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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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.
The authors declare no competing financial interests.
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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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