Synthesis of [¹²³I]-iodometomidate from a polymer-supported precursor with a large excluded volume

Abstract

We report on the synthesis of [¹²³I]-iodometomidate from a soluble polymer-supported precursor. The precursor was linked through a soluble polymer via a silyl linker polymer-supported precursor and released [¹²³I]-iodometomidate. The radiotracers were purified based on a combination of size-exclusive and normal-phase chromatography to provide [¹²³I]-iodometomidate in good quality.

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Nuclear imaging such as PET and SPECT visualizes target cells and organs using radioactive tracers that possess a suitable radionucleotide. It is used in clinics and in small animal research to noninvasively study the molecular basis of disease and to guide the development of novel molecular-based treatments.¹ The radioactive tracers are usually prepared using picomolar to nanomolar amounts of radioisotope and a large excess of non-radioactive precursors (in the micromolar to millimolar range). The reaction mixtures are usually purified via a reverse-phase HPLC system to remove structurally related, non-labeled products because they act as competitors to reduce the efficiency of the imaging. In addition, the synthesis of radioactive tracers must be achieved by automated synthesizers in order to protect operators from the toxicity of radioisotopes. The HPLC system is a useful technology for the automated purification of these radiotracers from reaction mixtures. However, it should not be easy to establish a standardized protocol for purification of the radioactive tracers from a large excess amount of the precursors. In addition, the complex HPLC apparatus must pass a validation cleaning procedure before use. Therefore, a rapid and robust methodology for separation of a small amount of the radioactive tracers from the reaction mixture by using a simple apparatus is required.

The phase-tag-assisted method of synthesis emerged as an effective method for the removal of unreacted precursors during the synthesis of radioactive tracers.² Supporting the precursors on a solid allows the separation of the precursors from the labeled product by filtration.³ Brown and co-workers reported on the synthesis of [¹⁸F]2-deoxy-2-fluoroglucose (2-FDG) from a solid-supported precursor.⁴ We recently reported the synthesis of 2-FDG using a solid-supported polymer as a precursor.⁵ However, immobilizing precursors on a solid frequently reduces the reactivity towards the incorporation reaction with radioisotopes. On the other hand, a light-fluorous tag enables compounds to be separated via solid-phase extraction with minimum effects on their reactivity.⁶ Gouverneur and co-workers reported on the fluorous assisted synthesis of radioactive compounds.⁷ The fluorous-labeled precursors were separated by fluorous solid-phase extraction. However, conventional phase-tag approaches are based on solid-phase extraction of the precursors and provide an eluate containing the radiotracers, reaction solvents, remaining radioisotopes and other reagents, which require additional purification involving concentration of the eluate, followed by column chromatography. However, concentration of the eluent containing water in an automated synthesizer is a time-consuming step that reduces the radiochemical yield.

To overcome these problems, we planned the synthesis of radiotracers involving purification based on solid-phase extraction of the radiotracers (Fig. 1). A soluble polymer with a large excluded volume was used as a precursor that could release the radiotracers via a labelling reaction. Size-exclusion chromatography released the polymer in a short retention time and enabled solid-phase extraction of the tracers. Elution of the radiotracer with evaporable organic solvents provided the radioactive tracers in high quality. In total, this method omits the concentration of the eluate and an additional purification in comparison with the previous methods and should provide an effective purification protocol for preparation of the radiotracer with high quality in an automated synthesizer without the use of HPLC purification.

Fig. 1 Concept for synthesis of a radiotracers based on the single-column purification using soluble polymer-supported precursors.

To demonstrate the utility of this method, we planned to synthesize radioiodinated iodometomidate (IMTO) 1 from the polymer-supported precursor 2 (Scheme 1). Iodine radioisotopes such as ¹²³I, ¹²⁴I, ¹²⁵I, and ¹³¹I are used as tracers and therapeutic agents in medicine.⁸ Metomidate (MTO) is a high-affinity ligand of adrenal steroidogenic enzymes.⁹ The [^123/131I]-IMTO was used as a radiotracer for the functional diagnosis of adrenal disease.¹⁰ In addition, [¹²⁴I]-IMTO recently appeared as an attractive PET tracer for the imaging of adrenals.¹¹ Conversional synthesis of IMTO involves iodination of a trialkylaryltin derivative as a precursor with a radioactive iodonium ion. Hydrophobicity of the triaklyltin groups makes a difference in the retention time between the precursors and IMTO and assists in the complete separation of the precursors from IMTO by HPLC. Our approach for the synthesis of IMTO uses the polymethacrylamide-supported MTO derivative linked though a silyl linker, which releases the radioactive IMTO 1 via radio-iodination.

Scheme 1 Synthesis of the radioiodinated IMTO 1 from the soluble polymer-supported precursor 2.

Scheme 2 shows the synthesis of the soluble polymer-supported precursor 2. The bromide 3 was treated with ⁿBuLi, followed by the addition of the silyl chloride 4.¹² Subsequent hydrolysis of the tetrahydropyranyl ether under acidic conditions afforded the alcohol 5 in 88% yield in 2 steps. Substitution of the benzyl alcohol 5 with imidazole 6 under Mitsunobu reaction conditions provided the N-alkylimidazole 7 in 63% yield. The bromide 7 was converted to the azide 8 via a nucleophilic substitution with sodium azide in 89% yield. Reduction of the azide followed by acylation of the resultant amine with acid chloride 9 provided the monomer 10 in 79% yield. The monomer 10 was polymerized via treatment with 5% AIBN at 65 °C. After hexane treatment of the product, the residue was purified via normal-phase column chromatography on silica gel with a pore size ranging from 5.4 to 7.4 nm. The polymer 2 was obtained in 68% yield as the first CH₂Cl₂ eluate. These results indicated that the normal-phase column chromatography acted as size-exclusion chromatography against the polymer 2 that could be used in purification of the radiotracer 1 after isotope labeling. The number-average molecular weight of the purified polymer 2 was estimated via size-exclusion chromatography and eluted using a 1.0 M LiCl DMF solution to a molecular weight of 1.02 × 10⁴. On the other hand, SEC analysis of the polymer 2 eluted with CH₂Cl₂ indicated that its molecular weight was >4.0 × 10⁵. In addition, proton signals of the ¹H NMR spectra of 2 in dichloromethane are broad in comparison with that in chloroform. These results suggested that the polymer 2 was likely to be aggregated in the dichloromethane solution. We next examined the synthesis of IMTO 1 from polymer 2. Treatment of the polymer 2 with sodium iodide and N-chlorosuccinimide (NCS) in trifluoroacetic acid (TFA) at 40 °C for 60 min provided a mixture of iodide 1 and chloride 11 in 80% yield. The polymer was easily removed via the solid-phase extractions of iodide 1 and chloride 11. The ratio (1 : 11) was estimated by ¹H NMR spectra of the mixture based on standard samples, and determined to be 78 : 2.

Scheme 2 Synthesis of the polymer-supported precursor 2 and the IMTO 1.

The success of the polymer-supported synthesis of [¹²³I]-IMTO 1 depends not only on the efficiency of iodination but also on the amount of undesired products released from the polymer 2 during the [¹²³I]-iodination. We first examined the [¹²³I]-iododesilylation of the silylated MTO 12 (Scheme 3 and Table 1). The silylated MTO 12 was treated with [¹²³I]-NaI and NCS in TFA for 60 min at room temperature and temperatures of 40 and 90 °C. Values for the radio chemical convergent (RCC) of the [¹²³I]-IMTO 1 were estimated at 73, 92 and 86%, respectively, using a Radio TLC analyser. On the other hand, the [¹²³I]-iododesilylation of 12 in AcOH at 90 °C did not proceed well, and provided the [¹²³I]-IMTO 1 in a low RCC (14%). Based on the examination, we concluded the reaction conditions (40 and 90 °C in TFA) were two candidates for [¹²³I]-iodination.

Scheme 3 Reaction of the silulated MTO 12.

Table 1 Synthesis of [¹²³I]-iodometomidate 1 from 12

Entry	Solvent	Temp (°C)	RCC (%)
1	TFA	rt	73
2	TFA	40	92
3	TFA	90	86
4	AcOH	90	14

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We next compared the two iodination conditions in terms of the side products (Scheme 3). To estimate the amount of the released side-products from the polymer 2, the silylated MTO 12 was exposed to the [¹²³I]-iodination conditions without iodine sources. Treatment of the silylated MTO 12 with 3.0 equivalents of NCS in TFA for 60 min at 40 and 90 °C provided a mixture of the precursor 12, the chloride 11 and the protonated product 13. The ratio (12 : 11 : 13) was estimated to be 0 : 16 : 84 and 92 : 4 : 4, respectively, via HPLC analysis based on UV absorbance at 254 nm. The precursor 12 disappeared at 90 °C for 60 min and was mainly converted to chloride 11 and protonated product 13. These results indicated that the reaction conditions would completely release the precursor as chloride 11 or the protonated product 13. On the other hand, most of the precursor 12 survived at 40 °C for 60 min. Based on the observation, we concluded that these conditions (TFA at 40 °C) would be suitable for [¹²³I]-iododesilylation of the polymer-supported precursor 2.

The synthesis of [¹²³I]-IMTO 1 from the polymer-supported precursor 2 under the established conditions was examined (Scheme 4). The [¹²³I]-iododesilylation of 2 was carried out via treatment with [¹²³I] NaI (325 MBq) and N-chlorosuccinimide (NCS) in TFA at 40 °C for 60 min. The reaction mixture was diluted with CH₂Cl₂, followed by neutralization of the TFA with a solid-supported amine. The resultant solution was subjected to a Sep-Pak column. The column chromatography was washed with CH₂Cl₂ to release the polymer-supported precursor 2. The neutralization of the reaction mixture with a solid-supported amine was important to hold the released radioactive tracer 1 in Sep-Pak Silica®. The [¹²³I]-iodometomidate was eluted using 10% MeOH in CH₂Cl₂. The decay corrected radiochemical yield (RCY) of the purified [¹²³I]-IMTO was 85%. These results clearly indicated that the reactivity of the soluble polymer-supported precursor 2 was comparable with that of the non-polymerized precursor 12. TLC analysis showed the radiochemical purity of the [¹²³I]-IMTO to be 94%. We quantified the impurity in the purified [¹²³I]-IMTO via HPLC analysis under UV at 254 nm (Fig. 2). The red and blue lines indicate the chromatograms of the purified [¹²³I]-iodometomidate (105 MBq) and iodometomidate (100 μg) in CH₃CN (2.0 mL), respectively. The gross area of the sample containing [¹²³I]-IMTO was based 50% on the pure IMTO (100 μg). Based on the results, we estimated the amount of the cold products in the purified [¹²³I]-IMTO to be 50 μg. These results indicated that this method provided 100 MBq of [¹²³I]-IMTO within 100 μg of the cold impurity.

Scheme 4 Synthesis of the radioiodinated IMTO 1 from soluble polymer-supported precursor 2.

Fig. 2 HPLC analysis of purified [¹²³I]-iodometomidate (a red line) and 100 μg of IMTO (a blue line) based on UV absorption.

Conclusions

In conclusion, we have reported an effective method for the synthesis of [¹²³I]-IMTO based on solid-phase extraction of the radio traces. The soluble polymer 2 with MTO attached via a tetraalkylsilyl linker underwent [¹²³I]-iododesilylation at 40 °C for 60 min to provide [¹²³I]-IMTO 1 in a good yield with a minimum amount of chlorinated and protonated products 11 and 13. The polymer 2 might be aggregated in dichloromethane and possessed a large excluded volume in the solvents. Normal-phase column chromatography on silica gel with a pore size ranging from 5.4 to 7.4 nm acted as a size-exclusion chromatography against the polymer and enabled a solid-phase extraction of the radiotracer 1. Using the polymer 2 as a precursor, we successfully prepared [¹²³I]-IMTO 1 (100 MBq) that contained less than 100 μg of the cold impurity without HPLC purification. These results clearly demonstrate the effectiveness of our method to the synthesis of the radiotracers.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00442c

‡ Current address: Faculty of Pharmaceutical Sciences, Yokohama University of Pharmacy, 601, Matano-chou, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan.

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