Structural and Functional Diversity in Rigid Thiosemicarbazones with Extended Aromatic Frameworks: Microwave-Assisted Synthesis and Structural Investigations

Fernando Cortezon-Tamarit, Kexin Song, Navaratnarajah Kuganathan, Rory L. Arrowsmith, Sara M. M. de Aguiar, Philip A. Waghorn, Adam Brookfield, Muralidharan Shanmugam, David Collison, Haobo Ge, Gabriele Kociok-Kohn, Charareh Pourzand, Jonathan R Dilworth, Sofia Pascu

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Abstract

The long-standing interest in thiosemicarbazones (TSCs) has been largely driven by their potential toward theranostic applications including cellular imaging assays and multimodality imaging. We focus herein on the results of our new investigations into: (a) the structural chemistry of a family of rigid mono(thiosemicarbazone) ligands characterized by extended and aromatic backbones and (b) the formation of their corresponding thiosemicarbazonato Zn(II) and Cu(II) metal complexes. The synthesis of new ligands and their Zn(II) complexes was performed using a rapid, efficient and straightforward microwave-assisted method which superseded their preparation by conventional heating. We describe hereby new microwave irradiation protocols that are suitable for both imine bond formation reactions in the thiosemicabazone ligand synthesis and for Zn(II) metalation reactions. The new thiosemicarbazone ligands, denoted HL, mono(4-R-3-thiosemicarbazone)quinone, and their corresponding Zn(II) complexes, denoted ZnL2, mono(4-R-3-thiosemicarbazone)quinone, where R = H, Me, Ethyl, Allyl, and Phenyl, quinone = acenapthnenequinone (AN), aceanthrenequinone (AA), phenanthrenequinone (PH), and pyrene-4,5-dione (PY) were isolated and fully characterized spectroscopically and by mass spectrometry. A plethora of single crystal X-ray diffraction structures were obtained and analyzed and the geometries were also validated by DFT calculations. The Zn(II) complexes presented either distorted octahedral geometry or tetrahedral arrangements of the O/N/S donors around the metal center. The modification of the thiosemicarbazide moiety at the exocyclic N atoms with a range of organic linkers was also explored, opening the way to bioconjugation protocols for these compounds. The radiolabeling of these thiosemicarbazones with 64Cu was achieved under mild conditions for the first time: this cyclotron-available radioisotope of copper (t1/2 = 12.7 h; β+ 17.8%; β– 38.4%) is well-known for its proficiency in positron emission tomography (PET) imaging and for its theranostic potential, on the basis of the preclinical and clinical cancer research of established bis(thiosemicarbazones), such as the hypoxia tracer 64Cu-labeled copper(diacetyl-bis(N4-methylthiosemicarbazone)], [64Cu]Cu(ATSM). Our labeling reactions proceeded in high radiochemical incorporation (>80% for the most sterically unencumbered ligands) showing promise of these species as building blocks for theranostics and synthetic scaffolds for multimodality imaging probes. The corresponding “cold” Cu(II) metalations were also performed under the mild conditions mimicking the radiolabeling protocols. Interestingly, room temperature or mild heating led to Cu(II) incorporation in the 1:1, as well as 1:2 metal: ligand ratios in the new complexes, as evident from extensive mass spectrometry investigations backed by EPR measurements, and the formation of Cu(L)2-type species prevails, especially for the AN-Ph thiosemicarbazone ligand (L–). The cytotoxicity levels of a selection of ligands and Zn(II) complexes in this class were further tested in commonly used human cancer cell lines (HeLa, human cervical cancer cells, and PC-3, human prostate cancer cells). Tests showed that their IC50 levels are comparable to that of the clinical drug cis-platin, evaluated under similar conditions. The cellular internalizations of the selected ZnL2-type compounds Zn(AN-Allyl)2, Zn(AA-Allyl)2, Zn(PH-Allyl)2, and Zn(PY-Allyl)2 were evaluated in living PC-3 cells using laser confocal fluorescent spectroscopy and these experiments showed exclusively cytoplasmic distributions.
Original languageEnglish
Pages (from-to)16047-16079
Number of pages33
JournalACS OMEGA
Volume8
Issue number18
Early online date25 Apr 2023
DOIs
Publication statusPublished - 9 May 2023

Bibliographical note

The authors thank the Royal Society, STFC, BBSRC, and MRC for funding (S.I.P.), as well as the EPSRC Crystallography Service (using Daresbury SRS, Diamond DLS and University of Southampton facilities). We also thank the EPSRC for access to the EPR NRF (NS/A000055/1, EP/W014521/1) and the EPSRC UK National Mass Spectrometry Facility at Swansea University for the acquisition of some mass spectra presented in this work. S.I.P., F.C.-T., and H.G. thank the European Commission for the funding through an ERC Consolidator Grant (O2SENSE Program, 2014–2020). F.C.-T. was funded by the European Commission FP7 Programme through the Marie Curie Initial Training Network PROSENSE (grant no. 317420, 2012–2016). The authors thank several previous research undergraduate research students and graduate researchers at Oxford and Bath Universities (especially T. Conry, L. Murray, J. Williams, E. Davis, O. Neil, S. Sarpaki, and G. Williams) for their contributions through helpful discussions and some early stage preliminary experiments. Dr. A. Cowley and Dr. A. Thompson (Oxford University) are thanked for their assistance with some of the earlier X-ray data collection. We also thanks Drs. Helen Betts, and Simon Bayly for assistance with 64Cu radiolabeling experiments and training at Siemens Oxford Molecular Imaging Laboratory (SOMIL). We would like to thank Mr. Paul Burke for generating the copper-64 and to Professor Franklin Aigbirhio for supplying 64Cu to SOMIL. We also thank Professors Franklin Aigbirhio (Wolfson Brain Imaging Centre, University of Cambridge) and Jason Lewis (MSKCC, US) for training in handling 64Cu, collaborative support and mentoring in radiochemistry and Professor Rex Tyrell for training in living cells assays.

The data that supports the findings of this study are available in the supplementary material of this article or from the authors.

Funding

The authors thank the Royal Society, STFC, BBSRC, and MRC for funding (S.I.P.), as well as the EPSRC Crystallography Service (using Daresbury SRS, Diamond DLS and University of Southampton facilities). We also thank the EPSRC for access to the EPR NRF (NS/A000055/1, EP/W014521/1) and the EPSRC UK National Mass Spectrometry Facility at Swansea University for the acquisition of some mass spectra presented in this work. S.I.P., F.C.-T., and H.G. thank the European Commission for the funding through an ERC Consolidator Grant (O2SENSE Program, 2014–2020). F.C.-T. was funded by the European Commission FP7 Programme through the Marie Curie Initial Training Network PROSENSE (grant no. 317420, 2012–2016). The authors thank several previous research undergraduate research students and graduate researchers at Oxford and Bath Universities (especially T. Conry, L. Murray, J. Williams, E. Davis, O. Neil, S. Sarpaki, and G. Williams) for their contributions through helpful discussions and some early stage preliminary experiments. Dr. A. Cowley and Dr. A. Thompson (Oxford University) are thanked for their assistance with some of the earlier X-ray data collection. We also thanks Drs. Helen Betts, and Simon Bayly for assistance with Cu radiolabeling experiments and training at Siemens Oxford Molecular Imaging Laboratory (SOMIL). We would like to thank Mr. Paul Burke for generating the copper-64 and to Professor Franklin Aigbirhio for supplying Cu to SOMIL. We also thank Professors Franklin Aigbirhio (Wolfson Brain Imaging Centre, University of Cambridge) and Jason Lewis (MSKCC, US) for training in handling Cu, collaborative support and mentoring in radiochemistry and Professor Rex Tyrell for training in living cells assays. 64 64 64 The authors thank the Royal Society, STFC, BBSRC, and MRC for funding (S.I.P.), as well as the EPSRC Crystallography Service (using Daresbury SRS, Diamond DLS and University of Southampton facilities). We also thank the EPSRC for access to the EPR NRF (NS/A000055/1, EP/W014521/1) and the EPSRC UK National Mass Spectrometry Facility at Swansea University for the acquisition of some mass spectra presented in this work. S.I.P., F.C.-T., and H.G. thank the European Commission for the funding through an ERC Consolidator Grant (O2SENSE Program, 2014-2020). F.C.-T. was funded by the European Commission FP7 Programme through the Marie Curie Initial Training Network PROSENSE (grant no. 317420, 2012-2016). The authors thank several previous research undergraduate research students and graduate researchers at Oxford and Bath Universities (especially T. Conry, L. Murray, J. Williams, E. Davis, O. Neil, S. Sarpaki, and G. Williams) for their contributions through helpful discussions and some early stage preliminary experiments. Dr. A. Cowley and Dr. A. Thompson (Oxford University) are thanked for their assistance with some of the earlier X-ray data collection. We also thanks Drs. Helen Betts, and Simon Bayly for assistance with 64Cu radiolabeling experiments and training at Siemens Oxford Molecular Imaging Laboratory (SOMIL). We would like to thank Mr. Paul Burke for generating the copper-64 and to Professor Franklin Aigbirhio for supplying 64Cu to SOMIL. We also thank Professors Franklin Aigbirhio (Wolfson Brain Imaging Centre, University of Cambridge) and Jason Lewis (MSKCC, US) for training in handling 64Cu, collaborative support and mentoring in radiochemistry and Professor Rex Tyrell for training in living cells assays.

FundersFunder number
EPSRC UK National Mass Spectrometry Facility at Swansea University
Wolfson Brain Imaging Centre
Medical Research Council
Engineering and Physical Sciences Research CouncilNS/A000055/1, EP/W014521/1
Biotechnology and Biological Sciences Research Council
Science and Technology Facilities Council
Royal Society
University of Cambridge
European Commission317420
European Research Council

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