Ruthenium (II) Complexes and Their Use as AntiCancer Agents

Abstract

The present invention relates to a compound of the following formula (I): (I) or a pharmaceutically acceptable salt and/or solvate thereof, notably for use as a drug, in particular in the treatment of cancer. The present invention also relates to a pharmaceutical composition comprising such a compound and to a method for the preparation of such a compound.

##STR00001##

Claims

1. A compound of formula (I-A): ##STR00050## wherein R.sup.1 to R.sup.6, R.sup.8 and R.sup.9 are each independently selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, CN, NO.sub.2, COR.sup.19, OR.sup.20 and NR.sup.21R.sup.22, R.sup.19 is selected in the group consisting of H, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.27 and NR.sup.28R.sup.29, R.sup.20, R.sup.21, R.sup.22, R.sup.27, R.sup.28 and R.sup.29 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.6alkyl, O═Y═O is a bidentate ligand, selected in the group consisting of: ##STR00051## in which R.sup.30, R.sup.31 and R.sup.32 each independently represent one or several substituent(s) independently selected in the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, CN, NO.sub.2, COR.sup.33, OR.sup.34 and NR.sup.35R.sup.36, R.sup.33 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, OR.sup.37 or NR.sup.38R.sup.39, R.sup.34 to R.sup.39 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.6alkyl, X.sup.− is a pharmaceutically acceptable anion, m is equal to 0 when O═Y═O is A and m is equal to +1 when O═Y═O is B or C, or a pharmaceutically acceptable salt and/or solvate thereof.

2. The compound according to claim 1, wherein R.sup.1 to R.sup.4 are each independently selected in the group consisting of H, halogen and optionally substituted aryl.

3. The compound according to claim 1, wherein R.sup.1 to R.sup.4 are identical.

4. The compound according to claim 1, wherein R.sup.5, R.sup.6, R.sup.8 and R.sup.9 each independently are H, halogen or OH.

5. The compound according to claim 1, wherein R.sup.1 to R.sup.4 are each a phenyl and R.sup.5, R.sup.6, R.sup.8 and R.sup.9 are each H.

6. The compound according to claim 1, wherein O═Y═O is selected in the group consisting of: ##STR00052## in which R.sup.30, R.sup.31 and R.sup.32 each independently represent one or several substituent(s) independently selected from the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, NO.sub.2 and OR.sup.34.

7. The compound according to claim 1, being selected from: ##STR00053## ##STR00054## ##STR00055##

8-10. (canceled)

11. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a compound according to claim 1.

12-13. (canceled)

14. A method for the preparation of a compound according to claim 1, comprising the following steps: (i) reacting a compound of formula (II-A) ##STR00056## in which R.sup.1 to R.sup.6, R.sup.8 and R.sup.9 are as defined in claim 1, P.sup.1 and P.sup.2 each independently represent halogen, OR.sup.40 or S(O)R.sup.41R.sup.42, R.sup.40 is H or C.sub.1-C.sub.6 alkyl, and R.sup.41 and R.sup.42 each independently represent a C.sub.1-C.sub.6 alkyl, with a compound of formula (III) ##STR00057## in which represents a single or a double bond, T is O when is a double bond and T is OH when is a single bond and Z is selected in the group consisting of: ##STR00058## in which R.sup.30, R.sup.31 and R.sup.32 are as defined in claim 1, in the presence of a base under inert atmosphere, (ii) placing the reaction mixture of step (i) in contact with an oxidant, (iii) when m is equal to +1, reacting the product resulting from step (ii) with a salt A.sup.+X.sup.−, wherein X.sup.− is as defined in claim 1 and A.sup.+ is a counter cation.

15. The method according to claim 14, wherein P.sup.1 and P.sup.2 are both halogen.

16. The method according to claim 14, wherein the oxidant is O.sub.2.

17. The method according to claim 14, wherein A.sup.+ is selected from H.sup.+, Na.sup.+, K.sup.+, Li.sup.+ and N.sup.+R′R″R′″R″″, with R′, R″, R′″ and R″″ independently being H or C.sub.1-C.sub.6 alkyl

18. The compound according to claim 1, wherein X.sup.− is selected in the group consisting of PF.sub.6.sup.−, Cl.sup.−, Br.sup.−, BF.sub.4.sup.−, (C.sub.1-C.sub.6 alkyl)-C(O)O.sup.−, (C.sub.1-C.sub.6 haloalkyl)-C(O)O.sup.−, (C.sub.1-C.sub.6 alkyl)-SO.sub.3.sup.− and (C.sub.1-C.sub.6 haloalkyl)-SO.sub.3.sup.−.

19. The compound according to claim 6, wherein R.sup.30, R.sup.31 and R.sup.32 each independently represent one or several substituent(s) independently selected from the group consisting of H, halogen, C.sub.1-C.sub.6 alkyl, NO.sub.2 and OR.sup.34, with R.sup.34 being a C.sub.1-C.sub.6 alkyl.

20. A method for treating a cancer comprising the administration to a person in need thereof of an effective dose of a compound according to claim 1.

21. The method according to claim 20, wherein the cancer is a lung cancer, an ovarian cancer, a colon cancer, a colorectal cancer, a stomach cancer, a testicular cancer, a head cancer, a neck cancer, a breast cancer, a kidney cancer, a liver cancer, a skin cancer, a tongue cancer, a pancreatic cancer, a gall bladder cancer, a bladder cancer or a leukemia.

22. A method for treating a cancer comprising the administration to a person in need thereof of an effective dose of a pharmaceutical composition according to claim 11.

23. The method according to claim 21, wherein the cancer is a lung cancer, an ovarian cancer, a colon cancer, a colorectal cancer, a stomach cancer, a testicular cancer, a head cancer, a neck cancer, a breast cancer, a kidney cancer, a liver cancer, a skin cancer, a tongue cancer, a pancreatic cancer, a gall bladder cancer, a bladder cancer or a leukemia.

Description

DESCRIPTION OF THE FIGURES

[0153] FIG. 1: Ruthenium cellular uptake (left) and fractionated subcellular uptake of ruthenium (right). The results were obtained by incubating 5 μM of the target ruthenium compound 1 for 2 h in HeLa cells.

[0154] FIG. 2: Ruthenium cellular accumulation in MRC5 healthy cells.

[0155] FIG. 3: Induction of apoptosis/necrosis in HeLa cells upon treatment with the target compound at different time frames. The white bar represents living cells while light grey displays early apoptotic events, dark grey late apoptotic events and black necrotic cells.

[0156] FIG. 4: Kaplan-Meier analysis of survival. The compounds were administered i.p. on days 1 and 7 after tumour inoculation, n=7 in each group.

[0157] FIG. 5: The weight of the solid Ehrlich tumour (in grams) on day 10 of mice injected on days 1 and 7 i.p. with pure vehicle, compound 1 or cisplatin. Values are the means±SEM (n=7 in each group). Control—tumour-bearing control treated with mixture of co-solvent and water; Pt—cisplatin 5 mg/kg i.p.; Ru 5-compound 15 mg/kg i.p.; Ru 10-compound 110 mg/kg i.p.; Ru 15-compound 115 mg/kg i.p. Significantly different from the controls (**P<0.01).

EXAMPLES

[0158] 1. Synthesis of Compounds According to the Present Invention Materials.

[0159] All chemicals were either of reagent or analytical grade and used as purchased from commercial sources without additional purification. Ruthenium trichloride hydrate was provided by I.sup.2CNS, 4,7-Diphenyl-1,10-phenanthroline, Lithium chloride (anhydrous, 99%), and catechol by Alfa Aesar, tetrabutylammonium hexafluorophosphate by Sigma-Aldrich. All solvents were purchased of analytical, or HPLC grade. When necessary, solvents were degassed by purging with dry, oxygen-free nitrogen for at least 30 min before use.

[0160] Instrumentation and Methods.

[0161] Amber glass or clear glassware wrapped in tin foil were used when protection from the light was necessary. Schlenk glassware and a vacuum line were employed when reactions sensitive to moisture/oxygen had to be performed under nitrogen atmosphere. Thin layer chromatography (TLC) was performed using silica gel 60 F-254 (Merck) plates with detection of spots being achieved by exposure to UV light. Column chromatography was done using Silica gel 60-200 μm (VWR). Eluent mixtures are expressed as volume to volume (v/v) ratios. .sup.1H and .sup.13C NMR spectra were measured on Bruker Avance 11 HD 400 MHz or Bruker Avance Neo 500 MHz spectrometers using the signal of the deuterated solvent as an internal standard..sup.9 The chemical shifts δ are reported in ppm (parts per million) relative to tetramethylsilane (TMS) or signals from the residual protons of deuterated solvents. Coupling constants J are given in Hertz (Hz). The abbreviation for the peaks multiplicity is br (broad). ESI-HRMS experiments were carried out using a LTQ-Orbitrap XLfrom Thermo Scientific (Thermo Fisher Scientific, Courtaboeuf, France) and operated in positive ionization mode, with a spray voltage at 3.6 kV. Sheath and auxiliary gas were set at a flow rate of 5 and 0 arbitrary units (a.u.), respectively. 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 200-2000 in profile mode. Spectrum was analysed using the acquisition software XCalibur 2.1 (Thermo Fisher Scientific, Courtaboeuf, France). 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 ρscan was acquired. 5 μL was injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific, Courtaboeuf, France) with a continuous infusion of methanol at 100 μL.Math.min-1. Electrospray Ionization-Mass Spectrometry (ESI-MS) experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific, Courtaboeuf, France) and operated in positive ionization mode, with a spray voltage at 3.6 kV. No sheath and auxiliary gas 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 an m/z range between 150 and 2000 in profile mode. Spectrum was analyzed using the acquisition software XCalibur 2.1 (Thermo Fisher Scientific, Courtaboeuf, France). The automatic gain control (AGC) allowed for the accumulation of up to 2×10.sup.5 ions for Fourier Transform Mass Spectrometry (FTMS) scans, maximum injection time was set to 300 ms and 1 μs scan was acquired. 10 μL was injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific, Courtaboeuf, France) with a continuous infusion of methanol at 100 μL-min-1. The ATR-FTIR spectra were collected with a dry-air-purged Thermo Scientific Nicolet 6700 FT-IR equipped with a MCT detector. Spectral resolution was 4 cm.sup.−1 and spectra were averaged from 256 scans. A horizontal diamond-coated ZnSe crystal with a single reflection and an angle of incidence of 45° was used. A small amount of solid was pressed using a dynanometric stainless steel pressure arm. Elemental analysis was performed at Science Centre, London Metropolitan University using Thermo Fisher (Carlo Erba) Flash 2000 Elemental Analyser, configured for % CHN. IR spectra were recorded with SpectrumTwo FTIR Spectrometer (Perkin-Elmer) equipped with a Specac Golden Gate™ ATR (attenuated total reflection) accessory; applied as neat samples; 1/λ in cm-1. Analytical HPLC measurement was performed using the following system: 2× Agilent G1361 1260 Prep Pump system with Agilent G7115A 1260 DAD WR Detector equipped with an Agilent Pursuit XRs 5C18 (100 Å, C18 5 μm 250×4.6 mm) Column and an Agilent G1364B 1260-FC fraction collector. The solvents (HPLC grade) were millipore water (0.1% TFA, solvent B) and acetonitrile (0.1% TFA, solvent A). The flow rate was 1 mL/min. Detection was performed at 215 nm, 250 nm, 350 nm, 450 nm, 550 nm and 650 nm with a slit of 4 nm. ICP-MS measurements were performed on an Agilent QQQ 8800 Triple quad ICP-MS spectrometer (Agilent Technologies) with a ASX200 autosampler (Agilent Technologies), equipped with standard nickel cones and a “micro-mist” quartz nebulizer fed with 0.3 ml/min analytic flow (as a 2% HNO.sub.3 aqueous solution).

[0162] Abbreviations.

[0163] DCM: dichloromethane

[0164] DIP: 4,7-diphenyl-1,10-phenanthroline

[0165] DMEM: Dulbecco's Modified Eagle Medium

[0166] DMF: dimethylformamide

[0167] DMSO: dimethyl sulfoxide

[0168] ESI: electrospray ionisation

[0169] HRMS: High resolution mass spectroscopy

[0170] ICP-MS: Inductively Coupled Plasma Mass Spectrometry

[0171] IR: Infrared

[0172] NMR: Nuclear magnetic resonance

[0173] r.t: room temperature

[0174] Synthesis and Characterization.

[0175] Ru(DMSO).sub.2Cl.sub.2. Ru(DMSO).sub.2Cl.sub.2 was synthesised following an adapted literature procedure (Brastos, I et al., 2010) Spectroscopic data (.sup.1H NMR) was in agreement with literature.

[0176] Ru(DIP).sub.2Cl.sub.2. The complex was synthesised following an adapted literature procedure (Caspar, R. et al., 2006). A mixture of Ru(DMSO).sub.2Cl.sub.2 (3.0 g, 6.19 mmol), DIP (4,7-diphenyl-1,10-phenanthroline) (4.11 g, 12.38 mmol) and LiCl (2.0 g, 47.18 mmol) dissolved in DMF (100 mL) was refluxed for 24 h. After cooling to room temperature, the solvent was reduced in vacuo to 8 mL and 350 mL of acetone were added. The mixture was then stored at −20° C. overnight before filtration with a Buchner funnel and washed with Acetone and Et.sub.2O to afford Ru(DIP).sub.2Cl.sub.2 as a deep purple solid (3.76 g, 4.49 mmol, 72%). Spectroscopic data (.sup.1H NMR) were in agreement with literature (Caspar, R. et al., 2006).

[0177] Compound 1 ([Ru(DIP).sub.2(Sq)](PF.sub.6))

##STR00042##

[0178] Ru(DIP).sub.2Cl.sub.2 (0.739 g, 0.88 mmol) and aq. NaOH (0.5 mL, 1 M) were dissolved in 2-propanol (40 mL). The solution was degassed for 15 min and catechol (0.155 g, 1.41 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in 2-propanol (7 mL) and H.sub.2O (56 mL) and NH.sub.4PF.sub.6 (0.700 g, 4.3 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product as the PF.sub.6 salt (0.70 g), which was chromatographed on silica (DCM/MeCN 20:1 Rf: 0.3). Evaporation of the solvent under vacuum provided 1 as a deep red solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the red solid was collected with DCM and dried under vacuum to afford a clean product (0.17 g, 0.167 mmol, 19%). IR (Golden Gate, cm.sup.−1): 3345w, 1710m, 1600w, 1520s, 1455s, 1335s, 1270s, 1125s, 820s, 760m. .sup.1H NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=8.79-8.20 (br, 5H, arom.), 8.09-7.88 (br, 5H, arom.), 7.73-7.42 (br, 14H, arom.), 7.26-6.92 (br, 10H, arom.), 6.92-6.63 (br, 2H, arom.). .sup.13C NMR (125 MHz, CD.sub.2Cl.sub.2): δ/ppm=149.84, 144.68, 136.10, 133.56, 130.36, 129.89, 129.53, 128.41, 126.21, 125.36, 121.47, 116.35. For the quaternary carbons, only two were observed in the .sup.13C NMR spectrum where five were expected. This could be explained by peak overlap or the signal being too weak to be detected within the acquisition time of the experiment which is common for quaternary carbons. HRMS (ESI+): m/z 874.1887 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.54H.sub.36F.sub.6N.sub.4O.sub.2PRu═C, 63.65; H, 3.56; N, 5.50. Found ═C, 63.62; H, 3.52; N, 5.45. HPLC: 0-3 minutes: isocratic 90% B (5% A); 3-25 minutes: linear gradient from 90% B (5% A) to 0% B (100% A); 25-30 minutes: isocratic 0% B (100% A), 30-35 minutes: linear gradient from 0% B (100% A) to 95% B (5% A), T.sub.R=31.304 min.

[0179] Compound 2 ([Ru(DIP).sub.2(Mal)](PF.sub.6))

##STR00043##

[0180] Ru(DIP).sub.2Cl.sub.2 (0.150 g, 0.18 mmol) and aq. NaOH (0.28 mL, 1 M) were dissolved in ethanol (18 mL). The solution was degassed for 30 min and maltol (3-Hydroxy-2-methyl-4H-pyran-4-one) (0.036 g, 0.29 mmol) was added. The mixture was heated to reflux for 3 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., H.sub.2O (200 mL) and NH.sub.4PF.sub.6 (1 g, 6 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. The solid was collected with DCM and dried under vacuum to deliver a clean product as the PF.sub.6 salt (0.17 g, 0.16 mmol, 90%). IR (Golden Gate, cm.sup.−1): 1590w, 1545w, 1490w, 1465w, 1445w, 1415w, 1400w, 1275w, 1205w, 1085w, 1025w, 915w, 830s, 765s, 735m, 700s. .sup.1H NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=9.49 (d, J=5.4 Hz, 1H), 9.33 (d, J=5.5 Hz, 1H), 8.23 (dd, J=9.4, 4.6 Hz, 2H), 8.13 (dd, J=9.4, 1.1 Hz, 2H), 8.04 (d, J=5.6 Hz, 2H), 7.98 (dd, J=13.5, 5.4 Hz, 2H), 7.78-7.60 (m, 11H), 7.59-7.47 (m, 10H), 7.33 (dd, J=5.6, 3.0 Hz, 2H), 6.54 (d, J=5.1 Hz, 1H), 2.37 (s, 3H). .sup.13C NMR (125 MHz, CD.sub.2Cl.sub.2): δ/ppm=185.00, 159.02, 155.49, 154.10, 154.01, 151.75, 151.47, 151.06, 150.64, 150.17, 149.98, 149.92, 147.66, 147.13, 146.01, 145.71, 136.50, 136.47, 136.23, 136.20, 130.15, 129.86, 129.81, 129.47, 129.44, 129.28, 129.21, 129.10, 128.78, 128.61, 128.55, 128.46, 125.96, 125.82, 125.77, 125.77, 125.69, 125.51, 125.25, 125.05, 112.40, 29.84. HRMS (ESI+): m/z 891.19042 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.54H.sub.39F.sub.6N.sub.4O.sub.4PRu═C, 61.54; H, 3.73; N, 5.32. Found=C, 61.53; H, 3.38; N, 5.17.

[0181] Compound 3 [Ru(DIP).sub.2(4-Methylsq)](PF.sub.6)

##STR00044##

[0182] Ru(DIP).sub.2Cl.sub.2 (0.170 g, 0.20 mmol) and aq. NaOH (0.3 mL, 1 M) were dissolved in 2-propanol (20 mL). The solution was degassed for 15 min and 4-methycatechol (0.05 g, 0.40 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in Ethanol (1 mL) and H.sub.2O (8 mL) and NH.sub.4PF.sub.6 (0.200 g, 1.2 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product as the PF.sub.6 salt (0.07 g), which was chromatographed on silica (DCM/MeCN 20:1 Rf: 0.3). Evaporation of the solvent under vacuum provided [Ru(DIP).sub.2(4-methylsq)](PF.sub.6) as a deep red solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the red solid was collected with DCM and dried under vacuum to afford a clean product (0.06 g, 0.06 mmol, 29%). IR (Golden Gate, cm.sup.−1): 3060w, 1620w, 1590, 1560, 1510w, 1420m, 1240m, 1120w, 1090w, 1030w, 912w, 827s, 762s, 735m, 698s. 1H NMR (500 MHz, CD.sub.2Cl.sub.2) δ 8.63-8.07 (br, 6H, arom.), 8.03-7.81 (br, 7H, arom.), 7.72-7.36 (br, 16H, arom.), 7.36-7.03 (m, 9H, arom.). .sup.13C NMR (125 MHz): δ/ppm=149.08, 147.51, 143.55, 142.50, 140.12, 140.01, 136.57, 136.18, 132.83, 132.35, 130.28, 130.19, 129.97, 129.60, 129.49, 128.89, 128.71, 128.37, 126.41, 124.88, 123.69. MS (ESI+): m/z 888.7 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.55H.sub.38F.sub.6N.sub.4O.sub.2PRu═C, 63.95; H, 3.71; N, 5.42. Found=C, 63.84; H, 3.62; N, 5.29. HPLC: 0-3 minutes: isocratic 65% B (35% A); 3-17 minutes: linear gradient from 65% B (35% A) to 0% B (100% A); 17-23 minutes: isocratic 0% B (100% A), T.sub.R=13.804 min.

[0183] Compound 4 ([Ru(DIP).sub.2(3-Methoxysq)](PF.sub.6))

##STR00045##

[0184] Ru(DIP).sub.2Cl.sub.2 (0.250 g, 0.29 mmol) and aq. NaOH (0.45 mL, 1 M) were dissolved in 2-propanol (5 mL). The solution was degassed for 15 min and 3-methoxycatechol (0.07 g, 0.5 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in 2-propanol (2.5 mL) and H.sub.2O (20 mL) and NH.sub.4PF.sub.6 (0.250 g, 1.5 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product as the PF.sub.6 salt (0.07 g), which was chromatographed on silica (DCM/MeCN 20:1 Rf: 0.3). Evaporation of the solvent under vacuum provided [Ru(DIP).sub.2(3-methoxysq)](PF.sub.6) as a deep red solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the red solid was collected with DCM and dried under vacuum to afford a clean product (0.072 g, 0.06 mmol, 23%). IR (Golden Gate, cm.sup.−1):3060w, 1620w, 1590w, 1540w, 1460m, 1400m, 1250m, 1160m, 1100m, 1030w, 827s, 764s, 735m, 700s. .sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2): δ/ppm=8.91-8.50 (br, 1H, arom.), 8.43-8.08 (br, 3H, arom.), 8.07-7.79 (br, 7H, arom.), 7.75-7.46 (br, 15H, arom.), 7.46-7.28 (br, 2H, arom.), 7.28-6.93 (br, 10H, arom.). .sup.13C NMR (125 MHz, CD.sub.2Cl.sub.2): δ/ppm=149.62, 146.57, 143.72, 140.55, 137.05, 136.03, 133.07, 132.47, 131.27, 130.31, 130.07, 130.00, 129.59, 129.56, 129.33, 128.97, 128.80, 128.57, 125.67, 125.46, 123.55. MS (ESI+): m/z 904.8 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.55H.sub.40F.sub.6N.sub.4O.sub.4PRu═C, 62.76; H, 3.64; N, 5.53. Found=C, 61.67; H, 3.63; N, 5.09. HPLC: 0-3 minutes: isocratic 65% B (35% A); 3-17 minutes: linear gradient from 65% B (35% A) to 0% B (100% A); 17-23 minutes: isocratic 0% B (100% A), T.sub.R=11.887 Min.

[0185] Compound 5 ([Ru(DIP).sub.2(Tetrabromocat)])

##STR00046##

[0186] Ru(DIP).sub.2Cl.sub.2 (0.270 g, 0.32 mmol) and aq. NaOH (0.50 mL, 1 M) were dissolved in 2-propanol (27 mL). The solution was degassed for 15 min and tetrabromocatechol (0.220 g, 0.5 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in 2-propanol (2.5 mL) and H.sub.2O (20 mL) and NH.sub.4PF.sub.6 (0.250 g, 1.5 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product (0.320 g), which was chromatographed on silica (DCM/Et.sub.2O 98:2 Rf: 0.8). Evaporation of the solvent under vacuum provided Ru(DIP).sub.2(tetrabromocat) as a blue solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the blue solid was collected with Ethanol and dried under vacuum to afford a clean product (0.209 g, 0.176 mmol, 54%). IR (Golden Gate, cm.sup.−1): 3060w, 1600w, 1430s, 1260m, 1080m, 1030w, 914m, 847m, 760m, 731m, 700s. 1H NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=8.84-8.44 (br, 6H, arom.), 8.16-8.01 (br, 4H, arom.), 7.76-7.62 (br, 8H, arom.), 7.62-7.41 (br, 11H, arom.), 7.41-7.28 (br, 3H, arom.). .sup.13C NMR (100 MHz, CD.sub.2Cl.sub.2): δ/ppm=141.93, 139.70, 131.49, 130.20, 129.81, 129.45, 126.33, 125.79, 125.33. MS (ESI+): m/z 1090.4 [M]+. Elemental Analysis: calcd. for C.sub.54H.sub.32Br.sub.4N.sub.4O.sub.2Ru═C, 54.52; H, 2.71; N, 4.71. Found=C, 54.56; H, 2.37; N, 4.89. HPLC: 0-3 minutes: isocratic 85% B (35% A); 3-7 minutes: linear gradient from 85% B (35% A) to 0% B (100% A); 7-9 minutes: isocratic 0% B (100% A), HPLC: T.sub.R=8.623 min.

[0187] Compound 6 [Ru(DIP).sub.2(3-Methylsq)](PF.sub.6)

##STR00047##

[0188] Ru(DIP).sub.2Cl.sub.2 (0.170 g, 0.20 mmol) and aq. NaOH (0.3 mL, 1 M) were dissolved in 2-propanol (20 mL). The solution was degassed for 15 min and 3-methycatechol (0.05 g, 0.40 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in Ethanol (1 mL) and H.sub.2O (8 mL) and NH.sub.4PF.sub.6 (0.200 g, 1.2 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product as the PF.sub.6 salt (0.06 g), which was chromatographed on silica (DCM/MeCN 20:1 Rf: 0.3). Evaporation of the solvent under vacuum provided [Ru(DIP).sub.2(4-methylsq)](PF.sub.6) as a deep red solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the red solid was collected with DCM and dried under vacuum to afford a clean product (0.05 g, 0.05 mmol, 24%). IR (Golden Gate, cm.sup.−1): 3060w, 1600w, 1540m, 1390m, 1250m, 1150m, 1100w, 1030w, 827s, 764s, 735s, 700s. 1H NMR (400 MHz, CD.sub.2Cl.sub.2) δ 8.79-8.11 (br, 6H, arom.), 8.08-7.82 (br, 6H, arom.), 7.60 (br, 15H, arom.), 7.40-6.81 (br, 11H, arom.). .sup.13C NMR (100 MHz): δ/ppm=148.62, 147.00, 142.78, 142.70, 142.59, 136.94, 136.22, 132.88, 130.54, 130.25, 130.13, 129.79, 129.61, 129.56, 128.64, 126.87, 126.52, 124.93, 124.47, 121.72. MS (ESI+): m/z 888.7 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.55H.sub.40F.sub.6N.sub.4O.sub.3PRu═C, 62.86; H, 3.84; N, 5.33. Found=C, 62.95; H, 3.69; N, 5.20. HPLC: 0-3 minutes: isocratic 65% B (35% A); 3-17 minutes: linear gradient from 65% B (35% A) to 0% B (100% A); 17-23 minutes: isocratic 0% B (100% A), T.sub.R=13.804 min.

[0189] Compound 7 [Ru(DIP).sub.2(4-Tert-Buthylsq)](PF.sub.6).

##STR00048##

[0190] Ru(DIP).sub.2Cl.sub.2 (0.250 g, 0.29 mmol) and aq. NaOH (0.45 mL, 1 M) were dissolved in 2-propanol (5 mL). The solution was degassed for 15 min and 4-tert-buthylcatechol (0.08 g, 0.5 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in 2-propanol (2.5 mL) and H.sub.2O (20 mL) and NH.sub.4PF.sub.6 (0.250 g, 1.5 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product as the PF.sub.6 salt (0.08 g), which was chromatographed on silica (DCM/MeCN 20:1 Rf: 0.3). Evaporation of the solvent under vacuum provided [Ru(DIP).sub.2(3-methylysq)](PF.sub.6) as a deep red solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the red solid was added dropwise to cold stirring Et.sub.2O (200 mL) filtrated, collected with DCM and dried under vacuum to afford a clean product (0.052 g, 0.05 mmol, 16%). IR (Golden Gate, cm.sup.−1): 3060w, 2960w, 1620w, 1580w, 1510m, 1450m, 1420m, 1220m, 1090w, 1030w, 827s, 764s, 735s, 700s. 1H NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=8.43-8.12 (br, 6H, arom.), 8.09-7.81 (br, 9H, arom.), 7.70-7.46 (br, 16H, arom.), 7.46-7.24 (br, 4H, arom.), 7.24-7.04 (br, 8H, arom.). .sup.13C NMR (125 MHz, CD.sub.2Cl.sub.2): δ/ppm=147.51, 147.31, 143.70, 136.70, 136.64, 132.77, 132.54, 130.21, 129.63, 129.45, 128.84, 128.75, 127.49, 126.60, 124.85, 124.45. MS (ESI+): m/z 930.8 [M-PF.sub.6].sup.+. Elemental Analysis: calcd. for C.sub.58H.sub.44F.sub.6N.sub.4O.sub.2PRu═C, 64.80; H, 4.13; N, 5.21. Found=C, 64.72; H, 4.13; N, 5.14.

[0191] Compound 8 (Ru(DIP).sub.2(4-Nitrocat)).

##STR00049##

[0192] Ru(DIP).sub.2Cl.sub.2 (0.270 g, 0.32 mmol) and aq. NaOH (0.50 mL, 1 M) were dissolved in 2-propanol (27 mL). The solution was degassed for 15 min and 4-nitrocatechol (0.073 g, 0.5 mmol) was added. The mixture was heated to reflux for 24 h under N.sub.2 atmosphere and protected from light. After cooling to r.t., the mixture was stirred opened to air for 2 h while still protected from light and the solvent was removed under vacuum. The residual solid was dissolved in 2-propanol (2.5 mL) and H.sub.2O (20 mL) and NH.sub.4PF.sub.6 (0.250 g, 1.5 mmol) were added. The mixture was stored in the fridge (4° C.) overnight. The precipitate was filtered with a Buchner funnel and washed with H.sub.2O (3×50 mL), Pentane (3×50 mL) and Et.sub.2O (3×50 mL). The solid was collected with DCM and dried under vacuum to deliver a crude product (0.120 g), which was chromatographed on silica (DCM/MeOH 96:4 Rf: 0.4). Evaporation of the solvent under vacuum provided Ru(DIP).sub.2(4-nitrocat) as a deep blue solid. Further wash with Et.sub.2O and Heptane were necessary in order to obtain clean product. The solid with the washing solvent (10 mL) was sonicated for 10 min and then centrifuged. This procedure was repeated three times for each solvent. Finally the blue solid was added dropwise to cold stirring Et.sub.2O (200 mL) filtrated, collected with DCM and dried under vacuum to afford a clean product (0.209 g, 0.176 mmol, 54%). IR (Golden Gate, cm.sup.−1): 3060w, 1550w, 1490m, 1410w, 1240s, 1120m, 1070s, 1030w, 949w, 910w, 845s, 762s, 733s, 698s. .sup.1H NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=9.65-9.21 (br, 3H, arom.), 8.24-7.85 (br, 8H, arom.), 7.81-7.40 (br, 17H, arom.), 7.41-7.18 (br, 2H, arom.), 6.87-6.59 (br, 1H, arom.), 6.60-6.27 (br, 2H, arom.), 6.11-5.84 (br, 2H, arom.). .sup.13C NMR (125 MHz, CD.sub.2Cl.sub.2): δ/ppm=153.27, 152.64, 152.53, 150.58, 146.76, 146.65, 144.72, 144.63, 137.40, 137.17, 130.79, 130.69, 130.19, 129.61, 129.50, 129.44, 128.86, 128.38, 126.17, 125.87, 125.49, 125.15, 124.99, 124.87. MS (ESI+): m/z 919.4 [M].sup.+. Elemental Analysis: calcd. for C.sub.54H.sub.39N.sub.5O.sub.6RuC, 67.92; H, 4.12; N, 7.33. Found=C, 68.04; H, 4.11; N, 7.28. HPLC: 0-3 minutes: isocratic 65% B (35% A); 3-17 minutes: linear gradient from 65% B (35% A) to 0% B (100% A); 17-23 minutes: isocratic 0% B (100% A), T.sub.R=15.907 min.

[0193] 2. Cytotoxicity Studies

[0194] The cytotoxicity of compounds according to the present invention toward the following cells: HeLa (human cervical), A2780 (human ovarian carcinoma), A2780 cis (human cisplatin resistant ovarian carcinoma), A2780 ADR (human doxorubicin resistant ovarian carcinoma), U87 (human brain glioblastoma), CT-26 (mouse colon adenocarcinoma), CT-26 LUC (mouse colon adenocarcinoma stably expressing luciferase) and RPE-1 (normal retina pigmented epithelial) cell lines was therefore investigated using a fluorometric cell viability assay (Frei et al., 2014). Cytotoxicity of cisplatin and doxorubicin was also determined in the same cell lines as positive controls.

[0195] Cell Culture

[0196] HeLa and CT-26 cell lines were cultured in DMEM media (Gibco) supplemented with 10% of fetal calf serum (Gibco). CT-26 LUC cell line was cultured in DMEM media (Gibco) supplemented with 10% of fetal calf serum (Gibco) and 1% Genticin. RPE-1 cell line was cultured in DMEM/F-12 media (Gibco) supplemented with 10% of fetal calf serum. A2780, A2780 cis, A2780 ADR cell lines were cultured in RPMI 1640 media (Gibco) supplemented with 10% of fetal calf serum (Gibco). The resistance of A2780 cis was maintained by cisplatin treatment (1 μM) for one week every month. The cells were used in the assays after one week from the end of the treatment in order to avoid interfered results. The resistance of A2780 ADR was maintained by doxorubicin treatment (0.1 μM) once a week. Cells were used in the assays after three days post doxorubicin treatment in order to avoid interfered results. U87 cell line was cultured in MEM media (Gibco) supplemented with 10% of fetal calf serum (Gibco) and 1% of non-essential amino acid mixture (Gibco). Cell lines were complemented with 100 U/ml penicillin-streptomycin mixture (Gibco) and maintained in humidified atmosphere at 37° C. and 5% of CO.sub.2.

[0197] Cytotoxicity Assay

[0198] Cytotoxicity of the tested Ru compounds according to the present invention was assessed by a fluorometric cell viability assay using Resazurin (ACROS Organics). Briefly, cells were seeded in triplicates in 96-well plates at a density of 4×10.sup.3 cells/well in 100 μL in medium. After 24 h, cells were treated with increasing concentrations of the compounds. Compounds dilutions were prepared as follows: 2.5 mM stock in DMSO was further diluted to 100 μM with media and then filtrated. After 48 h incubation, medium was removed, and 100 μl of complete medium containing resazurin (0.2 mg/ml final concentration) was added. After 4 h of incubation at 37° C., the fluorescence signal of resorufin product was read (ex: 540 nm em: 590 nm) in a SpectraMax M5 microplate Reader. IC.sub.50 values were then calculated using GraphPad Prism software.

[0199] Results and Discussion

TABLE-US-00001 TABLE 1 IC.sub.50 values for compounds of the invention in tested cell lines; cisplatin and doxorubicin were used as positive controls. A2780 A2780 CT-26 IC.sub.50 (μM) HeLa A2780 ADR cis CT-26 luc RPE-1 Cisplatin 9.28 ± 0.20 4.00 ± 0.76  8.32 ± 0.71 18.33 ± 2.92  2.60 ± 0.18 2.42 ± 0.23 30.24 ± 5.11 Doxorubicin 0.34 ± 0.02 0.19 ± 0.03  5.94 ± 0.58 0.54 ± 0.04 0.082 ± 0.003  0.18 ± 0.006  0.89 ± 0.17 Ru(DIP).sub.2Cl.sub.2 15.03 ± 0.4  4.69 ± 0.14 78.27 ± 4.9  6.36 ± 0.57 9.20 ± 1.22 6.65 ± 0.5   3.13 ± 0.07 1 0.50 ± 0.01 0.67 ± 0.04 4.13 ± 0.2 0.45 ± 0.03 1.00 ± 0.03 1.51 ± 0.14  0.90 ± 0.04 2 0.45 ± 0.04 0.74 ± 0.05 2.86 ± 0.3 0.42 ± 0.01  0.6 ± 0.02 0.72 ± 0.07  0.86 ± 0.04 3 0.61 ± 0.07 0.20 ± 0.01  1.45 ± 0.14 0.39 ± 0.03 0.65 ± 0.04 0.42 ± 0.01  0.58 ± 0.01 4 0.124 ± 0.004 0.0261 ± 0.0005  0.70 ± 0.05 0.076 ± 0.005 0.067 ± 0.004 0.269 ± 0.007 0.764 ± 0.23 5 10.46 ± 0.25  10.23 ± 0.14  15.01 ± 0.75 17.17 ± 1.4  10.49 ± 0.5  11.64 ± 0.7  23.15 ± 2.5  6 0.353 ± 0.006 0.18 ± 0.03  1.05 ± 0.22 0.39 ± 0.07 0.31 ± 0.02 0.24 ± 0.01 0.67 ± 0.2 7 2.11 ± 0.12 0.53 ± 0.03  1.91 ± 0.08 0.80 ± 0.03 1.167 ± 0.15  1.147 ± 0.224 2.965 ± 0.45 8 10.03 ± 0.44  12.4 ± 0.8  18.63 ± 2.02 16.37 ± 2.04  7.61 ± 0.11 9.01 ± 0.19 16.55 ± 0.98 catechol >100 22.36 ± 5.83  >100 55.98 ± 9.46  .sup. 16 ± 4.14 11.5 ± 0.4  >100 Maltol 74.01 ± 14.6  >100 >100 >100 >100 >100 >100 4-methylcatechol >100 15.16 ± 1.0  29.27 ± 1.96 34.56 ± 1.49  34.37 ± 1.41  33.33 ± 3.4  >100 3-methoxycatechol 56.19 ± 4.18  18.71 ± 1.17  30.07 ± 1.35 36.54 ± 1.94  45.72 ± 4.21  36.39 ± 6.28  >100 Tetrabromocatechol 29.95 ± 1.60  8.75 ± 0.20 14.39 ± 1.18 13.63 ± 0.88  5.53 ± 0.37 3.80 ± 0.34 13.5 ± 1.7 3-methylcatechol >100 9.99 ± 1.26  2.71 ± 0.82 14.68 ± 0.69  17.47 ± 0.73  12.13 ± 1.40  >100 4-tertbutylcatechol 93.14 ± 9.8  9.14 ± 0.7  12.89 ± 1.20 14.62 ± 0.81  9.94 ± 0.63 9.72 ± 0.67 55.05 ± 4.64 4-nitrocatechol >100 30.90 ± 2.05  61.45 ± 3.63 >100 17.46 ± 1.02  15.31 ± 1.0   45 ± 19

[0200] It is striking that the 1 compound has IC.sub.50 values (the half maximal inhibitory concentration) in the nanomolar range for most of the cell lines investigated in this study, even for cisplatin resistant cell line with a value of almost 100 times lower than cisplatin itself. On the other hand, cytotoxicity of doxorubicin and compound 1 against the doxorubicin resistant cell line appear to be in the same order of magnitude. The analogue carrying the maltol group (2) shows comparable activity against most of the cell lines tested. Comparing 1 with the derivatives carrying the EDG (3, 4, 6, 7), it is clear how the electron density on the organic moiety plays a role. In general compounds 3, 4, 6, 7 present higher cytotoxicity in most of the cell lines tested. Of particular interest is compound 4 with an IC.sub.50 in the low nanomolar range against the cisplatin resistant cell line which makes it 10 times more active than doxorubicin and around 200 times more active than cisplatin. 4, unlike 1, presents an IC.sub.50 of 0.7 μM against the doxorubicin resistant cell line which is 10 times lower than 1 and cisplatin. Overall, compounds 1, 3, 4, 6, 7 display a cytotoxicity which is comparable to doxorubicin and which is much higher than cisplatin, with 4 showing the best potential due to its exceptionally low cytotoxicity and its great activity towards resistant cell lines.

[0201] compounds carrying the EWG groups (5, 8) show much less cytotoxicity than their charged analogues mentioned above with IC.sub.50 in the micro molar range for all the cell lines tested.

[0202] Catechol, catechol derivatives, maltol and the precursor complex Ru(DIP).sub.2Cl.sub.2 were tested against the same cell lines as a control. From these results, it is possible to conclude that the great activity shown by compounds 1, 2, 3, 4, 6 and 7 is the consequence of the coordination of the electron rich organic ligands to the main Ru(Ill) polypyridyl core.

[0203] 3. Cellular Uptake

[0204] To have more insights about cellular uptake efficiency and cellular accumulation of compound 1, ICP-MS experiments on HeLa and MRC5 cells were performed.

[0205] Sample Preparation for In Vitro ICP-MS.t

[0206] HeLa and MRC5 cells were seeded a week before treatment at a concentration of 1×10.sup.6 cells/ml in 15 cm.sup.2 cell culture Petri dish, let grow until 80% of confluence and incubated with the target compound (previously dissolved in ethanol as vehicle, v/v<0.1%) at a concentration of 5.0 μM for 6 hours.

[0207] Working concentration and incubation time have been chosen in order to avoid extended cell mass lost due to the high cytotoxicity of the compounds but considering a Ru final amount that could afford a significant determination of the metal content. All the treatments were set up at the same concentration for comparative purposes. The medium was then removed, the cells washed with PBS and trypsinized. After re-suspension in PBS, the pellet was washed with ice cold PBS and collected per centrifugation (5910R, Eppendorf) at 500 g for 5 min at 4° C. The organelles were then isolated via differential centrifugation. To separate lysosomes, a Lysosome Isolation Kit (Cat. Nr: LYSISO1, Sigma Aldrich) as well as the manufacturer procedure with minor modification was used. Briefly, the collected pellets were redissolved in 2.0 ml of extraction buffer added with proteases cocktail (delivered with the kit) and incubated for 15 min on ice. The samples were then homogenized with a pre-chilled dounce homogenizer (7 ml, tight pestle A, 22 strokes) and centrifuged at 1000 g for 10 min at 4° C. After homogenization, the pellet obtained was redissolved in 2 ml of a sucrose solution (0.25 M sucrose, 10 mM MgCl.sub.2) and layered with 2 ml of a second hypertonic sucrose solution (0.55 M sucrose, 0.5 mM MgCl.sub.2). The suspension was centrifuged at 1450 g and 4° C. for 5 min. The pellet was resuspended in 3 ml of the second sucrose solution and centrifuged at 1450 g and 4° C. for 5 min to obtain the nuclear extract. These steps of the isolation procedure were monitored under phase contrast microscope on Menzel-Glaser coverslips (Olympus IX81 microscope).

[0208] The supernatant was transferred in a fresh tube and centrifuged at 14000 g for 30 min at 4° C. The pellet collected, dissolved in 1 mL of a 19% Optiprep Density Gradient Medium (present in the kit), added with a 250 mM solution of CaCl.sub.2 (final concentration 8 mM) and centrifuged at 5000 g for 15 minutes at 4° C. to give the isolated lysosomal suspension. The intact lysosomal content was proofed with Neutral-Red Reagent (delivered in the kit) with dual-wavelength absorbance mode at 460 and 510 nm over a kinetic of 3 minutes in a Spectramax M5 microplate reader (Molecular Devices).

[0209] Mitochondria fractions were isolated via further differential centrifugation from the pellet non-containing lysosomes using a mitochondria extraction buffer (Cat. Nr.: E2778, Sigma Aldrich) following the manufacturer instructions and a recent public procedure. The samples were re-dissolved in 1.5 mL of extraction buffer and centrifuged at 11000 g for 10 minutes at 4° C. to obtained final pellets represented pure mitochondrial fractions.

[0210] All the fractions (mitochondria, lysosomes and nucleus) were isolated from the same cellular sample for direct comparative purposes. The supernatant phases discarded during the isolation of nuclei, lysosomes and mitochondria procedures were collected and formed the “residual” fraction. An aliquot of crude lysate after homogenization, nuclear, mitochondrial (pellet lysed via freeze and thaw cycles followed by 20 minutes incubation in ultrasonic bath), lysosomal and residual fraction was each used for protein quantification using the Bradford method. The isolated samples were then lyophilized on an Alpha 2-4 LD plus (CHRIST). The resulting samples underwent chemical digestion with 10 mL of a 2% nitrohydrochloric acid solution for 24 h. The resulting suspensions were filtered on 0.20 μm non-pyrogenic sterile Filtropur filters (Sarstedt) and the obtained samples were injected in ICP-MS.

[0211] In Vitro ICP-MS Studies.

[0212] Ruthenium was measured against a Platinum single element standard (Merck 1703410100) and verified by a control (Agilent5188-6524 PA Tuning 2). Ruthenium content of the samples was determined by means of a 7-step serial dilution in the range between 0 and 300 ppb in Ru (R>0.99) with a background equivalent concentration of BEC: 6.3 ppt and a detection limit of DL: 17 ppt. Spiking the samples with untreated negative controls (to account for eventual carbon content from the biological samples) resulted in equivalent values within error ranges. A solution of Indium (500 ppb) and Tungsten (500 ppb) was used as internal standard. The results are expressed as ng Pt/mg protein (correction due to the different mass of the observed cellular compartments), as mean±standard deviation error of different independent experiments.

[0213] Results and Discussion

[0214] To have more insights about the possible mode of action of compound 1 and its different activity between healthy and cancer cells the, ICP-MS experiments on HeLa and MRC5 cells were performed. Near complete internalization was found to occur in HeLa after 2 h with preferential accumulation inside the nucleus (see FIG. 1, right). The uptake is consistent with similar studies on ruthenium polypyridyl complexes which suggests a passive diffusion mechanism due to the lipophilicity inferred by the large aromatic ligand scaffold (Puckett, C. A. et al., 2008). Furthermore, polypyridyl containing ruthenium complexes are capable of intercalating with the DNA (Puckett, C. A., 2009). Preferential accumulation inside the nucleus suggests that the mode of action is related to the damage of the DNA and/or prevention of replication as well as transcription. The same ICP-MS experiment was repeated in MRC-5 (normal lung fibroblasts) cell line (FIG. 2). In this case, less than 25% of the tested compound was taken up by healthy cells. This interesting behaviour strongly suggests a preference for compound 1 to accumulate into cancer cells.

[0215] 4. Cell Death Mechanism

[0216] To further understand the mode of action of compound 1, it was important to evaluate the type of cell death that it was causing. For this experiment, Annexin V and PI staining method were utilised on HeLa cells..sup.15 Staurosporin, a known inducer of apoptosis, was used as a positive control.

[0217] Material and Methods

[0218] Annexin V/PI Assay.

[0219] The apoptosis and necrosis induction in HeLa cells treated with 3 was evaluated via by means of the well-established AnnexinV/PI assay using flow cytometry and according to the manufacturer instructions with just minor changes (Sigma Aldrich, Cat. No:APOAF). Briefly, the cells were seeded in a 100×15 mm Petri dish at a concentration of 2×10.sup.5 and cultured at 37° C./6% CO.sub.2 for 24 h. The medium was then removed and replaced with fresh medium containing 10 μM solution of medium containing compound 1 and further incubated for 30 min, 4 h or 24 h. The cells were then trypsinized and pelleted, washed twice with ice cold PBS, centrifuged at 600 g for 5 minutes, resuspended in 500 μL binding buffer (provided with the kit) and transferred FACS culture tube. 7.5 μL of Annexin V FITC complex solution (provided with the kit) and 10 μL of propidium iodide (PI) solution (provided with the kit) were added. The samples were incubated for 15 minutes at room temperature (25° C.) in the dark, 1000 μL of binding buffer were added and the probes analyzed with a CynAn ADP9 flow cytometer with the FITC (for Annexin V-FITC, excitation=488 nm, emission=515-545 nm) and PE-Texas Red channels (for PI, excitation=488 nm, emission=564-606 nm) and. The data were analyzed with Summit v4.3 software. Positive controls of cells treated with 0.1 μM or 1.0 μM of staurosporine for 4 h or 24 h recovery respectively were performed.

[0220] Results and Discussion

[0221] As shown in FIG. 3, even 30 min after treatment with compound 1 induces apoptosis in treated cells. Further incubation of the cells with the Ru compound (4 h or 24 h) still points out for the apoptosis as a main type of the cell death.

[0222] 5. Metabolic Studies

[0223] The preference of compound 1 to accumulate in the nucleus and mitochondria in HeLa cells inspired further studies on metabolic pathways that could be affected by the compound. For this purpose, Seahorse XF Analyzer was used. Firstly, the effect of compound 1 on the mitochondrial metabolism (oxidative phosphorylation) in CT-26 cell line was investigated. The effect on the other metabolic pathways, i.e. glycolysis and the possible metabolic modulation of the three primary fuel pathways, i.e. glucose, glutamine and fatty acid were then examined.

[0224] Material and Methods

[0225] Mitostress Test.

[0226] CT-26 cells were seeded in Seahorse XFe96 well plates at a density of 30,000 cells/well in 80 μL. After 24 h, the media was replaced with fresh media and cisplatin (10 μM), catechol (10 μM), Ph.sub.2Phen (1 μM), complex Ru(DIP).sub.2Cl.sub.2 (10 μM) or compound 1 (1 μM) were added. After 24 h of incubation, the regular media was removed and the cells were washed thrice using bicarbonate and serum free DMEM, supplemented with glucose, 1.8 mg/mL; 1% glutamine and 1% sodium pyruvate and incubated in a non-CO.sub.2 incubator at 37° C. for 1 h. Mitostress assay was run using Oligomycin, 1 μM, FCCP 1 μM and mixture of Antimycin-A/Rotenone 1 μM each in ports A, B and C respectively using Seahorse XFe96 Extracellular Flux Analyzer.

[0227] Seahorse Glycolytic Stress Test.

[0228] CT-26 cells were seeded in Seahorse XFe96 well plates at a density of 30,000 cells/well in 80 μL. After 24 h, the media was replaced with fresh media and cisplatin (10 μM), catechol (10 μM), Ph.sub.2Phen (1 μM), complex Ru(DIP).sub.2Cl.sub.2 (10 μM) or compound 1 (1 μM) were added. After 24 h of incubation, the regular media was removed and the cells were washed thrice using bicarbonate, glucose and serum free DMEM, supplemented 1% glutamine and 1% sodium pyruvate and incubated in a non-CO.sub.2 incubator at 37° C. for an hour. Glycolytic stress test was run using glucose, 10 mM, Oligomycin, 1 μM and 2-Deoxyglucose, 50 mM in ports A, B and C respectively using Seahorse XFe96 Extracellular Flux Analyzer.

[0229] Fuel Flex Assay

[0230] CT-26 cells were seeded in Seahorse XFe96 well plates at a density of 30,000 cells/well in 80 μL. After 24 h, the media was replaced with fresh media and cisplatin (10 μM), catechol (10 μM), Ph.sub.2Phen (1 μM), complex Ru(DIP).sub.2Cl.sub.2 (10 μM) or compound 1 (1 μM) were added. After 24 h of incubation, the regular media was removed and the cells were washed thrice using bicarbonate, and serum free DMEM, supplemented with 1.8 mg/mL glucose, 1% glutamine and 1% sodium pyruvate and incubated in a non-CO.sub.2 incubator at 37° C. for an hour. Fuel flex assay for the different fuel pathways viz. glucose, glutamine and fatty acid was studied by measuring the basal oxygen consumption rates and that after addition of the inhibitor of the target pathway in port A and a mixture of the inhibitors of the other two pathways in port B. This gave a measure of the dependency of the cells on a fuel pathway. To study the capacity of a certain fuel pathway, the sequence of addition of the inhibitors was reversed. In port A was added the mixture of inhibitors for the other pathways and in port B was added the inhibitor for the target pathway. UK-5099 (pyruvate dehydrogenase inhibitor, 20 μM) was used as an inhibitor for the glucose pathway. BPTES (selective inhibitor of Glutaminase GLS1, 30 μM) was used as an inhibitor for the glutamine pathway. Etomoxir (O-carnitine palmitoyltransferase-1 (CPT-1) inhibitor, 40 μM) was used as an inhibitor for the fatty acid pathway.

[0231] Results and Discussion Mitochondrial respiration was found to be severely impaired in cells treated with compound 1 as opposed to the precursor of complex 1. This was clear from the low basal respiration, compared to the untreated cells. ATP production was also inhibited by compound 1. The mitochondrial membrane lost the capacity to restore the proton balance when treated with an uncoupling agent FCCP. All these effects point to the disrupted mitochondrial respiration in mouse colon carcinoma caused by the complex. In contrast, the glycolysis of the cells, which is a cytosolic process, is not affected by compound 1. Moreover, no direct effect on the 3-primary fuel pathways was shown by compound 1.

[0232] 6. In Vivo Studies

[0233] Next, in vivo studies were performed where both the effect on tumour growth and that on the survival of tumour-bearing mice of an immunocompetent strain were evaluated. The doses were selected according to the dose-finding study, which had revealed a maximum tolerated dose (MTD) of 15 mg/kg of body weight.

[0234] Material and Methods

[0235] Animals and Tumour Model.

[0236] Due to the poor solubility of compound 1 in water, dimethyl sulfoxide (DMSO), 1.81 ml/kg of body weight, had to be added to water for injections, for which reason the i.p. route of administration was chosen rather than i.v. Female outbred mice (NMRI) were used for this study, they were obtained from Masaryk University (Brno, Czech Republic). Animal care was conformed to EU recommendations and in accordance with the European convention for the protection of vertebrate animals used for experimental and other scientific purposes; it was approved by the Ethical Commission of the Medical Faculty in Hradec Kralove (Nr. MSMT-56249/2012-310). For the MTD assessment, two or three healthy mice per group were observed for weight loss (the limit was 10%) over 14 days after injection of the solution. For the in vivo activity study, 70 NMRI female mice weighting in the average 31.8 g (SD=1.27) were fed a standard diet and water ad libitum. A solid Ehrlich tumour was purchased from the Research Institute for Pharmacy and Biochemistry (VUFB) in Prague, and then maintained in NMRI mice by periodical transplantations. The homogenised tumour tissue was inoculated subcutaneously into all mice on day 0, using 0.2 ml of 1/1 (v/v) homogenate freshly prepared in isotonic glucose solution. The tumour-bearing mice were then divided into 5 groups of 14 animals as follows: a control group treated with the pure solvent (DMSO and water), 3 groups of animals treated with compound 1 in doses of 5, 10, and 15 mg/kg i.p. and a positive control receiving 5 mg/kg cisplatin i.p. (Cisplatin 50 ml/25 mg, EBEWE Pharma, Austria). The solutions were administered on days 1 and 7 in volumes of 0.2 ml per 20 g body weight. On the tenth day, half of the mice were sacrificed, and their tumours were weighed. The remaining animals were left in order to observe their survival.

[0237] Statistical Analysis.

[0238] One-Way Analysis of Variance with post-hoc Dunnetts's multiple comparison test was used to detect differences in tumour weight. Kaplan-Meier curves (FIG. 4) and logrank tests were used to compare survival times in groups. Here, the level of significance was α=0.05. MS Excel 2003 and NCSS software was used for the calculations and statistical evaluations.

[0239] Results and Discussion

[0240] During the study of the effect on the survival of tumour-bearing mice, it was observed that the geometric mean of the overall survival of tumour bearing mice without therapy was 20.6 days. Among the three doses of compound 1 tested, only 5 mg/kg prolonged the survival time significantly when compared with untreated tumour-bearing control mice (geom. mean=31.9 days, P=0.033). 10 mg and 15 mg/kg of compound 1 seemed to exceed the optimal dose, prolonging the survival insignificantly (P>0.05), with the geometric means of 30.2 and 25.5 days, respectively. The positive control cisplatin was effective too (geom. mean=33.7 days, P 0.014). An interesting and rare phenomenon was observed in all three groups of compound 1. Although the tumour was advanced in the later stage of the experiment, all mice treated with compound 1 showed active behaviour, little cachexia and unsuppressed food consumption.

[0241] Furthermore, the effect of compound 1 on tumour growth was also investigated. FIG. 5 shows the weight of tumours in mice treated with mixture of co-solvent and water, compound 1 at 5, 10 or 15 mg/kg, or cisplatin 5 mg/kg measured on day 10 and documents differences in the effect of the used drugs. Although only cisplatin exhibited a significant inhibitory effect on tumour growth (P=0.0011), there was a slight but insignificant suppression at 5 mg/kg compound 1 (P=0.108). As in the survival study, also here the optimum dose of compound 1 seems to be in the lower part of the range tested.

[0242] 7. Encapsulation

[0243] Formulation Protocol

[0244] Compound 4 was formulated in polysorbate 80 using the film rehydration method disclosed in Zeng, Z. W. et al., 2010. Briefly, compound 4 (2 mg, 2.2 μmol) and polysorbate 80 (100 mg) were dissolved in acetone (3 mL). The solvent was removed by rotary evaporation at 40° C. The red film was then resuspended in PBS (2 mL) at room temperature. The solution was finally sterile filtered on a 0.20 μm nylon membrane (Corning© 431224) to yield a clear red solution.

[0245] Compound Concentration Determination

[0246] 50 μL of the sample was diluted in 100 μL of acetonitrile and the absorbance was recorded at 480 nm in 96 wells plates from Corning© (Fisher Scientific 15329740) using a SpectraMax M5 microplate Reader. The measure was performed in triplicates and compound 4 concentration was determined using a calibration curve obtained in the same conditions (50 mg/mL polysorbate 80 in PBS/acetonitrile 1:3).

[0247] Encapsulation efficiency was calculated by comparing the absorbance of the solution before and after filtration using the following equation.

[00001]$\mathrm{Encapsulation}\mathrm{efficiency}=100*\frac{\mathrm{Absorbance}\mathrm{after}\mathrm{filtration}}{\mathrm{Absorbance}\mathrm{before}\mathrm{filtration}}$

[0248] To ensure its repeatability, the procedure was performed in triplicate.

[0249] Results

[0250] 0.84±0.06 mg/mL of compound 4 has been dissolved in 50 mg/mL of polysorbate 80, with an encapsulation efficiency of 95±3%.

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