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Metals in Medicine: Research

 

Photoactivated Anticancer Complexes

 

We are exploring the use of inert, nontoxic platinum complexes that can be activated locally in cancer cells by light. Our research on such photoactivated metallo-anticancer agents focuses on Pt(IV) complexes and makes use of the fact that they are kinetically inert under biological conditions and can be reduced to potentially reactive Pt(II) complexes upon irradiation.

Our studies involve the synthesis and full characterization of new Pt(IV) complexes, the study of their coordination chemistry and photochemical properties. In addition, we study their reactivity, both in the dark and after irradiation, towards biomolecules and determine their in vitro cytotoxicity. The principle techniques used to study these complexes are multidimensional, multinuclear NMR, X-ray crystallography, advanced mass spectrometry and cytotoxicity testing against various cancer cell lines.

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Photoactivated Platinum(IV) Complexes

Traditional platinum anticancer drugs, such as cisplatin, are one of most successful groups of chemotherapeutic agents in clinical use today. Their clinical use is, however, severely limited by dose-limiting side-effects and by the problem of inherent or acquired resistance. The development of nontoxic platinum(IV) prodrugs that can be activated locally and selectively to antitumour platinum(II) agents would provide an attractive alternative with the potential to alleviate these current limitations.[1,2]

Pt(IV)-Diazido Prodrugs

As a result, recent studies have focused on Pt(IV)-azido complexes, which are well-known to be photoactive. The photoreduction mechanism to Pt(II) is thought to involve the formation of azidyl radicals, which rapidly decompose in water into molecular nitrogen (N2). Most importantly, this photoactivation mechanism is not dependent on dioxygen. This distinguishes our approach from conventional photodynamic therapy, which typically uses photosensitizers such as porphyrins that require dioxygen for reactivity. Given the often hypoxic nature of many malignant and most aggressive tumours, photoactivatable complexes that do not require dioxygen for cytotoxic activity may be more effective.

We have reported the first crystal structures of the Pt(IV)-diazidodiam(m)ino complexes cis,trans,cis-[Pt(N3)2(OH)2(NH3)2] and cis,trans-[Pt(N3)2(OH)2(en)2] and shown that these complexes can be activated by light to give highly reactive Pt(II) species which bind rapidly to nucleotides, thereby forming known cis-platinum-nucleotide cross-links.[3,4] Both complexes were stable towards hydrolysis for more than 90 days, inert to reaction with nucleobases in the dark and, most significantly, react only very slowly with the ubiquitous cellular reductant glutathione over a period of several weeks. Based on these results, we anticipated that these complexes might reach the nucleus of cancer cells intact and exhibit cytotoxicity.

Recent studies on the UVA-induced photodecomposition pathways of the Pt(IV)-diazido complexes by 14N NMR spectroscopy revealed that the photoreaction not only results in reduction to Pt(II) with concomitant release of N­2, but also O2 evolution and the formation of nitrene intermediates was observed.[5] These results suggest that alternate photodecomposition pathways can operate even though the major photoproducts (Pt(II), dinitrogen) are consistent with the proposed photoreduction mechanism.

Cytotoxicity: Light-activated destruction of Cancer Cell Nuclei

The effects of the Pt(IV)-diazidodiam(m)ino complexes cis,trans,cis-[Pt(N3)2(OH)2(NH3)2] and cis,trans-[Pt(N3)2(OH)2(en)2] on the growth and morphology of human bladder cancer cells, both in the dark and in the light, were studied.[6] Cytotoxicity studies revealed that the complexes indeed are nontoxic to 5637 human bladder cancer cells in the dark, but are toxic to the cells upon irradiation with significantly decreased IC50 values of 49 and 63 micromolar (compared to > 300 micromolar in the dark). Photoactivation causes dramatic effects on the morphology of the cells. Shrinking of the cancer cells, loss of adhesion, packing of nuclear material and eventual disintegration of their nuclei was observed. No changes were observed in the absence of irradiation. The mechanism of action, therefore, appears to be different from cisplatin. Indeed, the diazido complexes were found to be equally toxic towards cisplatin-sensitive and cisplatin-resistant cell lines. The photolysis rates were furthermore found to closely parallel the rates of irreversible DNA platination by these two complexes, indicating direct reaction of the products with DNA.

A Photoactived trans-Diammine Complex as Cytotoxic as Cisplatin

Although it is well-known that transplatin itself is relatively nontoxic, it can be activated by light, whereupon it becomes as active as cisplatin.[7] Inspired by these results, we found that the inert trans,trans,trans-[Pt(N3)2(OH)2(NH3)2] isomer is as cytotoxic as cisplatin when photoactivated.[8] Advantages of the all-trans isomer are its increased aqueous solubility and a shift of the azide-to-Pt(IV) charge-transfer band to longer wavelengths compared to cis diazido isomer. Irradiation of trans,trans,trans-[Pt(N3)2(OH)2(NH3)2] in the presence of 5’-GMP led to rapid (>75% after 1 h) photoreduction and photosubstitution to give trans-[Pt(NH3)2 (5’-GMP-N7)2]. This reaction is much more efficient than that of the cis-diazido complexes.

We recently found that replacement of one of the ammine ligands in the all-trans isomer with a pi-acceptor pyridine ligand results in a significant increase in cytotoxicity.[9] The complex trans,trans,trans-[Pt(N3)2(OH)2(NH3)(py)] is not cytotoxic in the dark, but 13-80 x more toxic than cisplatin after photoactivation, and ca. 15 x more cytotoxic towards cisplatin-resistant human ovarian cancer cell lines. Although DNA platination levels in HaCaT skin cells were similar to those of cisplatin, different crosslinks form (trans G rather than cis G adducts). Moreover, the mechanism of action was found to differ significantly from that of cis complexes.

   
     

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Photoactivation of a Pt(IV)-diazido prodrug gives a reactive Pt(II) antitumour agent

     
   

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Formation of dinitrogen upon irradiation
of the Pt(IV)-diazido complexes

   

 

   

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A Pt(IV)-diazido complex with the azido ligands in cis positions

     
   

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Photoactivation of Pt(IV)-diazido complexes causes dramatic effects on the morphology of the cancer cell

 

Photoactivated Ruthenium(II)-arene anticancer complexes

 

Recently, we have also become interested in extending our family of ruthenium-arene anticancer complexes to organometallic complexes that can be photoactivated. The first results showed that dinuclear Ru(II)-arene complexes [ {h6-arene)RuCl}2(m-2,3-dpp)](PF6)2, constructed with different arenes, including the fluorophore indan, could be photoactivated to produce highly reactive ruthenium species that can bind to DNA.[10] Importantly, the mechanism of reactivity was again independent of oxygen. The concomitant release of indan also allows for fluorescence imaging of the location and efficiency of the photoactivation process, as the fluorescence of the unbound indan is 40 times greater than when it is complexed.

   

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Dinuclear Ru(II)-arene complexes can be photoactivated to produce highly reactive Ru(II) species


 

 

Selection of our recent publications on our photoactivated anticancer complexes:
 
1.     Bednarski PJ, Mackay FS, Sadler PJ: Photoactivatable Platinum Complexes. Anti-Cancer Agents in Medicinal Chemistry 2007, 7:75-93.
2.     Kratochwil NA, Parkinson JA, Bednarski PJ, Sadler PJ: Nucleotide platination induced by visible light. Angewandte Chemie-International Edition 1999, 38:1460-1463.
3.     Müller P, Schröder B, Parkinson JA, Kratochwil NA, Coxall RA, Parkin A, Parsons S, Sadler PJ: Nucleotide cross-linking induced by photoreactions of platinum(IV)-azide complexes. Angewandte Chemie-International Edition 2003, 42:335-339.
4.     Kasparkova J, Mackay FS, Brabec V, Sadler PJ: Formation of platinated GG cross-links on DNA by photoactivation of a platinum(IV) azide complex. Journal of Biological Inorganic Chemistry 2003, 8:741-745.
5.     Ronconi L, Sadler PJ: Unprecendented carbon-carbon bond formation induced by photoactivation of a platinum(IV)-diazido complex. Chemical Communications 2008:235-237.
6.     Bednarski PJ, Grunert R, Zielzki M, Wellner A, Mackay FS, Sadler PJ: Light-activated destruction of cancer cell nuclei by platinum diazide complexes. Chemistry & Biology 2006, 13:61-67.
7.     Heringova P, Woods J, Mackay FS, Kasparkova J, Sadler PJ, Brabec V: Transplatin is cytotoxic when photoactivated: Enhanced formation of DNA cross-links. Journal of Medicinal Chemistry 2006, 49:7792-7798.
8.     Mackay FS, Woods JA, Moseley H, Ferguson J, Dawson A, Parsons S, Sadler PJ: A photoactivated trans-diammine platinum complex as cytotoxic as cisplatin. Chemistry-a European Journal 2006, 12:3155-3161.
9.     Mackay FS, Woods JA, Heringova P, Kasparkova J, Pizarro AM, Moggach SA, Parsons S, Brabec V, Sadler PJ: A potent cytotoxic photoactivated platinum complex. Proc. Natl. Acad. Sci. USA 2007, 104:20743-20748
10.     Magennis SW, Habtemariam A, Novakova O, Henry JB, Meier S, Parsons S, Oswald IDH, Brabec V, Sadler PJ: Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex. Inorganic Chemistry 2007, 46:5059-5068.