Investigation of the role of the base in the synthesis of [18F]FLT

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Abstract

The role of the base in the synthesis of 3′-deoxy-3′-[18F]fluorothymidine, [18F]FLT, via nucleophilic substitution of the nosyl group with [18F]fluoride was investigated. The rate of 18F-incorporation into the molecule dramatically changed as a function of the precursor-to-base ratio. In the presence of excess base, the precursor was consumed by elimination before substitution was complete. When the precursor-to-base ratio was optimal, an overall [18F]FLT yield of 30–40% was achieved even if the precursor amount was as small as 8–13 mg.

Introduction

Recently, 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) has been developed as a radiopharmaceutical to image cellular proliferation in vivo with positron emission tomography (PET) (Vesselle et al., 2002; Been et al., 2004; Barthel et al., 2005). Intra-cellularly, [18F]FLT is phosphorylated by the enzyme thymidine kinase-1, which is expressed during the DNA synthesis phase of the cell cycle (Sherley and Kelly, 1988; Munch-Petersen et al., 1991). Subsequently, 18F-labeled thymidine monophosphate accumulates and is trapped within the cell. Unlike 11C-thymidine, [18F]FLT is not incorporated into DNA (Been et al., 2004). However, its accumulation into proliferating cells shows a high correlation with DNA replication. It has been shown that the uptake of [18F]FLT reflects the cell proliferation rate in tumors better than that of the widely utilized tumor imaging agent [18F]FDG (Chen et al., 2005; Reske and Deisenhofer, 2006). Furthermore, [18F]FLT images were demonstrated to differentiate tumor from inflammation (Buck et al., 2002; Van Waarde et al., 2004; Chen et al., 2005; Reske and Deisenhofer, 2006). The compound has also been evaluated in detecting tumor response to chemo- and radiotherapies (Sugiyama et al., 2004; Leyton et al., 2005; Dorow et al., 2006; Pio et al., 2006).

Synthesis of [18F]FLT via nucleophilic displacement of the nosyl functionality at the 3′ position with [18F]fluoride has been reported by Grierson et al. (1997), Grierson and Shields (2000), Martin et al. (2002), Yun et al. (2003) and Moon et al. (2006). Among them, the in-depth investigation by Yun et al. gives important information regarding optimization of the [18F]FLT preparation procedure. They report that their decay-corrected radiochemical yields reached 40–42% using 30–40 mg of the two precursors, 1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine and its 3-N-Boc analog. We initiated our [18F]FLT radiosyntheses based on their published method. However, we could not reproduce some of their results: (a) after heating the reaction mixture containing 10 mg of the N-Boc protected precursor, [18F]fluoride and Kryptofix 222/K2CO3 in acetonitrile at 100 °C for 5 min, 60–70% of 18F was found incorporated into the molecule whereas they report only 7% incorporation and (b) under the same reaction conditions, we discovered that 80–90% of the precursor was lost in 5 min. Furthermore, if the reaction was heated at 120 °C, the loss was greater than 99% while they report that an increase in temperature up to 120 °C resulted in higher 18F-incorporation. These problems prompted us to suspect that there might be some other critical factors determining the rate of the substitution reaction and ultimately the yield of [18F]FLT synthesis. We therefore undertook to further investigate the nucleophilic substitution reaction that produces [18F]FLT. Here we report our findings.

Section snippets

Chemicals

The precursors, 3-N-Boc-1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine (Precursor A) and 1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine (Precursor B) were purchased from ABX Advanced Biomedical Compounds (Radeberg, Germany). Kryptofix 222 (K222) and potassium carbonate were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and 2 N NaOH and 1 N HCl from Fisher Scientific. Anhydrous acetonitrile was obtained from Sigma-Aldrich and Pierce

Synthesis of [18F]FLT using Precursor A

The nucleophilic substitution reaction with [18F]fluoride in acetonitrile (Fig. 1, top) proceeded efficiently at 100 °C with 6–13 mg of the precursor (average: 8.8±2.2 mg, 11 μmol, n=11) in the presence of 5–6 mg of K222 (5.3±0.5 mg, 14 μmol) and 1.4 mg of K2CO3 (1.35±0.11 mg, 10 μmol). 18F-incorporation into the intermediate (Compound B, Fig. 1) was approximately 60–70% (average 64.4±10.5%) in 5 min. Two radioactive peaks were observed, one corresponding to the [18F]FLT intermediate (Compound B) and the

Discussion

This investigation reveals that the 18F-incorporation into the [18F]FLT intermediate (Compound B) is dramatically decreased with increase in the amount of the K222/K2CO3 complex (Fig. 3 and Table 1) suggesting that the basicity of the reaction plays a critical role in determining the rate of the nucleophilic substitution reaction with [18F]fluoride in [18F]FLT production. It is well known that base-induced elimination competes with nucleophilic substitution when the leaving group, such as a

Conclusion

The basicity of the reaction medium appears to play a critical role in determining the rate of nucleophilic substitution of the nosyl leaving group of Precursor A with [18F]fluoride. In the presence of an excess amount of the base vs that of the precursor, the elimination reaction appears to dominate, and the precursor is consumed rapidly before the nucleophilic substitution is complete. Under optimal conditions, where the precursor-to-base ratio is approximately 1.2–1.5, [18F]FLT can be

Acknowledgments

We thank Mr. Howard Sheh and Mr. Calvin Lom of Sloan-Kettering Institute for their careful management of the cyclotron facility and in particular for 18F production essential for this work. We also thank Dr. Alan A. Wilson, Chief Radiochemist of Vivian M Rokoff PET Imaging Center, University of Toronto, for kindly taking the time to discuss this work.

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