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Photochemistry and radiolabelling of gold(III) anticancer prodrugs

Periodic Reporting for period 1 - PhoRAu (Photochemistry and radiolabelling of gold(III) anticancer prodrugs)

Reporting period: 2018-03-01 to 2020-02-29

The social impact of cancer demands that medicinal inorganic chemists introduce innovative approaches and chemical tools for the treatment of cancer. Cisplatin and its approved derivatives continue demonstrating how transition metal complexes play a major role in the clinical treatment of cancer. In the last 15 years, the need to find an alternative to cisplatin and its analogues, enormously boosted the research on gold compounds as new valuable agents to treat cancer. Most of the studies on the anticancer activity of Au-NHCs concern Au(I) complexes. Reports on Au(III)-NHCs are more recent, and the stability of these compounds in biological environment is an issue not always exhaustively investigated. Knowledge on the fate of Au(III)-NHCs after their administration in vivo is still largely insufficient. The study of the biodistribution of Au(III) compounds in vivo and the evaluation of their metabolization to Au(I) species is a very challenging task that has not been thoroughly addressed so far, albeit this is a key step in drug development

PhoRAu aspires to:

• Develop new gold (pro)drug candidates based on the Au(III)-NHC scaffold;
• Label such Au(III)-NHC complexes with long-lived Iodine-124 (124I) for use as nuclear imaging agents.
• Study the biodistribution of one radio-labelled Au(III)-NHC complex via Postron Emission Tomography (PET).
Research objectives and work plan are organized in four Work Packages (WPs). Below a summary of the results obtained for each WP.

WP1: Within this WP, I prepared five AuI-NHC complexes and their corresponding new AuIII-NHC analogues in the cold lab. They have been fully characterized with standard techniques. All reactions have been performed and optimized in the cold chemistry using non-radioactive iodine. The most suitable compound (in terms of yield and stability in aqueous media) was then synthesized using the selected radioisotopes.

WP2: The dark stability of the synthesized compounds was investigated in buffer and in cell culture media (supplemented with fetal bovine serum). Two Au(III) compounds out of five were completely stable in buffer, while only one resulted moderately stable in biological relevant media. As a consequence, WP3 and WP4 have been focused on this lead compound, namely the Au(III) biscarbenic complex with 1-butyl-3-methyl-imidazol-2-ylidene NHC ligand (3). The photochemistry of such derivative in buffer was investigated with spectroscopic methods, however its moderate dark stability when solubilized in cell culture media, ruled out its use in advance biological studies. Therefore, the project has focused more on the Au(III) activation to Au(I) of 3 by biological agents in vitro and in vivo.

WP3: In order to investigate the behavior of the Au(III) derivative at the cellular level, a series of in vitro assays have been performed. Minor but important changes between the activity of the Au(III) and the Au(I) complexes in the different experimental conditions used have been observed indicating that the Au(III) compound can be considered as a drug per se and not a simple Au(I) prodrug. Furthermore, a clear-cut difference in their mode of action compared with the standard gold drug auranofin was observed, in contrast to recent literature where no particular divergences have been noted.

WP4: The lead Au(III) compound was synthesized using direct oxidation with radioactive I2 and its biodistribution in vivo was followed via PET imaging. Using this approach, we were able to dissect the reduction path of this compound once intravenously injected in healthy rats. The metal complex was rapidly, but not immediately, reduced to the Au(I) analogue as captured by PET imaging and ICP in comparison with [124I]KI control.

List of publications
1) F. Guarra, A. Terenzi*, C. Gabbiani, T. Biver, V. Gomez, J. Llop, C. Pirker, W. Berger, E. Zangrando, L. Salassa - “124I-radiolabeling of a Au(III)-NHC complex for in vivo biodistribution studies” - In Preparation (to be submitted to Angew. Chem. Int. Ed.)
2) G. Salassa, A. Terenzi* - Int. J. Mol. Sci. 2019, 20, 3483 DOI: 10.3390/ijms20143483
3) A. Terenzi*, H. Gattuso, A. Spinello, B.K. Keppler, C. Chipot, F. Dehez, G. Barone, A. Monari - Antioxidants 2019, 8, 472 DOI: 10.3390/antiox8100472

List of Conferences, workshops and and seminars in international institutes
1) 9th Asian Biological Inorganic Chemistry Conference (Asbic, Singapore 9-14 Dec 2018) “Award: Outstanding Oral Presentation”
2) 17th Congress of the International Union of Photobiology and 18th Congress of the European Society for Photobiology (Barcelona 25-30 Aug 2019)
3) 3rd Biennial Young Researchers Workshop on Biomaterials and Applications (bioMAPP19, Bilbao 4-5 Dec 2019)
4) Member of the organizing committee of the workshop “From Bioinorganic Chemistry to Catalysis 2019”, organized by DIPC (Donostia, 8 Nov 2019).
5) Invited lecture at Madrid Institute of Advanced Studies (IMDEA), Madrid (Spain) 15 Feb 2019

Outreach activities
1) Pint of Science 2018 (Donostia, 14-16 May 2018) - https://pintofscience.es/events/sansebastian
2) Eureka! Zienzia Museoa “A Life in Science” (Donostia, 5 November 2018) - https://www.eurekamuseoa.eus/es/encuentro-de-vidas-cientificas
3) Creativium Project (Donostia, 17 January 2019) - https://p4k.dipc.org/en/photographic-exhibition
We have described the synthesis and the characterization of new mono- and bis-NHC-Au(III) compounds. The biscarbene 3, resulting the most stable in biologically compatible media, was synthesized using direct oxidation with radioactive I2 in order to follow its biodistribution in vivo via PET imaging. Using this approach, we were able to dissect the reduction path of this compound once intravenously injected in healthy rats. Complex 3 was rapidly, but not immediately, reduced to the Au(I) analogue as captured by PET imaging and ICP in comparison with [124I]KI control. Instead, the immediate reduction of 3 to 1 place instead intracellularly in cancer cells, where there is no observable difference in activity between the two compounds. Yet, contrary to what reported in literature for 1 and for other similar NHC gold derivatives, their action strikingly differs from the one of auranofin, the reference gold compound to date. The present study highlight the potential of the radiolabeling strategy with 124I to unravel the fate of Au(III)-NHC drug candidates in vivo while, on the other hand, urges the bioinorganic chemistry community to synthesize stable Au(III) compounds.

We pursued and achieved significant innovative elements in fundamental disciplines such as inorganic chemistry, and radiochemistry, as well as in the application of metal-based chemotherapy. These can be summarized as follows:
1. The chemistry of the proposed Au derivatives allows adding imaging functionality to candidate anticancer (pro)drugs by labelling them with radioactive 124I isotopes (PET). Such ground-breaking agents have potential for image-guided metal-based therapies, overcoming an intrinsic limitation of metal complexes which can rarely be monitored in real time in vivo.
2. We have gained unprecedented insights in the thermodynamic, kinetic and redox properties Au(III)-NHC complexes in aqueous solution and in the presence of biological molecules. Since NHC ligands are widely used in catalysis, we envisage that this type of study might have an impact also in gold-based (photo)catalysis
Gold compound labelled with radioisotopes for PET imaging