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Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs

Nature Protocols volume 11, pages 781–794 (2016)Cite this article

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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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Figure 1: Attachment of the 4-nitrobenzyl group to the terminal hydroxyl group of the Chk1 inhibitor 6 affords the inactive compound 1.
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References

  1. Hammond, E.M. et al. The meaning, measurement and modification of hypoxia in the laboratory and the clinic. Clin. Oncol. (R. Coll. Radiol.) 26, 277–288 (2014).

    Article  CAS  Google Scholar 

  2. McKeown, S.R. Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response. Br. J. Radiol. 87, 20130676 (2014).

    Article  CAS  Google Scholar 

  3. Wilson, W.R. & Hay, M.P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 11, 393–410 (2011).

    Article  CAS  Google Scholar 

  4. Thomlinson, R.H. & Gray, L.H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9, 539–549 (1955).

    Article  CAS  Google Scholar 

  5. Vaupel, P. The role of hypoxia-induced factors in tumor progression. Oncologist 9, 10–17 (2004).

    Article  CAS  Google Scholar 

  6. Yasui, H. et al. Low-field magnetic resonance imaging to visualize chronic and cycling hypoxia in tumor-bearing mice 70, 6427–6436 (2010).

  7. Nordsmark, M. et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother. Oncol. 77, 18–24 (2005).

    Article  Google Scholar 

  8. Begg, A.C., Stewart, F.A. & Vens, C. Strategies to improve radiotherapy with targeted drugs. Nat. Rev. Cancer 11, 239–253 (2011).

    Article  CAS  Google Scholar 

  9. Cazares-Korner, C. et al. CH-01 is a hypoxia-activated prodrug that sensitizes cells to hypoxia/reoxygenation through inhibition of Chk1 and Aurora A. ACS Chem. Biol. 8, 1451–1459 (2013).

    Article  CAS  Google Scholar 

  10. O'Connor, L.J. et al. Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate evofosfamide (TH-302). Org. Chem. Front. 2, 1026–1029 (2015).

    Article  CAS  Google Scholar 

  11. Cavalleri, B., Ballotta, R. & Lancini, G. Synthesis of 1-alkyl-2-nitroimidazole-5-carboxaldehydes. J. Heterocycl. Chem. 9, 979–984 (1972).

    Article  CAS  Google Scholar 

  12. Matteucci, M. et al. Phosphoramidate alkylator prodrugs. Patent no. WO 2007/002931 A2 (2007).

  13. Parveen, I., Naughton, D.P., Whish, W. & Threadgill, M.D. 2-Nitroimidazol-5-ylmethyl as a potential bioreductively activated prodrug system: reductively triggered release of the PARP inhibitor 5-bromoisoquinolinone. Bioorg. Med. Chem. Lett. 9, 2031–2036 (1999).

    Article  CAS  Google Scholar 

  14. Damen, E.W.P., Nevalainen, T.J., van den Bergh, T.J.M., de Groot, F.M.H. & Scheeren, H.W. Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue. Bioorg. Med. Chem. 10, 71–77 (2002).

    Article  CAS  Google Scholar 

  15. Ferrer, S., Naughton, D.P. & Threadgill, M.D. H-1 NMR studies on the reductively triggered release of heterocyclic and steroid drugs from 4,7-dioxoindole-3-methyl prodrugs. Tetrahedron 59, 3445–3454 (2003).

    Article  CAS  Google Scholar 

  16. Zhang, Z., Tanabe, K., Hatta, H. & Nishimoto, S.-I. Bioreduction activated prodrugs of camptothecin: molecular design, synthesis, activation mechanism and hypoxia selective cytotoxicity. Org. Biomol. Chem. 3, 1905–1910 (2005).

    Article  CAS  Google Scholar 

  17. Hay, M.P., Wilson, W.R. & Denny, W.A. Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy. Bioorg. Med. Chem. 13, 4043–4055 (2005).

    Article  CAS  Google Scholar 

  18. Thomson, P. et al. Hypoxia-driven elimination of thiopurines from their nitrobenzyl prodrugs. Bioorg. Med. Chem. Lett. 17, 4320–4322 (2007).

    Article  CAS  Google Scholar 

  19. Granchi, C. et al. Bioreductively activated lysyl oxidase inhibitors against hypoxic tumours. ChemMedChem 4, 1590–1594 (2009).

    Article  CAS  Google Scholar 

  20. Zhu, R. et al. 4-Nitrobenzyloxycarbonyl derivatives of O(6)-benzylguanine as hypoxia-activated prodrug inhibitors of O(6)-alkylguanine-DNA alkyltransferase (AGT), which produces resistance to agents targeting the O-6 position of DNA guanine. J. Med. Chem. 54, 7720–7728 (2011).

    Article  CAS  Google Scholar 

  21. Lindquist, K.E. et al. Selective radiosensitization of hypoxic cells using BCCA621C: a novel hypoxia activated prodrug targeting DNA-dependent protein kinase. Tumor Microenviron. Ther. 1, 46–55 (2013).

    Article  Google Scholar 

  22. Couch, G.D., Burke, P.J., Knox, R.J. & Moody, C.J. Synthesis of 2-aryl-6-methyl-5-nitroquinoline derivatives as potential prodrug systems for reductive activation. Tetrahedron 64, 2816–2823 (2008).

    Article  CAS  Google Scholar 

  23. Cui, L. et al. A new prodrug-derived ratiometric fluorescent probe for hypoxia: high selectivity of nitroreductase and imaging in tumor cell. Org. Lett. 13, 928–931 (2011).

    Article  CAS  Google Scholar 

  24. Blanche, E.A., Maskell, L., Colucci, M.A., Whatmore, J.L. & Moody, C.J. Synthesis of potential prodrug systems for reductive activation. Prodrugs for anti-angiogenic isoflavones and VEGF receptor tyrosine kinase inhibitory oxindoles. Tetrahedron 65, 4894–4903 (2009).

    Article  CAS  Google Scholar 

  25. Naughton, D.P. Drug targeting to hypoxic tissue using self-inactivating bioreductive delivery systems. Adv. Drug Deliv. Rev. 53, 229–233 (2001).

    Article  CAS  Google Scholar 

  26. Wardman, P. Some reactions and properties of nitro radical-anions important in biology and medicine. Environ. Health Perspect. 64, 309–320 (1985).

    Article  CAS  Google Scholar 

  27. Foloppe, N. et al. Structure-based design of novel Chk1 inhibitors: insights into hydrogen bonding and protein-ligand affinity. J. Med. Chem. 48, 4332–4345 (2005).

    Article  CAS  Google Scholar 

  28. Larock, R.C. Comprehensive Organic Transformations (Wiley-VCH, 1999).

Download references

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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Author notes

  1. Cindy Cazares-Körner and Jaideep Saha: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK

    Liam J O'Connor, Cindy Cazares-Körner, Jaideep Saha, Charles N G Evans & Stuart J Conway

  2. Department of Oncology, Cancer Research UK (CRUK)/Medical Research Council (MRC) Institute for Radiation Oncology, University of Oxford, Oxford, UK

    Liam J O'Connor, Cindy Cazares-Körner, Charles N G Evans, Michael R L Stratford & Ester M Hammond

Authors
  1. Liam J O'Connor
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  4. Charles N G Evans
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  5. Michael R L Stratford
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  6. Ester M Hammond
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Contributions

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.

Corresponding authors

Correspondence to Ester M Hammond or Stuart J Conway.

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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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