Direct Incorporation of [11C]CO2 into Asymmetric [11C]Carbonates

A novel carbon-11 radiolabelling methodology for the synthesis of the dialkylcarbonate functional group has been developed. The method uses cyclotron-produced short-lived [11C]CO2 (half-life 20.4 min) directly from the cyclotron target in a one-pot synthesis. Alcohol in the presence of base trapped [11C]CO2 efficiently forming an [11C]alkylcarbonate intermediate that subsequently reacted with an alkylchloride producing the di-substituted [11C]carbonate (34% radiochemical yield, determined by radio-HPLC) in 5 minutes from the end of [11C]CO2 cyclotron delivery.


Introduction
Positron emission tomography (PET) is an imaging technique able to detect and monitor specific target proteins in vivo [1][2][3][4][5]. e use of PET imaging has advanced in the last few decades to become a valuable tool in clinical diagnostics, medical research, and drug discovery [6][7][8]. PET relies on the use of tracer amounts of imaging probes (radiotracers). e administration of radiotracers allows the biochemical process to be imaged and quantified in vivo without manifestation of pharmacological or toxicological effects [9][10][11][12][13].
Carbon-11 ( 11 C) is one of the most common radionuclides used for the synthesis of PET radiotracers. e short half-life of 11 C (20.4 min) makes it an attractive radionuclide as it enables the collection of a sufficient amount of PET data while keeping the subject radiation dose and exposure time to minimum. Furthermore, it allows orthologous substitution with carbon-12 in biologically active molecules with no alteration of the parent molecule's physicochemical and pharmacological properties. Carbon-11 is commonly produced in the form of [ 11 C]carbon dioxide ([ 11 C]CO 2 ) [14,15]. [ [16][17][18][19]. As these multistep conversion processes are time-consuming, the use of [ 11 C]CO 2 for directly radiolabelling functional groups is highly attractive.
[ 11 C]CO 2 is a weak electrophile with an affinity for electron-donating reagents such as amines and organometallics [20]. However, due to the thermodynamic and kinetic properties of [ 11 C]CO 2 , it has high activation energy which requires the use of highly reactive reagents, temperatures, pressures, or the presence of a catalyst [21][22][23]. Nevertheless, the primary synthon, [ 11 C]CO 2 , has been deployed successfully for the synthesis of 11 C-compounds that contain carbonyl groups such as [ 11 C]carbamates [24,25], amide [26], and [ 11 C]ureas [23,[27][28][29]. However, the radiolabelling of the carbonyl group of carbonates from [ 11 C]CO 2 has not yet been established. To date, the synthesis of [ 11 C]carbonates has relied on the use of [ 11 C]COCl 2 which is produced from a multistep process starting from cyclotronproduced [ 11 [30,31]. Although this 11 Ccarbonate reaction is rapid and efficient, routine production of [ 11 C]COCl 2 requires multistep syntheses and specialized equipment, thereby restricting its widespread use [30,31].
As the carbonate functional group is found in prodrug compounds as well as being an intermediate in organic synthesis [32][33][34][35], we aimed at developing a simple and robust radiolabelling methodology that uses [ 11 C]CO 2 for the synthesis of [ 11 C]carbonates. Here we present a rapid, one-pot radiosynthetic strategy using [ 11 C]CO 2 directly from the cyclotron, avoiding the need for specialized equipment and multistep syntheses.

Materials and Methods
All purchased chemicals were used without further purification. Chemicals were purchased in highest available purity from Sigma-Aldrich and Alfa Aesar and used as received (>99 % purity). All solvents were purchased as anhydrous in highest available purity (>99.8 % purity) from Sigma-Aldrich.
[ 11 C]CO 2 was produced by a Siemens RDS112 cyclotron (St omas' Hospital, London, United Kingdom) via the 14 N(p,α) 11 C nuclear reaction. Typical irradiation time for exploratory work was 1 minute, 10 µA, bombardment typically yielding ca. 300 MBq [ 11 C]CO 2 at end of cyclotron bombardment. Radiolabelling reactions were performed in a 1.5 mL screw top vial with a "V" internal shape. HPLC analysis was performed on an Agilent 2060 Infinity HPLC system with a variable wavelength detector (254 nm was used as default wavelength) [10] An Agilent Eclipse XDB-C18 reverse-phase column (4.6 × 150 mm, 5 µm) was used at a flow rate of 1 mL/min and H 2 O/MeOH (HPLC-grade solvents with 0.1 % TFA) gradient elution (flow rate: 1 mL/ min, 0-2 min: 5 % MeOH, 2-11 min: 5 to 95% MeOH linear gradient, 11-13 min: 90 % MeOH, 13-14 min: 90% to 5% MeOH linear gradient, and 14-15 min: 5 % MeOH). e RCY was estimated by radio-HPLC and defined as the area under the 11 C-product peak expressed as a percentage of the total 11 C labelled peak areas observed in the chromatogram. Molar radioactivity was calculated from analytical HPLC sample of 25 μL. A calibration curve of known mass quantity versus HPLC peak area (254 nm) was used to calculate the mass concentration of the 25 μL radiolabelled compound. e identity of the radiolabelled compound peak was confirmed by HPLC coinjection of a nonradioactive reference compound and yielded a single peak.

Results and Discussion
As the starting point, we selected the method developed by Salvatore et al. [21][22][23] (Figure 1) for the synthesis of carbonates. e established method used nonradioactive CO 2 , an alcohol derivative, and benzylchloride (BzCl) in the presence of Cs 2 CO 3 , TBAI in DMF to produce the corresponding carbonate derivative efficiently. By substituting CO 2 with [ 11 C]CO 2 and applying the same reaction conditions, the synthesis of di-substituted [ 11  e RCY is the nonisolated radiochemical yield determined by radio-HPLC analysis of the crude product of 24% (Table 1, entry 1). Interestingly, almost all the cyclotron-produced [ 11 C]CO 2 was trapped within the reaction mixture at room temperature (>95%); any unreacted radioactive [ 11 C]CO 2 was immobilized on an ascarite trap connected to the vial vent needle. e trapping efficiency is the amount of radioactivity trapped in the reaction vial as a percentage of the overall radioactivity produced by the cyclotron.
In an attempt to increase the RCY, Cs 2 CO 3 was replaced with Cs 2 SO 4 ( Table 1, entry 2). e trapping efficiency of [ 11 C]CO 2 dropped significantly from 95.2% to 1.5%. Since Cs 2 CO 3 contributed towards the trapping of [ 11 C]CO 2 efficiently, we investigated whether the Cs + or the CO 3 2ion was responsible for the high [ 11 C]CO 2 -trapping efficiency. Of a number of caesium bases explored (Table 1, entries 3-5), CsI and CsF trapped only minute amounts of [ 11 C]CO 2 (4% and 34%, respectively), indicating that the basicity of the reaction mixture had a major effect on trapping efficiency. ese results can be explained by the ability of a strong base to deprotonate the alcohol present in the reaction mixture enabling it to react with [ 11 C]CO 2 to form a 11 C radiolabelled intermediate. e importance of CO 3 2− was then explored by comparing Cs 2 CO 3 with other carbonate bases (K 2 CO 3 and CaCO 3 , Table 1, entries 6 and 7). e trapping efficiencies were extremely low for both reagents. High trapping in the reaction mixture with Cs 2 CO 3 is therefore most likely due to its superior solubility in organic solvents.
In a further attempt to increase the RCY of [ 11 C]1, a number of aprotic solvents were screened (CH 3 CN and DMSO, Table 1, entries 8 and 9). However, these solvents did not produce [ 11 C]1, and the trapping efficiency was poor (20% and 65%, respectively). Reaction dependency on temperature was subsequently examined. e RCY of [ 11 C]1 improved from 24% to 33% by increasing the reaction temperature from 25°C to 65°C (Table 1, entry 10). Increasing the temperature to 100°C promoted the product formation and resulted in the highest observed RCY (82%, Table 1, entry 11). is might be rationalised by an increase in Cs 2 CO 3 solubility at higher temperatures. However, due the presence of Cs 2 CO 3 as a reagent, low molar activities (A m ) were observed. e low A m (2 GBq/μmol in this case) is likely due to release of nonradioactive CO 2 from Cs 2 CO 3 . CO 3 2− deprotonates the alcohol to form HCO 3 − , which at high temperature has the potential to decompose releasing nonradioactive CO 2 causing isotopic dilution and low A m of  [26]. Replacing Cs 2 CO 3 with DBU ( Table 2, entry 1) resulted in [ 11 C]1 formation, but with low RCY (6%). e low RCY could be due to DBU being unable to deprotonate isopropyl alcohol efficiently. We opted for a stronger base, NaH, which was able to deprotonate the isopropyl alcohol. Using a ratio of 1 : 1 NaH : isopropanol (equiv.) at 100°C, [ 11 C]1 was obtained with an RCY of 26% (Table 2, entry 3). Decreasing the temperature from 100°C to 60°C slightly improved the RCY (31%, Table 2, entry 4). [ 11 C]1 was produced with a molar activity (A m ) of 10-20 GBq/µmol. is is because short cyclotron bombardments (1 minute) and low beam currents (5-10 µA) were used (0.3 GBq). In clinical productions at our facility, cyclotron bombardment times of 50 minutes and beam currents of 30 µA are used to produce higher amounts of radioactivity (typically 60 GBq). It is therefore estimated that this would increase the Am to > 50 GBq/µmol at end of synthesis. Decreasing the ratio of NaH : isopropanol (from 1 : 1 to 0.5 : 1) reduced the RCY further to 18% (Table 2, entry 5). Increasing the ratio NaH : isopropanol 2 : 1 did not produce the target product ( Table 2, entry 6). Increasing the amount of TBAI to 3 equiv. or removing it completely also did not improve the RCY (Table 2, entries 7 and 8).

Conclusions
In conclusion, we have developed a radiolabelling methodology for the synthesis of [ 11 C]carbonates using [ 11 C]CO 2 directly from the cyclotron. e carbonate [ 11 C]1 was synthesized by bubbling [ 11 C]CO 2 into a solution containing alkylchloride, alcohol, and a base in DMF. e choice of the base was critical for maximising the RCY and A m . e first protocol uses Cs 2 CO 3 and produces the target 11 C radiolabelled product in a high RCY and low A m . e second strategy, which uses NaH, produced [ 11 C]1 in high A m and moderate RCY. ese methodologies are a simple and practical alternative to 11 C-phosgene for the synthesis of 11 C-carbonates. 11 C-phosgene synthesis is technically challenging to implement and requires the use of specialist equipment.
e developed strategies described here use readily available labware and converts [ 11 C]CO 2 directly to [ 11 C]carbonates in rapid synthesis times.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.
Journal of Chemistry 3