Thermodynamics and Mechanism of Action of a Series of Organoruthenium Anticancer Reagents

International Journal of Advance in Medical Science (AMS) Volume 1 Issue 4, November 2013

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Thermodynamics and Mechanism of Action of a Series of Organoruthenium Anticancer Reagents Moussa Ali1, Bastien Boff1, Jean-Thomas Issenhuth1, Anne Boos2, Noëlle Potier1, Sébastien Harlepp3, JeanPhilippe Loeffler4 and Claude Sirlin1* Institut de Chimie de Strasbourg UMR 7177 CNRS/ Université de Strasbourg, 4, rue Blaise Pascal 67070 Strasbourg (France) 2ECPM, 25, rue Becquerel 67087 Strasbourg (France) 3IPCMS UMR 7504 CNRS/Université de Strasbourg 3, rue de l’Université 67084 Strasbourg (France) 4Laboratoire de Signalisations moléculaires et Neurodégénérescence U692 INSERM/Université de Strasbourg 11, rue Human 67000 Strasbourg (France) 1

sirlin@unistra.fr Abstract Antiproliferative effects on HCT-116 and U-87 cells by a series of organoruthenium complexes have been determined.

Redox properties have been measured as well. The highlighted binomial relationship between the biological activity and the oxidation potentials was interpreted as two competitive reactions of oxidation and reduction of the metallic centre. The oxidation of Ru (II) in Ru (III) complexes presumably by oxygen has been observed by UV-vis spectrophotometry and mass spectrometry. The reduction ofRu (III) in Ru (II) complexes is performed by reducing compounds from the cell (observed with glucose).

Keywords Organoruthenium; Anticancer; Redox

Introduction Cis-platin has become one of the three most widely used drugs in chemotherapy and is highly effective in the treatment of ovarian and testicular cancers.

However, cis-platin displays three major drawbacks that are severe neurotoxicity and nephrotoxicity, and limited applicability to a narrow range of tumours exhibiting natural or induced resistance (Wong 1999).

In the search for new cancer therapies, compounds that avoid these drawbacks are required. New platinum and non-platinum complexes including metallocenes (titanium (IV)), gold (I) complexes and gallium (III) salts have been considered as alternatives to cis-platin.

(Clarke 1999, Guo 1999) Special attention has been paid to ruthenium complexes. Ruthenium (III) complexes such as trans-[ruthenium(imidazole)(Me2SO)Cl4] (imidazole)+, NAMI-A, and trans-[ruthenium (indazole)2Cl4] -(indazole)+, KP1019, have recently entered phase II

clinical trials as antimetastatic drugs. (Hartinger 2006, Mestroni 1993, Sava 1999) Biological activities of ruthenium (II) compounds have been measured as well. Arene-ruthenium (II) complexes exhibit in vitro and in vivo anticancer activities. (Aird 2002, Habtemariam 2006, Morris 2001) Related complexes reduce the growth of lung metastases in mice bearing mammary carcinoma. (Dyson 2006, Scolaro 2005) High cytotoxicity of bis-(2-phenylazopyridine) ruthenium(II) complexes against carcinoma cells has also been established. (Hotze 2005, Velders 2005) Importantly, in all of these complexes N,O ligands are weakly bound to the metal. In contrast to these ruthenacycles, in which the ligand is strongly bound to the metal via a covalent bond, have been prepared and tested. (Fetzer 2011, Leyva 2007, Leyva 2008, Meng 2009, Vidlmar 2012) The aim of the present work was to identify the major factors governing the biological activities of the ruthenacycles. From our previous molecular library, it was not possible to establish a correlation between the anticancer activities and some physicochemical properties, like stereochemistry or simple electronic factors. It was, nevertheless, concluded that the main, but very important factor that contributed to the biological activity, was the presence of the rutheniumcarbon bond in the molecular structure. Consequently, chemical reactivity, i.e. redox properties, was thought to be involved in the biological activity and was measured. How redox properties of metal complexes are involved in anticancer activity has been questioned.

(Jungwirth 2011)

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International Journal of Advance in Medical Science (AMS) Volume 1 Issue 4, November 2013

N

N

N

Ru N PF6 N N

N

N

Ru N PF6 N N

N N Ru P N PF6 N N

N

N

N

N

N

N

N

Ru

N

Ru

PF6

N

N

N PF6

1

3

2

Br

Br N

N

N N

N

Ru

Ru

N

N

N 8

N

N O2N CF3SO3

N

N

10

9

N

N Ru

N CF3SO3

7

N

N N

N Ru

PF6

N

PF6 6

N

N N

N N

5

N

N

N

Ru N PF6 N N

4

N

N

N

Ru

Ru

N

Ru

N

N N

N

H2N CF3SO3 11

N N

N

13

Cl Ru N

19

N N

Ru Cl H2N PF6 NH2

PF6

N

14

Ph2 P

Ph2 P

N

Cl

N

PF6

Cl

21

20

Ru

Ru

P Ph2

N

N

N

N 2Cl

N

O

N

N

Au P Ph2

N

N

CF3SO3

N N

23

22

Chart 1

Results and Discussion A molecular library has been constructed that was completed by the three reference compounds, the bischloride bis-(2-phenylazo pyridine) ruthenium (II) 19, the chloride [(p-cymene)(ethylene-bis-amine)]ruthenium (II) 20, and the bis-[1,2-bis-(diphenyl-phosphane)ethane] gold (I) chloride 21.

TABLE 1

human adenocarcinoma, and of U-87, derived from a human glioblastoma. The viability of the cells was determined by measuring the activity of the mitochondrial succinic dehydrogenase of the remaining living cells. The results obtained with the two different cell lines are summarized in Table 1.

TABLE 2 OXIDATION POTENTIALS VERSUS SCE MEASURED BY CYCLIC VOLTAMPEROMETRY

Conditions: CH3CN, [nBu4NBF4] = 0.1 M, v = 100 mVs-1, rt

IC50 (μM) VALUES ON THE TWO CELL LINES FOR THE

Complexes 1 2 3 4 5 6 7 8 9 10 11 13 14 19 20 21 22 23

ORGANORUTHENIUM COMPLEXES AND REFERENCE COMPOUNDS

Complexes 1 2 3 4 5 6 7 8 9 10 11 13 14 19 20 21 22 23

IC50 in M (tumour cells) HCT-116 U-87 >50 >50 3±2 10±2 >50 >50 0.7±0.2 0.1±0.05 1±0.5 1±0.5 2.5±0.5 0.2±0.03 1±0.5 0.5±0.2 0.7±0.2 0.1±0.05 2.5±0.5 1±0.5 5±1 5±2 10±2 3±1 0.5±0.2 5±2 20±5 1±0.5 >250 >250 15±5 35±10

The anticancer properties of the constituents of the library were evaluated by measuring their effect on the cell proliferation of HCT-116, derived from a

46

a

E0 (RuL+/RuL0) V -1.63 -1.59 -1.63 -1.57 -1.58 -1.49 -1.23 -1.45 -1.61 -1.01 -1.73 -1.33 -1.51

E0 (RuIII /II) V +0.71 +0.56 +0.70 +0.59 +0.53 +0.62 +0.54 +0.43 + 0.41 + 0.69 + 0.20 +0.31 +0.49 +0.94 +1.50 +0.60a +1.35 +0.53

E0 (NR3+/0) V

+0.97

+1.02

+ 0.75 +1.36

redox couple AuIII/I

More complete results were obtained for the cell line HCT-116 that will be considered for discussion. Except 1, 3, 20, 22 and 23, IC50 values are in the 0.1 - 3 µM range. The IC50 measured for the compounds 22 and 23 without a ruthenium-carbon bond are 2,500 and 150

International Journal of Advance in Medical Science (AMS) Volume 1 Issue 4, November 2013

times higher than that of 9, respectively. As the redox properties of the complexes are thought to be involved in the biological activity, the electrochemical data of the complexes were measured (Table 2). The anodic region is dominated by reversible waves corresponding to the one-electron oxidation of the ruthenium (II) state, whereas the cathodic region exhibits poorly defined or irreversible waves resulting from the reduction of the ligands. The effect of the cyclometalation on the stability of the ruthenium (III) state is illustrated by a cathode shift of 0.94 V going from complex 22 to 9.

Unsubstituted complexes of the type[N^C, (N^N)2], [N^C^N, N^N^N] display redox potentials in the 0.41 - 0.53 V range. Substitution by an electron-donor like NH2 induced a cathode shift of 0.19 V while substitution by an electron-acceptor like NO2 induced an anode shift of 0.28 V. Oxidant 1

2 Ru II

Oxidant 2

Reductant 1

2 Ru III

Reductant 2

Scheme 1

As IC50 values range from 0.1 to over 250ÂľM, only the values lower than 15 ÂľM were retained for clarity.

Figure 1 presents a binomial relationship between the anticancer and electrochemical properties. Previous studies have determined redox potentials, but no correlation with IC50 was performed (Mestroni 1993,

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Sava 1999). The binomial function is interpreted as the existence of two competitive reactions of oxidation and reduction as seen in scheme 1.

Under air, aqueous solutions of ruthenium (II) complexes yield ruthenium (III) complexes characterized by UVvisible spectrophotometry and mass spectrometry; and the process is instantaneous in acid solutions. In a biological medium, Ru (II) complexes will be oxidized by oxygen. The reduction of Ru (III) complexes by reductants of the cell has been observed with glucose.

(Ryabov 2005, Ryabov 2001) As a result organoruthenium complexes act as a mediator of the cellular metabolism.

Membrane Crossing and Interaction with the Nucleic Acids To be active, the complexes must cross the cell membrane; and attention was paid to the lipophilicity of the series. Binomial relationships have been established between biological activities and lipophilicity.

(Clarke 1999, Sava 1999) Cellular uptake (and animal distribution) of organoruthenium complex 2 has been determined by ICPMS. Preliminary experiments were performed by mass spectrometry and showed that in contrast to platinum complexes, there was no covalent interaction between the complex 2 and DNA. Further experiments, performed by FRET and optical tweezer, were able to measure the dissociation constant of the complex between 2 and DNA. The organoruthenium complex intercalates between base pair. (Klajner 2010).

FIGURE 1 BINOMIAL RELATIONSHIP BETWEEN THE ANTICANCER PROPERTIES AND THE OXIDATION POTENTIAL

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International Journal of Advance in Medical Science (AMS) Volume 1 Issue 4, November 2013

Klajner M,Hébraud P, Sirlin C,Gaiddon C,Harlepp S J Phys

Conclusion Combining anticancer activities and physicochemical properties of a molecular library of organoruthenium complexes, it has been established that this class of compounds acts as a mediator of the cellular metabolism.

Furthermore membrane crossing and interaction with nucleic acids could be studied as the complexes are fluorescent.

Chem B 2010, 114, 14041.

Leyva L, Sirlin C, Rubio L,Franco C, Le Lagadec R, Spencer J, Bischoff P,Gaiddon C, Loeffler JP, Pfeffer M Eur. J. Inorg.

Chem. 2007, 3055.

Leyva L, Malek F,Benzina S, Denis JM , Guelette J, Dufour P, Gaiddon C, Loeffler JP, Sirlin C, Pfeffer M, Bischoff P Lett Drug Design & Discovery 2008, 5, 1.

Meng X, Leyva L,Benosman S, Fricker B, Harlepp S,Hebraud P,Boos A, Bischoff P, Sirlin C, Pfeffer M,Loeffler

ACKNOWLEDGEMENTS

JP,Gaiddon C Cancer Res 2009, 69, 5458.

Financial support from the CNRS, Region Alsace and ANR PhotoBioMet (ANR-09-BLAN-0191-01) is acknowledged.

Mestroni G, Alessio E, Sava G, Pacor S, Coluccia M,

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