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

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Abstract

[18F]-FBAU and [18F]-FCAU have been synthesized and evaluated in vivo as markers for HSV1-tk gene expression. At 2 hours, uptake of [18F]-FBAU and [18F]-FCAU in HSV1-tk–positive tumors was 7.9-fold and 6.0-fold higher than the control tumors, respectively. Micro-PET images also showed very high uptake in HSV-tk tumors. Compared to [14C]-FMAU, total uptake of [18F]-FBAU and [18F]-FCAU was similar in tk-positive cells, but the uptake ratio (tk+/wild) was higher. [18F]-FBAU and [18F]-FCAU appear to be potential PET imaging agents 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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