[18F]-EF5, a marker for PET detection of hypoxia: synthesis of precursor and a new fluorination procedure
Introduction
Tissue hypoxia has important biological and clinical implications in various pathological states. In tumors, the level of tumor oxygenation correlates with response to radiation therapy (Brizel et al., 1997, Hockel et al., 1993, Nordsmark et al., 1996). Therefore, the in vivo measurement of the level of tumor oxygenation is clinically important. We have previously developed a 2-nitroimidazole marker of hypoxia named EF5 (2-(2-nitroimidazol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide) (Lord et al., 1993, Evans et al., 1995, Koch et al., 1995). The reductive intracellular metabolism of EF5 leads to its covalent binding with cellular molecules. This process is inhibited by increasing oxygen concentration (Chapman et al., 1983, Koch et al., 1984, Varghese et al., 1976), providing specific drug binding in hypoxic tissue. Fluorescent labeled monoclonal antibodies allow the detection of the bound adducts and determination of the level and location of hypoxia (Evans et al., 1995, Koch et al., 1995).
Incorporation of 18F into 2-nitroimidazole molecules provides an opportunity to use these agents for the detection of hypoxia by positron emission tomography (PET) (Jerabek et al., 1986, Mathias et al., 1987). Several groups have developed 18F-labeled nitroimidazole-based PET assays, for example, [18F]-fluoromisonidazole (Rasey et al., 1987, Rasey et al., 1996, Grierson et al., 1989, Koh et al., 1992), [18F]-fluoroerythronitroimidazole (Yang et al., 1995) and [18F]-fluoroetanidazole (Tewson, 1997). The first described and most investigated compound of this type is [18F]-fluoromisonidazole (Rasey et al., 1987). This agent has been studied in several anatomic sites in humans including gliomas (Valk et al., 1992), lung cancer (Koh et al., 1994) and nasopharyngeal carcinoma (Yeh et al., 1996).
Despite the extensive investigations, none of the currently developed compounds is accepted clinically as a PET marker of hypoxia. A possible general problem is that the compounds described above are new products with relatively unknown pharmacological properties (Stöcklin, 1998), e.g. the structure of [18F]-fluoromisonidazole is substantially different from misonidazole, [18F]-fluoroetanidazole has the hydroxyl group of etanidazole substituted by a fluorine atom. It has only recently been shown that [18F]-fluoromisonidazole is not stable in vivo, and produces multiple radioactive products distinct from parent drug following renal clearance (Rasey et al., 1999). These differences and the use of very low drug concentrations for the PET studies may therefore lead to results not predicted from studies with the parent compounds or at much higher drug concentrations. In contrast, labeling of the EF5, molecule with 18F provides a unique opportunity to develop a true PET analog of a hypoxia marker with well documented pharmacological properties which has been shown to predict radiotherapy resistance in individual rodent tumors (Laughlin et al., 1996, Evans et al., 1996).
Previously, we described the preparation of a new PET hypoxia marker [18F]-EF1, [18F]-2-(2-nitroimidazol-1[H]-yl)-N-(3-fluoropropyl)-acetamide, with a structure similar to EF5 (Kachur et al., 1999). This compound was synthesized using nucleophilic substitution of the bromine atom of a precursor 2-(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)-acetamide, by [18F]-F−. [18F]-EF1 has shown good potential for labeling of hypoxic tumors and a relatively uniform biodistribution limited by slow equilibration with brain tissue (Evans et al., 2000). In the work to be described herein, we initially attempted to utilize the same technique. Unfortunately, nucleophilic substitution of a bromine substituted precursor to EF5 was not successful. Thus, we developed a new approach, namely an electrophilic fluorination of an allyl precursor. Here, we report the procedure for the preparation of [18F]-EF5 [18F]-2-(2-nitroimidazol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide by addition of [18F]-fluorine gas to 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,3,3-trifluoroallyl)-acetamide. Subsequent extraction and HPLC purification allow a facile synthesis which should be suitable for human use. Preliminary rodent biostability data demonstrated renal excretion of [18F]-EF5 with no evidence for breakdown products excreted in urine.
Section snippets
Experimental
Reagents and solvents were purchased from Aldrich and used without additional purification unless otherwise noted. 1H-NMR spectra were recorded on a Bruker-AMX-300 using CDCl3 or acetone-d6 as solvent and tetramethylsilane as an internal standard; 19F-NMR spectra were measured on a Varian XL at 282 MHz, referenced to external CF3COOH in D2O. HPLC was performed on a Waters system (with Waters UV detector and radioactivity detector from IN/US Service, Fairfield, NJ) using an Altima C-18 column (5
Discussion
Incorporation of 18F into 2-nitroimidazole molecules provides an opportunity to use these agents for hypoxia detection by non-invasive PET technique. Our early attempt to develop a PET agent [18F]-EF1 with a structure similar to EF5 (Kachur et al., 1999) was quite successful. This marker was found to give a tumor to muscle ratio of about 3 in hypoxic tumors, but only 1.2 in non-hypoxic tumors (Evans et al., 2000). However, we have almost no data on EF1 metabolism, while EF5 is well
Conclusion
We developed the procedure of fluorine addition to alkene double bond in trifluoroacetic acid at room temperature. This may be a useful synthetic method for the fluorination of multifunctional aromatic compound with basic function. We also demonstrated the possibility of the direct addition of fluorine gas to partially fluorinated double bond.
The proposed method may be used for the preparation of other PET agents. The results indicate our ability to prepare clinically useful amounts of [18
Acknowledgments
This work was supported by the Departments of Radiation Oncology and Radiology, School of Medicine, University of Pennsylvania and Department of Chemistry, University of Florida.
References (33)
- et al.
Synthesis of N-substituted aziridine-2-carboxylates
Tetrahedron
(1977) - et al.
Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck
International Journal of Radiation Oncology, Biology, Physics
(1997) - et al.
Reactions of an electrophilic glycine cation equivalent with Grignard reagents. A simple synthesis of β,γ-unsaturated amino acids
Tetrahedron Letters
(1986) - et al.
Synthesis of α-amino acids with β,γ-unsaturated side chains
Tetrahedron
(1988) - et al.
Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix
Radiotherapy and Oncology
(1993) - et al.
Synthesis and biodistribution of 18F-labeled fluoronitroimidazoles: potential in vivo markers of hypoxic tissue
International Journal of Radiation Applications and Instrumentation, Part A, Applied Radiation and Isotopes
(1986) - et al.
Synthesis of new hypoxia markers EF1 and [18F]-EF1
Journal of Applied Radiation and Isotopes
(1999) - et al.
Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole
International Journal of Radiation Oncology, Biology, Physics
(1992) - et al.
Radiolabeled hypoxic cell sensitizers: tracers for assessment of ischemia
Life Sciences
(1987) - et al.
Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck
Radiotherapy and Oncology
(1996)
Quantifying regional hypoxia in human tumors with positron emission tomography of [18F]fluoromisonidazole: a pretherapy study of 37 patients
International Journal of Radiation Oncology, Biology, Physics
Synthesis of [18F]fluoroetanidazole: a potential new tracer for imaging hypoxia
Nuclear Medicine and Biology
The Development of Radioactive Sensitizers as Markers for Hypoxic Cells in Tumors
Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide]
British Journal of Cancer
2-Nitroimidazole (EF5) binding predicts radiation resistance in individual 9L s.c. tumors
Cancer Research
Cited by (139)
Imaging Hypoxia
2021, Molecular Imaging: Principles and PracticeFluorine-18: A radionuclide with diverse range of radiochemistry and synthesis strategies for target based PET diagnosis
2020, European Journal of Medicinal ChemistryCitation Excerpt :Dolbier et al., demonstrated the synthesis of 18F-EF5, a marker for PET detection of hypoxia. The electrophilic radiofluorination of precursor 2-(2-nitro-1[H]- imidazole-1-yl)-N-(2,3,3-tri‾uoroallyl)-acetamide (fluorinated alkene bond) with fluorine gas (18F–F2) in trifluoroacetic acid offered target 18F-labeled compound [22] (Fig. 2B). Gerard W. M et al., optimized reaction conditions for the synthesis of 18F-labeled 5-fluorouracil (5-FU) using electrophilic fluorinating agents and evaluated in nude mice with colon 38 carcinoma (5-FU sensitive) and/or a rhabdomyosarcoma (5-FU resistant) tumor [23] (Fig. 2C).
NMR diffusion and relaxation studies of 2-nitroimidazole and albumin interactions
2018, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopySynthesis of F-18 labeled resazurin by direct electrophilic fluorination
2015, Journal of Fluorine ChemistryCitation Excerpt :Resazurin represents another group of complex organic molecules, which can be selectively fluorinated by diluted fluorine gas in acidic conditions with introduction of an 18F-label into the molecule. Although the yield of the products in this reaction (1–2.5%) is much lower in comparison with fluorine addition to double bonds (30%) [9] and fluorination of triarylmethane pH indicators (5–10%) [2–4], it is still high enough for preparation of the 18F-labeled compound at millicurie level, which is enough for practical application in any biological system. Introduction of the first fluorine atom into the molecule of resazurin occurs at ortho positions to the hydroxyl group.
Insight into Tumor Hypoxia: Radionuclide-based Biomarker as Diagnostic Tools
2023, Current Topics in Medicinal Chemistry