Automated synthesis of 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([18F]-FMAU) using a one reactor radiosynthesis module

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Abstract

2′-Deoxy-2′-[18F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([18F]-FMAU) is an established PET probe used to monitor cellular proliferation. For clinical applications, a fully automated cGMP-compliant radiosynthesis would be preferred. However, the current synthesis of [18F]-FMAU requires a multistep procedure, making the development of an automated protocol difficult and complicated. Recently, we have developed a significantly simplified one-pot reaction condition for the synthesis of [18F]-FMAU in the presence of Friedel-Crafts catalysts. Here, we report a fully automated synthesis of [18F]-FMAU based on a one reactor radiosynthesis module using our newly developed synthetic method. The product was purified on a semi-preparative high-performance liquid chromatography integrated with the synthesis module using 6% EtOH in 10 mM phosphate buffer or 8% MeCN/water. [18F]-FMAU was obtained in 12±3% radiochemical yield (decay corrected overall yield based on [18F]-F, n=4) with 383±33 mCi/μmol specific activity at the time of injection. The α/β anomer ratio was 4:6. The overall reaction time was about 150 min from the end of bombardment and the radiochemical purity was >99%. This automated synthesis should also be suitable for the production of other 5-substituted thymidine analogues.

Introduction

A number of radiolabeled 2′-deoxy-2′-fluoro-5-substituted-1-β-d-arabinofuranosyl-uracil derivatives have been evaluated as probes for imaging tumor proliferative activity and HSV1-tk reporter gene expression with positron emission tomography (PET) [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Among these, 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-d-arabinofuranosyl-uracil ([18F]-FMAU), 2′-deoxy-2′-fluoro-5-[11C]methyl-1-β-d-arabinofuranosyl-uracil and 2′-deoxy-2′-[18F]fluoro-5-bromo-1-β-d-arabinofuranosyl-uracil are markers for DNA synthesis through phosphorylation by human and other mammalian nucleoside kinases, including thymidine kinases TK1 and TK2 [3], [4], [13]. Although 18F-3′-deoxy-3′-fluorothymidine (18F-FLT) has been widely used for cell proliferation imaging by taking advantage of the pyrimidine salvage pathway [14], [15], 18F-FLT-triphosphate is not significantly incorporated into DNA [16], [17], [18], [19], [20] and the majority of 18F-FLT persists as mono- and triphosphates in the cytosol [15], [16]. Preclinical studies have shown that FMAU retention in tumors and nontumor tissues with rapid cell turnover (e.g., marrow and small intestine) reflects its incorporation into DNA [1], [2], [4], [13]. FMAU may be useful for imaging tumor cell proliferation with PET and that further clinical investigation of C-11 and F-18 FMAU, in comparison with 18F-FLT, is warranted. 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], [22], [23], [24]. Many of these 2′-fluoro-5-substitued arabinosyluracil derivatives were synthesized and evaluated earlier as antiviral agents [25], [26], [27]. The first radiochemical synthesis of FMAU with PET isotope [11C] was reported by us [28]. However, due to the short half-life of [11C] (t1/2=20 min), there was a need to develop an [18F]-labeled derivative. Subsequently, we reported the radiosynthesis of [18F]-labeled FMAU and other 5-substituted thymidine analogues [29], [30]. In this procedure, the radiosynthesis of [18F]-FMAU involves radiofluorination of 2-trifluoromethane-sulfonyl-1,3,5-tri-O-benzoyl ribofuranose to the 2-[18F]-fluoro-1,3,5-tri-O-benzoyl arabinofuranose derivative, followed by conversion to the 1-bromo-2-[18F]-fluoro-1,3,5-tri-O-benzoyl derivative, and then coupling of the 1-bromo-2-[18F]fluoro-2,3,-di-O-benzoylarabinofuranose with 2,4-bis-trimethylsilyluracil derivatives. Finally, hydrolysis of the protecting groups from the sugar moiety was performed and high-performance liquid chromatography (HPLC) purification yielded the desired products. Following our synthesis, another group of investigators also reported the [18F]-labeled synthesis of these pyrimidine nucleoside analogues [31]. Although we and other researchers in the field have demonstrated these reactions are very reliable and reproducible [32], [33], [34], the complexity of this method often requires significant modification of existing commercial automated modules, accompanied by frequent production failures. In order to find an efficient fully automated cGMP-compliant radiosynthesis methodology for the production of these probes, our group has been optimizing the reaction conditions in order to reduce synthetic time and simplify reaction conditions [35]. Recently, we reported the use of Friedel-Crafts catalysts for an improved synthesis of [18F]-FMAU, which also included a significantly simplified one-pot reaction condition (Scheme 1) [36], [37]. In this paper, we report for the first time an automated synthesis of [18F]-FMAU using a one-reactor radiosynthesis module. The method is also compatible with most commercially available modules typically used for production of cGMP-compliant radiotracers for clinical applications.

Section snippets

Reagents and instrumentation

All reagents and solvents were purchased from Aldrich Chemical (Milwaukee, WI, USA), and used without further purification. Solid-phase extraction cartridges were purchased from Waters. Ion exchange cartridges were purchased from ABX (Germany). 2-Trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-α-d-ribofuranose (precursor) and bis-2,4-trimethylsilyl-5-methyluracil were purchased from ABX (Germany). Non-radioactive FMAU anomers were prepared in house and used as HPLC standards. Analysis was

Results and discussion

In our previous research, we have developed a one-pot reaction condition for [18F]-FMAU synthesis [36], [37]. Once the one pot condition was developed, we incorporated it to an automated synthesis module. In the automated synthesis module, all reagents were stored in the reservoirs sequentially with the appropriate reagents and solvents under nitrogen prior to receiving the [18F]-fluoride from the target of the cyclotron. After receiving the radioactivity into the synthesis module,

Conclusion

A fully automated synthesis of [18F]-FMAU has been achieved in reasonable yields and high purity using a one-reactor synthesis module. The simplified and reliable synthetic method can be widely applied for the production of other 2′-[18F]fluoro-2′-deoxy-arabino-5-substituted pyrimidine nucleoside analogues and may make them more accessible for preclinical and clinical research.

Acknowledgments

This work was supported partially by research grant DE-SC0002353 from the Department of Energy, the USC Department of Radiology, and the Provost's Biomedical Imaging Science Initiative.

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