, 51.30, H, 3.59, N, 7.48; found: C, 51.06, H, 3.61, N, 7.38.
[Ru(bpy).SUB.2.((E,E′)-4,4′-Bis[o-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(13)
[0236] Under nitrogen, Ru(bpy).sub.2Cl.sub.2 (150 mg, 0.31 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[o-methoxystyryl]-2,2′-bipyridine (156 mg, 0.37 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) and the mixture was refluxed for 6 h. Then the solution was 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 with water and diethyl ether. The product was purified by silica gel chromatography using acetonitrile/potassium nitrate 0.3 M in water (9:1 v/v) as eluent. The fractions containing the product were collected and the solvent evaporated. The residue was dissolved in acetonitrile and filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in ethanol and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The precipitate was filtered, washed with water and diethyl ether, and dried under vacuum to yield 13 (225 mg, 0.20 mmol, 65%) as a dark red solid. 1H-NMR (400 MHz, CD.sub.3CN): δ 8.71 (d, J=1.9 Hz, 2H), 8.59-8.33 (m, 4H), 8.07 (m, 4H), 7.95 (d, J=16.5 Hz, 2H), 7.84 (d, J=4.3 Hz, 2H), 7.74 (ddd, J=5.6, 1.5, 0.8 Hz, 2H), 7.68 (dd, J=7.7, 1.7 Hz, 2H), 7.61 (d, J=6.0 Hz, 2H), 7.42 (m, 10H), 7.06 (m, 4H), 3.94 (s, 6H). .sup.13C-NMR (CDCl.sub.3, 101 MHz): δ=158.9, 158.2, 158.0, 152.6, 152.2, 148.3, 138.7, 132.7, 132.0, 129.0, 128.5, 125.8, 125.4, 125.2, 125.0, 121.9, 121.8, 112.6, 56.3. ESI-HRMS (pos. detection mode): calcd for C.sub.48H.sub.40N.sub.6O.sub.2Ru m/z [M].sup.2+ 417.1128; found: 417.1126. Elemental analysis calcd for C.sub.48H.sub.40F.sub.12N.sub.6O.sub.2P.sub.2Ru+H.sub.2O (%): C, 50.49, H, 3.71, N, 7.36; found: C, 50.27, H, 3.41, N, 7.66.
[Ru(bpy).SUB.2.((E,E′)-4,4′-Bis[p-fluorostyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(14)
[0237] Under nitrogen, Ru(bpy).sub.2Cl.sub.2 (242 mg, 0.50 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-fluorostyryl]-2,2′-bipyridine (238 mg, 0.60 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) and the mixture was refluxed for 6 h. Then the solution was 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 with water and diethyl ether. The product was purified by silica gel chromatography using acetonitrile/potassium nitrate 0.3 M in water (9:1 v/v) as eluent. The fractions containing the product were collected and the solvent evaporated. The residue was dissolved in acetonitrile and filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in ethanol and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The precipitate was filtered, washed with water and diethyl ether, and dried under vacuum to yield 14 (273 mg, 0.29 mmol, 57%) as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.72 (d, J=1.9 Hz, 2H), 8.53 (d, J=8.2 Hz, 4H), 8.07 (t, J=7.9 Hz, 4H), 7.85 (d, J=5.6 Hz, 2H), 7.80-7.68 (m, 8H), 7.65 (d, J=6.0 Hz, 2H), 7.52-7.36 (m, 6H), 7.26 (d, J=16.5 Hz, 2H), 7.19 (d, J=8.8 Hz, 4H). .sup.13C-NMR (CD.sub.3CN, 101 MHz): δ=164.3 (d, .sup.1J.sub.C-F=248 Hz), 158.2, 158.0, 158.0, 152.7, 152.4, 147.6, 138.8, 136.0, 133.4 (d, .sup.4J.sub.C-F=3 Hz), 130.5 (d, .sup.3J.sub.C-F=8 Hz), 128.6, 125.2, 125.0, 121.8, 116.9 (d, .sup.2J.sub.C-F=22 Hz). .sup.19F NMR (376 MHz, CD.sub.3CN) δ=−72.77 (d, J=706.7 Hz), −112.83. ESI-HRMS (pos. detection mode): calcd for C.sub.46H.sub.34N.sub.6F.sub.2Ru m/z [M].sup.2+ 405.0928; found: 405.0927. Elemental analysis calcd for C.sub.46H.sub.34F.sub.14N.sub.6O.sub.2P.sub.2Ru+H.sub.2O (%): C, 49.43, H, 3.25, N, 7.52; found: C, 49.76, H, 3.02, N, 7.47.
[Ru(bpy).SUB.2.((E,E′)-4,4′-Bis[p-hydroxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(15)
[0238] Under nitrogen, Ru(bpy).sub.2Cl.sub.2 (150 mg, 0.31 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-hydroxystyryl]-2,2′-bipyridine (145 mg, 0.37 mmol, 1.2 equiv.) were suspended in ethylene glycol (5 mL) and the mixture was heated at 130° C. for 24 h. Then the solution was 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 with water and diethyl ether and dried under vacuum to yield 15 (297 mg, 0.27 mmol, 87%) as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.66 (d, J=1.9 Hz, 2H), 8.50 (d, J=8.1 Hz, 4H), 8.05 (td, J=8.0, 1.5 Hz, 4H), 7.84 (d, J=5.1 Hz, 2H), 7.74 (d, J=5.0 Hz, 2H), 7.68 (d, J=16.4 Hz, 2H), 7.61-7.51 (m, 6H), 7.46-7.35 (m, 8H), 7.12 (d, J=16.5 Hz, 2H), 6.90 (d, J=8.6 Hz, 4H). .sup.13C-NMR (CD.sub.3CN, 101 MHz): δ=159.5, 158.01, 157.95, 152.6, 152.1, 148.2, 138.6, 137.3, 130.2, 128.7, 128.5, 125.1, 124.9, 122.1, 121.2, 116.9. ESI-HRMS (pos. detection mode): calcd for C.sub.46H.sub.36N.sub.6O.sub.2Ru m/z [M].sup.2+ 403.0972; found: 403.0970. Elemental analysis calcd for C.sub.46H.sub.36F.sub.12N.sub.6O.sub.2P.sub.2Ru+H.sub.2O (%): C, 49.60, H, 3.44, N, 7.55; found: C, 49.46, H, 3.35, N, 7.31.
[Os(bpy).SUB.2.((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(16)
[0239] Under nitrogen, Os(bpy).sub.2Cl.sub.2 (100 mg, 0.17 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (84 mg, 0.20 mmol, 1.2 equiv.) were suspended in ethylene glycol (5 mL). The mixture was degassed by nitrogen bubbling for 15 min and heated at 130° C. for 24 h. Then the solution was 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 with water and diethyl ether and dried under vacuum to yield 20 (189 mg, 0.16 mmol, 94%) as a dark brown solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.64 (d, J=2.0 Hz, 2H), 8.49 (d, J=8.0 Hz, 4H), 7.86 (ddt, J=9.7, 8.2, 1.7 Hz, 4H), 7.74 (d, J=5.4 Hz, 2H), 7.71-7.59 (m, 8H), 7.50 (d, J=6.1 Hz, 2H), 7.38-7.26 (m, 6H), 7.16 (d, J=16.3 Hz, 2H), 7.02 (d, J=8.8 Hz, 4H), 3.84 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 101 MHz): δ=162.0, 159.97, 159.92, 159.87, 151.8, 151.7, 151.3, 147.5, 138.0, 137.2, 130.1, 129.3, 129.0, 125.4, 125.2, 122.2, 121.4, 115.4. ESI-HRMS (pos. detection mode): calcd for C.sub.48H.sub.40N.sub.6O.sub.2Os m/z [M].sup.2+ 462.1414; found: 462.1408. Elemental analysis calcd for C.sub.48H.sub.40F.sub.12N.sub.6O.sub.2OsP.sub.2+H.sub.2O (%): C, 46.83, H, 3.44, N, 6.83; found: C, 46.92, H, 3.29, N, 6.34.
[Ru(bpy).SUB.2.((E,E′)-4,4′-Bis[p-aminostyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(17)
[0240] Compound 17 was synthesized following a procedure described in the literature (Storrier, G. D. and Colbran, S. B., 1997). Elemental analysis calcd for C.sub.46H.sub.38F.sub.12N.sub.8P.sub.2Ru+3H.sub.2O (%): C, 48.13, H, 3.86, N, 9.76; found: C, 48.09, H, 3.37, N, 10.05.
[Ru(bpy).SUB.2.(4,4′-(E,E′)-2-(4-methoxyphenyl)prop-1-en-1-yl)-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(18)
[0241] Under nitrogen, Ru(bpy).sub.2Cl.sub.2 (63 mg, 0.13 mmol, 1.0 equiv.) and 4,4′-bis((E)-2-(4-methoxyphenyl)prop-1-en-1-yl)-2,2′-bipyridine (65 mg, 0.15 mmol, 1.1 equiv.) were suspended in dry ethanol and the mixture was refluxed for 15 h. Then the solution was 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 with water and diethyl ether. The product was purified by silica gel chromatography using acetonitrile/potassium nitrate 0.3 M in water (9:1 v/v) as eluent. The fractions containing the product were collected and the solvent evaporated. The residue was dissolved in acetonitrile and filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in ethanol and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The precipitate was filtered, washed with water and diethyl ether, and dried under vacuum to yield 18 (124 mg, 0.11 mmol, 85%) as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=8.55-8.41 (m, 6H), 8.07 (tdd, J=7.8, 4.7, 1.5 Hz, 4H), 7.91-7.82 (m, 2H), 7.79-7.73 (m, 2H), 7.66-7.53 (m, 6H), 7.50-7.35 (m, 6H), 7.06-6.95 (m, 4H), 6.95-6.87 (m, 2H), 3.94-3.74 (m, 6H), 2.41-2.24 (m, 6H). .sup.13C NMR (101 MHz, CD.sub.3CN) δ=161.10, 160.60, 157.94, 157.69, 152.66, 152.60, 151.83, 148.61, 145.49, 138.61, 135.58, 129.86, 128.45, 127.64, 125.14, 124.79, 122.58, 114.86, 56.00, 18.11. ESI-HRMS (pos. detection mode): calcd for C.sub.50H.sub.44O.sub.2N.sub.6Ru m/z [M].sup.2+ 431.1285; found: 431.1282. Elemental analysis calcd for C.sub.50H.sub.44F.sub.12N.sub.6O.sub.2P.sub.2Ru+2H.sub.2O (%): C, 50.55, H, 4.07, N, 7.07; found: C, 50.73, H, 3.79, N, 7.21.
[Ru(bpz).SUB.2.((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)][PF.SUB.6.].SUB.2 .(19)
[0242] Under nitrogen, Ru(bpz).sub.2Cl.sub.2 (100 mg, 0.21 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (104 mg, 0.25 mmol, 1.2 equiv.) were suspended in dry ethylene glycol (5 mL) and the mixture was heated at 130° C. for 24 h. Then the solution was 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 with water and diethyl ether. The product was purified by silica gel chromatography using acetonitrile/potassium nitrate 0.3 M in water (9:1 v/v) as eluent. The fractions containing the product were collected and the solvent evaporated. The residue was dissolved in acetonitrile and filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in ethanol and a sat. aqueous solution of NH.sub.4PF.sub.6 was added. The precipitate was filtered, washed with water and diethyl ether, and dried under vacuum to yield 19 (43 mg, 0.04 mmol, 19%) as a dark red solid. .sup.1H-NMR (CD.sub.3CN, 400 MHz): δ=9.76 (t, J=1.5 Hz, 4H), 8.71 (d, J=1.9 Hz, 2H), 8.61 (dd, J=9.8, 3.2 Hz, 4H), 7.90 (dd, J=3.3, 1.2 Hz, 2H), 7.87 (dd, J=3.2, 1.2 Hz, 2H), 7.76 (d, J=16.4 Hz, 2H), 7.66 (d, J=8.9 Hz, 4H), 7.54 (d, J=6.0 Hz, 2H), 7.48 (dd, J=6.1, 1.8 Hz, 2H), 7.19 (d, J=16.3 Hz, 2H), 7.03 (d, J=8.8 Hz, 4H), 3.85 (s, 6H). .sup.13C-NMR (CD.sub.3CN, 101 MHz): δ=162.2, 157.2, 152.6, 151.7, 149.7, 149.34, 149.30, 147.9, 147.3, 146.1, 138.0, 130.2, 129.2, 125.3, 122.2, 121.6, 115.5, 56.1. ESI-HRMS (pos. detection mode): calcd for C.sub.44H.sub.36N.sub.10O.sub.2Ru m/z [M].sup.2+ 419.1033; found: 419.1030. Elemental analysis calcd for C.sub.44H.sub.36F.sub.12N.sub.10O.sub.2P.sub.2Ru+4H.sub.2O (%): C, 44.04, H, 3.70, N, 11.67; found: C, 44.11, H, 2.95, N, 11.60.
[Ru(4,7-Diphenyl-1,10-phenanthroline).SUB.3.][PF.SUB.6.].SUB.2.([Ru(bphen).SUB.3.][PF.SUB.6.]2) (Comparative Example)
[0243] [Ru(bphen).sub.3][PF.sub.6].sub.2 was synthesized as previously published (Crosby, G. A. and Watts, R. J., 1971) using RuCl.sub.2dmso.sub.4 precursor. Purity of the sample was assessed by HPLC and elemental analysis. Elemental analysis calcd for C.sub.72H.sub.48F.sub.12N.sub.6P.sub.2Ru (%): C, 62.30, H, 3.49, N, 6.05; found: C, 62.28, H, 3.44, N, 5.92.
[0244] 2. Photophysical Properties
[0245] Photophysical measurements were performed to evaluate the potential of the complexes as photosensitizers.
[0246] Spectroscopic Measurements
[0247] The absorption of the samples was measured 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. As a detector, a XPI-Max 4 CCD camera (Princeton Instruments) has been used.
[0248] Luminescence Quantum Yield Measurements
[0249] For the determination of the luminescence quantum yield, the samples were prepared in an CH.sub.3CN solution with an absorbance of 0.1 at 355 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla) at 355 nm. The emission signal was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. As a detector a XPI-Max 4 CCD camera (Princeton Instruments) has been used. The luminescence quantum yields were determined by comparison with the reference [Ru(bipy).sub.3]Cl.sub.2 in CH.sub.3CN (Φ.sub.em=5.9%) (Nakamaru, K., 1982) applying the following formula:
Φ.sub.em, sample=Φ.sub.em, reference*(F.sub.reference/F.sub.sample)*(I.sub.sample/I.sub.reference)*(n.sub.sample/n.sub.reference).sup.2
F=1-10.SUP.−A
[0250] Φ.sub.em=luminescence quantum yield, F=fraction of light absorbed, I=integrated emission intensities, n=refractive index, A=absorbance of the sample at irradiation wavelength.
[0251] Lifetime Measurements
[0252] For the determination of the lifetimes, the samples were prepared in an air saturated and in a degassed CH.sub.3CN solution with an absorbance of 0.2 at 355 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla) at 355 nm. The emission signal was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. As a detector a R928 photomultiplier tube (Hamamatsu) has been used.
TABLE-US-00001 TABLE 1 Spectroscopic properties of characterised compounds 1-19 in CH.sub.3CN at room temperature. τ/ns λ.sub.abs/nm (ε/M.sup.−1 cm.sup.−1 * 10.sup.−3) λ.sub.em/nm ϕ.sub.em air degassed 1 300 (81.6), 385 (111.6), 515 677 0.019 86 385 (47.8) 2 305 (109.5), 425 (133.4), 495 709 <0.001 48 222 (111.8) 3 305 (96.6), 370 (152.3), 495 682 0.011 76 338 (63.9) 4 290 (57.0), 425 (93.5), 485 703 0.004 69 417 (82.6) 5 295 (76.9), 360 (99.8), 475 674 0.014 36 231 (39.5) 6 290 (79.0), 415 (57.3), 460 697 0.005 54 405 (61.4) 7 290 (95.7), 365 (64.8), 465 664 0.028 96 542 (34.4) 8 265 (99.6), 305 (30.7), 420 698 0.004 136 334 (57.0), 460 (57.9) 9 265 (101.4), 290 (50.2), 360 663 0.019 193 669 (61.9), 465 (36.6) 10 280 (125.9), 305 (56.6), 425 698 0.006 72 339 (64.2), 475 (70.4) 11 280 (147.7), 305 (81.4), 360 663 0.027 108 679 (69.8), 475 (55.1) 12 290 (71.5), 330 (37.8), 400 — — — — (13.3), 460 (20.5), 480 (18.2), 550 (1.5), 600 (0.2) 13 290 (85.9), 330 (34.4), 360 — — — — (43.5), 410 (18.4), 460 (27.2), 480 (23.9), 550 (1.9), 600 (0.1) 14 290 (87.3), 330 (33.1), 410 — — — — (16.7), 460 (26.1), 480 (23.2), 550 (1.9), 600 (0.2) 15 290 (66.2), 360 (44.8), 420 — — — — (17.9), 460 (24.5), 480 (21.9), 550 (1.9), 600 (0.2) 16 290 (60.1), 320 (31.2), 350 — — — — (38.6), 420 (18.3), 450 (21.5), 480 (19.0), 550 (7.4), 600 (5.1), 690 (3.5), 720 (1.6) 17 290 (48.9), 330 (13.5), 390 — — — — (25.8), 440 (21.9), 470 (22.2), 480 (21.2), 550 (2.5), 600 (0.3) 18 290 (64.6), 320 (24.6), 340 — — — — (28.4), 410 (9.6), 460 (18.4), 480 (14.2), 550 (1.0), 600 (0.06) 19 300 (87.2), 330 (59.4), 350 — — — — (69.6), 410 (37.1), 430 (41.2), 480 (19.2), 550 (2.1), 600 (0.5) λ.sub.abs = Absorption maximum, λ.sub.em = Emission maximum, ϕ.sub.em = Luminenscene Quantum Yield, τ = Lifetime.
[0253] Results
[0254] The photophysical properties of the prepared complexes were systematically investigated to evaluate their potential as PSs (Table 1). One crucial parameter in a PDT treatment is the penetration depth of the light and therefore the used wavelength. Based on this, the one-photon absorption spectra of the compounds were determined in CH.sub.3CN (
[0255] Afterwards, the emission in CH.sub.3CN has been determined for compounds 1-11 upon excitation at 355 nm. The emission of the compounds was measurable between 550-900 nm with a maximum between 664-709 nm. Worthy of note, the complexes which show a high red shift of the MLCT transition, have accordingly also the emission maximum at higher wavelengths. This large Stokes shift for all investigated compounds implies minimal inference between excitation and emission. Following this, the luminescence quantum yields were measured upon excitation at 355 nm. The comparison between the values shows luminescence quantum yield between 0.028-<0.001.
[0256] Consequently, the luminescence lifetimes were determined in degassed and air saturated CH.sub.3CN upon excitation at 355 nm to investigate the influence of oxygen on the excited state. The measured lifetimes of the compounds 1-11 were found to be in the nanosecond range in a degassed solution between 222-542 ns and in an aerated solution between 36-96 ns. Importantly, for all Ru(II) polypyridyl complexes a decrease of the lifetime could be observed in an aerated solution in comparison to a degassed solution. This indicates that the excited state of the complex (.sup.3MLCT) is able to interact with a component in the air.
[0257] 3. Singlet Oxygen Generation
[0258] The ability to generate reactive oxygen species upon light exposure was investigated
[0259] for compounds 1-11.
[0260] Electron Spin Resonance (ESR) Measurements
[0261] For verification of the reactive species formed upon light exposure of the compounds, the respective ESR spectra were recorded. The samples with a final concentration of 10 μM were dissolved in CH.sub.3CN or PBS containing 20 mM TEMP (2,2,6,6-tetramethylpiperidine) as a .sup.1O.sub.2 scavenger or 20 mM DMPO (5,5-dimethyl-1-pyrroline N-oxide) as a *OH radical scavenger. Capillary tubes were filled with the solution and sintered by fire. EPR spectra were recorded on a Bruker A300 spectrometer with 1 G field modulation, 100 G scan range and 20 mW microwave power. The samples were measured in exclusion from light and after irradiation for 60 s (450±10 nm, 21.8 mW cm.sup.−2).
[0262] Singlet Oxygen Measurements
[0263] Direct Evaluation
[0264] 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. A longpass glass filter was placed in front of the monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. 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 Rose Bengal (Φ=76%) (Kochevar, I. E. and Redmond, R. W., 2000) was used and the singlet oxygen quantum yields were calculated using the following formula:
Φ.sub.sample=Φ.sub.reference*(S.sub.sample/S.sub.reference)*(I.sub.reference/I.sub.sample)
[0265] I=I.sub.0*(1-10.sup.−A)
[0266] Φ=singlet oxygen quantum yield, S=slope of the linear regression of the plot of the areas of the singlet oxygen luminescence peaks against the irradiation intensity, I=absorbance correction factor, 10=light intensity of the irradiation source, A=absorbance of the sample at irradiation wavelength.
[0267] Indirect Evaluation
[0268] 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 0.2 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 (A.sub.0−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.
TABLE-US-00002 TABLE 2 Singlet oxygen quantum yields in CH.sub.3CN and aqueous solution. Average of three independent measurements. n.d. = not detectable. direct direct indirect indirect indirect indirect 450 nm 450 nm 450 nm 450 nm 540 nm 540 nm CH.sub.3CN D.sub.2O CH.sub.3CN PBS CH.sub.3CN PBS 1 65% n.d. 66% 5% 61% 3% 2 18% n.d. 25% 3% 16% 1% 3 52% n.d. 48% 6% 49% 5% 4 34% n.d. 40% 2% 37% 3% 5 54% n.d. 51% 8% 46% 6% 6 46% n.d. 53% 2% 38% 2% 7 75% n.d. 77% 11% 68% 10% 8 59% n.d. 62% 3% 57% 2% 9 75% n.d. 82% 9% 79% 8% 10 51% n.d. 48% 2% 43% 2% 11 66% n.d. 73% 7% 72% 5% [Ru(phen).sub.3].sup.2+ 25% n.d. 23% 2% 22% 2% (PF.sub.6.sup.−).sub.2 (*) [Ru(bphen).sub.3].sup.2+ 44% n.d. 47% 4% 45% 3% (PF.sub.6.sup.−).sub.2 (*) (*) comparative examples
[0269] For identification of the type of ROS produced upon light exposure, electron spin resonance (ESR) spectroscopy in CH.sub.3CN as well as in phosphate-buffered saline (PBS) was employed. As a singlet oxygen (.sup.1O.sub.2) scavenger 2,2,6,6-tetramethylpiperidine (TEMP) and as a .OOH or .OH radical scavenger 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used. While no signal for the formation of a .OOH or .OH radicals were detected, the formation of .sup.1O.sub.2 in CH.sub.3CN and PBS was confirmed by observation of the characteristic .sup.1O.sub.2-induced triplet signal in the ESR spectrum. Following this, the amount of generated .sup.1O.sub.2 was quantitatively determined by two methods, namely 1) direct measurement of the phosphorescence of .sup.1O.sub.2 and 2) indirect measurement of the variation in absorbance of a 102 scavenger and monitoring its change in absorbance. The singlet oxygen quantum yields (Table 2) were found to be between 16-82% in CH.sub.3CN and 1-11% in an aqueous solution. Therefore, the compounds of the invention 1-11 are able to produce 102 in an efficient way. Overall, compound 7 was found to have an impressive singlet oxygen production (i.e., CH.sub.3CN: 68-77%, aqueous solution: 10-11%).
[0270] 4. Stability
[0271] As a crucial parameter in view medicinal applications, the stability of the compounds in a biological environment was investigated as previous investigations have shown that this could be problematic for metal complexes.
[0272] Stability in Human Plasma
[0273] The stability of the complexes 1-11 was evaluated with caffeine as an internal standard, which has already been shown to be suitable for these experiments (Guy, P. A. et al., 2009). The pooled human plasma was obtained from Biowest and caffeine from TCI Chemicals. Stock Solutions of the compounds (20 μM) and caffeine (40 μM) were prepared in DMSO. One aliquot of the solutions was added to 975 μL of human plasma to a total volume of 1000 μL. Final concentrations of the compounds of 0.25 μM and caffeine of 0.5 μM were achieved. The resulting solution was incubated for 48 h at 37° C. with continuous gentle shaking (ca. 300 rpm). The reaction was stopped after the incubation time by addition of 3 mL of methanol. The mixture was centrifuged for 60 min at 3000 rpm at 4° C. The methanolic solution was filtered through a 0.2 μm membrane filter. The solvent was evaporated under reduced pressure and the residue was dissolved in 1:1 (v/v) CH.sub.3CN/H.sub.2O 0.1% TFA solution. The solution was filtered through a 0.2 μm membrane filter and analyzed using a HPLC System. The solvents (HPLC grade) were millipore water (0.1% TFA, solvent A) and acetonitrile (solvent B). Method M1: 0-3 minutes: isocratic 50% A (50% B); 3-17 minutes: linear gradient from 50% A (50% B) to 0% A (100% B); Method M2: 0-3 minutes: isocratic 95% A (5% B); 3-17 minutes: linear gradient from 95% A (5% B) to 0% A (100% B); 17-23 minutes: isocratic 0% A (100% B). The flow rate was 1 mL/min and the chromatogram was detected at 250 nm.
[0274] Photostability
[0275] The samples were prepared in an air saturated CH.sub.3CN solution. To measure the photostability, the samples were irradiated at 450 nm in 96 well plates with an Atlas Photonics LUMOS BIO irradiator during time intervals from 0-10 min. The absorbance spectrum from 350-700 nm was recorded with a SpectraMax M2 Microplate Reader (Molecular Devices) after each time interval and compared. As a positive control [Ru(bipy).sub.3]Cl.sub.2 and as a negative control Protoporphyrin IX has been used.
[0276] Results
[0277] To assess the compatibility of the here reported compounds under biological conditions, their stability in human pooled plasma was tested. For this purpose, the compounds were incubated in human plasma at 37° C. for 48 h and after this time extracted from the plasma. The compounds were analysed before and after giving them into the human plasma via HPLC. As an internal standard, caffeine has been used. The comparison of the HPLC chromatograms shows no change before and after the incubation for the compounds 1-11 which indicates the stability of the compounds under biological conditions. Next to the stability in human plasma, the stability upon irradiation was investigated. Importantly, currently approved photosensitizers are associated with a low stability upon irradiation. For this purpose, the compounds were exposed to irradiation at 450 nm and their UV/Vis absorption monitored in constant intervals. [Ru(bipy).sub.3]Cl.sub.2 has been used as a positive control and Protoporphyrin IX has been used as a negative control. The results show that the photobleaching effect is correlating with the coordinated ligand. The complexes 1-11 show little to no photobleaching in comparison to the photosensitizer Protoporphyrin IX, which completely changed in the investigated time interval.
[0278] 5. Dark and (Photo)-Cytotoxicity in Monolayer Cells
[0279] The compounds 1-19 were tested in various cell lines to determine their ability to act as a photosensitizer.
[0280] Cell Culture
[0281] The human glioblastoma astrocytoma (U373) cell line was cultured in MEM medium supplemented with 10% FBS, 1% NEAA (non-essential amino-acids) and 1% penicillin/streptomycin. The human cervical carcinoma (HeLa), doxorubicin-resistant human colon adenocarcinoma (SW620/AD300) and the mouse colon carcinoma (CT-26) cell lines were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. The human retinal epithelial cells (RPE-1, non-cancerous cells immortalized with hTERT) were cultured in DMEM-F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were obtained from the American Type Culture Collection (ATCC) and cultured at 37° C. and 5% CO.sub.2. Before an experiment, the cells were passaged three times.
[0282] (Photo-)Cytotoxicity on 2D Cell Monolayers
[0283] The cytotoxicity of the compounds was assessed by measuring cell viability using a fluorometric resazurin assay. The cultivated cells were seeded in triplicates in 96 well plates with a density of 4000 cells per well in 100 μL of media. After 24 h, the medium was removed and the cells were treated with increasing concentrations of the compound diluted in cell media achieving a total volume of 200 μL. The cells were incubated with the compound for 4 h. After this time, the media was removed and replaced with 200 μL of fresh medium. For the phototoxicity studies, the cells were exposed to light with an Atlas Photonics LUMOS BIO irradiator. Each well was constantly illuminated with either a 480 nm or 540 nm irradiation. During this time, the temperature was maintained constantly at 37° C. The cells were grown in the incubator for additional 44 h. For the determination of the dark cytotoxicity, the cells were not irradiated and after the medium exchange directly incubated for 44 h. After this time, the medium was replaced with fresh medium containing resazurin with a final concentration of 0.2 mg/mL. After 4 h incubation, the amount of the fluorescent product resorufin was determined upon excitation at 540 nm and measurement its emission at 590 nm using a SpectraMax M2 Microplate Reader (Molecular Devices).
[0284] The obtained data was analyzed with the GraphPad Prism software.
TABLE-US-00003 TABLE 3 IC.sub.50 values in μM in the dark and upon irradiation at 480 nm for compounds 1-18 in comparison to [Ru(phen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, [Ru(bphen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, cisplatin and Protoporphyrin IX (PpIX) in non-cancerous retinal pigment epithelium (RPE-1) and nm for compounds 1-19 in comparison to [Ru(phen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, [Ru(bphen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, cisplatin and Protoporphyrin IX (PpIX) in human cervical carcinoma (HeLa) cells. Compounds 1-11 were also tested at 540 nm. Average of three independent measurements. RPE-1 HeLa 480 nm 540 nm 480 nm 540 nm (10 min, (40 min, (10 min, (40 min, dark 3.1 J/cm.sup.2) PI 9.5 J/cm.sup.2) PI dark 3.1 J/cm.sup.2) PI 9.5 J/cm.sup.2) PI 1.sup.a) >100 30.9 ± 0.8 >3.2 31.7 ± 1.2 >3.2 >100 29.5 ± 1.2 >3.4 48.7 ± 4.5 >2.1 2.sup.a) >100 38.4 ± 8.1 >2.6 49.0 ± 6.2 >2.0 >100 29.4 ± 8.3 >3.4 52.3 ± 6.1 >1.9 3.sup.a) >100 53.6 ± 3.2 >1.9 44.9 ± 2.9 >2.2 >100 15.3 ± 1.4 >6.5 11.3 ± 2.2 >8.8 4 >100 10.9 ± 2.7 >9.2 14.7 ± 3.1 >6.8 >100 8.5 ± 1.2 >11.8 10.6 ± 2.2 >9.4 5 >100 10.2 ± 2.1 >9.8 12.7 ± 2.9 >7.9 >100 6.4 ± 1.7 >16.7 7.9 ± 1.3 >12.7 6 >100 7.3 ± 1.2 >13.7 8.1 ± 1.6 >12.3 >100 5.3 ± 0.6 >18.9 5.8 ± 0.9 >17.2 7 >100 3.1 ± 0.8 >32.3 8.4 ± 1.3 >11.9 >100 1.2 ± 0.4 >83.3 1.5 ± 0.5 >66.7 8 >100 9.2 ± 1.6 >10.9 15.6 ± 2.3 >6.4 >100 12.6 ± 1.3 >7.9 11.4 ± 1.5 >8.8 9 >100 2.4 ± 0.9 >41.7 2.1 ± 0.7 >47.6 >100 1.5 ± 0.6 >66.7 1.2 ± 0.7 >83.3 10 15.6 ± 1.3 1.8 ± 0.3 8.7 2.1 ± 0.4 7.4 10.7 ± 1.4 1.6 ± 0.3 6.7 1.7 ± 0.4 6.3 11 20.8 ± 1.5 0.9 ± 0.4 23.1 0.7 ± 0.3 29.7 16.5 ± 0.8 0.6 ± 0.3 27.5 0.7 ± 0.5 23.6 12 >100 1.6 ± 0.2 >63 — — >100 0.6 ± 0.3 >167 — — 13 >100 1.52 ± 0.09 >66 — — 62 ± 10 0.5 ± 0.1 >124 — — 14 >100 1.47 ± 0.06 >68 — — >100 0.8 ± 0.1 >125 — — 15 >100 11 ± 2 >9 — — >100 1.0 ± 0.3 >100 — — 16 >100 30 ± 7 >3 — — >100 27 ± 6 >4 — — 17 >100 8 ± 1 >13 — — >100 1.4 ± 0.1 >71 — — 18 >100 33 ± 4 >3 — — >100 16.7 ± 0.7 >6 — — 19 — — — — — >100 59 ± 8 >2 — — [Ru(phen).sub.3].sup.2+ >100 >100 n.d. >100 n.d. >100 >100 n.d. >100 n.d. (PF.sub.6.sup.−).sub.2 [Ru(bphen).sub.3].sup.2+ 12.1 ± 0.6 0.9 ± 0.2 13.4 1.2 ± 0.3 10.1 8.5 ± 0.4 0.7 ± 0.2 12.1 1.1 ± 0.2 7.7 (PF.sub.6.sup.−).sub.2 PpIX >100 3.8 ± 0.1 >26.3 3.3 ± 0.1 >30.3 >100 2.5 ± 0.1 >40.0 2.1 ± 0.3 >47.6 cisplatin 29.3 ± 1.4 — — — — 10.5 ± 0.8 — — — — .sup.a)due to solubility limitations the compounds were investigated as chloride salts.
TABLE-US-00004 TABLE 4 IC.sub.50 values in μM in the dark and upon irradiation at 480 and 540 nm for compounds 1-11 in comparison to [Ru(phen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, [Ru(bphen).sub.3].sup.2+ (PF.sub.6.sup.−).sub.2, cisplatin and Protoporphyrin IX (PpIX) in mouse colon carcinoma (CT-26) and human glioblastoma astrocytoma (U373) cells. Average of three independent measurements. CT-26 U373 480 nm 540 nm 480 nm 540 nm (10 min, (40 min, (10 min, (40 min, dark 3.1 J/cm.sup.2) PI 9.5 J/cm.sup.2) PI dark 3.1 J/cm.sup.2) PI 9.5 J/cm.sup.2) PI 1.sup.a) >100 20.3 ± 1.8 >4.9 33.4 ± 3.5 >3.0 >100 51.7 ± 3.2 >1.9 83.1 ± 6.7 >1.2 2.sup.a) >100 42.6 ± 3.8 >2.3 62.5 ± 8.2 >1.6 >100 49.1 ± 6.0 >2.0 61.4 ± 5.6 >1.6 3.sup.a) >100 19.3 ± 2.1 >5.2 23.0 ± 2.6 >4.3 >100 41.0 ± 3.5 >2.4 51.3 ± 3.4 >1.9 4 >100 7.3 ± 1.4 >13.7 9.6 ± 1.9 >10.4 >100 14.3 ± 2.4 >7.0 19.0 ± 2.8 >5.3 5 >100 5.1 ± 1.1 >19.6 6.2 ± 1.0 >16.1 >100 15.3 ± 2.2 >6.5 17.8 ± 3.2 >5.6 6 >100 2.4 ± 0.5 >41.7 3.1 ± 0.3 >32.3 >100 8.3 ± 1.1 >12.0 10.7 ± 1.9 >9.3 7 >100 0.7 ± 0.4 >142.9 0.9 ± 0.3 >111.1 >100 10.5 ± 1.7 >9.5 13.5 ± 1.6 >7.4 8 >100 8.2 ± 1.1 >12.2 7.8 ± 0.9 >12.8 >100 11.7 ± 1.5 >8.5 13.3 ± 2.4 >7.5 9 >100 1.1 ± 0.5 >90.9 0.9 ± 0.4 >111.1 >100 2.5 ± 0.9 >40.0 2.1 ± 0.7 >47.6 10 5.2 ± 0.8 1.3 ± 0.3 4.0 1.1 ± 0.4 4.7 13.7 ± 1.1 1.8 ± 0.3 7.6 2.1 ± 0.4 6.5 11 8.6 ± 0.9 0.5 ± 0.2 17.2 0.6 ± 0.2 14.3 18.5 ± 1.0 0.9 ± 0.2 20.6 0.7 ± 0.3 26.4 [Ru(phen).sub.3].sup.2+ >100 >100 n.d. >100 n.d. >100 >100 n.d. >100 n.d. [Ru(bphen).sub.3].sup.2+ 2.8 ± 0.5 0.4 ± 0.2 7.0 0.5 ± 0.2 5.6 10.1 ± 0.7 1.3 ± 0.2 7.8 1.2 ± 0.3 8.4 PpIX 2.8 ± 0.5 0.4 ± 0.2 7.0 0.5 ± 0.2 5.6 10.1 ± 0.7 1.3 ± 0.2 7.8 1.2 ± 0.3 8.4 cisplatin 6.5 ± 1.1 — — — — 17.6 ± 1.7 — — — — .sup.b) due to solubility limitations the compounds were investigated as chloride salts.
[0285] Results
[0286] To investigate the toxicity of the compounds, the IC.sub.50 values in the dark as well as upon light exposure at 480 (10 min, 3.1 i/cm.sup.2) and 540 nm (40 min, 9.5 i/cm.sup.2) were determined. For this as a control of non-cancerous cells retinal pigment epithelium (RPE-1) was chosen and as cancerous cells human cervical carcinoma (HeLa), mouse colon carcinoma (CT-26) and human glioblastoma astrocytoma (U373) were chosen. Importantly for a PS, in all investigated cell lines (Table 3 and 4) the compounds 1-9 and 12-19 were found to be non-toxic in the dark (IC.sub.50, dark>100 IM, IC.sub.50, dark=62 l-M for compound 13), and the bphen coordinated compounds (10-11) showed little dark cytotoxicity which does not prevent their use as PS. The results show that all compounds are able to act as a PS with PI values from >1.2->167. Compounds 7, 9 and 12-15 display a phototoxicity on HeLa cells in the low micromolar range while being non-toxic in the dark. More particularly, the impressive ability of compounds 7 and 12 with PI values of >142.9 and >167 in CT-26 and HeLa cells respectively could have been unveiled.
[0287] 6. Dark and (Photo)-Cytotoxicity in 3D Multicellular Tumour Spheroids
[0288] The abilities of compounds 1-11 were investigated in 3D multicellular tumour spheroids (MCTS) as this presents a model which is closer to clinically treated tumours.
[0289] Generation and Analysis of 3D Multicellular Tumor Spheroids (MCTS) A suspension of 0.75% agarose in PBS buffer was heated inside a high-pressure
[0290] autoclave. The hot emulsion was transferred into wells (50 μL per well) of a 96 cell culture well plate. The plates were exposed for 3 h to UV irradiation to ensure the sterility and allow the agarose solution to cool down. After this time, the agarose was overlayed with a HeLa cell suspension at a density of 3000 cells per well in 150 μL of media. The MCTS were cultivated and maintained at 37° C. in a cell culture incubator at 37° C. with 5% CO.sub.2 atmosphere. The culture media was replaced every two days. Within two-three days MCTs were formed from the cell suspension. The formation as well as integrity, diameter and volume of the MCTs was monitored by an Axio Observer Z.sub.1 (Carl Zeiss) phase contrast microscope. The volume was calculated using the following formula: V=4/3πr.sup.3. The luminescence images along the z-axis were captured by a one-(λ.sub.ex=458 nm, λ.sub.em=600-750 nm) or two-photon (λ.sub.ex=800 nm, λ.sub.em=600-750 nm) excitation in the z-stack mode with a an LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with Argon and a Coherent Chameleon 2-Photon laser and a GaAsP detector.
[0291] Results
[0292] After analysis of the effect that the compounds of the invention have on monolayer cells, their ability in 3D multicellular tumour spheroids (MCTS) has been investigated for compounds 1-11. MCTS are an employed tissue model for the assessment of the delivery of drugs as it is closer to clinically treated tumours. Worthy of note, many investigated anticancer agents have failed at the transition of a cancer monolayer cell to in vivo studies. Partly this is been attributed to the failed drug delivery through the penetration of extracellular barriers. It has been shown that small MCTS with diameters of 200 μm are able to simulate intercellular interactions and therefore investigate the drug delivery. Recent studies have shown that the investigation of larger MCTS can also mimics the pathological conditions found in solid tumours as hypoxia in the tumour centre and its proliferation gradients. Consequently, we have chosen to investigate MCTS with diameters of 600-800 μm as an in vivo model. For this purpose, HeLa MCTS were incubated with the compounds 1-11 and the distribution of the compounds analysed via one and two-photon Z-stack imaging microscopy. After incubation for 12 h shows that the compounds 6-11 show a strong luminescence signal at every section depth corresponding with a complete penetration of the compounds in the MCTS. This is illustrated on
[0293] 3D Multicellular Tumor Spheroids (MCTS) Growth Inhibition Assay
[0294] MCTS were treated with the corresponding compounds (20 μM 1-7, 20 μM tetraphenylporphyrin H.sub.2TPP, 10 μM cisplatin, 30 μM cisplatin, 2% DMSO, v %) by replacing 50% of the media with drug supplemented media in the dark for three days. After this time, the MCTS were exposed to a two-photon irradiation (800 nm, 10 J/cm.sup.2) with a section interval of 5 μm using a LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture media was replaced every two days. The integrity and diameter of the MCTs was monitored with an Axio Observer Z.sub.1 (Carl Zeiss) phase contrast microscope every 24 h.
[0295] Results
[0296] MCTS with a diameter of about 800 μm were incubated with compounds 1-7 (20 μM, 2% DMSO, v %), tetraphenylporphyrin (H.sub.2TPP) (20 μM, 2% DMSO, v %), cisplatin (10 μM and 30 μM) for three days strictly in the dark. The MCTS were then exposed to 1P irradiation (500 nm, 10 J/cm.sup.2) or 2P irradiation (800 nm, 10 J/cm.sup.2, section interval of 5 μm) on day 3. During the whole time period, the shape and volume of the MCTS was constantly monitored. As expected, the volume of the MCTS in the control group which was treated purely with cell media/DMSO as well as of compounds 1-7 and H.sub.2TPP (
[0297] 3D Multicellular Tumor Spheroids (MCTS) Viability Assay
[0298] MCTS were treated with the corresponding compounds (20 μM 1-7, 2% DMSO, v %) by replacing 50% of the media with drug supplemented media in the dark for three days. After this time, the MCTS were exposed to a two-photon irradiation (800 nm, 10 J/cm.sup.2) with a section interval of 5 μm using a LSM 880 (Carl Zeiss) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture media was replaced every two days. Two days after the irradiation the MCTS viability was tested using a Viability/Cytotoxicity Kit for mammalian cells (Invitrogen). Living cells can be identified from dead cells through the presence of ubiquitous intracellular esterase activity which can be monitored by the enzymatic conversion of the non-fluorescent calcein AM to the intensely fluorescent calcein (λ.sub.ex=495 nm, λ.sub.em=515 nm). As the spectroscopic properties of the dead cell probe EthD-1 overlaps with the one of the investigated compounds, this probe was not used and only calcein AM as a probe for living cells was used. MCTS were incubated with calcein AM (2 μM) for 30 min and images of the MCTS taken with an Axio Observer Z1 (Carl Zeiss, Germany) inverted fluorescence microscope.
[0299] Results
[0300] To further study the effect the complexes on tumour survival, the treated MCTS were stained with calcein AM (
[0301] (Photo-)Cytotoxicity on 3D Multicellular Tumor Spheroids (MCTS)
[0302] The cytotoxicity of the compounds in 3D multicellular tumor spheroids (MCTS) was assessed by measurement of the ATP concentration. MCTS were treated with increasing concentrations of the compound (2% DMSO, v %) by replacing 50% of the media with drug supplemented media and incubation for 12 h. After this time, the MCTS were divided in three identical groups. The first group was strictly kept in the dark. The second group was exposed to a one-photon irradiation (500 nm, 10 J/cm.sup.2) using a LED and the third group was exposed to a two-photon irradiation (800 nm, 10 J/cm.sup.2) with a section interval of 5 μm using a LSM 880 (Carl Zeiss, Germany) laser scanning confocal microscope equipped with a Coherent Chameleon 2-Photon laser. After the irradiation, all groups were incubated additional 48 h. The ATP concertation was measured using a CellTiter-Glo 3D Cell Viability kit (Promega) by measuring the generated chemiluminescence with an infinite M200 PRO (Tecan) plate reader. The obtained data was analyzed with the GraphPad Prism software.
TABLE-US-00005 TABLE 5 IC.sub.50 in μM values in the dark and upon 1-Photon irradiation (500 nm, 10 J/cm.sup.2) irradiation or 2-Photon irradiation (800 nm, 10 J/cm.sup.2, section interval of 5 μm) for compounds 1-11 in comparison to cisplatin and tetraphenylporphyrin (H.sub.2TPP) in HeLa MCTS. Average of three independent measurements. 1-Photon 2-Photon irradiation irradiation (500 nm, (800 nm, dark 10 J/cm.sup.2) PI 10 J/cm.sup.2) PI 4 >100 78.3 ± 5.1 >1.3 63.0 ± 4.2 >1.6 5 >100 19.3 ± 2.7 >5.2 13.4 ± 3.9 >7.5 6 >300 33.8 ± 3.4 >8.9 26.5 ± 2.9 >11.3 7 >300 6.8 ± 0.2 >44.1 1.4 ± 0.2 >214.3 8 >300 32.6 ± 2.5 >9.2 27.8 ± 3.1 >10.8 9 >300 7.5 ± 0.2 >40.0 1.2 ± 0.3 >250.0 10 27.8 ± 3.6 8.9 ± 0.7 3.1 3.1 ± 0.6 9.0 11 29.3 ± 2.9 3.8 ± 0.4 7.7 0.8 ± 0.5 36.6 H.sub.2TPP >100 >100 n.d. >100 >100 cisplatin 18.6 ± 1.3 — — — — .sup.a) due to solubility limitations the compounds were investigated as chloride salts.
[0303] Results
[0304] To quantify the photodynamic effect that the compounds have on HeLa MCTS, the (photo-)cytotoxicity (Table 5) was determined in the dark as well as upon 1 Photon (500 nm, 10 J/cm.sup.2) and 2 Photons irradiation (800 nm, 10 J/cm.sup.2, section interval of 5 μm) by measurement of the ATP concentration of living cells through conversion into chemiluminescence. Importantly, no measurable cytotoxicity in the dark could be observed in HeLa MCTS for all compounds. The compounds 4-6, 8, 10 were found to be phototoxic in the micromolar range with PI values from >1.1 to >11.3. In comparison, compounds 7, 9 and 11 were found with much higher phototoxicity. These results confirm the (photo-)cytotoxicity evaluation on 2D monolayer cells. As a promising compound, 7 was identified. Importantly, no significant toxicity in the dark (IC.sub.50, dark>300 μM) and high phototoxicity in the low micromolar range (IC.sub.50, 500 nm>6.8±0.2 μM, IC.sub.50, 800 nm>1.4±0.2) with a PI value of >44.1 or >214.3, respectively were determined.
[0305] These results are very promising in comparison to H.sub.2TPP which did not have a cytotoxic effect (IC.sub.50, dark=IC.sub.50, 500 nm=IC.sub.50, 800 nm>100 μM) under identical experimental conditions. The comparison between 1 Photon and 2 Photons irradiation demonstrates a stronger 2 Photons phototoxicity, which is likely attributed to the deeper penetration depth of the longer wavelength as the intensity of the light is declining with tissue penetration. Overall, the highly phototoxic compound 7 in MCTS, which is able to act efficiently upon 1 Photon and 2 Photon irradiation could be unveiled.
[0306] 7. In Vivo Evaluation
[0307] Based on the remarkable properties of compound 7, this compound was investigated inside a mouse model.
[0308] In Vivo Experiment
[0309] 6 weeks age nu/nu female mice were purchased from Charles River. Compound 7 was dissolved into physiological saline at first. Tumor xenograft: 6×10.sup.6 SW620/AD300 cells were subcutaneously (s.c.) injected in the nude mice, the cells were suspension in 150 μL Matrigel (Corning) and saline (1:1, v/v). After a week, the tumor volumes of the mice reached approximately 80 mm.sup.3.
[0310] In Vivo (Photo-)Cytotoxicity
[0311] 30 SW620/AD300 nude tumour-bearing nude mice were randomly separated into 6 groups and five mice for each group.
[0312] Group 1: injected 7 (2 mg/Kg 50 μL) intravenously and irradiate under 800 nm laser (50 mW, 1 kHz, pulse width 35 fs, 5 s/mm) 1 h after injection;
[0313] Group 2: injected 7 (2 mg/Kg 50 μL) intravenously and irradiate under 500 nm light (10 mW/cm.sup.2, 60 min);
[0314] Group 3: injected by physiological saline (50 μL) and treated with 800 nm laser (50 mW, 1 kHz, pulse width 35 fs, 5 s/mm) 1 h after injection;
[0315] Group 4: injected by physiological saline (50 μL) and treated with 500 nm light (10 mW/cm.sup.2, 60 min);
[0316] Group 5: intravenous injected 7 (2 mg/Kg 50 μL);
[0317] Group 6: injected 50 μL physiological saline.
[0318] The mice were anesthetized by the injection of 4% chloral hydrate aqueous solution (0.2 mL/20 g) before treatment. The tumour volume and body weight were measured and record for each two days. Tumor volume was calculated by the following formula
[0319] Histological Examination
[0320] After the treatment, the mice were euthanized. The tumour was collected and fixated by 4% paraformaldehyde, and then obtained as paraffin-embedded samples and stained with hematoxylin and eosin (H&E). A Carl Zeiss Axio Imager Z2 microscope was used to observe the tissue structure and cell state of the sections.
[0321] Results
[0322] In vivo PDT experiments were prepared on SW620/AD300 (doxorubicin-selected P-gp-overexpressing human colon cancer cell) tumour-bearing nude mice. After the tumours volume reached 80 mm.sup.3, the mice were randomly separated into six groups and treated (day 1); group 1: injection of 2 mg/Kg 7 intravenously and 2P irradiation at 800 nm, group 2: injection of 2 mg/Kg 7 intravenously and 1P irradiation at 500 nm, group 3 and 4: were injection of physiological saline and treated with 1P and 2P irradiation, group 5: injection of 2 mg/Kg 7 intravenously and group 6 injection of the same volume of physiological saline. Importantly, the animals treated with 7 behave normally, without signs of pain, stress or discomfort. The body weight and tumour volume were recorded every two days (
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