Preparation of NIn-Methyl-6-[18F]fluoro- and 5-Hydroxy-7-[18F]fluorotryptophans as Candidate PET-Tracers for Pathway-Specific Visualization of Tryptophan Metabolism

Tryptophan (Trp) is an essential proteinogenic amino acid and metabolic precursor for several signaling molecules that has been implicated in many physiological and pathological processes. Since the two main branches of Trp metabolism—serotonin biosynthesis and kynurenine pathway—are differently affected by a variety of neurological and neoplastic diseases, selective visualization of these pathways is of high clinical relevance. However, while positron emission tomography (PET) with existing probes can be used for non-invasive assessment of total Trp metabolism, optimal imaging agents for pathway-specific PET imaging are still lacking. In this work, we describe the preparation of two 18F-labeled Trp derivatives, NIn-methyl-6-[18F]fluorotryptophan (NIn-Me-6-[18F]FTrp) and 5-hydroxy-7-[18F]fluorotryptophan (5-HO-7-[18F]FTrp). We also report feasible synthetic routes for the preparation of the hitherto unknown boronate radiolabeling precursors and non-radioactive reference compounds. Under optimized conditions, alcohol-enhanced Cu-mediated radiofluorination of the respective precursors afforded NIn-Me-6-[18F]FTrp and 5-HO-7-[18F]FTrp as application-ready solutions in radiochemical yields of 45 ± 7% and 29 ± 4%, respectively. As such, our work provides access to two promising candidate probes for pathway-specific visualization of Trp metabolism in amounts sufficient for their preclinical evaluation.


Introduction
Tryptophan (Trp) is an essential proteinogenic amino acid with an indole ring in the side chain.It is the least abundant amino acid in animal proteins (approx.1.4%) and serves mainly as a metabolic precursor for a plethora of bioactive molecules (Figure 1) [1,2].
Under physiological conditions, over 95% of dietary Trp enters the kynurenine (KYN) pathway and is metabolized through cleavage of the indole ring by the rate-limiting enzymes tryptophan-2,3-dioxygenase (TDO) in the liver or indoleamine-2,3-dioxygenase (IDO) in other tissues [1,2].Depending on the exact metabolic route, subsequent steps can produce an array of bioactive intermediates that may exert either neuroprotective or neurotoxic effects (Figure 1).While the concentration of toxic Trp metabolites in the normal brain is low, local upregulation of IDO or changes in the activity of downstream enzymes can result in significant built up of KYN and other intermediates in neuroinflammatory, neurodegenerative, and neuropsychiatric disorders [1][2][3][4].In addition, accelerated Trp metabolism due to upregulation of IDO has been linked to tumor immune escape and has been shown to correlate with a poor prognosis in several types of cancer [5,6].
escape and has been shown to correlate with a poor prognosis in several types of cancer [5,6].A minor fraction (1-2%) of dietary Trp enters the serotonin pathway and is converted into the neurotransmitter serotonin (5-hydroxytryptamine or 5-HT), which is mainly (≈90%) formed in the gastrointestinal tract but also present in the brain (≈5%) [7].Serotonin is biosynthesized through 5-hydroxylation of the indole ring in Trp by the rate-limiting enzyme tryptophan hydroxylase (TPH) and subsequent decarboxylation by aromatic amino acid decarboxylase (AADC).In the pineal gland, serotonin may be further transformed into melatonin (Figure 1).Serotonergic signaling is well known for its multifaceted role in emotional and cognitive processing under physiological conditions.Furthermore, a number of neuropsychiatric and neurodegenerative disorders have been shown to be associated with dysfunction or loss of serotonergic neurons in the brain [8][9][10][11].In addition, neuroinflammation-driven changes in KYN pathway activity in patients with A minor fraction (1-2%) of dietary Trp enters the serotonin pathway and is converted into the neurotransmitter serotonin (5-hydroxytryptamine or 5-HT), which is mainly (≈90%) formed in the gastrointestinal tract but also present in the brain (≈5%) [7].Serotonin is biosynthesized through 5-hydroxylation of the indole ring in Trp by the rate-limiting enzyme tryptophan hydroxylase (TPH) and subsequent decarboxylation by aromatic amino acid decarboxylase (AADC).In the pineal gland, serotonin may be further transformed into melatonin (Figure 1).Serotonergic signaling is well known for its multifaceted role in emotional and cognitive processing under physiological conditions.Furthermore, a number of neuropsychiatric and neurodegenerative disorders have been shown to be as-sociated with dysfunction or loss of serotonergic neurons in the brain [8][9][10][11].In addition, neuroinflammation-driven changes in KYN pathway activity in patients with neuropsychiatric disorders can reduce serotonin synthesis by lowering the Trp availability and suppressing the activity of TPH [12].
Given that several pathological conditions are associated with simultaneous and often opposite changes in KYN pathway activity and serotonin synthesis, selective visualization of the different branches of Trp metabolism could help in the understanding, prognosis and therapy of many disorders.However, optimal PET-tracers for pathway-specific imaging of Trp metabolism are still lacking.Thus, while 11 C-labeled tracers like α-[ 11 C]methyltryptophan ([ 11 C]AMT) and 5-hydroxy-[β- 11 C]tryptophan ([ 11 C]5-HTP) (Figure 1) have proven useful for imaging Trp metabolism and/or serotonin synthesis [13][14][15][16], their application is limited by several inherent shortcomings, such as the short half-life of carbon-11 (t 1/2 = 20 min) and cumbersome production routes.Fluorine-18 possesses more favorable characteristics, like a longer half-life (t 1/2 = 109 min) and lower kinetic energy of emitted positrons.Although the first 18 F-labeled racemic fluorotryptophans (rac-5-and rac-6-[ 18 F]fluorotryptophan, Figure 1) were already described in 1972, their preparation suffered from low molar activities and radiochemical yields (RCYs), long reaction times and complex synthetic procedures with dangerous diazonium salts [17].A more recent example of an 18 F-labeled tracer that is thought to preferentially target the KYN pathway is 1-(2-[ 18 F]fluoroethyl)-tryptophan ([ 18 F]FETrp, Figure 1), which accumulates in IDO1-expressing cells and IDO1-positive tumor xenografts [18,19].However, the radiosynthesis of [ 18 F]FETrp is elaborate and requires separation of enantiomers by chiral preparative HPLC [19].
Being interested in easily accessible PET-tracers for imaging of Trp metabolism, we previously developed an efficient route for the preparation of 4-7-[ 18 F]fluorotryptophans (4-7-[ 18 F]FTrps) via alcohol-enhanced Cu-mediated radiofluorination and subjected them to a preclinical evaluation [20,21].Whereas 4-6-[ 18 F]FTrps suffered from rapid defluorination in vivo, 7-[ 18 F]FTrp (Figure 1) was stable and showed preferential uptake in serotonergic brain regions and the melatonin-producing pineal gland of healthy rats.However, there is still an unmet need for PET-tracers that could be used to selectively address the different branches of Trp metabolism.
The aim of the present work was to prepare N In -methyl-6-[ 18 F]fluorotryptophan (N In -Me-6-[ 18 F]FTrp) and 5-hydroxy-7-[ 18 F]fluorotryptophan (5-HO-7-[ 18 F]FTrp) as candidate PET-tracers for selective visualization of either the KYN pathway or serotonin synthesis, respectively (Figure 2).Based on the exceptionally broad substrate specificity of amino acid transporters like LAT1, which are highly expressed in the blood-brain-barrier and have been shown to accept a range of structurally diverse unnatural amino acids [22][23][24], both probes should be efficiently transported into the brain.Additionally, N In -Me-6-[ 18 F]FTrp was chosen based on previous findings that both N In -methyltryptophan and 6-fluorotryptophan are substrates of IDO [18,25,26], suggesting that probes derived from these compounds could be used for visualization of the KYN pathway in the brain.Furthermore, N In -methyltryptophan has been shown to not be a substrate of AADC [27] or TDO [18,28], and the electron-withdrawing fluorine substituent in 6-position of the indole ring should destabilize the cationic transition state formed during 5-hydroxylation of tryptophans by TPH [29,30], suggesting that N In -Me-6-[ 18 F]FTrp will neither enter the serotonin pathway nor be subject to significant peripheral metabolism via the KYN pathway in the liver.Conversely, 5-HO-7-[ 18 F]FTrp was chosen based on the low affinity of 5-hydroxytryptophanes for IDO [18] and the fact that 7-fluorotryptophan is neither a good substrate of IDO nor of TDO [26,31].Furthermore, 5-hydroxytryptophan is a known endogenous AADC substrate and aromatic fluorine substituents have been shown to be well tolerated by this enzyme [32][33][34][35][36], which suggests that this candidate could be used to target the second step in serotonin biosynthesis while minimizing metabolism via the KYN pathway.In addition to the preparation of the two radiolabeled amino acids, we describe the synthesis of the non-radioactive reference compounds and the corresponding radiolabeling precursors.

Chemistry
The synthesis of the radiolabeling precursor for N In -Me-6-[ 18 F]FTrp was started from commercially available 6-bromoindole (1).First, 1 was methylated with iodomethane (MeI) using sodium hydride as a base, which afforded intermediate 2 [37] in a quantitative yield (Scheme 1).Next, 2 was borylated to give 6-Bpin-N-methylindole (3) [38].The latter was introduced into a Mannich-type reaction [39], furnishing 6-Bpin-N In -methylgramine (4) in a yield of 88%.Subsequent addition of MeI at low temperature resulted in the quaternized gramine 5 as hydroiodide salt, which was used for stereoselective alkylation of an (S)-Ni-BPB-Gly complex [40].In the latter reaction, the N-benzylproline moiety directed the diastereoselective Michael addition of deprotonated (S)-Ni-BPB-Gly to an 1-methyl-3-methylidene-3H-indolium species (generated in situ from 5 via elimination of trimethylamine followed by isomerization), resulting in selective formation of (S,S)-Ni-BPB-N In -Me-6-Bpin-Trp (6) [41].The yield for this reaction step was fair (13%), and the product had to be purified via successive normal-and reversed-phase chromatography, so that precursor 6 was obtained in a total yield of 6% over 5 steps (Scheme 1).However, given that presence of the N In -methyl group hindered formation of the reactive intermediate, this was considered to be acceptable.
The initial steps for preparation of the reference compound N In -Me-6-FTrp were similar to those for preparation of 6 described above (Scheme 2).In particular, 6-fluoroindole (7) was subjected to a Mannich-type reaction (92% yield) followed by quaternization of the resulting intermediate (8) with MeI to furnish 1-(6-fluoro-1H-indol-3-yl)-N,N,Ntrimethylmethanaminium iodide (9) in 82% yield.Initial attempts to utilize this quaternized gramine for Michael addition with an achiral Ni-BPA-Gly complex [42] were unsuccessful owing to an extremely low solubility of the complex in MeCN used as reaction solvent.Since direct production of racemic intermediates through this method was not feasible, we instead opted to synthesize (R)-and (S)-N In -Me-6-FTrp separately.To this end, either (R)-or (S)-Ni-BPB-Gly were alkylated with 9, furnishing (R,R)-or (S,S)-10 in a yield of 56%.These complexes were then decomposed with (Et 4 N) 2 DTPA [43], followed by in situ N α -Boc protection to give the respective protected tryptophans, (R)-and (S)-11, in 56% and 59% yield, respectively.Subsequent N In -methylation and deprotection of the resulting intermediates (R)-and (S)-12 yielded the desired reference compounds (R)-and (S)-N In -Me-6-FTrp [(R)-and (S)-13] as hydrochloride salts in total yields of 1% and 6% over six steps, respectively (Scheme 2).
reaction with methyl 2-acetamidoacrylate.The latter furnished methyl 2-a acetoxy-7-fluoro-1H-indol-3-yl)propanoate (24) in a yield of 14%, which protected at both nitrogen atoms to obtain 25 (63% yield).Subsequent twotion via the intermediate 26 afforded the reference compound racemic 5-TFA salt (27 × TFA) in a total yield of 1% over 7 steps.Due to the difficulty of introducing a hydroxy group into the indole ring together with the low stability of substituted 5-hydroxyindoles and -tryptophans, synthesis of the reference compound 5-HO-7-FTrp proved to be particularly challenging (for details on various synthetic routes evaluated, see supporting information).However, we eventually obtained the desired product using a combination of oxidation via potassium nitrosodisulfate (Fremy's Salt) [49] and Friedel-Crafts alkylation [50] (Scheme 4).To this end, 7-fluoroindole (20) was initially reduced to 7-fluoroindoline (21) using sodium cyanoborohydride in acetic acid (66% yield) as described by Lohray et al. [51].Indoline 21 was then utilized as substrate for regioselective introduction of a hydroxy group in the 5-position of the indole ring using Fremy's salt [49].This step was performed in phosphate buffer at pH = 7 for 30 min, which provided the best conversions with minimal formation of side-products.The yield of isolated 5-hydroxy-7-fluoroindole (22) thus obtained amounted to 31%, and subsequent Oacylation of 22 [52]
Spectroscopic/analytical data were identical for both isomers.Only data for (R,R)-10 are reported. 1  (R)-11: A solution of DTPA (4.13 g, 10.5 mmol) in 25% Et 4 NOH (12.1 mL) was added to a solution of (R,R)-10 (0.62 g, 1 mmol, 1 eq) in MeOH (30 mL), and the deeply red-colored mixture was stirred at 85 • C for 24 h.The resulting faintly green colored solution was cooled to ambient temperature and MeOH was removed under reduced pressure.The remaining aqueous solution was cooled to 0 • C, and the precipitated (R)-BPB was filtered off and washed with cold H 2 O.The filtrate was basified to pH = 9.0-9.5 with 1 M NaOH and washed with Et 2 O (3 × 20 mL).Saturated NaHCO 3 (4.5 mL), followed by Boc 2 O (0.66 g, 3 mmol, 3 eq) and MeOH, was added to the aqueous solution until the mixture was homogeneous.The mixture was stirred for 3 d at ambient temperature, and MeOH was removed under reduced pressure.The remaining aqueous phase was washed with Et 2 O (3 × 20 mL), acidified to pH = 2 with solid NaHSO 4 , and extracted with Et 2 O (2 × 30 mL).The organic phase was washed with 1 M NaHSO 4 (3×), H 2 O (3×), and brine (2×); dried; and concentrated under reduced pressure to obtain (R)-11 as a colorless solid (0.18 g, 0.56 mmol, 56%).
Spectroscopic/analytical data were identical for both isomers.Only data for (R)-11 are reported.NMR spectra showed the presence of two rotamers.Only data for the major rotamer are reported. 1

General
No-carrier-added aqueous [ 18 F]fluoride was produced via the 18 O(p,n) 18 F nuclear reaction through the bombardment of enriched [ 18 O]H 2 O with 17 MeV protons in a BC1710 cyclotron (The Japan Steel Works, Tokyo, Japan) or a GE PETtrace (GE Healthcare, Chicago, IL, USA), both at the INM-5 (Forschungszentrum Jülich).Radioactivity was measured using a CRC-55tR Dose Calibrator from Capintec, Inc. (Florham Park, The Netherlands) and/or a Curiementor 2 (PTW, Freiburg, Germany).QMA carbonate light plus cartridges (130 mg, Waters GmbH, Eschborn, Germany) were preconditioned with 2 mL H 2 O. SepPak C18 light cartridges (130 mg, Waters GmbH, Eschborn, Germany) were preconditioned with 5 mL EtOH followed by 5 mL H 2 O.

Semi-Preparative HPLC
Semi-preparative HPLC was performed on a dedicated semi-preparative HPLC system consisting of a Knauer K-100 pump (Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany), a Knauer K-2501 UV Detector (Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany), a Rheodyne 6 port injection valve equipped with a 2 ml injection loop, and a custom-made Geiger counter.HPLC columns were purchased from Phenomenex (Aschaffenburg, Germany) and Merck KGaA (Darmstadt, Germany).The identity of 18 F-labeled tracers and their enantiomeric excess (ee) were determined via co-injection of an authentic excess was determined according to method E {R t ([ 18 F]13): 9.7 min}.The product [ 18 F]13 was obtained as a ready-to-use solution in RCYs of 45 ± 7% within 83 ± 8 min with a radiochemical purity (RCP) of >99% and an ee of >99%.Molar activities (A m ) amounted to 66 ± 33 GBq/µmol (1-1.5 GBq tracer).[ 18 F]Fluoride (0.5-10 GBq) in [ 18 O]H 2 O was loaded (from the male side) onto a QMA carbonate light plus cartridge.The cartridge was washed with MeOH (2 mL), dried with air (10 mL), and eluted (from the female side) with a solution of Et 4 NHCO 3 (1 mg, 5.23 µmol) in MeOH (0.5 mL).If the QMA cartridge was loaded, flushed, and eluted from the female side only, a significant amount of [ 18 F]fluoride sometimes remained on the resin (probably because QMA-light cartridges have a single frit on the male side but four frits on the female side).MeOH was removed within 5 min at 80 • C under reduced pressure (300-400 mbar) in a stream of argon.A solution of precursor 19 (5.4 mg, 10 µmol) and Cu(OTf) 2 (3,4-Me 2 -Py) 4 (6.8 mg, 10 µmol) in DMI (0.75 mL) was added, and the reaction mixture was stirred under air at 110 • C for 15 min.The reaction mixture was then diluted with H 2 O (7 mL), and the resulting suspension was loaded (from the female side) onto a SepPak C18 light cartridge.The cartridge was washed with H 2 O (5 mL) and dried with air (20 mL), and the radiolabeled intermediate (S)-Boc-5-AcO-7-[ 18 F]FTrp-OtBu ([ 18 F]29) was eluted (from the female side) with MeOH (1 mL).MeOH was removed within 5 min at 80 • C under reduced pressure (300-400 mbar) in a stream of argon.The residue was taken up in 2 M HCl (0.3 mL) and stirred at 110 • C for 10 min to deprotect [ 18 F]29 into [ 18 F]27.2 M NaOH (0.25 mL) was added, the mixture was diluted with HPLC mobile phase (8% EtOH in acetate buffer, 1 mL), and the radiolabeled product was isolated using semi-preparative HPLC (method B, product fraction at 13-14 min).The purity of the isolated product was analyzed according to method D {R t ([ 18 F]27): 7.9 min}.Enantiomeric excess was determined according to method E {R t ([ 18 F]27): 7.7 min}.The product [ 18 F]27 was obtained as a ready-to-use solution in RCYs of 29 ± 4% within 81 ± 3 min, with an RCP of >99% and ee of >99%.A m amounted to 46 ± 17 GBq/µmol (1.05-1.35GBq tracer).

Conclusions
In summary, N In -Me-6-[ 18 F]FTrp ([ 18 F]13) and 5-HO-7-[ 18 F]FTrp ([ 18 F]27) were efficiently prepared in RCYs of 45 ± 7% and 29 ± 4% via Cu-mediated radiofluorination of the respective pinacol boronate precursors and subsequent decomposition/deprotection of the resulting radiolabeled intermediates.In addition, feasible synthetic routes for preparation of the hitherto unknown radiolabeling precursors and non-radioactive reference compounds were developed.The latter proved to be especially challenging in the case of 5-HO-7-FTrp, synthesis of which ultimately succeeded starting from 7-fluoroindole using a route that involves oxidation with Fremy's salt and Friedel-Crafts alkylation as key steps.As such, our work provides access to two promising new 18 F-labeled Trp derivatives (and the corresponding non-labeled reference compounds) in amounts sufficient for their preclinical evaluation as candidate PET-tracers for pathway-specific visualization of Trp metabolism.

Figure 2 .
Figure 2. Candidate PET-tracers for pathway-specific imaging of tryptophan metabolism prepared in the present study.

Figure 2 .
Figure 2. Candidate PET-tracers for pathway-specific imaging of tryptophan metabolism prepared in the present study.

23 Figure 2 .
Figure 2. Candidate PET-tracers for pathway-specific imaging of tryptophan metabolism prepared in the present study.