18F-Labelled Intermediates for Radiosynthesis by Modular Build-Up Reactions: Newer Developments

This brief review gives an overview of newer developments in 18F-chemistry with the focus on small 18F-labelled molecules as intermediates for modular build-up syntheses. The short half-life (<2 h) of the radionuclide requires efficient syntheses of these intermediates considering that multistep syntheses are often time consuming and characterized by a loss of yield in each reaction step. Recent examples of improved synthesis of 18F-labelled intermediates show new possibilities for no-carrier-added ring-fluorinated arenes, novel intermediates for tri[18F]fluoromethylation reactions, and 18F-fluorovinylation methods.


Introduction
The positron emitter fluorine-18 is a commonly used radionuclide in molecular imaging with positron emission tomography (PET) due to its advantageous nuclear properties. Thus, it finds wide application as noninvasive, quantitative, and versatile modality in medical diagnosis, research, and drug development [1]. Fluorine-18 has a short half-life of 109.7 min which only allows time-limited syntheses and study protocols. The methods for introducing this shortlived radionuclide into organic molecules thus require fast chemistry, and it is desirable to introduce the 18 F-label during the last possible synthetic step.
A further aspect is the stoichiometry of 18 F-chemistry that differs from "cold" fluorinations. The radionuclide is produced in low (nano-to picomolar) amounts and its concentration in reaction mixtures is several orders of magnitude lower than the precursor concentration. Furthermore, the syntheses of the radiotracers have to be performed in closed, lead-shielded hot cells, which necessitates an easily applicable and remote-controlled process. Thus, besides the development of more efficient and flexible 18 F-labelling methods new technological approaches have been examined, especially in the field of microfluidic chemistry [2][3][4][5][6][7][8][9][10]. The development of a reliable 18 F-labelling technique together with an automatic synthesis module is a major prerequisite of routine production of 18 F-labelled PET radiopharmaceuticals [11][12][13].
Methods for the introduction of [ 18 F]fluorine into organic molecules can be divided into two groups, namely, direct and indirect. The direct method entails introduction of [ 18 F]fluorine without changing the carbon skeleton structure of the molecule. However, in many cases this necessitates the protection of functional groups or requires other transformations like reduction or oxidation of functional groups after introduction of radiofluorine [14].
The indirect method involves build-up syntheses, that is, changing the carbon skeleton structure and starting from small molecules which themselves can be easily 18 Ffluorinated by nucleophilic substitution. Such small 18 Flabelled alkyl [15] or aryl [16] groups bear typically reactive functional groups for further transformation reactions. Those intermediates are used to synthesize more complex biological molecules which cannot be labelled with fluorine-18 due to mechanistic reasons or are not stable enough to tolerate direct 18 F-fluorination conditions.
In the case of 18 F-labelling of macromolecules like peptides, proteins, and antibodies, these small 18 F-labelled intermediates are commonly called "prosthetic groups. " In the last decade progress has been made regarding the 18 F-labelling of macromolecules [17,18]. Besides the use of prosthetic groups

Aliphatic Intermediates
Aliphatic 18 F-fluorination is certainly the most prominent method for 18 F-labelling [32], and important PET-radiotracers for clinical use are aliphatically 18 F-labelled compounds which fulfill these requirements, for example,  [39], most often in aqueous solution.

Intermediates for Nucleophilic Substitution and Other
Coupling Reactions. The synthesis of intermediates for 18 Ffluoroalkylation is characterized by a two-or three-step procedure (cf. Figure 3) [40]. First, [ 18 F]fluoride is introduced into a molecule using precursors containing a good leaving group. The 18 F-labelled precursor is then isolated and purified before coupling with a further molecule.
In the first step the [ 18 F]fluoride has to be separated from the target water and activated for a nucleophilic substitution reaction. The standard conditions of these basic methods are described in several reviews [11,32,41]. A simplification of this approach was achieved by water removal on a strong anion-exchange resin [42] or by use of strong organic bases as additives replacing the inorganic bases or salts classically used in the resin eluent [43][44][45][46]. Instead of trapping on anionexchange resins n.c.a. [ 18 F]fluoride can also be separated by electrochemical methods which are useful to minimize the reaction volume especially for the use in microfluidic systems [47][48][49][50][51]. The use of mixtures of nonpolar tert-alcohols with acetonitrile as a reaction medium enhanced the reactivity of cesium [    to those conventionally obtained only with dipolar aprotic solvents [52,53]. Bromine and iodine and several sulfonate derivatives serve generally as leaving groups for a nucleophilic aliphatic radiofluorination [15,40,54,55]. Alternatively, in the case of preparation of O-[ 18 F]fluoromethylated aliphatic and aromatic ethers, the 1,2,3-triazolium triflate group serves as a very good nucleofuge for displacement by the [ 18 F]fluoride ion [56].
The purification of 18 F-fluorinating agents is performed by HPLC, solid phase extraction (SPE), or distillation. The main challenge is the complete separation of the 18 F-labelled intermediate from the precursor which also would act as reaction partner in the following coupling step. This leads to unwanted side reactions which could lower the radiochemical yield (RCY) or necessitate a higher concentration of the precursor for the subsequent coupling reaction. A purification of the 18 F-fluorinating agent via HPLC (or GC) is very effective and is often used [57][58][59], but it is more inconvenient for automatization [60,61]. The use of SPE [62][63][64] or a distillation process for purification is principally easier to automate [40]. For instance, 1-bromo-3-(nitrobenzene-4sulfonyloxy)-propane as starting precursor will be retained in the reaction vessel during the distillation process of 1-bromo-3-[ 18 F]fluoropropane, due to its very high boiling point, thus eliminating the risk of formation of pseudocarrier [65]. In a few cases the direct coupling of the 18 F-labelled intermediate was performed without former separation and purification [66].
Another possibility for simplified workup is the use of fluorous solid phase extraction (FSPE). A nucleophilic 18 F-fluorination of fluorous-tagged precursors can easily be purified by FSPE regardless of the affinity of the untagged substrate for the stationary phase. FSPE-purified labelled compounds can then be used in subsequent reactions or more easily purified by HPLC before administration [67,68]. A similar approach was performed using molecular imprinted polymers [69].
Coupling reactions of the 18 F-fluorination agent with the desired target molecule are performed either by the use of a further leaving group, by the click chemistry approach [70], by Staudinger ligation [71][72][73], or by Pd(0) mediated reactions [74].
A series of arylsulfonates were prepared as nucleophile assisting leaving groups (NALG) in which the metal chelating unit is attached to the aryl ring by an ether linker. Under microwave irradiation and without the assistance of a cryptand, such as Kryptofix 2.2.2, primary substrates with selected NALGs led to a 2-3-fold improvement in radiofluorination yields over traditional leaving groups [75].

Tri[ 18 F]fluoromethyl
Group. The CF 3 group has an electronegativity similar to that of oxygen [76] and is characterized by a large hydrophobic parameter as measured by the relative partition coefficient [77]. The trifluoromethyl group is an important pharmacophore present in many biologically active pharmaceutical and agrochemical drugs. The increased lipophilicity and a superior metabolic stability compared to that of the trifluoromethyl analogues often account for an improved activity profile [78]. Thus, radiolabelled trifluoromethyl groups are of potential interest to facilitate drug discovery. Earlier attempts to synthesize an 18 F-labelled trifluoromethyl group were also characterized by low RCY and low specific activity due to decomposition of the target material [79][80][81].
The recently published developments can be divided in aliphatic and aromatic tri[ 18 F]fluoromethylation reactions (cf. Figure 3, method B).
A novel, one-step method for nucleophilic radiosynthesis of aliphatic tri[ 18 F]fluoromethyl groups using the n.c.a. [ 18 F]fluoride ion under relatively mild conditions was developed by incorporation of the radiolabel by an equivalent nucleophilic addition of H[ 18 F]F to the 1-tosyl-2,2difluorovinyl group (cf. Figure 4). The tosylate function then serves as leaving group in a subsequent coupling step [82,83]. The specific activity of the tri[ 18 F]fluoromethylether was determined to be 86 MBq/nmol. The need of a double bond to achieve the addition of the [ 18 F]fluoride limits this reaction to aliphatic tri[ 18 F]fluoromethylations.
Aromatic tri[ 18 F]fluoromethyl groups were formerly synthesized using hardly accessible aromatic-CF 2 Br groups [84]. Two new approaches were published quite recently (cf. Figure 5). Both methods start with an aliphatic precursor which is first labelled with fluorine-18 and then coupled to the benzene ring. In a two-step procedure tri[ 18 F]fluoromethane ([ 18 F]fluoroform) available from difluoroiodomethane and [ 18 F]fluoride [85] is coupled in a copper(I) mediated reaction to aromatic halides using potassium tert-butoxide as base. The RCY was determined up to 65% with a specific activity of up to 50 GBq/ mol [86]. This method has recently been improved performing a one-pot synthesis in the presence of copper(I)bromide, N,N-diisopropyl-N-ethylamine, and the corresponding iodoarene without separation of the [ 18 F]fluoroform intermediate [87]. The RCYs of the desired tri[ 18 F]fluoroarenes were determined with up to 90%, but no information on the specific activities was given. An alternative method is used in a one-pot process. The trifluoromethylation agent [ 18 F]CF 3 Cu, generated in situ from methyl chlorodifluoroacetate, CuI, TMEDA, and [ 18 F]fluoride, is coupled to (hetero)aryl iodides in RCYs ranging from 17 to 87% [88]. A drawback of this procedure is still the relative low specific activity of 0.1 GBq/ mol exemplified so far only for 4-tri[ 18 F]fluoromethyl nitrobenzene. However, the method enables an efficient tri-[ 18 F]fluoromethylation of complex molecules like [ 18 F]fluoxetine. N-Boc protected [ 18 F]fluoxetine was readily prepared in 37% RCY. The subsequent N-Boc deprotection delivered [ 18 F]fluoxetine with 95% yield. A more detailed review on the scope and limitations of the radiosynthesis of tri[ 18 F]fluoromethyl groups is provided as part of this special issue [89]. 18 F-Fluorovinylation. Transition metal catalyzed allylic substitution is a powerful method for carbon-carbon and carbonheteroatom bond formation (cf. Figure 3 above, method C). These reactions encompass a wide variety of heteroatoms (N, O, and S) as nucleophiles [90]. In the field of 18 F-chemistry a palladium catalyzed allylic fluorination reaction was developed and transferred to n.c.a. conditions yielding 18 F-labelled cinnamyl fluoride starting from [ 18 F]TBAF, cinnamyl methyl carbonate, [Pd (dba) 2 ], and triphenylphosphine in anhydrous acetonitrile [91].

Palladium, Managnese and Iridium Catalyzed
Further, a rapid allylic fluorination method utilizing trichloroacetimidates in conjunction with an iridium catalyst has been developed. The reaction is performed at room temperature without the need of inert gas atmosphere and relies on the Et 3 N⋅3HF reagent to provide branched allylic fluorides with complete regioselectivity. This high-yielding reaction can be carried out on a multigram scale and shows considerable functional group tolerance. The use of Kryptofix 2.2.2/K 2 CO 3 allowed an incorporation of fluorine-18 within 10 min [92]. The RCY of allylic [ 18 F]fluoride was determined to be 38%. A specific activity for the aforementioned reactions, however, was not reported.
A new method enables the facile n.c.a. 18 F-labelling of aliphatic C-H bonds in benzylic position using manganese salen catalysts with RCY ranging from 20% to 72% within 10 min without the need for preactivation of the labelling precursor [93].

18 F-Labelled Aromatic and Heteroaromatic
Intermediates by Classic Approaches. Historically, the use of the Balz-Schiemann or Wallach reaction was the first attempt to synthesize 18 F-labelled aromatic rings starting from [ 18 F]fluoride (cf. Figure 6, method A) [94,95]. However, the thermal decomposition of the corresponding aryl diazonium salts and of the aryl triazenes is characterized by low RCY, a low specific radioactivity, and extensive byproduct formation [95]. The use of tetrachloroborate or 2,4,6triisopropylbenzenesulfonate as counterions led to improvements of the Balz-Schiemann reaction which enables the synthesis of [ 18 F]fluoroarenes in 39% RCY at the n.c.a. level, exemplified for 4-[ 18 F]fluorotoluene [96]. In a recently published study these nucleophilic 18 F-labelling methods were reinvestigated using polymer bound aryl diazonium salts and aryl triazenes [97]. The solid phase supported dediazofluorination using arenediazonium cations, ionically bound to a sulfonate functionalised ion exchange resin, was, however, not suitable for nucleophilic 18 F-labelling of aromatic compounds, whereas the solid supported triazene yielded the 18 F-labelled product in a reasonable RCY of 16%.
Most successful for the introduction of fluorine-18 into aromatic rings is the conventional aromatic nucleophilic substitution (S N Ar) reaction using the [ 18 F]fluoride anion to displace a suitable leaving group from an electron deficient benzene ring. As leaving groups serve halides, the nitro and the trimethylammonium function. The activation of the aromatic ring is usually achieved by suitable functional groups with a-M effect like the carbonyl, carboxyl, cyano, and nitro group [32]. These highly activating groups especially enable the efficient introduction of [ 18 F]fluoride into aromatic rings to label small 18 F-intermediates for build-up syntheses. The activating functionality is then converted by reduction, oxidation, or hydrolysis to nucleophilic groups for subsequent coupling reactions. The n.c.a. inter-   [101].
The azocarbonyl unit is a new group for activation of the arene ring by an S N Ar mechanism. The aromatic core of phenylazocarboxylic esters is highly activated towards nucleophilic aromatic 18 F-substitution (cf. Figure 8) [102]. This kind of compounds was converted in a radical arylation reaction into biaryl compounds or in substitutions at its carbonyl unit to produce azocarboxamides. Because of the high reactivity of the aryl radical, side products like [ 18 F]fluorobenzene and 4-[ 18 F]fluorophenol were also formed.
The conventional nucleophilic aromatic substitution reaction can principally be used for the n.c.a. 18 F-labelling of aromatic rings in complex molecules [14]. However, the direct introduction of [ 18 F]fluoride is often hampered by a lack of activation and further functional groups, especially those which have acidic protons. In the case of free amino, hydroxyl, or carboxylic acid functions the use of protecting groups is indispensable which have to be removed at the end of synthesis. Generally, the direct 18 F-labelling of complex molecules enables the establishment of one-pot syntheses which is advantageous of being better introduced in a remote controlled synthesizer. In a multistep synthesis the intermediates have often to be purified (e.g., [103]) which hampers the installation in a synthesis module. Thus, one-pot syntheses are normally preferable over the build-up synthesis using several reactor vessels. There are exceptions to this rule, for example, when the build-up synthesis gives substantially higher RCYs [104].
In contrast to benzene, some heteroarenes like pyridine efficiently support the S N Ar reaction and can directly be used to prepare 18 F-labelled heteroarenes in the 2-or 4-position [105][106][107]. Because of its straightforward feasibility, this method was even applied for radiofluorination of complex structures containing an azabenzoxazole [108], a 1,3-thiazole [109], a fluoropurine [110], a pyridine [111][112][113][114][115][116][117][118], a quinolinol [119], or a pyrimidine moiety [120].  Figure 6, Method D, and Figure 9). The classical approach of n.c.a. nucleophilic aromatic 18 Fsubstitution reactions is limited to activated arene rings. The use of diaryliodonium salts enables the introduction of n.c.a. [ 18 F]fluoride into aromatic rings without further activation of strong electron withdrawing groups, which was first demonstrated in 1995 [121]. The reaction via an S N Ar mechanism leads to n.c.a. [ 18 F]fluoroarenes and the corresponding iodoarenes. The nucleophilic attack on the diaryliodonium salt occurs preferably at the more electrondeficient ring and a steric influence of substituents, especially  of ortho-substituted, could be observed [95]. Further studies have recently been performed to examine the possibilities and limitations of this reaction, with a focus on the synthesis of ortho-and meta-substituted arenes and the use of microreactors [122][123][124].

Iodonium Salts (See
An interesting aspect here is that the reaction of diaryliodonium salts with [ 18 F]fluoride is feasible in the presence of water, however, depending on the substituents present on the arene ring. Iodonium salts bearing a para-or orthoelectron-withdrawing group (e.g., p-CN) reacted rapidly (∼ 3 min) to give the expected major [ 18 F]fluoroarene product in useful, albeit moderate radiochemical yields even when the solvent had a water content of up to 28%. Iodonium salts bearing electron-withdrawing groups in metaposition (e.g., m-NO 2 ) or an electron-donating substituent (p-OMe) gave low radiochemical yields under the same conditions. The finding that [ 18 F]fluoroarenes, that having an ortho-alkyl substituent or an ortho-or a para-electron withdrawing group, can be synthesized without the need to remove irradiated water or to add a cryptand, could be attractive in some radiotracer production settings, particularly as this method saves time, avoids any need for automated drying of cyclotronproduced [ 18 F]fluoride, and also avoids substantial loss of radioactivity through adsorption onto hardware surfaces [125]. In order to control the attack of the [ 18 F]fluoride ion on the diaryliodonium salts it is important that one arene ring be more electron-rich than the ring to be labelled with fluorine-18. Here, the use of symmetrically substituted diaryliodonium salts [126] or the use of aryl(heteroaryl) iodonium salts [127] is an alternative to direct the 18 F-labelling to the desired ring. More recently, the use of aryiodonium ylides became of interest for this purpose. The electron-rich status of the ylides, made, for example, from dimedone (5,5dimethylcyclohexane-1,3-dione), even enables the synthesis of electron-rich arenes in high RCY [128]. This type of precursor has recently been demonstrated to be even suitable for complex molecules [129].
Some special intermediates like azide-containing diaryliodonium salts bearing an azidomethyl group on one aryl ring and with a 4-methoxy group on the second one enable the synthesis of click-labelling synthons up to 50 % RCY, even in the presence of a high fraction of water in the reaction solvent [130].
Halopyridinyl-(4 -methoxyphenyl)iodonium tosylates were used to rapidly produce [ 18 F]fluorohalopyridines and in useful RCYs, including the otherwise difficult to access 3-[ 18 F]fluorohaloisomers [131]. Figure 6 and Methods E and F). Another newer method for the 18 F-labelling of nonactivated aromatic compounds makes use of triarylsulfonium salts. The method is applicable to a wide range of substituted aryl systems including amides [132].

Sulfur Activated Systems (See
A new radiosynthetic method for producing n.c.a. [ 18 F]fluoroarenes is based on the reactions of diaryl sulfoxides bearing electron-withdrawing paragroups with the [ 18 F]fluoride ion. These reactions are relatively mild, rapid, and efficient. However, this reaction is limited to aromatic rings bearing an electron withdrawing function like the nitro, cyano, or trifluoromethyl group [133]. Figure 6 and Methods G and H). New concepts to synthesize 18 F-labelled aromatic rings try to achieve fluoride-derived electrophilic n.c.a. fluorination reagents by fluoride umpolung [134,135]. A preliminary realization of this concept was achieved by using a n.c.a. [ 18 F]fluoride capture by a Pd(IV) complex to form an electrophilic 18 F-fluorination reagent followed by a subsequent n.c.a. 18 F-fluorination of palladium aryl complexes [136,137]. Another kind of palladium catalyzed fluoride activation enables the synthesis of 18 F-labelled 1-[ 18 F]fluoronaphthalene in 33% RCY but only in the presence of fluoride carrier [138]. Another advanced method for a transition metal catalyzed late-stage radiofluorination relies on a one-step oxidative 18 Ffluorination using a nickel aryl complex and a strong oxidation agent [139].

Umpolungs Reactions (See
[ 18 F]Fluoride can also be introduced into organic molecules by electrochemical oxidative fluorination via an aryl cation that undergoes rearomatization by loss of a proton. Oxidation of benzene in an electrolysis cell, using Et 3 N⋅3HF and Et 3 N⋅HCl in acetonitrile as the electrolyte, gave c.a. [ 18 F]fluorobenzene in 17% RCY [140] and  [ 18 F]fluorophenylalanine in 10.5% RCY with a specific activity of 1.2 GBq/mmol [141]. However, the aim of all these methods is the late stage 18 F-fluorination of electron-neutral and electron-rich aromatic compounds to simplify the synthesis of radiotracers. Regarding the palladium and nickel reactions, the precursor synthesis is often complex, has to be carefully handled under inert atmosphere, and needs high synthetic experience. This method is far from ideal, given the many reagents and demanding reaction conditions necessary, which hamper to fulfill a "good manufacturing practice" (GMP) pharmaceutical production [142,143]. Thus, although principally applicable, their limitation and complexity do not warrant usefulness for the syntheses of build-up intermediates, as there are more efficient methods available for those molecules.  [144]. These methods required either the preparation of an anhydrous tetrachloroborate or a two-step synthesis from [ 18 F]fluoroaniline and were not established for radiotracer production.

N.c.a. 4-[
A more reliable preparation of n.c.a. 2-and 4-[ 18 F]fluorophenol was achieved using the Baeyer-Villiger reaction on 18 F-labelled benzaldehyde, acetophenone, or benzophenone derivatives. Total radiochemical yields of about 25% were received using m-chloroperbenzoic acid as oxidant in the presence of trifluoroacetic acid [145]. The Baeyer-Villiger reaction of 18 F-labelled benzophenone derivatives containing further electron withdrawing groups yielded up to 65% of 4-[ 18 F]fluorophenol within 60 min with a high radiochemical purity. However, a considerable disadvantage of this method is the somewhat cumbersome work-up of the aqueous reaction mixture in order to isolate the product for its further use [146]. The formation of 18 F-labelled 4-phenol derivatives by Baeyer-Villiger oxidation was, for example, applied to the direct 18 F-fluorination of 6-[ 18 F]fluoro-L-dopa [147].
A novel radiochemical transformation by an oxidative 18 F-fluorination of tert-butylphenols uses the concept of an aryl umpolung (cf. Figure 10) and is also applicable to other O-unprotected phenols. The reaction is performed at room temperature by applying a one-pot protocol and can also be performed in a commercially available microfluidic device [148].
Furthermore, aryl(thienyl) iodonium salts [149] and bis(4-benzyloxyphenyl) iodonium salts [150] have successfully been employed for the preparation of [ 18 F]fluorophenol in a two-step procedure. Compared with the Baeyer Villiger method using benzophenone derivatives, this pathway saves 20 to 45 min of preparation time and delivers [ 18 F]fluorophenol in an organic solution. So these methods are more useful for subsequent coupling reactions under anhydrous conditions. In contrast to the aryl umpolung reaction, the iodonium strategy, however, necessitates a deprotection step after the 18 F-exchange.

Conclusion
The lack of universally useful methods for direct n.c.a. radiofluorination of complex molecules causes the wide use of 18 F-labelled intermediates for the build-up synthesis of radiotracers. Nevertheless, multistep build-up syntheses of 18 F-labelled radiotracers are confronted with several fundamental challenges, which often hamper a remotely controlled, large scale production by this type of reactions. Time consuming separation steps and the use of moisture or even air sensitive reagents complicate the automation of these build-up syntheses. Their application is therefore limited to specialized laboratories with the suitable equipment and experienced staff. The use of build-up reactions then often enables the only way to achieve the synthesis of new radiotracers. Once proven that a radiotracer has the potential to be a useful radiopharmaceutical for molecular imaging, most often ways can be found to establish its routine production via an alternative, simpler synthetic concept and/or by optimisation. Here, the novel developments in umpolung reactions or the improvements in iodonium chemistry in 18 F-labelling of arenes are promising methods, which might also be effective for the late-stage 18 F-fluorination of complex precursors. However, their suitability for daily routine GMP-production of radiopharmaceuticals remains to be elucidated.

Conflict of Interests
The author declares that there is no conflict of interests regarding the publication of this paper.