Original articleSynthesis and evaluation of a thymidine analog for positron emission tomography study of tumor DNA proliferation in vivo
Introduction
Positron emission tomography (PET) is a unique, non-invasive imaging technique which is finding greater utility in oncology and is becoming an increasingly valuable method for the imaging and staging of known and suspected tumors [1]. PET can provide images based on tumor metabolism and does not have to rely on morphological identity comparisons to detect tumors [2]. This implies that PET can be used to identify tumors not easily seen using more widespread imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), or ultrasonography [3]. Importantly, PET can also be used for monitoring tumor response to therapy [4]. As PET measures the total radioactivity retained in tissue at any point in time, regardless of the chemical species associated with the isotope, it is necessary for the imaging agent used to be stable in vivo for the duration of the scan [5].
As part of an ongoing research project into the synthesis of a novel nucleoside based radio imaging agent, we were attracted to 1 as a potential lead compound. Initially designed as a non-polar shape mimic for natural thymidine, 1 was used to investigate the role of hydrogen bonding within the DNA helix [6], [7]. It has been shown that 1 is isosteric with thymidine and that its triphosphate is readily accepted by various DNA polymerases [8], being inserted opposite adenine in oligonucleotide templates [9]. We planned to incorporate the fluorine-18 radionuclide at the benzylic methyl position of 1, thereby potentially creating a new radiotracer for imaging cell proliferation.
Section snippets
Synthetic studies toward the radiolabelling precursor 3
We propose that a simple modification to the synthetic strategy used by Kool and co-workers would afford a suitable precursor for radiolabelling studies. Thus, the substitution of a suitably protected difluorobenzyl alcohol 2 for the difluorotoluene utilized by Kool and co-worker for the Grignard reaction [7] should provide an avenue toward the desired precursor 3, Scheme 1.
Unfortunately, our attempts at the bromination of the silyl protected 2,4-difluorobenzyl alcohol following conditions
Radiosynthesis and formulation
The radio-fluorination of the precursor 3 was achieved by treatment with 18F-fluoride ion generated via the 18O(p,n)18F nuclear reaction with 10 MeV proton beam using an IBA Cyclone 10/5 cyclotron. The dried K[18F]kryptofix 2.2.2 complex was reacted with a solution of 3 in acetonitrile and allowed to reflux for 5 minutes to form the radio-fluorinated intermediate 12. Saponification of the toluoyl esters of 12 was achieved by treating with a 1 M solution of sodium hydroxide for 10 minutes,
Biological evaluation
In vitro stability of radiotracer 13 was first evaluated at 37°C in both saline solution and blood serum. Samples were taken from each vial at regular intervals and analyzed by radio-TLC. Radio-defluorination was not evident, with only a single radio-peak observed at Rf 0.34. Further studies showed that 13 was stable in saline solution up to temperatures of 70°C.
The biological evaluation of 13 was then undertaken, beginning with a simple cell incorporation study. Incubation of 13 in the
Experimental
Melting point determinations were made using a Gallenkamp Capillary melting point apparatus and are uncorrected. 1H Nuclear magnetic resonance (NMR) spectra were recorded at 300 MHz using a Varian Unity 300 spectrometer and at 400 MHz using a JEOL GX-400 spectrometer. 13C Nuclear magnetic resonance spectra were recorded at 75 MHz using a Varian Unity 300 spectrometer and at 100 MHz using a JEOL GX-400 spectrometer. Tetramethylsilane (TMS) was used as the nominal standard for all proton and
Conclusions
We have successfully synthesized and radiolabelled the titled compound 13. Although initial cell studies were promising and showed a linear relationship between uptake and cell concentration, the de-fluorination observed in vivo, and the lack of uptake in the DNA incorporation study led us to conclude that 13 and its direct analogs were unsuitable as radiotracers for imaging cellular proliferation. Further work is required to identify a thymidine analog with physiochemical characteristics
Acknowledgements
The authors are grateful for the financial support provided by the NHMRC (Dora Lush Scholarship), and for the Maintenance Grant obtained from the AHMRF. They also wish to thank Dr. F. T. Lee and Ms. Cathrine Hall for their excellent technical assistance.
References (21)
- et al.
Clinical imaging of cancer metastasis
Eur J Cancer
(2000) - et al.
What does positron emission tomography offer oncology?
Eur J Cancer
(2000) - et al.
An efficient method for the synthesis of aromatic C-nucleosides
Tetrahed Lett
(1995) Current status of positron emission tomography in oncology
Intern Med J
(2001)- et al.
PET in clinical oncology
Cancer Metast Rev
(1988) - et al.
Contribution of labeled carbon dioxide to PET imaging of carbon-11-labeled compounds
J Nucl Med
(1992) - et al.
Aromatic nonpolar nucleosides as hydrophobic isosteres of pyrimidines and purine nucleosides
J Org Chem
(1994) - et al.
Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication
J Am Chem Soc
(1997) - et al.
Solution structure of a nonpolar, non-hydrogen-bonded base pair surrogate in DNA
J Am Chem Soc
(2000) - et al.
Synthesis of fluorinated aromatic monomers. V. Preparation of fluorine containing isophthalic and terephthalic acids
Kogyo Kagaku Zasshi
(1970)