A concise method for fully automated radiosyntheses of [18F]JNJ-46356479 and [18F]FITM via Cu-mediated 18F-fluorination of organoboranes

A modified alcohol-enhanced 18F-fluorodeboronation has been developed for the radiosyntheses of [18F]JNJ-46356479 and [18F]FITM. Unlike the [18F]KF/K222 approach, this method tolerates the presence of sensitive heterocycles in Bpin precursors 4 and 8 allowing a one-step 18F-fluorodeboronation on the fully automated TRACERlab™ FXFN platform.


Introduction
Fluorine-containing functionalities, especially (hetero)aryl uorides, are widely present among important drugs and bioactive molecules. 1 In medicinal chemistry, uorine is introduced into a compound to enhance chemical and biological properties, such as activity, metabolism stability and bioavailability. 2,3 Fluorine atom substitution by 18 F enables the use of positron emission tomography (PET) to image biological processes at the molecular level. [4][5][6][7][8] Radiolabeling of (hetero)aryl uorides has improved signicantly, allowing incorporation of 18 F on precursors of aryliodonium salts, 9 aryliodonium ylides, 10 aryl-Pd/Ni complexes, 11,12 triarylsulfonium salts/diaryl sufoxides, 13,14 and aryl boronic acid/ ester/tin species. 15 Among these methods, the Cu-mediated 18 F-uorination of organoboranes has been used for the preparation of various radiopharmaceuticals under manual and automated settings, [16][17][18] including eight clinically relevant radioligands 19 and the recent publications on [ 18 F]TRACK, 20 a tropomyosin receptor kinase inhibitor, and [ 18 F]olaparib, 21 the poly(ADP-ribose) polymerase inhibitor (Fig. 1A). However, in cases of challenging heteroarenes, optimal retro-radiosynthetic routes 22 and alternative radiolabeling strategies 23 must be implemented to maximize radiouorination. For example, the radiolabeling of 4-[ 18 F]uoro-N- [4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2yl]-N-methylbenzamide ([ 18 F]FITM) and [ 18 F]Risperidone with this method is carried out in a two-step manner by rst radiolabeling the heterocycle containing fragments, followed by a subsequent coupling step with their corresponding counterparts. 23 Application of Cu-mediated 18 F-uorination in automated platforms is essential in producing large-scale radioligands to support preclinical and clinical studies. Successful automated radiosyntheses with this method are the preparation of [ 18 (Fig. 1B). 24 Moreover, addition of alcohols, such as methanol and n-butanol, as co-solvents, seemed to enhance the functionality tolerance of this 18 F-uorodeboronation method to include indoles, phenols, and anilines radiolabeled via unprotected precursors. However, the authors investigated this approach manually where extensive physical operations were required. For example, the aqueous cartridge, an anion-exchanger to enrich 18 F from [ 18 O]H 2 O, from the male side instead of the female side followed by an alcohol wash and air ush from the female side to remove the [ 18 O] water and increase the recover yield of 18 F. Air was also introduced into the reactor to promote reaction. These special and tedious operations hinder its application toward the fully automated synthetic modules such as GE TRACERlab™ FX FN platform. Nevertheless, this strategy has been recently utilized by Bernard-Gauthier et al. for the radiosynthesis of [ 18 F]TRACK using semiautomated radiosynthesis module Scintomics GRP (Germany). 20 To investigate the suitability of [ 18 F]JNJ-46356479 as a PET imaging ligand for metabotropic glutamate receptor 2 (mGluR2) in the brain, we modied this alcohol-enhanced Cumediated 18 F-uorination to (a) utilize its potential in tolerating sensitive organoboranes that are not compatible with the [ 18 F]KF/K 222 system, and (b) avoid manual manipulations to allow a fully automated radiosynthesis on a GE TRACERlab™ FX FN platform ( Fig. 1). Aer a thorough investigation of radio-uorination conditions for [ 18 F]JNJ-46356479, a fully automated method was developed, which was also applicable to the automated one-step radiosynthesis of a mGluR1 negative allosteric modulator (NAM) [ 18 F]FITM.

Results
Chemistry JNJ-46356479 was reported by Cid et al. as a potent mGluR2 positive allosteric modulator (PAM) (i.e., EC 50 ¼ 78 nM, E max ¼ 256%) with favorable physiochemical and pharmacological properties as well as CNS-penetrant. 25 JNJ-46356479 has also been established as a selective blocking reagent to characterize the mGluR2 radioligand of [ 11 C]JNJ42491293 in rats and nonhuman primates. 26 Hence, 18 F-radiolabeled JNJ-46356479 is an attractive mGluR2 PET radiotracer if produced in high molar activity using a fully automated platform.
The synthesis of non-labeled JNJ-46356479 was achieved via the method reported by Cid et al., starting from 2,4-dichloro-3-(triuoromethyl) pyridine using over 6 steps to allow nal reductive coupling reaction between aldehyde 1 and 1-(2,4-diuorophenyl) piperazine 2 (Scheme 1). 25 The para-aryl uoride of JNJ-46356479 was selected as a radiolabeling site to avoid steric hinderance when introducing 18 F or bulky leaving groups. Traditional nucleophilic S N Ar substitution of nitro-or iodoleaving groups was assumed not to be feasible due to the poor activating effect of adjacent uoride. The sulfonium or iodonium salt or iodonium ylide radiolabeling methods, however, required oxidative reaction conditions that might not be tolerated by the heterocycle containing JNJ-46356479.
The 18 F-uorodeboronation precursor 4 was synthesized in a similar manner as that of JNJ-46356479 with the boronic ester 3. However, direct application of the same reductive amination reaction conditions, using HOAc and NaBH(OAc) 3 , resulted in substantial side products that were in close vicinity with the desired product 4, including the deboronation product (aryl-H) and boronic species 5. Interestingly, this decomposition did not occur to the structurally simpler fragment 3, which was prepared via the de-Boc protection of compound 6 under strong acidic condition at room temperature (Scheme 2). To improve the yield of compound 4, the basic reductive amination conditions with triethylamine (TEA) and NaBH(OAc) 3 were applied, resulting in much less side products and an increased yield of compound 4.

Radiosynthesis of [ 18 F]JNJ-46356479
Initial radiouorination of compound 4 with this 18 F-uorodeboronation using K 2 CO 3 as a base and K 222 as a phase transfer reagent, however, failed to give the desired [ 18 23 In this case, a multistep radiolabeling protocol is required by radiolabeling the heterocycle 6 rst, followed by N-deprotection and N-alkylation coupling with aldehyde 1. Indeed, under the same conditions, fragment 6 could be radiolabeled to give [ 18 F]7 with a radiochemical conversion (RCC) of 60 AE 5% (n ¼ 2). It is therefore reasonable to assume the [1,2,4]triazolo[4,3-a]pyridine core structure of JNJ-46356479 is vulnerable under the suggested [ 18 F]KF/K 222 radiouorination conditions. Before resorting to the complicated and tedious two-step strategy, we intended to explore the alcohol-enhanced protocol reported by Zischler and coworkers 24 to nd solutions for the one-step radiosynthesis of The original manual radiolabeling protocol was not directly applied for [ 18 F]JNJ-46356479 due to the reproducibility issue witnessed during the radiouorination of compound 6 and incomplete removal of [ 18 O]H 2 O from QMA cartridge by air would inhibit the reaction. Instead, the labeling protocol was comprehensively investigated to reveal critical inuential factors that would affect the reaction outcomes. More importantly, efforts were focused on simplifying the manual handlings of this method to apply it to the fully automated synthetic modules.
As shown in Table 1, based on the original reaction conditions, the reaction temperature, time as well as the amounts of TEAB, precursor 4 and catalyst [Cu(OTf) 2 py 4 ] were optimized. The anhydrous dimethylacetamide (DMA) was used as a solvent. The optimal reaction temperature was 130 C and further elevation of reaction temperature did not signicantly increase the RCCs (entries 1-3). Increasing the amount of base was detrimental to the RCCs (entry 4), though it could improve the elution efficiency of 18 F from QMA cartridge ($95-99%). On the other hand, increased precursor amount was benecial to enhance the RCCs (entries 5-8). These results indicated that boronic pinacol ester (Bpin) 4 was vulnerable to the excess amount of base. This sensitivity of organoboranes was also noted by Zhang et al., where use of Py-OTf or DMAP-OTf as 18 F eluting agent and base alleviated this issue. 27 To maintain a balanced recovery of 18 F (85-90%) and to limit precursor use, 3.5 mg (6.25 mmol) of 4 and 2.7 mg (14.1 mmol) of TEAB were considered as optimal amounts (entry 8). The reaction time showed little effect on the resulting RCCs (entries 9-11), thus a shorter reaction time was preferred. The amount of [Cu(OTf) 2 (py) 4 ] was also optimized, where reduction of the catalyst by half (i.e., 9 mg, 13.3 mmol) led to similar RCCs (entries [12][13][14]. In addition, the boronic acid precursor 5 was also examined for the synthesis of [ 18 F]JNJ-46356479 with the same method. Although MeOH was claimed as an accompanying alcohol for boronic acid precursors by Zischler and coworkers, 24 it failed to give the desired product in this radio-uorination reaction (Table 1, entry 15; Table S2, † entries 1 and 2). However, n-BuOH led to the formation of [ 18 F]JNJ-46356479 (Table 1, entry 16; Table S2, † entries 3-6). The RCCs ranged from 5% to 13% with elevated temperature and increased amount of precursor favored the radiochemical conversion (Table S2, † entries 3-6). Therefore, it was reasonable to conclude that the boronic pinacol ester 4 was more reactive than the boronic acid 5 for this radiouorination method.
Noteworthy, the 18 F in [ 18 O]water was loaded onto the QMA cartridge from the female side and eluted out from the male side in the normal manner. The [ 18 F]TEAF complex was obtained aer conventional azeotropic dryings and no air was ushed into the reactor during reaction. These modications facilitated the subsequent utilization of fully automated synthetic modules.
Automated radiosynthesis of [ 18 F]JNJ-46356479 was performed with the optimal conditions depicted in Table 1

Radiosynthesis of [ 18 F]FITM
To further test the applicability of this method to sensitive heterocycle containing molecules, the automated radiosynthesis of [ 18 F]FITM was also investigated. The Bpin precursor 8 was synthesized in 3 steps from the 4-(6-chloropyrimidin-4-yl)-N-methylthiazol-2-amine via the literature method (Scheme 3). 23 As reported by the Taylor et al., direct radiouorination of 8 with the [ 18 F]KF/K 222 system failed to give the desired product. 23 Alternatively, [ 18 F]FTIM was prepared by a three-step one-pot Table 1 Optimization of the one-step Cu-mediated 18

Discussion
The alcohol enhanced Cu-mediated radiouorination reported by Zischler  In fact, the presence of sensitive heterocycles also made compounds 4 and 8 chemically unstable under certain conditions. They slowly decomposed to their aryl boronic acid analogues during ash column purication with either silica gel or alumina. Initial purications of 4 via a preparative C-18 column with a gradient elution (mobile phase: CH 3 CN/0.1% formic acid in water, ow rate: 15 min mL À1 ) resulted in a 75% degradation of 4 to its boronic acid analog 5 aer the collected product fractions were dried in lyophilizer overnight. Although the boronic pinacol esters are generally considered more stable than boronic acids under protodeboronation conditions, 29 the protodeboronation side product was minor (<5%). In addition, slight decompositions were also noticed for 4 and 8 during NMR characterizations in deuterated solvents of CDCl 3 , CD 3 OD and dimethyl sulfoxide-d 6 . In practice, the Bpin precursors were puried via ash column chromatography immediately aer workup and characterized by LC-MS and NMR for their purities and identities. Fortunately, the boronic acid analogues were also suitable for the radiouorination method as tested here with compound 5. Moreover, once isolated, both precursors are quite stable at room temperature. During the reaction, compounds 4 and 8 maintained a high purity of >95%, with marginal amount of their boronic acid analogues. Although pure compound 5 was not directly tested under the [ 18 F]KF/K 222 conditions, a mixture containing 62% of 4, 35% of 5 and 3% of their protodeboronation side product did not lead to the desired [ 18 F]JNJ-46356479, whereas under the optimized reaction conditions shown in Table 1  Switching the base from K 2 CO 3 to TEAB together with the addition of n-BuOH had alleviated the harshness of the reaction conditions and led to the desired products from their Bpin precursors. Even under this modied protocol, the amount of TEAB had to be limited to ensure an optimal RCC (Table 1, entry 4). The n-BuOH was positioned separately in vial 2 and added to reaction vessel before the precursor solution in the FX FN platform, which proved to be crucial, otherwise only negligible [ 18

Conclusions
The modied alcohol-enhanced Cu-mediated 18 F-uorination here is succinct, robust, and highly producible. It allows the large-scale production of [ 18 F]JNJ-46356479 in a range of 1.1 GBq to 3.0 GBq from a single batch with sufficiently high molar activity (180 AE 102 GBq mmol À1 ) to satisfy preclinical and clinical usage. Considering the important role of mGluR2 in various psychiatric and neurological disorders [30][31][32][33] and the lack of efficient mGluR2 PET imaging ligands to visualize and quantify mGluR2 in the brain, 26 the impact of this work is substantial. Moreover, the successful one-step radiosynthesis of [ 18 F]FTIM by this method demonstrates its broader organoborane compatibility. Along with this work, the preclinical characterization of [ 18 F]JNJ-46356479 as a mGluR2 PET imaging ligand in rodents and monkey brains has been performed and will be disclosed in due course.

Conflicts of interest
There are no conicts to declare.