Elsevier

Bioorganic & Medicinal Chemistry

Volume 24, Issue 21, 1 November 2016, Pages 5326-5339
Bioorganic & Medicinal Chemistry

Synthesis and preclinical characterization of 1-(6′-deoxy-6′-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-6′-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia

https://doi.org/10.1016/j.bmc.2016.08.053Get rights and content

Abstract

Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6′-deoxy-6′-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-[18F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12 ± 8% (n = 10, based on [18F]fluoride starting activity) in a total synthesis time of 60 min with a specific activity at end of synthesis of 218 ± 58 GBq/μmol (n = 10). Both radiolabeling precursor β-6 and unlabeled reference compound β-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of β-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of β-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice β-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13 ± 0.22 (n = 4) at 2 h after administration of β-[18F]1. In ex vivo autoradiography experiments β-[18F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, β-[18F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

Introduction

A variety of solid tumors exhibit oxygen deficiency as a result of their rapid growth and/or insufficient tumor angiogenesis.1 Due to this shortage in blood supply, the distribution of oxygen within a tumor diminishes towards the less vascularized center of the tumor, ultimately resulting in hypoxia.2, 3 Tumor hypoxia is associated with an aggressive phenotype, poor prognosis, increased risk of invasion and metastasis, and finally resistance to chemo- and radiation therapy.4 Positron emission tomography (PET) utilizing fluorine-18 labeled 2-nitroimidazole radiotracers has been shown to be a suitable imaging technique due to its non-invasive nature and the possibility to accurately quantify hypoxic regions within tissues. These radiotracers pass the cell membrane through passive diffusion and are reduced intracellularly in all viable cells to free nitro-radical anions which are further reduced to nitroso- and hydroxylamine compounds if hypoxic conditions are present.5, 6, 7 The binding of the such formed hydroxylamine compounds to macromolecules as well as the formation of glutathione conjugates has been recently shown to be the major contributor to radiotracer accumulation in hypoxic regions of tumors.8

Fluorine-18 labeled misonidazole ([18F]FMISO, Fig. 1) was one of the first PET radiotracers used clinically showing a wide applicability for many cancer types.9, 10, 11, 12, 13 However, [18F]FMISO accumulates slowly in hypoxic (target) tissue and the slow washout of unbound (hypoxia unrelated) tracer results in modest hypoxic-to-normoxic tissue ratios and therefore images with moderate contrast. Moreover, its predominately hepatic clearance results in a high radiation dose delivered to the liver and the presence of radiolabeled metabolites in blood and urine further limits its utility.14 To overcome these limitations, efforts mainly focused on synthesizing compounds exhibiting a careful balance between sufficiently high diffusion into target cells and rapid clearance from non-target cells.6, 15 In particular 2-nitroimidazole compounds containing sugar-moieties showed great promise such as the nucleoside analogue 1-(5′-deoxy-5′-[18F]fluoro-α-d-arabinofuranosyl)-2-nitroimidazole (α-5′-[18F]FAZA, Fig. 1), which displays a hypoxia-specific uptake with tumor-to-background ratios superior to [18F]FMISO due to its faster clearance from blood.16, 17, 18, 19, 20

However, efforts to improve the imaging characteristics of hypoxia radiotracers are limited by the passive diffusion-driven cellular uptake. It has been suggested, that the exploitation of naturally occurring transmembrane transporter systems, such as glucose transporters (e.g., SLC2A family) or nucleoside transporters (e.g., SLC28A and SLC29A families), may lead to a faster entry of the radiotracer into viable cells. In particular bidirectional transporters like SLC29A1 would provide excellent targets for this concept, as they facilitate transfer of their substrates into both directions, either into the cell, which would increase radiotracer entry into cells, or out of the cell, which would increase clearance of non-bound radiotracer from non-target tissues. Consequently, when retained under hypoxic conditions, this may result in an improved imaging contrast especially at earlier time frames. These considerations have led to the development of 1-(2′-deoxy-2′-[18F]fluoro-β-d-glucopyranosyl)-2-nitroimidazole (β-2′-[18F]FDG-2-NIm, Fig. 1) which was shown to be transported by glucose transporters but failed to show retention in hypoxic tissue.21 To utilize nucleoside transporters, β-analogues of α-5′-[18F]FAZA have been synthetized, such as 1-(5′-deoxy-5′-[18F]fluoro-β-d-ribofuranosyl)-2-nitroimidazole (β-5′-[18F]FAZR), 1-(2′-deoxy-2′-[18F]fluoro- β-d-arabinofuranosyl)-2-nitroimidazole (β-2′-[18F]FAZA) and 1-(3′-deoxy-3′-[18F]fluoro-β-d-xylofuranosyl)-2-nitroimidazole (β-3′-[18F]FAZL, Fig. 1).22, 23 However, these radiotracers have not yet been characterized with respect to their ability to image tumor hypoxia in vivo.

The C(3′)-OH, C(2′)-OH and C(5′)-OH of the sugar moiety were shown to be structural determinants of nucleoside interaction with SLC29A1/2 and SLC28A1/2/3 transporters.24 With the aim to leave these positions unchanged for such a transporter interaction we designed 1-(6′-deoxy-6′-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-6′-[18F]FAZAL; β-[18F]1, Fig. 1) and herein report the precursor synthesis, radiosynthesis and preclinical evaluation as a potential hypoxia radiotracer.

Section snippets

Chemistry and radiolabeling

The synthesis of 1-(2′,3′,5′-tri-O-acetyl-6-O-p-toluenesulfonyl-β-d-allofuranosyl)-2-nitroimidazole (β-6), the radiolabeling precursor of β-[18F]1, is shown in Scheme 1. First, commercially available 1,2:5,6-di-O-isopropylidene-α-d-allofuranose was converted into known 5,6-diol 2 in 85% yield by acetylation and removal of the 5,6-O-isopropylidene group, using literature procedures.25 Derivative 2 was selectively monotosylated at the C-6 hydroxyl group with p-toluenesulfonyl chloride in pyridine

Conclusions

We described the radiosynthesis of the 18F-labeled 2-nitroimidazole derivative β-[18F]1 as a potential hypoxia specific PET tracer, which shows interaction with human nucleoside transporters suggesting that uptake of β-[18F]1 into tumor cells is mediated. β-[18F]1 was shown to have hypoxia sensitive uptake in murine and human tumor cell lines. In vivo studies showed a fast and homogenous distribution of β-[18F]1 in healthy tissues and negligible radiotracer metabolism. PET studies revealed high

General

Unless otherwise stated, all chemicals were of analytical grade and obtained from Sigma–Aldrich Chemie GmbH (Schnelldorf, Germany), Acros (distributed by Fisher Scientific, Schwerte, Germany) or Merck (Darmstadt, Germany) and used without further purification. 1,2:5,6-Di-O-isopropylidene-α-d-allofuranose was obtained from Carbosynth Limited (Compton, UK). Isoflurane was obtained from Abbott Laboratories Ltd. (Maidenhead, UK). All cell culture media (Waymouth’s MB752/1, RPMI) as well as heat

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding sources

The research leading to these results has received funding from the Lower Austria corporation for research and education (NFB) under grant agreement number LS11-002 awarded to C. Kuntner.

Acknowledgments

The authors thank the radiochemistry staff (Seibersdorf labor GmbH) for their skillful assistance, Andreas Eger (IMC University of Applied Science Krems) for helpful advice regarding cell culture and viability assays, Susanne Felsinger for recording NMR spectra and Johannes Theiner for combustion analysis.

References and notes (45)

  • P. Kumar et al.

    Sem. Nucl. Med.

    (2015)
  • K. Hendrickson et al.

    Radiother. Oncol.

    (2011)
  • Z. Lin et al.

    Int. J. Radiat. Oncol. Biol. Phys.

    (2008)
  • J.S. Rasey et al.

    Int. J. Radiat. Oncol. Biol. Phys.

    (1996)
  • D. Thorwarth et al.

    Radiother. Oncol.

    (2006)
  • G. Reischl et al.

    Appl. Radiat. Isot.

    (2005)
  • D. Sorger et al.

    Nucl. Med. Biol.

    (2003)
  • M. Patt et al.

    Appl. Radiat. Isot.

    (2002)
  • J.D. Young et al.

    Mol. Aspects Med.

    (2013)
  • O. Schulze et al.

    Carbohydr. Res.

    (2005)
  • Y. Akiyama et al.

    J. Fluorine Chem.

    (2006)
  • J.E. Moulder et al.

    Int. J. Radiat. Oncol. Biol. Phys.

    (1984)
  • M. Haga et al.

    Carbohydr. Res.

    (1972)
  • J.D. Chapman

    Cancer

    (1984)
  • M. Hockel et al.

    Cancer Res.

    (1991)
  • P. Vaupel et al.

    Cancer Res.

    (1991)
  • J.M. Brown

    Cancer Res.

    (1999)
  • A. Nunn et al.

    Eur. J. Nucl. Med.

    (1995)
  • G. Mees et al.

    Eur. J. Nucl. Med. Mol. Imaging

    (2009)
  • Y. Masaki et al.

    Sci. Rep.

    (2015)
  • L.S. Mortensen et al.

    Acta Oncol.

    (2010)
  • J.S. Rasey et al.

    J. Nucl. Med.

    (1999)
  • Cited by (0)

    Deceased.

    View full text