Periodic Reporting for period 1 - CylcoRu4PACT (Cyclometallated ruthenium complexes for photo-activated chemotherapy) Reporting period: 2018-04-01 to 2020-03-31 Summary of the context and overall objectives of the project In chemotherapy the primary question is that of the selectivity of the drug, as the toxicity of the compound should affect only the tumour, not the healthy cells. Selective activation of a prodrug by an external signal such as light has appeared as a promising alternative to biological targeting, as it allows for delivery of the toxicity of the compound with great spatial and time selectivity. The optimal wavelengths for activating prodrugs in vivo is the region where light penetration is maximal, i.e. in the red region of the spectrum. The results obtained in this project represent a significant contribution to the design and development of new photoactivated drugs. Namely, in the literautre, cyclometallated ruthenium complexes absorb in the red region of the spectrum but they cannot be activated with such light. Compounds synthesized in this project not only absorbs in the red region of the spectrum, but also they exchange a ligand upon red light irradiation. One of the compounds prepared in this project showed twice higher toxicity towards A431 (skin cancer) cells after red light irradiation. The main problem of the complexes studied in this project is their low thermal stability in the dark and their insolubility in water. Follow-up research is now taking place to introduce ligands that improve the dark stability and water solubility of these complexes. With these improvements, new complexes will be at hand that will show much higher light-induced toxicity in vitro and can be further tested in animal models. Overall, the present project has opened new prospects for the design of light-activated anticancer drugs. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this project three organoruthenium precursor complexes and three cyclometallated complexes with strerically hindered ligands were synthesized and fully characterized, by standard techniques (NMR, mass spectrometry, UV-Vis spectroscopy, elemental analysis and X-ray for some of them). Photochemical and photophysical properties of the new complexes were also investigated.The photosubstitution of the monodentate ligand(s) by a solvent molecule was investigated by UV-vis and 1H NMR spectroscopy. The stability of the compounds was monitored in the dark and after light irradiation. This has been done using 1H NMR, UV-vis spectroscopy, and mass spectrometry. Cyclometallated compounds were stable in the dark up to 6 hours, while organoruthenium compounds were stable in the dark for at least 24 hours. When organoruthenium complexes were irradiated with white light, they substituted 1 or 2 ligands by solvent molecule, which makes them very good candidates for photoactivated chemotherapy (PACT). The potential of new compounds to be used in photodynamic therapy (PDT) was further investigated using singlet oxygen measurements. Obtained results suggest that the PDT properties of the new complexes are low, which is expected for PACT complexes.The antiproliferative activity of the ruthenium complexes on commercially available human cancer cell lines has been tested first in the dark and then upon visible light irradiation using a colorimetric SRB assay. The cytotoxicity was tested on A431 (skin) and A549 (lung) cancer cells for the organoruthenium complexes, and on A431 (skin) for cyclometallated compounds, in both normoxic (95% air and 5% CO2) and hypoxic (1% O2 and 5% CO2) conditions. The biological experiments showed that the organoruthenium compounds have a low dark cytotoxicity, with EC50-values above 100 µM, and a low phototoxicity. This indicates that either there was not enough photo-activation of these complexes in the irradiation conditions of the biological experiment (blue light λ = 455 nm), or that the photoactivated products were not cytotoxic.The cytotoxicity of the cyclometallated compounds was overall very high, with effective concentrations (EC50) values around 0.5 µM and photoindexes around 2 in normoxic conditions, and around 1 for hypoxic condition. The low photo index comes from the toxicity in the dark, which is already quite high, because these compounds are highly hydrophobic. Overall the cyclometallated complexes have shown more potential for PACT than the organoruthenium complexes, but their phototoxicity index was still low. Their toxicity in the dark needs to be reduced. A possible way to do this would be to attach a hydrophilic group to the ligand that undergoes light-mediated photosubstitution. This would decrease the lipophilicity and hence cellular uptake and therefore decrease the toxicity before irradiation, without jeopardizing the light toxicity, for which the hydrophilic ligand would be detached. This strategy could not be investigated due to the early termination of the contract. During the project various trainings were followed by the researcher. In May 2018 a course on lab safety and fire extinguishing were offered by the Faculty of Science. Trainings on physical and health hazards in the work area, along with applicable exposure control measures were provided. Within the research group the researcher followed 1 day training related to using a glove box for handling air sensitive compounds or reactions. Also, training on photosubstitution quantum yield and singlet oxygen measurement by members of the Prof. Bonnet group has been provided. Training on cancer cell cytotoxicity measurements and cell irradiation, using their specific training protocols, were organised in November and December 2018, also by members of the Bonnet group.The results of this research will be disseminated via 2 scientific publications in peer-reviewed international journals such as Angewandte Chemie and Dalton Transaction. Some of results were presented in national conference CHAINS Dutch Chemistry meeting and HRSMC annual symposium. The supervisor Bonnet will also show the results of this work in international scientific conferences planned in 2020. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Photoactive ruthenium compounds are known, but cyclometallated derivatives of them are usually recognised as non-photoreactive. Green ruthenium complexes are also known, but they are based on ruthenium(III), and are not photo substitutionally active. By combining cyclometallation to statically hindered bidentate ligands, we re-installed photoreactivity, and obtained green-colored, ie red light-absorbing, photosubstitutionally active ruthenium(II) complexes, which is going beyond the state of the art in ruthenium photochemistry. This project has been stopped on request of the researcher, 6 months before the end (31 March 2020). Further research in the host group focuses on introducing a ligand which improves thermal stability and water solubility of these cyclometallated complexes.