Synthesis of 13 N-labelled polysubstituted triazoles via Huisgen cycloaddition †

The use of the positron emitter nitrogen-13 ( 13 N) has been historically restricted due to its short half-life ( T 1/2 ¼ 9.97 min). However, its stable isotopes (nitrogen-14 and nitrogen-15) are present in many biologically active molecules; therefore, the incorporation of 13 N in the toolbox of PET chemists might be a valuable option for the preparation of new labelled compounds or incorporation of the label in di ﬀ erent positions. Here we present the unprecedented radiosynthesis of 13 N-labelled polysubstituted triazoles via Huisgen cycloaddition by reaction of 13 N-labelled aromatic azides with alkyne derivatives and aldehydes. Six di ﬀ erent 13 N-labelled triazoles were successfully synthesized. After automatization of the synthetic process and optimization of experimental conditions, one selected triazole could be prepared with high radiochemical purity and decay-corrected radiochemical yields of 11 (cid:2) 2%. The amount of activity obtained should be su ﬃ cient to approach future in vitro and in vivo studies. The novel methodology might open new avenues for the preparation of radiotracers which cannot be labelled using other more conventional positron emitters. unreacted 13 N-labelled 1-azido-4-iodobenzene, were found; (b and c) chromatographic pro ﬁ les (UV-Vis and radioactive detectors, respectively) corresponding to ﬁ ed 13 N] 6 . No chemical nor


Introduction
Positron Emission Tomography (PET) is a minimally invasive molecular imaging technique that enables the determination of the spatiotemporal distribution of a radiolabelled molecule (radiotracer) aer administration to a living organism. Due to the high sensitivity of PET, the radiotracer is usually administered in the sub-micromolar range, facilitating the investigation of biological or physiological processes in vivo without having any toxicological, pharmacological and/or undesired side effects.
Among all positron emitters, uorine-18 ( 18 F) and carbon-11 ( 11 C) have been the most widely used. 1 Fluorine-18 has a relatively long half-life (T 1/2 ¼ 109.7 min), facilitating the centralised production of radiotracers and distribution to surrounding centres. It decays almost quantitatively by positron emission and has a short positron range (E bmax ¼ 0.64 MeV, maximum range in water ¼ 2.4 mm), which ultimately results in higher resolution images. Carbon-11 has a shorter half-life (T 1/2 ¼ 20.4 min) and a longer positron range (E bmax ¼ 0.96 MeV, maximum range in water ¼ 3.8 mm); however, it can be produced in different chemical forms in biomedical cyclotrons; additionally, because all organic molecules contain stable carbon atoms in their structure, the use of carbon-11 enables the preparation of radiotracers identical to the non radioactive molecule to be investigated.

Synthetic procedures: chemistry
Synthesis of 1-6 via (3 + 2) azide-alkyne cycloaddition. The preparation of compounds 1-6 was carried out following a previously published method. 11 In brief, to a stirred solution of azide (1.69 mmol), alkyne (2.54 mmol), CuSO 4 $5H 2 O (0.169 mmol) and an aqueous solution of sodium ascorbate (0.845 mmol in 1 mL) were added. The vial was capped and submitted to microwave heating (80 C, 125 W max, 10 min) using a Biotage® Initiator 2.0, 400 W. Ultrapure water (25 mL) was added and the precipitate was ltered and washed with water (2 Â 10 mL) and petroleum ether (40-60, 2 Â 10 mL). Aer complete drying under vacuum, the pure triazoles 1-6 were obtained. Characterization was performed using 1 H-and 13 C-NMR and mass spectrometry. Characterization data was compared to literature. [11][12][13] Synthetic procedures: radiochemistry General. All procedures were carried out under EU standards in terms of radioprotection and following internal procedures. Initial experiments to set up experimental conditions were performed manually in a lead-shielded hot cell. Aer optimization of the experimental conditions, the whole synthetic procedure for the preparation of the selected labelled compound was carried out using an automatic synthesis module (see below and ESI † for experimental details).
Production of the primary labelling agent [ 13 N]NO 2 À . Nitrogen-13 was produced in an IBA Cyclone 18/9 cyclotron by irradiation of puried water (1.8 mL) via the 16   Teknokroma, Spain) was used as stationary phase, and a solution containing additive for ionic chromatography (15 mL) in a mixture water/acetonitrile (86/14, V ¼ 1 L) basied to pH ¼ 8.6 with 1 M sodium hydroxide solution was used as the mobile phase at a ow rate of 1 mL min À1 . Simultaneous UV (l ¼ 254 nm) and isotopic detection were used. Retention times for [ 13  Synthesis of 13 N-labelled triazoles by reaction of 13 N-labelled azide with aldehyde using liquid-liquid extraction for intermediate purication ([ 13 N]1). The 13 N-labelled azide was prepared following the above mentioned procedure, extracted with dichloromethane (1 mL) and the organic fraction was evaporated to dryness under a stream of nitrogen. The catalyst (DBU, 0.021 mmol in 0.2 mL of DMSO) and phenylacetaldehyde (0.136 mmol) were then added and the reaction was allowed to occur for 10 minutes at different temperatures (25 C or 60 C). The reaction was quenched by addition of aqueous ammonium formate (pH ¼ 3.9), and radiochemical conversion was determined by radio-HPLC using the same methodology as above.
Synthesis of 13 N-labelled triazoles by reaction of 13 N-labelled azide with alkynes using solid phase extraction for intermediate purication ([ 13 N]1 and [ 13 N]6). The procedure for the preparation of 13 N-labelled azide was the same as described above, but the reaction mixture containing the azide was puried by solid phase extraction. With that aim, 7 mL of 0.5 M sodium acetate solution ([ 13 N]1) or ultrapure water ([ 13 N]6) was added to the reaction mixture and the resulting solution was passed through a C-18 Cartridge (Sep-Pak C18 Plus, Waters). The cartridge was rinsed with ultrapure water (2 mL) and subsequently ushed with helium gas for 1 minute. Finally the C-18 Cartridge was eluted with acetonitrile (1 mL), the liquid was collected in a vial containing the corresponding alkyne and the catalyst and the resulting mixture was allowed to react for 10 min at 50 C. The reaction mixture was nally diluted with mobile phase and analyzed by HPLC using the conditions mentioned above.
Fully automated synthesis of [ 13 N]6. The automated synthesis of [ 13 N]6 was carried out using a Tracerlab™ FX FE synthesis module (GE Healthcare) with modications on its original conguration (see ESI: Fig. S1 for schematic representation of the module and Table S1 † for description of the remote-controlled sequence). Reaction time, reaction temperature and the amount of catalyst were varied in order to nd optimal experimental conditions.

Results and discussion
Nitrogen-13 can be produced in different chemical forms in biomedical cyclotrons and offers a wide variety of synthetic possibilities for the preparation of PET radiotracers. However, its short half life demands for the development of fast, efficient and robust synthetic processes. This was especially relevant in the work reported here, because the synthetic strategy was envisioned through a 4-steps process plus one intermediate and one nal purication steps.
In our previous work, 10 we demonstrated that two approaches could be used for the synthesis of 13 N-labelled aryl azides: (i) reaction of aniline with sodium nitrite to yield the non-labeled diazonium salt and subsequent reaction with 13 Nlabelled azide ion (prepared by reaction of hydrazine hydrate with [ 13 N]NO 2 À in acidic media); and (ii) reaction of aniline with [ 13 N]NO 2 À to yield the 13 N-labelled diazonium salt and subsequent reaction with azide ion (prepared by reaction of hydrazine hydrate with sodium nitrite in acidic media). Our results showed that the radiolabel was almost quantitatively transferred to the azide under route (ii). Consequently, this route was considered as the most appropriate to achieve optimal results and the experimental conditions of this part of the reaction were not further optimised. It is worth mentioning that during the preparation of labelled azides it is paramount to work under no-carried-added conditions, in order to achieve high specic activity (amount of radioactivity per unit mass) values in the nal labelled triazoles. In other words, the preparation of the labelled diazonium salt had to be conducted without addition of non-radioactive NO 2 À . This, ultimately, led to the situation in which the labelled specie in the (3 + 2) cycloaddition reaction for the formation of the 13 Nlabelled triazoles is the limiting reagent, as its concentration is much lower than any other reagent. Taking this into account, we rst tackled the optimization of the experimental conditions for the preparation of 13 N-labelled triazoles by reaction of the labelled azides with alkynes (see Fig. 1). During this process, the work was conducted manually to have a better control of the individual steps. In all cases, control analysis performed aer the reduction step showed that 70-80% of the radioactivity eluted from the cadmium column was due to [ 13  salt) were found in the organic phase, although the presence of the starting aniline could be detected. This impurity was anticipated not to interfere in the following catalytic reaction. To conduct the (3 + 2) cycloaddition under hot conditions, our rst attempts were performed using CuSO 4 $H 2 O as the catalyst, mimicking the reaction conditions used during preparation of the non-radioactive analogues. With that aim, the preparation of [ 13 N]1 was approached by reaction of [ 13 N]phenylazide (redissolved in acetonitrile aer evaporation of dichloromethane) with phenylacetylene in the presence of the catalyst (2 mg) for 10 min at RT. Unfortunately, the formation of the desired labelled triazole could not be detected by radio-HPLC. Similar results were obtained with copper(II) oxide (CuO) and copper(I) iodide as the catalyst. In view of these results, we decided to use [(Icy) 2 Cu]PF 6 , which has proven efficient in a wide variety of (3 + 2) cycloaddition reactions. 14 In this case, the reaction at RT for 10 minutes offered radiochemical conversion values (RCC, calculated from radiochromatographic proles, expressed as the ratio between the area under the peak corresponding to [ 13 N]triazole and the sum of the areas for all the peaks in the chromatogram, in percentage) of 15 AE 2% (Table 1, entry 1).
Aer these encouraging results, we decided to extend the methodology to the preparation of other functionalized triazoles ([ 13 N]2-[ 13 N]6), and different reaction temperatures were assayed. As it can be seen in Table 1, our method enabled the preparation of all labelled triazoles. Radiochemical conversion values increased with temperature to reach acceptable values (38-94%) when the reaction was carried out for 10 minutes at 50 C ( Table 1,  Interestingly, high conversion values were obtained when aliphatic alkynes were used. This result is not in agreement with previous works reported in the literature, as aliphatic alkynes are known for their lower reactivity when compared to aromatic ones. 16,17 However, long reaction times (hours) were used in Table 1 Radiochemical conversion values for the preparation of 13 Nlabelled triazoles by reaction of 13   these previous works. Additionally, because we conducted the reactions under no-carrier-added conditions, the 13 N-labelled azide is the limiting reagent and its concentration is extremely low when compared to that of the alkyne. Despite further investigation would be required, we hypothesize that the low RCC values obtained with aromatic azides might be due to the kinetics of the reaction. Longer reaction times may lead to quantitative conversion, as observed in the case of aliphatic triazoles.
In view of the promising results, we decided to expand the scope of our work by tackling the preparation of [ 13 N]1 by reaction of the labelled azide with phenylacetaldehyde. In our rst attempts, [(Icy) 2 Cu]PF 6 was used as the catalyst, but the formation of [ 13 N]1 could not be detected by radio-HPLC. The formation of substituted triazoles by reaction of azides with aldehydes has been previously reported in the literature. 18 In this previous work, 1,8-diazabicycloundec-7-ene (DBU) offered excellent results when the reaction was carried out in DMSO. In our hands, this catalyst offered good radiochemical conversion values (23 AE 5%, Table 1, entry 7) when the reaction was conducted in DMSO at 25 C. The reaction was almost quantitative at T ¼ 60 C (see Fig. 2c and d for examples of chromatographic proles).
Liquid-liquid extraction as an intermediate purication step is a very well established procedure for reactions that are conducted under non-radioactive conditions. However, such experimental step is sub-optimal in radioactive conditions because automatisation is extremely challenging. Hence, and moving towards the development of a fully automated process, other alternatives were explored. In previous works, our group has used solid phase extraction cartridges as a suitable tool to switch from aqueous to organic solvent during the synthesis of 13 Nlabelled azo compounds. 7 Here, we anticipated that dilution of the reaction crude with water aer formation of the labelled azide and subsequent elution through a C-18 cartridge would result in quantitative trapping of the labelled azide. However, rst attempts performed with [ 13 N]phenylazide were unsuccessful, and no trapping could be observed. Because the reaction for the formation of the 13 N-labelled azide is carried out under strong acidic conditions, we postulated that the lack of retention might be due to the formation of the protonated form (positively charged) of the azide, which has low affinity for the C-18 phase. As expected, replacement of water by sodium acetate solution and elution through the C-18 cartridge resulted in quantitative trapping of the labelled azide, as revealed by radio-HPLC analysis of the eluate. In the case of [ 13 N]p-iodophenylazide, used in the preparation of compound [ 13 N]6, dilution with water led to quantitative trapping, probably due to the presence of the iodine atom, which confers a stronger hydrophobic character to the molecule, regardless of the net charge.
In all cases, ushing of the C-18 cartridge with helium gas for 1 min was sufficient to remove the majority of the water; subsequent elution of the trapped labelled species with acetonitrile and reaction with the corresponding alkyne under catalytic conditions led to the formation of the desired labelled compounds ([ 13 N]1-[ 13 N]6) with RCC values equivalent to those shown in Table 1.
Incorporation of the solid phase extraction process as an intermediate purication step enabled the automatisation of the whole process (including nal purication by semipreparative HPLC) using the Tracerlab™ FX FE synthesis module, with appropriate adaptation (see Fig. S1 and ESI † for further experimental details). The experimental conditions for the preparation of compound [ 13 N]6 were further optimized at this step. This triazole was selected for full automatisation because its non radioactive analogue has shown promising properties to target b-amyloid aggregates, and hence it may nd application in the early diagnose or evaluation of response to treatment in Alzheimer's disease. 12 The amount of catalyst, the reaction temperature and the reaction time for the formation of the labelled triazole were varied ( Table 2).
As it can be seen in the table, no reaction was observed when the reaction time was set to 2 min, irrespective of the amount of catalyst (entries 1-3). It is worth mentioning that heating of the vial where the reaction is carried out starts at t ¼ 0. Hence, 2 min might not be sufficient to reach the desired temperature in the reaction mixture. In general terms, higher temperature, longer reaction times and higher amount of catalyst led to higher RCC values, as expected. At optimal reaction temperature (110 C) and amount of catalyst (10 mg), the reaction time of 10 min proved to be less desirable than the 5 min period, because the gain in RCC (approximately 10%, from 85 to 96%, see entries 12 and 13 in Table 2) was negligible compared with the decay of nitrogen-13 during the extra 5 min gap (close to 50%). Hence, T ¼ 110 C, t ¼ 5 min and amount of catalyst ¼ 10 mg were established as optimal conditions among the investigated scenarios.
These experimental conditions were applied for the fully automated preparation of pure [ 13 N]6 (see ESI † for experimental details). Aer purication by semi-preparative HPLC (see Fig. 3a for example of chromatographic prole) isolated radiochemical yields of 11 AE 2%, related to the starting amount of 13 N Table 2 Radiochemical conversion values for the preparation of [ 13 N] 6 by reaction of 13  produced in the cyclotron and decay corrected to the end of the irradiation process, were obtained in overall production times of 25 min (non-decay corrected radiochemical yield ¼ 2 AE 0.4%). Specic activities at the end of the synthesis were 4.6 AE 0.2 GBq mmol À1 , and radiochemical purity was >99% in all cases, as determined by radio-HPLC. No chemical impurities were detected by HPLC (Fig. 3c). Despite non-decay corrected radiochemical yields are low, an irradiation of 4 mA h yields around 6.6 GBq of 13 N in the cyclotron target. Hence, around 130 MBq (3.6 mCi) of pure radiotracer could be obtained at the end of the preparation process. Typical injected doses in mice and rats are around 100 and 500 mCi, respectively. Hence, the here reported methodology should enable subsequent in vitro or in vivo studies in small animal species.

Conclusions
In conclusion, we present here an unprecedented and fully automated methodology for the preparation of chemically and radiochemically pure 13 N-labelled substituted triazoles by reaction of 13 N-labelled azides with alkynes. By accurate selection of the catalyst, the methodology could be extended to the preparation of triazoles using aldehydes instead of alkynes in the (3 + 2) cycloaddition step. Sufficient amount of radiotracer to approach subsequent in vitro and in vivo studies in small animal species could be obtained aer optimization of the experimental conditions. Radiolabelling with 13 N may nd interesting applications, especially in those occasions in which incorporation of other positron emitters into the target molecule is not feasible, or to tackle the incorporation of the label in different positions to enable accurate metabolic studies.