Synthesis of 2′-deoxy-2′-[18F]fluoro-5-bromo-1-β-D-arabinofuranosyluracil ([18F]-FBAU) and 2′-deoxy-2′-[18F]fluoro-5-chloro-1-β-D-arabinofuranosyl-uracil ([18F]-FCAU), and their biological evaluation as markers for gene expression
Introduction
Herpes simplex virus type-1 thymidine kinase (HSV1-tk) is being used as a suicide gene for gene therapy of cancer [10], [24], [25], [31]. In animal models malignant tumors have been successfully treated with suicide gene therapy using HSV1-tk gene and ganciclovir [11], [22]. However, clinical results with this method have suggested that gene delivery to the tumor cell in human is not sufficient [24], [25] for therapy. An in vivo method to assess the HSV1-tk enzyme activity after gene transfer is desirable to monitor gene expression as an indicator of gene delivery. Imaging probes can be used to assess vector targeting, the level of suicide gene (HSV1-tk) expression, and quantitatively monitor the level of enzyme in gene therapy [3], [12], [29]. Nuclear medicine techniques, such as positron emission tomography (PET) can provide repeated, noninvasive and quantitative assessment of the expression of genes in tissues and organs [12], [13], [15], [27], [28], [34].
Imaging of the HSV1-tk reporter gene along with various reporter probes is of current interest [3], [12], [15]. In contrast to the mammalian kinase, which phosphorylates thymidine preferentially, HSV1-tk is less discriminative and phosphorylates a wide range of nucleoside analogues such as acycloguanosines and 2′-fluoro-2′-deoxyuridine derivatives that are not phosphorylated efficiently by the native enzyme [3], [8], [14], [26], [27], [31]. More specifically, 5-substituted 2′-fluoro-2′-deoxy-arabinofuranosyluracil nucleosides are efficiently phosphorylated by HSV-tk. This property, together with the presence of fluorine in the 2′-arabino-position, endows the 2′-fluoro-2′-deoxyuridines with antiviral activity against HSV type 1 and type 2 [32].
Therefore, a number of radiolabeled nucleoside derivatives are under investigation as reporter probes for HSV-tk. Earlier we developed the acycloguanosine derivatives [18F]-FHPG and [18F]-FHBG for PET imaging of HSV1-tk gene expression [1], [2], [3], [4] and demonstrated that [18F]-FHBG is more useful than [18F]-FHPG for this purpose [2], [4]. Acyclovir (FACV), ganciclovir (FGCV), and pencyclovir (FPCV) labeled with 18F at the C-8 position have also been reported [8], [9], [14], [23], with FPCV being a better reporter probe than FGCV [14].
Compared to the acycloguanosine analogues FHPG and FHBG, the pyrimidine nucleoside derivatives 2′-deoxy-2′-fluoro-5-iodo-1-β-D-arabinofuranosyluracils (FIAU) and 2′-deoxy-2′-fluoro-5-methyl-1-β-D-arabinofuranosyluracils (FMAU) have been found to be much more sensitive probes (higher cellular accumulation) for PET imaging of HSV1-tk gene expression [5], [30]. Radio-iodinated FIAU has been shown to be superior to the acycloguanosine derivatives FHPG and FHBG in some cell lines [30] in terms of total uptake and uptake ratio (tk+/control), although it is susceptible to deiodination in vivo.
The syntheses of [18F]-FMAU and other [18F]-labeled 5-substituted pyrimidine nucleoside analogues [6], [7], as well as imaging studies with [124I]-FIAU [30], lead to an increased interest in the development of other related analogues as PET imaging agents. 2′-Deoxy-2′-fluoro-5-bromo-1-β-D-arabinofuranosyluracil labeled with 76Br ([76Br]-FBAU) and its 3′,5′-dibenzoate were developed for PET imaging of tumor proliferation [16], [17], [18], [19]. However, it has been noted that the use of FBAU labeled with 76Br has disadvantages on the account of the cumbersome production of the isotope, and the potentially high radiation dose to the patient [19]. On the other hand 18F-fluoride production is relatively straightforward and readily available today. Most recently a synthesis of [18F]-FBAU has been reported [21], which is similar to the general synthesis of 5-substituted pyrimidine nucleoside published in the literature [7]. Although a significant amount of work has been devoted to radiolabeled FBAU, the 5-chloro analogue, FCAU has received very little attention [33]. To explore FBAU and FCAU as markers for HSV1-tk gene expression we have synthesized [18F]-FBAU and [18F]-FCAU and studied them in vivo, including biodistribution and micro-PET imaging in tumor-bearing nude mice, and compared results with those of [14C,18F]-FMAU in the same cell line.
Section snippets
Methods and materials
[18F]-FBAU and [18F]-FCAU were synthesized following a general method developed in our laboratory [6], [7] as shown in Fig. 1.
Non-radioactive compounds were prepared with 19F-fluoride for characterization by spectroscopic methods and HPLC standards. 2-Deoxy-2-fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofuranose was prepared by the reaction of the triflate 1 with tetrabutylammoniumfluoride at 80°C. The fluorosugar was converted to its 1-bromo-derivative 2 by reaction with hydrogen bromide in acetic
Results and discussion
Synthesis of [18F]-FBAU or [18F]-FCAU, performed following the method developed in our laboratory [7], is represented in Fig. 1. The coupling reaction between the compound 2 and protected 5-bromouracil produced low yield (25%) and that between 2 and 5-chloro-uracil produced relatively higher yield (50%) in non-radioactive preparations. The coupled pyrimidine benzoate esters 3a and 3b were characterized by 1H and 19F NMR spectroscopy. 1H NMR spectra of these compounds were consistent with the
Acknowledgments
This work was supported in part by National Cancer Institute Grant CA 72896.
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An investigation on stereospecific fluorination at the 2′-arabino- position of a pyrimidine nucleoside: Radiosynthesis of 2′-deoxy-2′- [<sup>18</sup>F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil
2012, TetrahedronCitation Excerpt :Finally hydrolysis of the protecting groups with a strong base and purification produced the desired 2′-fluoro-1-β-d-arabino-pyrimidine nucleoside.2,4,11–13 Because of the anticancer and antiviral properties, some of these fluorinated pyrimidine nucleoside analogues have been radiolabeled with 18F for positron emission tomography (PET) of tumor proliferation14–16 and herpes simplex virus type 1-thymidine kinase (HSV1-tk) reporter gene expression.17–22 Alauddin et al. developed 18F-labeled 2′-fluoro-arabinofuranosyluracil derivatives, such as 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([18F]FMAU) for the first time23 and some other 2′-deoxy-2′-[18F]fluoro-5-substituted-1-β-d-arabinofuranosyluracil derivatives,24 by modification of the multistep process, such as radiofluorination of 1,3,5-tri-O-benzoyl-α-d-ribofuranose-2-trifluoromethylsulfonate ester, followed by bromination of the radiolabeled sugar and coupling of the radiolabeled 1-bromosugar with pyrimidine-bis-trimethylsilyl ether.
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2011, Nuclear Medicine and BiologyCitation Excerpt :Previously, we have demonstrated that good β anomer selectivity was maintained by passing the fluorinated sugar motif through a silica cartridge. In this newly developed method, the multistep radiosynthesis was performed in one reaction vial (one-pot) without extra purification, which may make the reaction system more complicated compared with the reported methods (mostly two reaction vials and/or two purifications) [11,12,20,24,33–35,36]. Moreover, in order to push the reaction to be done quickly, higher temperature was employed.
Automated synthesis of 2′-deoxy-2′-[<sup>18</sup>F]fluoro-5-methyl-1-β-d-arabin ofuranosyluracil ([<sup>18</sup>F]-FMAU) using a one reactor radiosynthesis module
2011, Nuclear Medicine and BiologyCitation Excerpt :FMAU is undergoing preclinical and clinical studies for imaging tumor proliferation in a variety of cancer types [3,4,13,21]. The other uracil derivatives, such as 2′-deoxy-2′-[18F]-fluoro-5-iodo-1-β-d-arabinofuranosyluracil, 2′-deoxy-2′-[18F]fluoro-5-fluoro-1-β-d-arabinofuranosyl-uracil and 2′-deoxy-2′-[18F]-fluoro-5-chloro-1-β-d-arabinofuranosyl-uracil are excellent substrates for the viral kinases such as herpes simplex virus Types 1 and 2, and 2′-deoxy-2′-[18F]-fluoro-5-iodo-1-β-d-arabinofuranosyluracil (FIAU), is also a substrate for hepatitis B virus and Epstein Barr virus thymidine kinase [7,8,21-24]. Many of these 2′-fluoro-5-substitued arabinosyluracil derivatives were synthesized and evaluated earlier as antiviral agents [25-27].