Abstract
Regions of insufficient oxygen supply—hypoxia—occur in diverse contexts across biology in both healthy and diseased organisms. The difference in the chemical environment between a hypoxic biological system and one with normal oxygen levels provides an opportunity for targeting compound delivery to hypoxic regions by using bioreductive prodrugs. Here we detail a protocol for the efficient synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which is a key intermediate that can be converted into a range of 1-methyl-2-nitro-1H-imidazole–based precursors of bioreductive prodrugs. We outline methods for attaching the bioreductive group to a range of functionalities, and we discuss the strategy for positioning of the group on the biologically active parent compound. We have used two parent checkpoint kinase 1 (Chk1) inhibitors to exemplify the protocol. The PROCEDURE also describes a suite of reduction assays, of increasing biological relevance, to validate the bioreductive prodrug. These assays are applied to an exemplar compound, CH-01, which is a bioreductive Chk1 inhibitor. This protocol has broad applications to the development of hypoxia-targeted compounds.
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Acknowledgements
S.J.C., E.M.H. and L.J.O.C. thank the UK Medical Research Council (MRC) for the award of a studentship to L.J.O.C. S.J.C., E.M.H. and C.C.-K. thank Cancer Research UK (CRUK) for the award of a studentship to C.C.-K. S.J.C., E.M.H. and C.N.G.E. thank CRUK and the UK Engineering and Physical Sciences Research Council (EPSRC) for the award of a studentship to C.N.G.E., through the CRUK and EPSRC Cancer Imaging Centre in Oxford (OCIC). S.J.C. and J.S. thank the European Commission for the award of a Marie Curie Fellowship to J.S. (PIIF-GA-2012-331327, Hypoxprobe). E.M.H. thanks CRUK for research funding. S.J.C. thanks St. Hugh's College, Oxford, for research funding.
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The study was conceived and planned by S.J.C. and E.M.H., and they also supervised the in vitro bioreduction studies. The chemical synthesis was conducted by L.J.O.C., C.C.-K., J.S. and C.N.G.E. The chemical synthesis was supervised by S.J.C. The in vitro bioreduction studies and the cellular bioreduction studies were performed by L.J.O.C., C.C.-K. and M.R.L.S. The cellular bioreduction studies were supervised by E.M.H. All authors interpreted the data. The manuscript was written by L.J.O.C., J.S., E.M.H. and S.J.C. All authors commented on the manuscript.
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O'Connor, L., Cazares-Körner, C., Saha, J. et al. Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs. Nat Protoc 11, 781–794 (2016). https://doi.org/10.1038/nprot.2016.034
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DOI: https://doi.org/10.1038/nprot.2016.034
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