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Metals in Medicine: Research

Organometallic anticancer complexes

 

 

We are studying the design and mechanism of action of ruthenium and osmium arene anticancer complexes. Our research involves the synthesis and full characterization of new organometallic complexes, the study of their coordination chemistry and the determination of relevant kinetic and thermodynamic parameters. In addition, we study their reactivity towards biomolecules and determine their cytotoxicity both in vitro and in vivo. The principal techniques used to study these complexes are multidimensional, multinuclear NMR, (protein) X-ray crystallography, advanced mass spectrometry and cytotoxicity testing against various cancer cell lines.

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Ruthenium-Arene Complexes


The organometallic anticancer complexes typically have a half-sandwich “piano-stool” structure, but full-sandwich complexes are studied as well. We found that such half-sandwich ruthenium complexes often possess good aqueous solubility (an advantage for clinical use) and the arene is relatively inert towards displacement under physiological conditions. The arene furthermore stabilizes the +2 oxidation state, which is thought to be the relevant one for anticancer activity.


Structure and Activity

The complexes consist of three main building blocks, the arene, a monodentate ligand and a bidentate, chelating ligand. In aqueous media, the monodentate ligand acts as a leaving group to form the more reactive aqua complex. This hydrolysis is thought to be essential for anticancer activity, as complexes that do not hydrolyse tend to be inactive or weakly active. The rate of hydrolysis can span several orders of magnitude, depending on the nature of the monodentate ligand. Understanding the thermodynamics and kinetics of this aquation is therefore of prime importance. In addition, a bidentate chelating ligand seems of key importance for achieving high cytotoxicity. Ethylenediamine (en) is usually employed as the bidentate ligand, and the N-H bonds were found to be essential, indicating their involvement in hydrogen bonding interactions. Intriguingly, replacement of en by acac or a bipyridine diol, for instance, results in complexes with markedly increased cytotoxicity. These results illustrate that correlating activity with the structure of the chelating ligand is quite complex. Finally, the size of the arene ligand can be changed and it was found that activity appears to increase with the size of the coordinated arene. This observation fits to their presumed involvement in stacking interactions with DNA bases (see below). The systematic variation of these main building blocks thus allows for the fine-tuning the pharmacological properties of the ruthenium-arene anticancer complexes. Establishing such a structure-activity relationship is one of the main goals of this research.

Cytotoxicity

The ruthenium-arene complexes show reproducible cytotoxic activity against A2780 human ovarian cancer cells in vitro. A recent study of evaluated the activity of [(eta6-bip)Ru(en)Cl]+ in a 13-cell line panel. Particular sensitivity was noted in breast cancer and non-small cell lung cancer cell lines. These in vitro activity patterns were mirrored to a large degree in vivo with the compound effecting significant growth delays against both cisplatin-sensitive and –resistant human ovarian A2780 tumours grafted on mice (xenografts).

Interactions with Biomolecules: DNA

In biological systems, the ruthenium-arene anticancer complexes will encounter a vast array of biomolecules with which they could potentially interact. Hence, it is of great importance to gain a detailed understanding of such interaction with ligands ranging from nucleobases, oligonucleotides, DNA, amino acids, proteins and other cellular components. The study of these interactions forms a major component of our research in this field.

Binding studies with nucleobases are of special interest since DNA is the primary target of cisplatin and the ruthenium-arene anticancer drugs could operate through the same mechanism of action. Nucleoside binding studies show a strong preference of [(eta6-bip)Ru(en)]2+ for binding to N7 of guanine (G), a selectivity that seems to be enhanced by the specific hydrogen bonding interactions of the NH2-groups of en with exocyclic oxygens of G. Similar observations (strong preference for G over A) were made for the mononucleotides as for nucleosides. The binding preference, however, does strongly depend on the chelating ligand, as replacement of en by acac resulted in equal binding to guanosine and adenosine. Interestingly, the acac complex does retain its in vitro activity. These results illustrate that direct coordination to the bases, intercalation, and stereospecific H-bonding are useful features to incorporate into the design of ruthenium-arene complexes to optimise the recognition of DNA.
Indeed, comparative studies have shown that [(eta6-arene)Ru(en)Cl]+ complexes bind relatively rapidly to calf thymus DNA, preferentially at the G sites, and effectively inhibits DNA-directed RNA synthesis in vitro. Circular dichroism and flow linear dichroism studies, combined with melting temperature measurements provided strong evidence for combined intercalative and coordinative binding modes with the biphenyl and anthracene complexes. Oligonucleotide binding studies using a 14-mer DNA duplex also revealed unique modes of binding of the anticancer complex, including coordinative binding to G sites and intercalation. The specificity is guided by hydrogen bonding and stacking interactions. Further DNA binding studies in collaboration with Victor Brabec’s group have provided support for a different mechanism for antitumour activity of the ruthenium-arene complexes compared to cisplatin.

Interaction with Biomolecules: Proteins

The multitude of proteins that the anticancer drugs might encounter also constitutes an important target and may also play a role in the mechanism of action of the ruthenium-arene complexes. The interaction of [(eta6-arene)Ru(en)Cl]+ complexes with, for instance, cytochrome c and lysosyme has been studied. In the latter case, a X-ray crystal structure showed selective binding of the complex to a single histidine residue. In comparison, however, proteins seem to have a lower reactivity towards the ruthenium complexes than DNA. This may account for the low toxic side-effects and may aid in transport and delivery of the anticancer agent.

The catalytic properties of the Ru-arene complexes have also been explored and the complexes were found to catalyse regioselective reduction of NAD+ under biological conditions.

   
     

Ru-Arene Anticancer Complex

A typical half-sandwich ruthenium(II) anticancer complex

     
    Multiple Interactions in 9-EtG Binding
Multiple Interactions in 9-EtG binding to a Ruthenium-Arene
     
   

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A molecular model of a ruthenated 14-mer DNA duplex illustrates the coordinative binding and intercalation

   

 

   

Ru-Arene Lysozyme Complex
Crystal structure of a half-sandwich arene Ru-enzyme complex

     

 


Osmium-Arene Complexes

 

We have recently extended this chemistry to osmium, the heavier congener of ruthenium. Completely different reaction kinetics are to be expected for the Os(II) arene complexes. The aqueous and hydrolytic chemistry of osmium analogues of the half-sandwich ruthenium(II) complexes were studied by systematic variation of the chelating ligand on osmium and was found to differ substantially from the analogous ruthenium complexes. The rational control of the chemical reactivity of the osmium complexes, however, did allow us to design cytotoxic osmium-arene complexes with promising activity against human A549 and A2780 ovarian cancer cells.

   
     

Osmium -Nucleobase Adduct

Nucleobase adduct of an osmium-arene half-sandwich complex

 

Selection of our recent publications on our organometallic anticancer complexes:
 
 1.     Yan YK, Melchart M, Habtemariam A, Sadler PJ: Organometallic chemistry, biology and medicine: ruthenium arene anticancer complexes. Chemical Communications 2005:4764-4776
 2.     Morris RE, Aird RE, Murdoch PD, Chen HM, Cummings J, Hughes ND, Parsons S, Parkin A, Boyd G, Jodrell DI, et al.: Inhibition of cancer cell growth by ruthenium(II) arene complexes. Journal of Medicinal Chemistry 2001, 44:3616-3621.
 3.     Habtemariam A, Melchart M, Fernandez R, Parsons S, Oswald IDH, Parkin A, Fabbiani FPA, Davidson JE, Dawson A, Aird RE, et al.: Structure-activity relationships for cytotoxic ruthenium(II) arene complexes containing N,N-, N,O-, and O,O-chelating ligands. Journal of Medicinal Chemistry 2006, 49:6858-6868.
 4.     Wang FY, Habtemariam A, van der Geer EPL, Fernandez R, Melchart M, Deeth RJ, Aird R, Guichard S, Fabbiani FPA, Lozano-Casal P, et al.: Controlling ligand substitution reactions of organometallic complexes: Tuning cancer cell cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America 2005, 102:18269-18274.
 5.     Chen HM, Parkinson JA, Parsons S, Coxall RA, Gould RO, Sadler PJ: Organometallic ruthenium(II) diamine anticancer complexes: Arene-nucleobase stacking and stereospecific hydrogen-bonding in guanine adducts. Journal of the American Chemical Society 2002, 124:3064-3082.
 6.     Chen HM, Parkinson JA, Morris RE, Sadler PJ: Highly selective binding of organometallic ruthenium ethylenediamine complexes to nucleic acids: Novel recognition mechanisms. Journal of the American Chemical Society 2003, 125:173-186.
 7.     Liu HK, Berners-Price SJ, Wang FY, Parkinson JA, Xu JJ, Bella J, Sadler PJ: Diversity in guanine-selective DNA binding modes for an organometallic ruthenium arene complex. Angewandte Chemie-International Edition 2006, 45:8153-8156.
 8.     Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, Sadler PJ, Jodrell DI: In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer. British Journal of Cancer 2002, 86:1652-1657.
 9.     Guichard SM, Else R, Reid E, Zeitlin B, Aird R, Muir M, Dodds M, Fiebig H, Sadler PJ, Jodrell DI: Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds. Biochemical Pharmacology 2006, 71:408-415.
 10.     Novakova O, Chen HM, Vrana O, Rodger A, Sadler PJ, Brabec V: DNA interactions of monofunctional organometallic ruthenium(II) antitumor complexes in cell-free media. Biochemistry 2003, 42:11544-11554.
 11.     Novakova O, Kasparkova J, Bursova V, Hofr C, Vojtiskova M, Chen HM, Sadler PJ, Brabec V: Conformation of DNA modified by monofunctional Ru(II) arene complexes: Recognition by DNA binding proteins and repair. Relationship to cytotoxicity. Chemistry & Biology 2005, 12:121-129.
 12.     McNae IW, Fishburne K, Habtemariam A, Hunter TM, Melchart M, Wang FY, Walkinshaw MD, Sadler PJ: Half-sandwich arene ruthenium(II)-enzyme complex. Chemical Communications 2004:1786-1787.
 13.     Yan YK, Melchart M, Habtemariam A, Peacock AFA, Sadler PJ: Catalysis of regioselective reduction of NAD(+) by ruthenium(II) arene complexes under biologically relevant conditions. Journal of Biological Inorganic Chemistry 2006, 11:483-488.
 14.     Peacock AFA, Parsons S, Sadler PJ: Tuning the hydrolytic aqueous chemistry of osmium arene complexes with N,O-chelating ligands to achieve cancer cell cytotoxicity. Journal of the American Chemical Society 2007, 129:3348-3357.