4-[18F]Fluorophenylpiperazines by Improved Hartwig-Buchwald N-Arylation of 4-[18F]fluoroiodobenzene, Formed via Hypervalent λ3-Iodane Precursors: Application to Build-Up of the Dopamine D4 Ligand [18F]FAUC 316

Substituted phenylpiperazines are often neuropharmacologically active compounds and in many cases are essential pharmacophores of neuroligands for different receptors such as D2-like dopaminergic, serotoninergic and other receptors. Nucleophilic, no-carrier-added (n.c.a.) 18F-labelling of these ligands in an aromatic position is desirable for studying receptors with in vivo molecular imaging. 1-(4-[18F]Fluorophenyl)piperazine was synthesized in two reaction steps starting by 18F-labelling of a iodobenzene-iodonium precursor, followed by Pd-catalyzed N-arylation of the intermediate 4-[18F]fluoro-iodobenzene. Different palladium catalysts and solvents were tested with particular attention to the polar solvents dimethylformamide (DMF) and dimethylsulfoxide (DMSO). Weak inorganic bases like potassium phosphate or cesium carbonate seem to be essential for the arylation step and lead to conversation rates above 70% in DMF which is comparable to those in typically used toluene. In DMSO even quantitative conversation was observed. Overall radiochemical yields of up to 40% and 60% in DMF and DMSO, respectively, were reached depending on the labelling yield of the first step. The fluorophenylpiperazine obtained was coupled in a third reaction step with 2-formyl-1H-indole-5-carbonitrile to yield the highly selective dopamine D4 ligand [18F]FAUC 316.


Introduction
Since their first uses in the1960s, cross-coupling reactions using Pd(0) complexes play a major role in organic syntheses to form carbon-carbon or carbon-heteroatom bonds [1]. This was recently also recognized by awarding the Nobel Prize to Richard F. Heck, Ei-ichi Negishi, and Akira Suzuki in 2010 [2]. Therefore, it is remarkable that application of this method in radiosyntheses using short-lived radionuclides like 11 C (t1/2 = 20.4 min) or 18 F (t1/2 = 109.7 min) is still an exception and only a few examples of its use are described [3][4][5]. Cross-coupling reactions allow for a quick synthesis of many structure motives of biomolecules relevant for non-invasive diagnostic imaging techniques such as positron emission tomography (PET). Reasons for the rare use of palladium-mediated coupling methods in radiochemistry with positron-emitters may be the following: • Whenever possible, multi-step reactions are avoided in radiosyntheses with short-lived nuclides.
• Syntheses with many reactants often lead to problems of reproducibility and optimization under no-carrier-added (n.c.a.) conditions and complicate a rapid on-line separation of products.
• The typical use of non-polar solvents and dry reaction conditions for the coupling reaction necessitate an extensive pre-separation of labelled intermediates which is often time and yield consuming.
• Insoluble compounds (mixture of organic and inorganic reactants) complicate the applicability of the method in remotely controlled synthesis devices.
Otherwise none of these problems are insuperable, and new technical developments, like microfluidic systems, may help to solve the problem with solubility [6,7]. Since diaryliodonium salts allow to access n.c.a. [ 18 F]fluorohalobenzenes in high yields and in one reaction step [8][9][10] the availability of 18 F-labelled electrophilic coupling partners is not any longer a hindrance to apply such coupling methods [5,[11][12][13][14].
One basic cross-coupling reaction is the Hartwig-Buchwald N-arylation (HBC), which leads to arylamines, important structure elements of a lot of bioactive compounds. Although alternative pathways for generating fluoroarylamines have been used including direct fluorination, these proved not to be practical for n.c.a. 18 F-labelled arylamine compounds. Para-alkylamine substituents such as piperidine or piperazine cause a maximum slow-down of reaction kinetics of SNAr reactions, what is reflected by very low Hammett σ constants of about −1 of those substituents [15]. Direct labelling of arylamines with [ 18 F]fluoride was therefore impossible so far. Alternatively, three-step syntheses of n.c.a. [ 18 F]fluorophenylpiperazine have been developed starting from dinitrobenzene derivatives [16,17].
Thus, the HBC offers the possibility of a two-step procedure to 18 F-labelled arylpiperazine or piperidines. The choice of the solvent is the main challenge is of this reaction. Dimethylformamide (DMF) which is commonly used for 18 F-labelling of iodonium salts failed as solvent for HBC in previous studies [18,19]. The aim of this work, therefore, was the optimization of HB-coupling conditions with n.c.a. 18 F-labelled iodobenzene to allow for the use of polar media with regard to facilitate a one-pot synthesis of 1-(4-[ 18 F]fluorophenyl)piperazine. Such reaction conditions avoid both pre-separation and pre-drying, and may result in a definite increase of efficiency of HBC in radiochemistry.
1-(4-Fluorophenyl)piperazine is itself a neuropharmacologically active substance [20] and thus interesting as an imaging probe, but it was not 18 F-labelled so far. The actual benefit of this molecular structure for the development of pharmaceuticals lies in the possibility of easy-to-perform coupling by reductive amination (mild conditions, toleration of moisture and of many functional groups, high yields and reproducibility) of different aldehyde moieties in order to synthesize various phenylbenzyl-piperazines. These are important structure motives for different neuroligands, including ligands for D2-like dopamine receptors, serotonin receptors, sigma receptors [21], adrenergic receptors [22] and calcium channels [23]. In many cases, an aromatic fluorine substituent increases the affinity and/or selectivity of a ligand, which is important for its use in radiodiagnostics [24,25].
One very interesting ligand of this group is the highly affine (Ki(D4) = 1 nM) and selective (Ki(D1-3) = 8600-28,000 nM) dopamine D4 ligand FAUC 316 (1) (see Figure 1) [26]. A direct radiolabelling of this compound by fluorine-18 or carbon-11 was not carried out so far. However, in order to achieve an efficient radiolabelling of FAUC analogues, two fluoroethoxy-substituted derivatives, namely 2-[4-(2-(2-fluoro-ethoxy)phenyl)-piperazin-1-ylmethyl]indole-5-carbonitrile and 2-[4-(4-(2-fluoroethoxy)-phenyl)-piperazin-1-ylmethyl]indole-5-carbonitrile, have been developed which were 18 F-labelled with radiochemical yields of 80% and 47%, respectively [27]. Their respective Ki values of 2.1 and 9.9 nM for the dopamine D4 receptor subtype are comparable with the Ki value of 1 nM of FAUC 316. However, the D4 selectivity within the D2 family, best displayed by a 420-fold D4-selectivity over D2 receptors by 2-[4-(4-(2-fluoroethoxy)-phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile, is much lower compared to the factor of 19,000 for FAUC 316. Furthermore, a new series of FAUC derivatives, derived from aminomethyl-substituted pyrazolo[1,5-a]pyridine (FAUC 113 and 213), have been pharmacologically evaluated and labelled with fluorine-18 [28]. The best derivative of this series, labelled by 18 F-fluoroethylation with 88% RCY, showed a Ki value of 13 nM which again is by a factor of about 10 lower than that of FAUC 316. The dopamine D4 receptor exhibits a real challenge with regard to its in vivo determination due to the extremely low concentration (Bmax(brain) ≈ 9-30 fmol/g(tissue)) of this receptor subtype in the mammalian brain [29]. Therefore, a suitable radioligand for PET or an alternative molecular imaging method does not exist until now [30,31]. Due to the strong and multiple effects of dopamine on the human body it is difficult to differentiate between the individual influences of the 5 dopamine subtypes since they show a very similar binding behavior [32,33]. This similarity poses a high demand on the selectivity of ligands for imaging of an individual subtype of dopaminergic receptors.

Palladium-Mediated N-Arylation
The synthesis of an n.c.a. 4-[ 18 F]fluorohalobenzene as electrophilic coupling compound for HBC was conducted by using hypervalent λ 3 -iodane (also called iodonium) species as 18 F-labelling precursor. Although the exact labelling mechanism of the nucleophilic substitution on iodo(III) arene compounds with fluoride is not known until now [34], it lead to [ 18 F]fluoroaryls also with poorly activating and even electron withdrawing substituents in high yields. Ideally, it is desirable that all [ 18 F]fluoroarenes are synthesized by direct labelling of, e.g., a corresponding iodonium compound. In reality, however, the synthesis of those precursors is limited since it requires harsh oxidative conditions with a rather poor tolerance of functional groups, and extremely low labelling yields are often obtained from more complex iodonium precursors. Especially nitrogen-containing aryliodonium salts are difficult to synthesize by classical routes for diaryliodonium salts [35]. New modifications were developed to overcome this challenge [36,37], but only very few examples of complex nitrogen-containing iodonium salts can be found in the literature [38][39][40]. The fact that all of the complex iodonium precursors used for a successful 18 F-labelling do not contain an amine group, is only an empirically based information. However, it was verified in our study with the example of 4-(4-benzylpiperazin-1-yl)phenyl(4-methoxyphenyl)iodonium triflate. No 18 F-labelling could be observed using this precursor; that is why a direct one-step synthesis of [ 18 F]FAUC 316 ([ 18 F]1) failed. Therefore, multi-step reactions are still necessary for 18 F-fluorination of these electron rich aryls, where hypervalent λ 3 -iodane species, nevertheless, serve as excellent starting material. In this work, 1-[ 18 F]fluoro-4-iodobenzene ([ 18 F]4) was used rather than the bromine-analogue because higher labelling yields were found and preliminary examinations showed even slightly higher coupling rates. For comparison, an 18 F-for-N(CH3)3 exchange on 4-iodophenyltrimethylammonium triflate (2) yielded only 12% ± 3% in DMSO at 160 °C, while the unsymmetrical iodonium salt, 4-iodophenyl-(4′methoxyphenyl)-iodonium triflate (3a), yielded [ 18 F]4 at 130 °C in DMF with 40% ± 7% (Figure 2). Since a convenient synthesis of the symmetrical bis(4-iodophenyl)iodonium salt (3b) was accessible [41], highest labelling yields of 60% ± 9% could be reached with this iodonium precursor under the same conditions using the Kryptofix ® 2.2.2./potassium carbonate system, without any additive or scavenger which was recommended by Carroll et al. [42]. The solvents DMSO and mixtures of DMSO/DMF proved less suitable for the 18 F-fluorination of the corresponding diaryliodonium salts.
N-Arylation was performed using different Buchwald co-ligands of the fourth generation [46], i.e., dialkylbiaryl phosphine ligands which were optimized for different substrates. For example, RuPhos is typically used for coupling of secondary amines in high yields [47]. Therefore, the used catalyst system was confined on one group.
Typically, for Pd-mediated N-arylation a non-polar solvent like toluene is used together with a strong base like sodium or potassium tert-butoxide. Normally, weak bases are only used when excellent tolerance of functional groups is required. With toluene and potassium tert-butoxide high RCYs of about 70% were reached with the ligands DavePhos and RuPhos within 10-15 min reactions time. This is in agreement with the findings of Wüst et al. [18] who obtained similar yields by coupling an indole derivative under conditions given in Table 1 under entry 1.
The necessity of a separation and purification of [ 18 F]6 led to a considerable loss of product especially when starting with the bis(4-iodophenyl)iodonium salt (3b) in DMF as 18 F-labelling precursor which produced a colloidal precipitate upon dilution with water. Furthermore, the bad miscibility of toluene and water causes problems in cartridge-based separation-processes. For an optimal performance of the reaction the same solvent should be used for both, labelling and the cross-coupling process, in order to avoid those problems and to simplify the work-up procedure. DMF is the solvent to consider in the first place for this purpose since it is typically used for 18 F-fluorination of iodonium-precursors. However, catalyst/base systems which result in best coupling yields within short reaction time in toluene show no conversion when applied in DMF, even after extension of the reaction time to 1 h (see Table 1). This is due to a possible side reaction of the HBC during the catalytic circle. While normally a reductive amination takes place as last step to produce the desired N-aryl compound, a β-hydride-elimination can lead to the dehalogenated product and a tetrahydropyrazine. As Christensen et al. [48] describe, this path becomes dominant when deprotonation is slow which normally occurs in a polar solvent like DMF, DMSO or dimethylacetamide (DMAA) under the employed conditions including NatOBu as base.
Otherwise the successful use of polar solvents [49,50] and even water [51] in HBC is known. In fact, a lot of catalyst/base systems could be found which lead to high coupling yields in DMF up to 88% within 15 min which is an acceptable reaction time for multi-step 18 F-chemistry. The pivotal point is the use of a weak base like K3PO4 or Cs2CO3. Here small but clearly higher yields are obtained with K3PO4 in DMF. In DMSO even higher yields were observed with a reverse trend concerning the use of K3PO4 and Cs2CO3 (Table 1, entry [10][11][12][13][14][15][16][17][18][19]. The system Pd2(dba)3, RuPhos and Cs2CO3 in DMSO lead to a nearly complete conversion within 15 min at 100 °C. In all solvents, a double coupling of n.c.a. 4-[ 18 F]fluoroiodobenzene with piperazine was not observed, which is probably due to the low concentration of the n.c.a. radiotracer [ 18 F]4 present in the reaction mixture. Another possible side reaction, the coupling of 4-[ 18 F]fluorophenylpiperazine with excessive precursor, which was not separated from the reaction mixture, was also not found. The necessity of a weaker base is surprising since a deprotonation process is estimated to be inferior under this condition. Otherwise, the strength of the base does not seem to have a considerable influence on the coupling reaction. The anion activator Kryptofix ® 2.2.2./K2CO3 used for 18 F-fluorination is still present in the DMF solution, which is strongly and basically due to the chelated potassium. Here, deiodination is still comparatively low, but the fact that in general highest yields are obtained in DMSO can be a result of the weaker basic conditions during labelling, when tetrabutyl-ammonium bicarbonate was used as anion activator as was done with precursor 3c. The results do not allow for a definite conclusion on the mechanism during this step of the HBC. However, the reason for slow deprotonation in polar media may be a steric hindrance of base due to coordination with the solvent. Carbonate and phosphate which can also coordinate to palladium may have better kinetics during this step due to their multidentate character.

Synthesis and Evaluation of the D4 Agonist [ 18 F]FAUC 316
The synthesis of [ 18 F]FAUC 316 was performed by a reductive amination of 1-(4-[ 18 F]fluorophenyl)piperazine ([ 18 F]6) with 2-formyl-1H-indole-5-carbonitrile (7) (see Figure 3). A pre-purification of 1-(4-[ 18 F]fluorophenyl)piperazine ([ 18 F]6) is essential to use it for application in a radiopharmaceutical synthesis following the HBC. The liquid-liquid extraction of [ 18 F]6 with hydrochloric acid (2 mol/L) allowed a selective separation of the reaction mixture containing amines as well as water soluble substances. The progress of extraction could easily be traced following the distribution of radioactivity between the two phases. After addition of sodium hydroxide until pH 9, organic compounds were fixed on a Sep-Pak C18 cartridge and eluted with DMSO as solvent of the following reaction. The liquid-liquid extraction was almost quantitative in contrast to a direct solid phase extraction on the weak cation exchanger Strata X-CW which is a faster process, but only provides a product yield of 20% ± 4%.    The synthesis of the cyanoindole precursor 7 for coupling was performed according to methods from the literature [27]. The standard compound FAUC 316 was prepared by reductive amination of commercially available 1-(4-fluorophenyl)piperazine (2) with the aldehyde 7 and acidic sodium cyanoborohydride in methanol obtaining a RCY of about 50%. Compound 7 was also used for radioactive coupling of the 18 F-labelled amine ([ 18 F]6) obtaining radiochemical yields of 85% ± 5% of [ 18 F]1 after 15 min using DMSO as solvent. In order to study whether the strong electron withdrawing effect of the cyano group of compound 7 has an influence on the reductive amination reaction, the coupling was also studied with the unsubstituted indole-2-carbaldehyde 8. However, no significant difference in yield was observed in this case (see Figure 3). Obviously, the cyano substituent does not influence this reaction. The overall RCY of [ 18 F]1 was about 10% and 18% within 80 and 120 min, respectively, depending if a separation of the aryliodide intermediate [ 18 F]4 was necessary. Maximum molar activities of the product reached 90 GBq/µmol and the radiochemical purity was determined by HPLC to be >98%. In Table 2, the whole reaction sequence under optimized conditions is summarized.
For evaluation of the pharmacological properties of the highly affine D4 radioligand [ 18 F]FAUC 316 for later in vivo application its accumulation behavior on rat brain slices was examined by in vitro autoradiography. Thereby, the binding of the radioligand was competed with spiperone and the inactive standard compound FAUC 316 1. Spiperone, an unselective but high affine dopaminergic ligand, was chosen because no applicable gold standard for D4 exists. The extent of competition is lower than 10% for spiperone and even less than 5% for 1. Non-specific binding refers to the binding of a ligand to other sites in the tissue, e.g., other receptors, enzymes or membrane transporters, or to distribution of the ligand into the lipid cell membrane [52]. Therefore, in spite of the excellent affinity and selectivity reported for the inactive ligand, the preliminary in vitro rat brain studies with [ 18 F]FAUC 316, exhibiting a comparatively high molar activity, the high non-specific binding of both radiofluorinated tracers in rat brain does not encourage to recommend them as in vivo PET imaging agents for dopamine D4 receptors in humans.
Sep-Pak C-18 plus-cartridges were purchased from Waters (Eschborn, Germany), Li-Chrolut EN cartridges and glass columns (65 × 10 mm) from Merck (Darmstadt, Germany). Thin layer chromatography (TLC) was done on precoated plates of silica gel 60F254 (Merck) or Alumina N. The compounds were detected by UV at 254 nm. HPLC was performed on the following system from Dionex (Idstein, Germany): an Ultimate 3000 LPG-3400A HPLC pump, an Ultimate 3000 VWD-3100 UV/VIS-detector (272 nm), a UCI-50 chromatography interface, an injection valve P/N 8215. Reversed-phase HPLC was carried out using a Gemini 5 µm C18 110 Å column from Phenomenex (Aschaffenburg, Germany), with a dimension of 250 mm × 4.6 mm (flow 1 mL/min) for analytical separations and 250 mm × 10 mm (flow 5 mL/min) for semi-preparative applications. 1 H, 13 C and 19 F NMR spectra were recorded on a Bruker DPX Avance 200 spectrometer with samples dissolved in CDCl3 or DMSO-d6. All shifts are given in δ ppm using the signals of the corresponding solvent as a reference. Mass spectra were obtained with a Finnigan Automass Multi mass spectrometer with an electron beam energy of 70 eV. High resolution electron spray mass spectra were recorded on a LTQ FT Ultra (Thermo Fischer). Melting points are uncorrected and were determined on a Mettler FP-61 apparatus in open capillaries.

Chemistry
2- ((4-(4-Fluorophenyl)piperazine-1-yl)methyl)-1H-indole-5-carbonitrile, FAUC 316 (1) (alternative to literature method [26]). 2-Formyl-1H-indole-5-carbonitrile (7) [57]. The solvent was evaporated under a stream of argon at 80 °C and 600 mbar. This azeotropic drying was repeated twice using each time 1 mL of dry acetonitrile, followed by evaporation at 8-15 mbar for 5 min. After 10 min reaction time 100 mg of Celite 503 suspended in 20 mL of water was added to the reaction mixture and the total solid was removed by a LiChrolut cartridge (Merck) with a 10 µm PTFE strainer. After washing with 1 mL of water the almost clear solution was passed through a Sep-Pak C18 cartridge, which was previously conditioned with 8 mL of ethanol and 8 mL of water. After washing the cartridge with 5 mL of water and drying for 5 min by a stream of argon the product was subsequently eluted through an unconditioned Alumina N cartridge with 2 mL of anhydrous toluene into a second reaction vial.
Incubation conditions for in vitro autoradiography of all tested substances were similar to those previously described by Zhang et al. [59]. All incubations were performed at 22 °C in Tris-HCl buffer (50 mmol/L, pH 7.4). After pre-incubation in buffer for 10 min rat brain slices were incubated in 10 nmol/L of [ 18 F]FAUC 316 for 30 min either with 10 µmol/L of competitor (spiperone or standard 1) or with the same amount of DMSO, respectively. They were washed twice for 5 min in ice-cold Tris-HCl buffer, rapidly rinsed with ice-cold distilled water, and placed under a stream of dry air for rapid drying. Object slides were exposed to γ-sensitive phosphor-imaging plates for 15-30 min and then laser scanned by a phosphor imager BAS 5000 (Fuji). The analysis of receptor autoradiography was processed according to standard image analysis software (AIDA 2.31; Raytest Isotopenmeßgeräte, Germany). Non-specific binding was defined as the residual activity in the presence of cold standard. Thus, specific binding was calculated as the difference between total and non-specific binding.

Conclusions
Hartwig-Buchwald coupling is traditionally performed in toluene together with a strong base like sodium or potassium tert-butoxide. This Pd-mediated reaction was chosen in order to synthesize the intermediate n. When using DMF or DMSO as solvent for the coupling, this separation step could be omitted as those solvents are typically also used for the 18 F-labelling reaction. Thus, biarylphosphonium ligands together with the multidental inorganic bases K3PO4 or Cs2CO3 were best suited for the arylation step and led to conversion rates of more than 70% in DMF, which is comparable to those in toluene. Even in DMSO, quantitative conversation was also observed. Overall radiochemical yields of 1-(4-[ 18 F]fluorophenyl)piperazine of up to 40% and 60% were reached in DMF and DMSO, respectively, depending on the labelling yield of the first step. An additional reaction step led to a quick radiosynthesis of the D4 agonist [ 18 F]FAUC 316 in good radiochemical yields and high molar activity. The radioligand showed, however, a high non-specific binding in first in vitro autoradiographic studies.