Synthesis and evaluation of 6-[18F]fluoro-3-(pyridin-3-yl)-1H-indole as potential PET tracer for targeting tryptophan 2, 3-dioxygenase (TDO)

Article abstract of DOI:10.1016/j.nucmedbio.2019.12.007

Introduction: The increase in expression of tryptophan 2, 3-dioxygenases (TDO) and indoleamine 2,3-dioxygenase (IDO) have been reported as potential tumor biomarkers. TDO and IDO are enzymes that catalyze the first and rate-limiting step of the kynurenine pathway. Positron emitting tomography (PET) tracers investigating the kynurenine pathway may allow for the detection of different disease pathologies in vivo including cancer. However, current PET tracers being developed for TDO and IDO have suffered from either multi-step low yielding syntheses or de-fluorination of the tracer in vivo. Results: TDO inhibitors based on 6-fluoroindole with C3 substituents are a class of small molecules that have been shown to bind to TDO effectively, restore tryptophan concentration and decrease the production of immunosuppressive metabolites. The compound 6-fluoro-3-(pyridine-3-yl)-1H-indole has been reported to have high in vitro affinity for TDO. Herein we report the fully automated radiosynthesis of 6-[¹⁸F]fluoro-3-(pyridine-3-yl)-1H-indole [¹⁸F]4 using a copper-mediated nucleophilic ¹⁸F-fluorination resulting in a non-corrected yield of 5 to 6% of the tracer with a radiochemical purity of >99% after 4 h. Small animal dynamic PET/CT imaging of [¹⁸F]4 intravenously injected into normal C57BL/6 mice revealed rapid accumulation in heart and brain, reaching maximum occupancy in heart (10.9% ID/g) and brain (8.1% ID/g) at 1.75 min and 2.25 min, respectively. Furthermore, these in vivo studies revealed no de-fluorination of the tracer, as evidence by the absence of [¹⁸F]fluoride accumulation in bone. Conclusion: In vitro studies demonstrate that 4 has good affinity for hTDO and the radiolabeled analogue [¹⁸F]4 can be synthesized with suitable radiochemical yields. [¹⁸F]4 demonstrates good uptake in the brain and the radiolabeled compound shows no de-fluorination in vivo in C57BL/6 mice.

Full text of DOI:10.1016/j.nucmedbio.2019.12.007

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Synthesis and evaluation of 6-[18F]fluoro-3-(pyridin-3-yl)-1H-
indole as potential PET tracer for targeting tryptophan 2,
3-dioxygenase (TDO)
Zheng Qiao, Karine Mardon, Damion Stimson, Mary-anne
Migotto, David C. Reutens, Rajiv Bhalla
PII:
S0969-8051(19)30507-4
DOI:
https://doi.org/10.1016/j.nucmedbio.2019.12.007
NMB 8108
Reference:
To appear in:
Nuclear Medicine and Biology
Received date:
Revised date:
Accepted date:
7 October 2019
17 December 2019
17 December 2019
Please cite this article as: Z. Qiao, K. Mardon, D. Stimson, et al., Synthesis and evaluation
of 6-[18F]fluoro-3-(pyridin-3-yl)-1H-indole as potential PET tracer for targeting
tryptophan 2, 3-dioxygenase (TDO), Nuclear Medicine and Biology(2019), https://doi.org/
10.1016/j.nucmedbio.2019.12.007
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Synthesis and evaluation of 6-[18F]fluoro-3-(pyridin-3-yl)-1H-indole as potential
PET tracer for targeting tryptophan 2, 3-dioxygenase (TDO).
Zheng Qiao,1 Karine Mardon,1,2 Damion Stimson, Mary-anne Migotto,1 David C. Reutens1 and
Rajiv Bhalla1,*
1Centre for Advanced Imaging, The University of Queensland, St Lucia, Brisbane, Queensland
4072, Australia
2National Imaging Facility, The University of Queensland, Brisbane, Australia
* Corresponding author
E-mail: r.bhalla@uq.edu.au

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Abstract
Introduction: The increase in expression of tryptophan 2, 3-dioxygenases (TDO) and indoleamine
2,3-dioxygenase (IDO) have been reported as potential tumor biomarkers. TDO and IDO are
enzymes that catalyze the first and rate-limiting step of the kynurenine pathway. Positron emitting
tomography (PET) tracers investigating the kynurenine pathway may allow for the detection of
different disease pathologies in vivo including cancer. However, current PET tracers being
developed for TDO and IDO have suffered from either multi-step low yielding syntheses or de-
fluorination of the tracer in vivo.
Results: TDO inhibitors based on 6-fluoroindole with C3 substituents are a class of small molecules
that have been shown to bind to TDO effectively, restore tryptophan concentration and decrease the
production of immunosuppressive metabolites. The compound 6-fluoro-3-(pyridine-3-yl)-1H-indole
has been reported to have high in vitro affinity for TDO. Herein we report the fully automated
radiosynthesis of 6-[18F]fluoro-3-(pyridine-3-yl)-1H-indole [18F]4 using a copper-mediated
nucleophilic 18F-fluorination resulting in a non-corrected yield of 5 to 6 % of the tracer with a
radiochemical purity of > 99% after 4 hours. Small animal dynamic PET/CT imaging of [18F]4
intravenously injected into normal C57BL/6 mice revealed rapid accumulation in heart and brain,
reaching maximum occupancy in heart (10.9% ID/g) and brain (8.1% ID/g) at 1.75 min and 2.25
min, respectively. Furthermore, these in vivo studies revealed no de-fluorination of the tracer, as
evidence by the absence of [18F]fluoride accumulation in bone.
Conclusion: In vitro studies demonstrate that 4 has good affinity for hTDO and the radiolabeled
analogue [18F]4 can be synthesized with suitable radiochemical yields. [18F]4 demonstrates good
uptake in the brain and the radiolabeled compound shows no de-fluorination in vivo in C57BL/6
mice.
Keywords: TDO inhibitor, 6-fluoroindole, PET, biodistribution, fluorine-18

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1. Introduction
Tryptophan is an essential amino acid which is metabolized predominantly via the kynurenine
pathway by the enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase
(IDO). These enzymes are overexpressed in a wide variety of tumors especially in glioblastoma.1
TDO and IDO catalyze the first and rate-limiting step of the kynurenine pathway in which the
oxidative indole ring cleavage reaction of tryptophan to N-formyl-kynurenine is achieved by
employing molecular oxygen or reactive oxygen species across the C2-C3 bond (Figure 1).2
Inhibition of tryptophan metabolism using TDO or IDO inhibitors and the subsequent restoration of
tryptophan concentration by could prove to be a beneficial strategy in the context of disease
progression in cancer.3, 4
Monitoring the upregulation of IDO and TDO using PET tracers has been the focus of several
research groups for tumor detection. The majority of these PET tracers are radiolabeled derivatives
of tryptophan including α-[11C]methyl-L-tryptophan ([11C]AMT)5, 1-N-[11C]methyl-L/D-tryptophan
(L/D-[11C]1MTrp)6, 1-N-[18F]fluoroethyl-L/D-tryptophan (1-L/D-N-[18F]FETrp)7, 4, 5, 6-
[18F]fluorotryptophan (4, 5, 6-[18F]FTrp)8, 9 (Figure 2). However only [11C]AMT has been widely
investigated in clinical studies imaging IDO-upregulated tumors, especially for patients with
glioblastoma.10, 11 However, the wider utility of [11C]AMT has been limited because of complex
radiosynthesis of this radiotracer and short half-life of carbon-11 (20 minutes).12, 13 The other tracers
have either (i) demonstrated poor uptake of the tracers in the brain or (ii) involved multistep
radiosyntheses or (iii) shown defluorination of the tracer in vivo.6-9 These results indicate that the
need for the development of PET tracers (ideally fluorine-18 tracers) which show good affinity to
IDO / TDO and are easy to synthesize and show minimal defluorination in vivo.
The 6-fluoroindole moiety has been demonstrated to be a potential useful building block for the
design and synthesis of IDO and TDO inhibitors. Whilst 6-fluoro-tryptophan exhibits high in vitro
affinity to TDO,14 recent studies of the fluorine-18 labeled analogue have demonstrated de-
fluorination of the radiotracer in vivo. Indole derivatives with substituents at the C3 position exhibit

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pico- to nanomolar inhibitory actions against a wide range of biological targets and processes.15, 16
Consequently C3 substituents of 6-fluoroindole have been synthesized and evaluated in terms of
their solubility and in vitro activity to TDO.
In this present study, we report the synthesis and initial pre-clinical evaluation of a radiolabeled
TDO inhibitor. This radiolabeled agent 6-[18F]]fluoro-3-(pyridine-3-yl)-1H-indole [18F]4 contains a
6-fluoroindole moiety with substituents on the C3 position. The non-radioactive analogue 6-fluoro-
3-(pyridine-3-yl)-1H-indole has an IC50 of 850 nM in an enzymatic assay.16
2. Experimental
2.1. Materials and instruments
All chemicals and solvents were purchased from Sigma-Aldrich and used as received without
further purification. Thin-layer chromatography (TLC) was performed on pre-coated silica gel 60
F254 M (Merck, Darmstadt, Germany) with UV detection at 254 nm . Milli-Q water was used
throughout the experiments. Human TDO with a concentration of 2.87 mg/mL was obtained from
BPS Bioscience. Sep-Pak® Accel Plus QMA Carbonate Light Cartridge (46 mg sorbent/cartridge,
40 μm particle size, p/n 186004540, Waters) was purchased and used as received. Sep-Pak® C18
Plus Light Cartridge (130 mg sorbent/cartridge, p/n WAT023501) was also purchased from
Waters® and conditioned before use with 10 mL of ethanol, then 10 mL of water and finally 20 mL
of air.
13
1H and C NMR spectrum were recorded on Bruker-500 or 900 Hz spectrometers at ambient
temperature, the chemical shifts (δ) were reported as ppm in CDCl3 or DMSO-d6. Mass spectra
(MS) data were acquired on Bruker Autoflex Speed MALDI-TOF MS in positive mode, using α-
cyano-4-hydroxycinnamic acid (CHCA) as the matrix (matrix-free for radiolabelling precursor 8).
The UV-vis adsorption at specific wavelength were recorded on Thermal Scientific Nanodrop
2000/2000c. No-carried-added [18F]fluoride was generated using an 18 MeV cyclotron (Cyclone
“Twin” 18/18 MeV, IBA). The fully automated radiosynthesis was carried out on commercially

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available Synthra RNplus (Synthra, GmbH) module. Analytical HPLC studies were performed using
Shimadzu and Agilent systems equipped with UV and radiation detection.
2.2. Chemistry
2.2.1. Synthesis of tert-butyl 6-fluoro-3-iodo-1H-indole carboxylate (1)
The Boc protection to form compound 1 was carried out using a procedure analogous to that used
for the synthesis of tert-butyl 3-iodo-1H-indole-1-carboxylate.17 A solution of iodine (13.2 mmol,
3.35 g) in 20 mL of DMF was dropped to the solution of 6-fluoroindole (12 mmol, 1.62 g) and
KOH (30 mmol, 1.68 g) in 20 mL of DMF at room temperature. The mixture was stirred overnight,
followed by adding to 300 mL of cold H2O containing sodium metabisulphite (6 g, 2%) and
ammonia (3 g, 1%). The mixture was extracted with Et2O and dried over Na2SO4, filtered and
concentrated. The obtained concentrate was suspended in dichloromethane (100 mL). DMAP (2.4
mmol, 0.29 g), triethylamine (2.4 mmol, 0.24 g) and (Boc)2O (14.4 mmol, 3.14 g) were added to the
reaction mixture. The reaction was allowed to stir at room temperature overnight. Saturated NH4Cl
and dichloromethane were added to the reaction mixture and the organic layer extracted. The
combined organic layers were dried over Na2SO4, filtered and concentrated. Chromatographic
purification (40% EtOAc in hexane) afforded desired compound as a brown solid (1.30 g, 79%
yield, Rf = 0.88). 1H NMR (500 MHz, CDCl3): δ 7.87 (d, J = 9.8 Hz, 1H), 7.69 (s, 1H), 7.32 (dd, J
= 8.7, 5.3 Hz, 1H), 7.06 (td, J = 8.9, 2.4 Hz, 1H), 1.66 (s, 9H). 13C NMR (125 MHz, CDCl3) δ
161.7, 148.6, 130.6, 128.6, 122.6, 122.5, 111.9, 111.7, 102.6, 102.4, 84.9, 64.9, 28.3.
2.2.2. Synthesis of 3-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (2)
The title compound was prepared as described by Andersen et al.18 A solution of 3-bromopyridine
(10 mmol, 1.58 g) in 5 mL of Et2O was slowly added to a 2.5 M hexane solution of n-BuLi (12
mmol, 4.8 mL) in 50 mL of Et2O at -78°C. The reaction mixture was stirred for 30 min and then
trimethoxyborane (12 mmol, 1.34 mL) was slowly added to the reaction mixture at -78°C. The
reaction mixture was warmed to room temperature and left under stirring for 24 h. Pinacol (10.5

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mmol, 1.24 g) was added to the solution and stirred for 1 h. Acetic acid (2.2 mmol, 1.26 mL) was
added to the reaction mixture and left stirring for a further 6 h. The mixture was then concentrated
and the extracted with dichloromethane. The crude product was concentrated to afford an off-white
solid. The resultant solid was washed with CH3CN to afford desired compound (1.65 g, 80% yield).
1H NMR (500 MHz, CDCl3): δ 8.95 (s, 1H), 8.67 (dd, J = 4.9, 1.9 Hz 1H), 8.05 (dt, J = 7.5, 1.7 Hz
1H), 7.30 – 7.27 (m, 1H), 1.36 (s, 12H). 13C NMR (125 MHz, CDCl3): δ 155.6, 152.1, 142.3, 123.2,
84.3, 25.0.
2.2.3. Synthesis of tert-butyl 6-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1
carboxylate (3)
The incorporation of the boronate ester was performed using two different synthetic routes using
chemical methodologies as described by Jia et al.19 (synthetic route 1) and Sigala et al20 (synthetic
route 2) with minor modification. Synthetic route 1: 1 (3 mmol, 1.08 g), bis(pinacolato)diboron (6
mmol, 4.52 g), KOAc (9 mmol, 0.88 g) and Pd(dppf)Cl2 (0.09 mmol, 0.07 g) were mixed under
nitrogen. 30 mL of 1, 4-dioxane was added to the flask under nitrogen. The resultant mixture was
heated at 80°C overnight. The solvent was removed after a crude purification by silica gel column
to remove the catalyst. Chromatographic purification (10% Et2O in hexane) afforded desired
compound as white solid (0.21 g, 19.5% yield, Rf = 0.37). 1H NMR (500 MHZ, CDCl3): δ 7.96 (s,
1H), 7.88 (dd, J = 8.6, 5.7 Hz, 2H), 7.00 (td, J = 9.0, 2.4 Hz, 1H), 1.36 (s, 12H). 13C NMR (125
MHz, CDCl3): δ 159.7, 148.1, 134.3, 128.5, 122.2, 110.1, 109.9, 101.3, 101.1, 83.2 (2C), 82.6, 27.1
(3C), 23.8 (4C).
Synthetic route 2: 1 (3 mmol, 1.08 g) and Pd(dppf)Cl2 (0.09 mmol, 0.07 g) were mixed under
nitrogen. Degassed toluene (15 mL) was added, and the mixture was degassed for another 5 min
with N2. Et3N (11.4 mmol, 1.15 g) and pinacolborane (4.57 mmol, 0.58 g) were successively added
to the reaction mixture. The mixture was heated at 80°C for 3 h. The solvent was removed to leave
the crude compound which was then purified by column chromatography (10% Et2O in hexane) to
1
give the desired compound as a white solid (0.87 g, 81% yield, Rf = 0.37). H NMR (500 MHz,

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CDCl3): δ 7.96 (s, 1H), 7.88 (dd, J = 8.6, 5.7 Hz, 2H), 7.00 (td, J = 9.0, 2.4 Hz, 1H), 1.36 (s, 12H).
13C NMR (125 MHZ, CDCl3): δ 159.7, 148.1, 134.3, 128.5, 122.2, 110.1, 109.9, 101.3, 101.1, 83.2
(2C), 82.6, 27.1 (3C), 23.8 (4C).
2.2.4. Synthesis of 6-fluoro-3-(pyridin-3-yl)-1H-indole (4)
6-fluoro-3-(pyridin-3-yl)-1H-indole was prepared by two different Pd-catalyzed cross-coupling
methodologies as described by Song et al21 (synthetic route 1) and Okada et al22 (synthetic route 2).
Synthetic route 1: to a suspension of 3 (0.75 mmol, 0.27 g) in degassed co-solvent composed of 1,
4-dioxane (15 mL) and H2O (5 mL), 3-bromopyridine (0.75 mmol, 0.12 g) and K3PO4 (1.5 mmol,
0.32 g) were added under nitrogen. And then Pd(dppf)Cl2 (0.08 mmol, 0.06 g) was added under
nitrogen. The reaction was heated at 100°C for 18 h. The reaction mixture was then extracted with
EtOAc, dried over Na2SO4, filtered and concentrated. Chromatographic purification (50% EtOAc in
1
hexane) afforded desired compound as a brown solid (0.02 g, 12.5% yield, Rf = 0.17). H NMR
(500 MHz, CDCl3): δ 8.89 (d, 1H), 8.60 (s, 1H), 8.53 (dd, J = 4.9, 1.6 Hz 1H), 7.99 (dt, J = 7.9, 1.9
Hz 1H), 7.77 (dd, J = 8.8, 5.2 Hz 1H), 7.44 – 7.39 (m, 2H), 7.15 (dd, J = 9.3, 2.3 Hz 1H), 6.99 (td, J
= 9.1, 2.3 Hz 1H). 13C NMR (125 MHz, CDCl3): δ 160.2, 147.1, 145.9, 136.7, 135.3, 131.7, 124.0,
122.7, 121.9, 120.1, 114.3, 109.7, 98.0. MS (MALDI-TOF, [M + H]+) calcd for C13H10FN2 213.23,
found 213.10.
Synthetic route 2: 1 (3 mmol, 1.08 g), 3-(4, 4, 5, 5,-tetramethyl-1, 3, 2-dioxaborolan-2-yl)pyridine
(2.5 mmol, 0.51 g), K2CO3 (12.6 mmol, 1.74 g), and Pd(dppf)Cl2 (0.09 mmol, 0.07 g) were mixed
under nitrogen. 20 mL of degassed DMSO was added to the flask under nitrogen. The reaction
mixture was heated at 90°C for 3 h and subsequently H2O was added to quench the reaction. The
reaction mixture was extracted with EtOAc, dried over Na2SO4, filtered and concentrated. The
residue was purified by silica gel column chromatography (50% EtOAc in hexane) to afford the
1
product as a brown solid (0.19 g, 30% yield, Rf = 0.17). H NMR (500 MHz, CDCl3): δ 8.89 (d,
1H), 8.60 (s, 1H), 8.53 (dd, J = 4.9, 1.6 Hz 1H), 7.99 (dt, J = 7.9, 1.9 Hz 1H), 7.77 (dd, J = 8.8, 5.2
13
Hz 1H), 7.44 – 7.39 (m, 2H), 7.15 (dd, J = 9.3, 2.3 Hz 1H), 6.99 (td, J = 9.1, 2.3 Hz 1H). C NMR

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(125 MHz, CDCl3): δ 160.2, 147.1, 145.9, 136.7, 135.3, 131.7, 124.0, 122.7, 121.9, 120.1, 114.3,
109.7, 98.0. MS (MALDI-TOF, [M + H]+) calcd for C13H10FN2 213.23, found 213.10.
2.2.5. Synthesis of tert-butyl 6-bromo-3-iodo-1H-indole carboxylate (5)
The boc protection to form compound 5 was carried out using an analogous procedure for that used
in the synthesis of tert-butyl 3-iodo-1H-indole-1-carboxylate as described by Witulski et al.17
A
solution of iodine (4.4 mmol, 1.12 g) in DMF (30 mL) was added to a solution of 6-bromoindole (4
mmol, 0.78 g) and KOH (10 mmol, 0.56 g) in DMF (30 mL) at room temperature. The mixture was
stirred overnight, followed by addition to 300 mL of cold H2O containing sodium metabisulphite (6
g, 2%) and ammonia (3 g, 1%). The mixture was extracted by Et2O and dried over Na2SO4, filtered
and concentrated. This was suspended in dichloromenthane (100 mL) and then DMAP (0.8 mmol,
0.09 g), Et3N (0.8 mmol, 0.08 g) and (Boc)2O (4.8 mmol, 1.05 g) were added. The reaction was
allowed to stir at room temperature overnight. Saturated NH4Cl and dichloromethane were added to
the reaction mixture and the organic layer extracted. The combined organic layers were dried over
Na2SO4, filtered and concentrated. Chromatographic purification (40% EtOAc in hexane) afforded
1
desired compound as a brown solid (1.18 g, 70% yield, Rf = 0.88). H NMR (500 MHZ, CDCl3): δ
13
8.35 (s, 1H), 7.67 (s, 1H), 7.42 (dd, J = 8.4, 1.7 Hz 1H), 7.25 (d, 1H), 1.66 (s, 9H). C NMR (125
MHZ, CDCl3) δ 147.1, 130.0, 129.5, 125.6, 121.6, 118.3, 117.2, 83.9, 63.8, 28.7, 27.0 (3C).
2.2.6. Synthesis of 6-bromo-3-(pyridin-3-yl)-1H-indole (6)
Compound 6 was prepared using Pd-catalyzed cross coupling methodologies as described by Okada
et al.22 5 (2.5 mmol, 1.05 g), 3-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (2.1 mmol,
0.43 g), and K2CO3 (10.4 mmol, 1.44 g) were mixed under nitrogen. 30 mL of degassed DMSO was
added to the flask under N2. The reaction mixture was heated at 90°for 3 h. H2O was added to
quench the reaction. The reaction was extracted with EtOAc, dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chromatography (50% acetone in
1
hexane) to afford product as a brown solid (0.15 g, 22% yield, Rf = 0.56). H NMR (500 MHZ,
DMSO-d6): δ 11.63 (s, 1H), 8.89 (dd, J = 2.4, 0.9 Hz 1H), 8.43 (dd, J = 4.7, 1.6 Hz 1H), 8.05 (ddd,

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J = 7.9, 2.4, 1.6 Hz 1H), 7.85 (s, 1H), 7.81 (d, J = 8.5 Hz 1H), 7.64 (d, J = 1.8 Hz 1H), 7.43 (ddd, J
13
= 7.9, 4.8, 0.9 Hz 1H), 7.22 (dd, J = 8.5 Hz 1H). C NMR (125 MHZ, DMSO-d6): δ 147.9, 147.2,
138.4, 134.1, 131.6, 125.8, 124.5, 124.4, 123.4, 121.3, 115.2, 114.9, 113.1. MS (MALDI-TOF,
[M]+) calcd for C13H9BrN2 273.13, found 273.01.
2.2.7. Synthesis of 3-(pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (7)
The incorporation of the boronate ester was carried out using analogous methods to those described
by Taylor et al.23 6 (0.26 mmol, 0.07 g), bis(pinacolato)diboron (0.51 mmol, 0.13 g), KOAc (0.77
mmol, 0.075 g), and Pd(dppf)Cl2 (0.026 mmol, 0.02 g) were mixed under N2. 3 mL of degassed
toluene was added and the reaction mixture was heated at 90°C for 24 h. H2O was added to quench
the reaction. The reaction mixture was extracted with EtOAc, dried over Na2SO4, filtered and
concentrated. The catalyst was removed by silica gel column chromatography (50% acetone in
hexane) to afford crude product and used for the next step.
2.2.8. Synthesis of tert-butyl 3-(pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
indole-1-carboxylate (8)
Boc protection was carried out using a process analogous to that described by Tasch et al.24
7
(0.09 mmol, 0.03 g) was suspended in dichoromethane (3 mL), followed by the addition of DMAP
(0.02 mmol, 0.002 g), Et3N (0.02 mmol, 0.002 g) and (Boc)2O (0.11 mmol, 0.02 g). The reaction
mixture was stirred at room temperature for 1 h. The solvent was removed by rotary evaporator to
leave the crude compound which was purified by HPLC to give the desired compound as a white
solid (0.037 g, 0.09 mmol, 98% yield). Eclipse XDB-C18 Semi-Prep 5µm, 9.4 × 250mm, p/n
1
990967-202). Mobile phase: 70% MeCN in water with a constant flow rate of 4 mL/min. H NMR
(500 MHZ, CDCl3): δ 9.02 (s, 1H), 8.80 (s, 1H), 8.66 (m, 1H), 8.57 (d, 1H), 7.98 (s, 1H), 7.91 (s,
13
1H), 7.81 (dd, J = 7.9, 1.0 Hz, 1H), 7.75 (m, 1H), 1.36 (s, 12H), 1.66 (s, 9H). C NMR (225 MHz,
DMSO-d6): δ 148.7, 148.3, 148.3, 135.1, 134.9, 130.2, 128.9, 128.8, 125.3, 123.4, 121.4, 119.1,
117. 8, 84.3, 83.7 (2C), 54. 9, 27.6 (3C), 24.7 (4C). MS (MALDI-TOF, [M + H]+) calcd for
C24H30BN2O4 421.32, found 421.25.

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2.3. Final optimized radiosynthesis of [18F]4
[18F]Fluoride in oxygen-18 enriched water (98%) was transferred using helium pressure to the hot-
cell where it was measured in a dose calibrator and then transferred into the “V-vial” of the Synthra
RNPlus synthesis module. The aqueous [18F]F- was transferred under vacuum via valve V1 to the
18
QMA cartridge with the enriched water directed to a H2 O recovery vial via V13. The [18F]F- was
eluted from the cartridge via V13, and V14 and collected in the glassy carbon reaction vessel using
a 1 mL solution containing 4 mg Kryptofix® (10.6 µmol) and K2CO3 (0.69 mg, 5 µmol) in 1 mL
(50:50 water : MeCN). Drying of the fluoride was performed using helium flow (V18) with
vacuum applied (V19, V22) at a temperature of 68°C for 5 minutes, then at 95°C for a further 10
minutes. Helium flow ceases and the reaction vessel was cooled to 30°C before the vacuum was
stopped. A solution of the precursor 8 (7 µmol) and [Cu(py)4](OTf)2 (9 µmol) in 1 mL of DMA was
transferred from vial A3 into the reaction vessel, followed by addition of 1 mL of air from the
syringe. Radiolabeling was carried out at 120°C for 10 minutes, followed by thermal deprotection
by heating at 150°C for 5 minutes. The reaction vessel was then cooled to 30°C and water (1mL )
was added from vial A4. The diluted crude reaction mixture was drawn-up into the syringe unit
using position 3 of the syringe valve. The syringe unit was used to load the 5mL volume HPLC
loop with the diluted crude reaction mixture via position 2 of the syringe valve and injected onto the
HPLC column using V26. Semi-preparative HPLC purification was carried out using isocratic
conditions with an Eclipse XDB-C18 Semi-Prep 5µm (9.4 mm × 250 mm; p/n 990967-202). Mobile
phase: 40% MeCN / 60% 0.1 M NH4OAc (with 10 mg / mL of sodium ascorbate in the mobile
phase) with a flow rate of 4 mL/min.
The purified product was cut to a collection vial containing 40 mL of water (containing 10 mg /
mL of sodium ascorbate) via V27 and stirred. Helium pressure was applied to the vial to transfer
the contents through a manually preconditioned (10 mL EtOH, 10 mL water then 20 mL air) C18
cartridge (Sep-Pak® C18 light Cartridge, Waters, p/n WAT023501) to waste via V35 and V36.

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The loaded C18 Sep-Pak cartridge was then washed with 10mL of water from vial C3 directly to
waste. [18F]4 was eluted from the C18 cartridge with 1 mL ethanol from C2 to the vented product
collection vial containing 9 mL of 0.9% saline (containing 100 mg of sodium ascorbate). The total
synthesis time (which has not been optimized) from 18F delivery to reformulated product collection
was under 70 minutes.
2.4. Manual labeling experiments
Methods and equipment used to test the labelling at low activity have recently been described by
Stimson et al.25
2.5. Quality control (QC) of [18F]4
[18F]4 was injected on analytical HPLC connected with a gamma-detector for the determination of
radiochemical yield (RCY), and radiochemical stability and purity. Injection with reference
compound 4 was conducted to determine the identity of [18F]4. The radiochemical stability was
checked up to 4 hours after the end of synthesis (EOS) at room temperature.
All analytical HPLC methods in this paper have used an Eclipse Plus C18 column, 5 µm (46 ×
150 mm, Agilent, Part NO. 959993-902) using 0.1M ammonium acetate as solvent A and MeCN as
solvent B at a flow rate of 1 mL/ min. The method for the analysis for the HPLC traces in Figure 3
(HPLC Method 1) was 0 to 1 min (30% B), 1 to 25 min (30 to 95% B), 25 to 30 min (95% B), 30 to
31 min (95 to 30% B), 31 to 33 min (30% B). The method for the final QC analysis for the HPLC
traces (HPLC Method 2) in Figure 6 was 0 to 20 min (40% B), 20 to 21 min (40 to 90% B), 21 to
26 min (90% B), 26 to 27 min (90 to 30% B), 27 to 30 min (40% B).
2.6. Determination of lipophilicity (logD) of 4
In order to measure the distribution coefficient, 2 mg of 4 was simply dispersed into 2 mL of n-
octanol / 0.2 M sodium phosphate buffer system pH 7.5 (1:1, v:v). The vial was strongly vortexed
for 5 min and centrifuged at 13000 rpm for another 5 min. An aliquot of each layer (100 μL) was

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taken off and measure under UV-vis. LogD was calculated as the decimal logarithm of the ratio
between the UV-vis absorption in the n-octanol phase and the buffer phase.
2.7. In vitro enzymatic essay
2.7.1. Establishment of kynurenine standard curve
The kynurenine standard was established according to the literature with minor modification.26 The
standard assay mixture (200 μL) contained 76 μL of 1 M potassium phosphate buffer (pH = 6.5), 20
μL of 0.4 M sodium ascorbate, 4 μL of 1 mM methylene blue, 40 μL of 1 mg/mL catalase, and
different amount of 1 mM kynurenine (25 to 45 μL). 40 μL of 30% trichloroacetic acid was added
to each vial. After centrifugation at 13000 rmp for 15 min, aliquots of 125 μL of supernatant was
mixed with 125 μL of 2% p-dimethylaminobenzaldehyde and measured under UV-vis of Nanodrop
2000/2000c at 480 nm.
2.7.2. In vitro enzymatic essay of TDO
The in vitro enzymatic essay of TDO was performed according to the literature with minor
modification.27 A standard reaction vial (200 μL) contained 86 μL of 1 M potassium phosphate
buffer (pH = 6.5), 20 μL of 0.4 M sodium ascorbate, 4 μL of 1 mM methylene blue, 40 μL of 1
mg/mL catalase, 20 μL of different concentrations of L-tryptophan (0.4, 0.8, 1.6, 3.2, and 6.4 mM),
10 μL of 1 mM 6-fluoro-3-(pyridine-3-yl)-1H-indole and 20 μL of 0.5 μM of TDO. Each vial was
o
incubated at 37 C for 60 min and then 40 μL of 30% trichloroacetic acid was added, followed by
further incubation at 65 oC for 20 min. After centrifugation at 13000 rmp for 15 min, aliquots of 125
μL of supernatant was mixed with 125 μL of 2% p-dimethylaminobenzaldehyde and measured
under UV-vis of Nanodrop 2000/2000c at 480 nm.
2.8. In vivo PET/CT imaging studies
All animal experiments were approved by the University of Queensland Ethics committee and
carried out at the Centre for Advanced Imaging, UQ. PET/CT images were acquired using an
Inveon multimodality PET/CT scanner (Siemens). On the experimental days, 6 healthy female

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C57BL/6 mice were anaesthetized with 2-3% isoflurane (IsoFlo, Abbott Laboratories) in oxygen at
a flow of 2 L/min. A catheter was inserted into the lateral tail vein before the animal was placed in
the scanner. Mice were maintained under 1 – 2% isoflurane in air-oxygen mixture at a flow rate of
2L/min for the duration of the imaging session. Physiological monitoring (respiratory using a sensor
probe) was achieved throughout all experiments using an animal monitoring system (BioVet
system, m2m Imaging, Australia). A single intravenous injection of the tracer was performed with a
total injected volume not greater than 200 μL, containing not greater than 15 MBq of tracer in a
solution of 10% EtOH in saline. A 60 min dynamic PET scan was started simultaneously with the
tracer injection and was followed by a 15 min CT attenuation scan.
The CT images of the mice were acquired through an X-ray source with the voltage set to 80 kV
and the current set to 500 μA. The scans were performed using 360o rotation with 120 rotation steps
with a low magnification and a binning factor of four. The exposure time was 23 ms with an
effective pixel size of 106 μm. The total CT scan time process took approximately 15 minutes. The
CT images were reconstructed using a Feldkamp conebeam back-projection algorithm provided by
an Inveon Acquisition Workstation (IAW 2.1, Siemens). For the dynamic PET data acquisition, the
emission data were normalized and corrected for radioactive time decay. The list-mode data were
sorted into 41 frames (10 × 30 sec, 25 × 60 sec, 6 × 300 sec time frames). The resulting sinograms
were reconstructed with FBP (filtered back-projection) and an ordered-subset expectation
maximization (OSEM2D) algorithm and analysed using the Inveon Research Workplace software
(IRW 4.1, Siemens) which allows fusion of CT and PET images and definition of region of interest
(ROIs). For each PET image, 3D ROIs were drawn over the brain and other organs of interest
guided by the CT using the IRW 4.1 software. Activity per voxel were converted to nci/cc using a
conversion factor obtained by scanning a cylindrical phantom filled with a known activity of
[18F]fluoride to account for PET scanner efficiency. Activity concentrations were then expressed as
percent of the decay-corrected injected activity per cm3 of tissue that can be approximated as

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percentage injected dose/g (%ID/g). Time activity curves (TACs) were drawn for each organ of
interest including brain, heart, liver, kidney, guts and bladder.
3. Results and Discussion
3.1. Synthesis of reference compound 4
An overview of the synthetic route of reference compound 4 is presented in Scheme 1. Compound
1 was synthesized in two steps by iodination at the 3-position of 6-fluoroindole, followed by
protection of the 1-N-position of the indole moiety with a Boc group. Initial attempts to make
compound 3 by refluxing 1 with bis(pinacolato)diboron, KOAc, and Pd(dppf)Cl2 in 1,4-dioxane at
80 oC for 5 h resulted in significant de-iodination. Compound 3 was subsequently synthesized using
a method recently reported by Sigala et al.20 Refluxing 1: with pinacolborane, Et3N and Pd(dppf)Cl2
in toluene at 80°C for 3 h resulted in an 81% yield of 3 of with significantly less formation of de-
iodinated compound.
Two different synthetic strategies were investigated to construct the C-C bond between the indole
and pyridyl motifs to form compound 4. The first approach which utilized Suzuki coupling of 3
with 3-bromopyridine on resulted in a 12% yield of compound 4. Given the challenging purification
of 3 and low yield of 4 via this synthetic route, an alternative synthesis for compound 4 was
developed. Our alternative strategy involved the cross coupling of compound 1 with 3-(4,4,5,5,-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (compound 2). However the initial attempted
synthesis of compound 2 by refluxing 3-bromopyridine with bis(pinacolato)diboron, KOAc and
Pd(dppf)Cl2 in 1,4-dioxane at 80°C for 20 h was unsuccessful. Increasing the amount of B2pin2,
prolonging reaction time, elevating temperature, and changing solvent were investigated and these
did not result in the synthesis of 2. After further exploration of the literature, we utilized the method
reported by Andersen et al in 201618 using n-BuLi and trimethoxyborane. By careful control of the

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reaction temperature, 2 was obtained in 80% yield without the need for silica gel column
purification. Reference compound 4 was synthesized via coupling 1 and 2 in DMSO at 90 oC for 3
h using K2CO3 as the base and Pd(dppf)Cl2 as the catalyst. A yield of 30% was obtained using this
method, which was almost 2.5 times greater than the previous synthetic routes attempted. It should
be noted that the Boc group at indole 1-N-position was removed during the Suzuki coupling
reaction.28
3.2. Radiochemistry strategy and the synthesis of the radiolabeling precursor 8
The 6-fluoro-indole position of our target compound 4 is not chemically reactive to traditional
nucleophilic aromatic substitution and consequently attempts to form the corresponding 6-nitro or
6-trimethlyammonum derivative would result in no (or very low) radiochemical yields with
fluorine-18. Recently Gouverneur et al29 have demonstrated that aryl-boronate esters can be readily
fluorinated in the presence of [Cu(py)4](OTf). This led us to synthesize the labelling precursor tert-
butyl 3-(pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate 8
(Scheme 2). Since the reference compound 4 had already been synthesized, we initially decided to
use 4 as a substrate to synthesize 7 via C-F bond cleavage. Methodology recently reported by Niwa
et al30 aroused our interest to transform 4 into 7 via Ni/Cu-catalyzed defluoroborylation reaction.
Unfortunately, the desired product 7 was not obtained via this Ni/Cu-catalyzed defluoroborylation
reaction. Switching to the 6-bromo derivative (6) proved a more successful approach since the
bond energy of the C-Br bond is lower than that of the C-F bond. Compound 5 was synthesized in
two steps via iodination of the 3-position of 6-bromoindole, followed by protection of the indole 1-
N-position with a Boc group. Suzuki coupling of 5 with 2 in the presence of K2CO3 and
Pd(dppf)Cl2 gave compound 6 in 22% yield. Condensing 6 with bis(pinacolato)diboron in the
o
presence of KOAc and Pd(dppf)Cl2 at 90 C for 24 h, followed by silica gel column afforded 7.
Finally, indole 1-N-position of compound 7 was protected with Boc group to afford compound 8
which was purified using HPLC. Since the Boc group can be thermally deprotected, in order to

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avoid deprotection (and avoiding formation of 7) the solvent was removed under vacuum without
heating.
3.3. Radiochemistry
3.3.1 Radiochemistry development
Prior to radiolabelling compound 8, pinacol phenylboronate was selected as a model compound to
develop the experimental conditions required for the copper-mediated radiosynthesis (Scheme 3).
Our initial manual radiosynthesis of [18F]fluorobenzene indicated pinacol phenylboronate and
[Cu(py)4](OTf)2 with molar ratio of 7 µmol: 9 µmol in 1 mL of DMA with air gave desired product
with a decay corrected RCY of 22%. Further investigations demonstrated increasing amount of
pinacol phenylboronate (50 µmol) and [Cu(py)4](OTf)2 (70 µmol) resulted in improving the RCY
to 52%. However, since the current synthesis of precursor 8 has not currently been fully optimized,
we have utilized lower quantities of the radiolabeling precursor 8 (7 µmol) and [Cu(py)4](OTf)2 (9
µmol) in our initial studies described in this paper since this should provide sufficient radiochemical
yields for pre-clinical imaging assessment.
The initial radiolabeling of 8 (Scheme 4) at 120 oC revealed the presence of two radio-peaks (in
addition to [18F]fluoride) in the HPLC trace that eluted with retention times of 8.7 min and 18.7
minutes (Figure 3a) which have been attributed to [18F]4 and [18F]9 (the Boc-protected derivative
o
of 4) respectively. The predominant radiolabeled species at 120 C is the Boc protected compound
[18F]9 with a RCY of 18% (a small amount of [18F]4 with a RCY of 2% was also present). Given

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the evidence of deprotection occurring during the labeling, we were concerned that the deprotection
of the Boc-group from the precursor may also occur at 120°C and this may have had a negative
impact on the radiochemical yield (since the indole N-H has the potential to hydrogen bond with the
fluoride). In fact labelling the deprotected precursor (7) at 120°C resulted in a RCY of only 3%.
Decreasing the labelling temperature of 8 to 100 °C eliminated deprotection, however resulted in a
reduced radiochemical incorporation of fluorine-18 of only 8% at this temperature. Increasing the
labelling temperature to 150 °C resulted in an increased level of deprotection of the Boc-group but
resulted in a decreased overall incorporation of fluorine-18 to only 10% (Figure 3b), Zlatopolskiy
et al. reported labelling in their studies was improved by using tert-butanol as a co-solvent (50:50
DMA: tert-butanol).9 However, this did not significantly modify the radiochemical incorporation
(RCY = 20%). Consequently the labelling temperature for the automation studies was fixed at 120
°C and the reaction were performed in 100% DMA.
3.3.2 Radiochemistry Automation
Researchers using copper-mediated nucleophilic [18F]fluorination of aryl-boronate esters have
indicated the importance of adding air during the radiolabeling step.31 Whilst the introduction of air
was relatively trivial for the manual labelling procedure (addition of air manually via a syringe) this
initially presented a challenge for the automated platform (Synthra) that we use for our high activity
radiosynthetic productions. This was primarily due to the fact that (i) the compressed air linked into
the Synthra synthesis module is only linked into the cooling of the heater and (ii) helium gas is used
to transfer reagents from the reagent vials (eg A1, A3, A4 of Figure 4) into the reaction vessel.
Consequently, after the drying step, the system is essentially under inert (helium atmospheric)
conditions. In order to facilitate the introduction of air, we have modified the automation setup.
Figure 4 highlights that we have disconnected the line connection from position 1 of the synthesis
module (see highlighted section in yellow). Line 1 of the syringe system is now open to air and it is
now possible to draw in air (from the hot cell environment) into to the syringe and then into the
reaction vessel (after the addition of the radiolabeling precursors). However, since this syringe is

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still normally wet after the clean-up of the synthesis module, we have now had to add an additional
step to the protocol for the setup of the module. This now requires that, prior to synthesis, the
syringe and the associated inlet ports (1 and 3) are dried thoroughly with helium. Additionally,
since the air is delivered by the line from port 3 to the reaction vessel, the line connecting the
syringe to the reaction vessel must also be thoroughly dried. The first part of the automation
sequence (when air is required) now involves the syringe drawing up 1 mL of air from position 1.
This means that by the time the sequence reaches the point when the labelling precursor and
[Cu(py)4](OTf)2 are added from vial A3 to the reaction vessel, the syringe is primed to deliver the 1
mL of air into the reaction.
For the purposes of the testing this automation and to explore the benefits of the addition of air
during the labelling step, we have tested this using the model compound pinacol phenylboronate
(Scheme 3). This involved writing two sequences - one that involved the addition of the air and one
that did not have air added. After the labelling, 1 mL of water was added to the reaction vessel (in
order to re-dissolve any unreacted [18F]fluoride) and the crude product was transferred into a vial
and the reactions were analysed by HPLC. Our experiments have demonstrated that we can improve
the RCY of the model compound from 22 ±2% (n = 2) to 29 ±3% (n=2) simply by the addition of
air into the reaction vessel using the above modification of the synthesis module.
Following the positive impact of this modification of the Synthra automated module, we
utilized this sequence for the automation of the automated radiolabeling of precursor 8. During our
manual labelling investigations, we observed that (i) the best radiochemical incorporation of
fluorine-18 into our target compound was achieved at 120°C and (ii)
the Boc-group is readily
removed at 150 °C. Combining these two observations, for the automated radiosynthesis of [18F]4,
we have incorporated a labelling step of 120°C for 10 minutes (with the addition of 1 mL of air via
the syringe) and included a thermal deprotection step after the labelling (5 minutes at 150°C). After
labelling and deprotection, the reaction was allowed cool to 40°C before 1mL of water was added to
dilute the crude reaction mixture. This diluted crude reaction mixture was the purified by HPLC.

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Figure 5 shows an example of purification of the tracer using a semi-preparative HPLC column.
After HPLC purification, the purified fraction was diluted into 60 mL of water and this was then
subsequently trapped onto a C18 light Waters 130 mg solid phase extraction (SPE) cartridge. [18F]4
was eluted from the cartridge using 1 mL of ethanol into 9 mL of 0.9% saline.
However, the initial automated productions demonstrated evidence of radiolysis of the radiolabeled
product even at relatively low radioactivite concentrations of 95 MBq/mL. To minimize this
radiolysis, sodium ascorbate was added to the product saline solution as a stabilizer in subsequent
radiosyntheses. Despite addition of 50 mg of sodium ascorbate in the saline, radiolysis was still
observed in the final product. The HPLC trace of [18F]4 at t = 1 hour (after production) revealed that
the radiochemical purity (RCP) of [18F]4 drops from 97% at t=0 to only 88% at t= 2 hour (for a
production with a radioactive concentration of 159 MBq /mL). Addition of more sodium ascorbate
(100 mg) in the saline and addition of sodium ascorbate in the mobile phase and in the collection
vial used for dilution (both at 10 mg / mL) were then explored. These further modifications resulted
in the formation of a stable product. Using this final method, [18F]4 was produced in isolated yields
of 1.6 to 1.7 GBq (n = 3) with a radiochemical concentration of 160 to 170 MBq / mL. The non-
corrected yield of these productions ranged from 5 to 6% and the RCP remained greater than 99%
up to 4 h after end of synthesis (EOS) at room temperature (Figure 6). The molar activity of [18F]4
ranged from 214 to 261 GBq / µmol and the apparent molar activity (including other chemical
impurities in the formulation ranged from 76 to 111 GBq / µmol). Figure S1 confirms identity by
19
HPLC and shows the co-injection of [18F]F4 with the F reference standard. Figure S2 displays
the “zoomed in section” the UV trace of the HPLC from one of the stable final formulations [18F]4.
The area of the peak at approximately 6.3 minutes was used to calculate the molar activity and the
total area of peaks at 5.9, 6.3 and 8.3 minutes were used to calculate the apparent molar activity of
[18F]4. Figure S3 shows the calibration curve which was used to determine the quantity of chemical
in the production of [18F]4. For the purpose of the apparent molar activity, we have assumed that the

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chemical species with peak retention times at 5.9 and 8.3 have similar absorption coefficients (and
molecular weights) to 4.
3.3. Determination of lipophilicity (LogD) of 4
The lipophilicity is a physical and chemical property of compound which provides an approximate
reflection of its adsorption and distribution in vivo. A LogD value above 1 indicates that the
compound probably can pass BBB just by diffusion. Secondly, non-specific binding is known to be
higher with increasing lipophilicity among structurally relevant compound,32 which revealed that
the upper limit for LogD is estimated to 3.33 The value of 1.8 found for 4 (n = 3) indicates that 4
possesses a suitable lipophilicity for the compound to cross the BBB.
3.4. Kinetic parameters for TDO
The kinetic parameters of compound 4 for TDO were determined using initial rate analysis. Initial
rate data were fitted to Michaelis-Menten model. The inhibitory kinetics were evaluated by plotting
1/[S] against 1/[V], where [S] represents substrate concentration and [V] represents reaction rate.
The Michaelis-Menten constant (Km) of TDO is 41.7 × µM and the binding constant is Ki=735 nM
(see Supplementary for graph).
3.5. In vivo PET/CT imaging studies
In vivo dynamic PET/CT imaging studies were performed to evaluate the overall biodistribution of
[18F]4 in normal healthy mice. Following intravenous injection of [18F]4 into C57BL/6 mice (n = 6),
the tissue biodistribution was measured by PET/CT and summarized in Figure 7. [18F]4 localized
rapidly to the heart and brain immediately after injection, reaching to the maximum occupancy in
heart (10.9% ID/g) and brain (8.1% ID/g) at 1.75 min and 2.25 min, respectively. The radiolabeled
compound was then steadily cleared from heart and brain, and taken up in liver, with 14% ID/g
observed in the liver within the first 30 min. High levels of radioactivity were detected in kidney
and bladder, suggesting excretion through kidney and bladder. A constant level of [18F]4 was found

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in guts throughout the duration of the scan. Notably, tracer [18F]4 was not defluorinated in vivo on
the time scale of the scan, as evidenced by the absence of [18F]fluoride accumulation in bone
(Figure 8). During the course of our investigations, the evaluation of several [18F]-labelled
IDO/TDO tracers have been reported. Henrottin et al34 reported that 1-L-N-[18F]FETrp showed
higher affinity to hIDO compared to 1-D-N-[18F]FETrp and 1-L/D-N-[18F]FETrp. Small animal
PET/CT imaging studies reported by Xin et al7 revealed that tumour uptake of 1-L-N-[18F]FETrp
was 4.6 ± 04 %ID/g; in contrast 1-D-N-[18F]FETrp uptake only 1.0 ± 0.2 %ID/g. In a recent report,
Zlatopolskiy et al9 radiosynthesized 4, 5, and 6-[18F]FTrp from their corresponding pinacol
boronate precursors. Whilst the 4, 5 and 6-[18F]FTrp derivatives were stable in vitro, these
radiolabelled tryptophan derivatives suffered from rapid defluorination in vivo. Tang et al8 also
highlighted failure of 5-L-[18F]FTrp to cross the BBB in their study which they proposed was due to
defluorination of this tracer in vivo. In contrast, [18F]4 showed high uptake in the liver and the brain
and no evidence of defluorination in vivo, highlighting benefits of investigating this tracer in the
future in tumor-burdened disease models.
4. Conclusion
We have radiosynthesized 6-[18F]fluoro-3-(pyridine-3-yl)-1H-indole using a copper-mediated
nucleophilic [18F]fluorination of arenes method in a fully automated radiosynthesis module with
non-corrected yield of 5 to 6% and a molar activity of 214 to 261 GBq / µmol. This study has
demonstrated the need to include sodium ascorbate during the purification process and in the final
formulation of [18F]4 to minimize radiolysis, which has resulted in the radiochemical purity of our
final formulation of > 99% up to 4 hours post synthesis. Using dynamic PET/CT imaging studies
we have demonstrated that 6-[18F]fluoro-3-(pyridine-3-yl)-1H-indole has the ability to permeate the
BBB and show no defluorination in vivo up to 1 hour following administration. Thus we propose

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that [18F]4 warrants further exploration in oncology models as potential as a PET probe for imaging
tryptophan metabolism in vivo.
5. Acknowledgements
The research was funded by the Australian Research Council Linkage grant #LP130100703. The
authors acknowledge the facilities and scientific and technical assistance of the National Imaging
Facility, a National Collaborative Research Infrastructure Strategy (NCRIS) capability, at the
Centre for Advanced Imaging, University of Queensland. The authors would like to thank Dr Greg
Pierens, Dr Brett Hamilton and Dr Lorine Wilkinson for additional support for the NMR, MS
analysis and enzymatic essay, respectively. We would like to express gratitude to Dr Takashi Niwa
(Chemical biology team, division of bio-function dynamics imaging, RIKEN centre for life science
technologies) and Dr Masahiro Okada (Graduate school of pharmaceutical science, the university of
Tokyo) for their warmly e-mail reply when acquiring experiment details of their publications.
Reference
1.
Ott, M.; Litzenburger, U. M.; Rauschenbach, K. J.; Bunse, L.; Ochs, K.; Sahm, F.; Pusch, S.; Opitz,
C. A.; Blaes, J.; von Deimling, A.; Wick, W.; Platten, M., Suppression of TDO-mediated tryptophan
catabolism in glioblastoma cells by a steroid-responsive FKBP52-dependent pathway. Glia 2015, 63 (1), 78-
90.
2.
Breda, C.; Sathyasaikumar, K. V.; Sograte Idrissi, S.; Notarangelo, F. M.; Estranero, J. G.; Moore,
G. G. L.; Green, E. W.; Kyriacou, C. P.; Schwarcz, R.; Giorgini, F., Tryptophan-2,3-dioxygenase (TDO)
inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites. Proc. Natl.
Acad. Sci. U. S. A. 2016, 113 (19), 5435-5440.
3.
4.
5.
Siegel, R. L.; Miller, K. D.; Jemal, A., Cancer statistics, 2018. CA-Cancer J. Clin. 2018, 68 (1), 7-30.
QUESNEL, B., Tumor dormancy and immunoescape APMIS 2008, 116 (7‐8), 685-694.
Huang, X.; Xiao, X.; Gillies, R. J.; Tian, H., Design and automated production of 11C-alpha-methyl-l-
tryptophan (11C-AMT). Nucl. Med. Biol. 2016, 43 (5), 303-308.
6. Xie, L.; Maeda, J.; Kumata, K.; Yui, J.; Zhang, Y.; Hatori, A.; Nengaki, N.; Wakizaka, H.; Fujinaga,
M.; Yamasaki, T.; Shimoda, Y.; Higuchi, M.; Suhara, T.; Wang, F.; Zhang, M.-R., Development of 1-N-11C-
Methyl-l- and -d-Tryptophan for pharmacokinetic imaging of the immune checkpoint inhibitor 1-Methyl-
Tryptophan. Sci Rep 2015, 5, 16417.
7.
Xin, Y.; Cai, H., Improved Radiosynthesis and Biological Evaluations of L- and D-1-[18F]Fluoroethyl-
Tryptophan for PET Imaging of IDO-Mediated Kynurenine Pathway of Tryptophan Metabolism. Mol.
Imaging. Biol. 2017, 19 (4), 589-598.

Journal Pre-proof
8.
Tang, T.; Gill, H. S.; Ogasawara, A.; Tinianow, J. N.; Vanderbilt, A. N.; Williams, S.-P.;
Hatzivassiliou, G.; White, S.; Sandoval, W.; DeMent, K.; Wong, M.; Marik, J., Preparation and evaluation of
L- and D-5-[18F]fluorotryptophan as PET imaging probes for indoleamine and tryptophan 2,3-dioxygenases.
Nucl. Med. Biol. 2017, 51, 10-17.
9.
Zlatopolskiy, B. D.; Zischler, J.; Schäfer, D.; Urusova, E. A.; Guliyev, M.; Bannykh, O.; Endepols,
H.; Neumaier, B., Discovery of 7-[18F]Fluorotryptophan as a Novel Positron Emission Tomography (PET)
Probe for the Visualization of Tryptophan Metabolism in Vivo. J. Med. Chem. 2018, 61 (1), 189-206.
10.
Juhász, C.; Chugani, D. C.; Muzik, O.; Wu, D.; Sloan, A. E.; Barger, G.; Watson, C.; Shah, A. K.;
Sood, S.; Ergun, E. L.; Mangner, T. J.; Chakraborty, P. K.; Kupsky, W. J.; Chugani, H. T., In Vivo Uptake and
Metabolism of α-[11C]Methyl-l-Tryptophan in Human Brain Tumors. J. Cereb. Blood Flow Metab. 2005, 26
(3), 345-357.
11.
Juhász, C.; Muzik, O.; Chugani, D. C.; Chugani, H. T.; Sood, S.; Chakraborty, P. K.; Barger, G. R.;
Mittal, S., Differential kinetics of α-[11C]methyl-l-tryptophan on PET in low-grade brain tumors. J. Neuro-
Oncol. 2011, 102 (3), 409-415.
12.
tryptophan for PET studies. Nucl. Med. Biol. 1995, 22 (3), 303-307.
13. Chakraborty, P. K.; Mangner, T. J.; Chugani, D. C.; Muzik, O.; Chugani, H. T., A high-yield and
Mzengeza, S.; Venkatachalam, T. K.; Diksic, M., Asymmetric radiosynthesis of α-[11C]methyl-l-
simplified procedure for the synthesis of α-[11C]Methyl-l-tryptophan. Nucl. Med. Biol. 1996, 23 (8), 1005-
1008.
14.
Rafice, Sara A.; Chauhan, N.; Efimov, I.; Basran, J.; Raven, Emma L., Oxidation of <span
class="sc">L</span>-tryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and
indoleamine 2,3-dioxygenase. Biochem. Soc. Trans. 2009, 37 (2), 408-412.
15.
Hu, W.; Guo, Z.; Chu, F.; Bai, A.; Yi, X.; Cheng, G.; Li, J., Synthesis and biological evaluation of
substituted 2-sulfonyl-phenyl-3-phenyl-indoles: a new series of selective COX-2 inhibitors. Bioorg. Med.
Chem. 2003, 11 (7), 1153-1160.
16.
derivatives, pharmaceutical compositions and methods for use. WO2015121812A1, 2015.
17. Witulski, B.; Buschmann, N.; Bergsträßer, U., Hydroboration and Suzuki–Miyaura Coupling
Crosignani, S.; Cauwenberghs, S.; Driessens, G.; Deroose, F. Novel 3-(indol-3-yl)-pyridine
Reactions with the Electronically Modulated Variant of an Ynamine: The Synthesis of (E)-β-Arylenamides.
Tetrahedron 2000, 56 (43), 8473-8480.
18.
Andersen, T. L.; Frederiksen, M. W.; Domino, K.; Skrydstrup, T., Direct Access to α,α-
Difluoroacylated Arenes by Palladium-Catalyzed Carbonylation of (Hetero)Aryl Boronic Acid Derivatives.
Angew. Chem.-Int. Edit. 2016, 55 (35), 10396-10400.
19.
Jia, Y.; Bois-Choussy, M.; Zhu, J., Synthesis of DEFG Ring of Complestatin and Chloropeptin I:ꢀ Highly
Atropdiastereoselective Macrocyclization by Intramolecular Suzuki-Miyaura Reaction. Org. Lett. 2007, 9
(12), 2401-2404.
20.
Sigala, I.; Ganidis, G.; Thysiadis, S.; Zografos, A. L.; Giannakouros, T.; Sarli, V.; Nikolakaki, E.,
Lynamicin D an antimicrobial natural product affects splicing by inducing the expression of SR protein
kinase 1. Bioorg. Med. Chem. 2017, 25 (5), 1622-1629.
21.
Butyloxycarbonyl (Boc)-Protected Indoles. Asian J. Org. Chem. 2013, 2 (10), 877-881.
22. Okada, M.; Sugita, T.; Wong, C. P.; Wakimoto, T.; Abe, I., Identification of Pyridinium with Three
Indole Moieties as an Antimicrobial Agent. J. Nat. Prod. 2017, 80 (4), 1205-1209.
23. Taylor, N. J.; Emer, E.; Preshlock, S.; Schedler, M.; Tredwell, M.; Verhoog, S.; Mercier, J.;
Song, J.; Wang, Q.; Fu, H.; Fu, S.; Li, S.; Shi, F.; Wu, C., Copper-Catalyzed N-Alkynylation of N-tert-
Genicot, C.; Gouverneur, V., Derisking the Cu-Mediated 18F-Fluorination of Heterocyclic Positron Emission
Tomography Radioligands. J. Am. Chem. Soc. 2017, 139 (24), 8267-8276.
24.
Tasch, B. O. A.; Antovic, D.; Merkul, E.; Müller, T. J. J., One-Pot Synthesis of Camalexins and 3,3′-
Biindoles by the Masuda Borylation–Suzuki Arylation (MBSA) Sequence. Eur. J. Org. Chem. 2013, 2013 (21),
4564-4569.
25.
Stimson, D.; Qiao, Z.; C Reutens, D.; Venkatachalam, T.; Bhalla, R., Investigation on the impact of
three different QMA cartridges on the radiosynthetic yields of [18F]fluoromethyl tosylate. Journal of
Labelled Compounds and Radiopharmaceuticals 2019, 62.
26.
Giglio, B. C.; Fei, H.; Wang, M.; Wang, H.; He, L.; Feng, H.; Wu, Z.; Lu, H.; Li, Z., Synthesis of 5-
[(18)F]Fluoro-α-methyl Tryptophan: New Trp Based PET Agents. Theranostics 2017, 7 (6), 1524-1530.

Journal Pre-proof
27.
Huang, Q.; Zheng, M.; Yang, S.; Kuang, C.; Yu, C.; Yang, Q., Structure–activity relationship and
enzyme kinetic studies on 4-aryl-1H-1,2,3-triazoles as indoleamine 2,3-dioxygenase (IDO) inhibitors. Eur. J.
Med. Chem. 2011, 46 (11), 5680-5687.
28.
Gan, T.; Liu, R.; Yu, P.; Zhao, S.; Cook, J. M., Enantiospecific Synthesis of Optically Active 6-
Methoxytryptophan Derivatives and Total Synthesis of Tryprostatin A1. J. Org. Chem. 1997, 62 (26), 9298-
9304.
29.
Tredwell, M.; Preshlock, S. M.; Taylor, N. J.; Gruber, S.; Huiban, M.; Passchier, J.; Mercier, J.;
Génicot, C.; Gouverneur, V., A General Copper-Mediated Nucleophilic 18F Fluorination of Arenes.
Angewandte Chemie 2014, 126 (30), 7885-7889.
30.
for Diverse C–F Bond Functionalizations. J. Am. Chem. Soc. 2015, 137 (45), 14313-14318.
31. Taylor, N. J.; Emer, E.; Preshlock, S.; Schedler, M.; Tredwell, M.; Verhoog, S.; Mercier, J.;
Niwa, T.; Ochiai, H.; Watanabe, Y.; Hosoya, T., Ni/Cu-Catalyzed Defluoroborylation of Fluoroarenes
Genicot, C.; Gouverneur, V., Derisking the Cu-Mediated 18F-Fluorination of Heterocyclic Positron Emission
Tomography Radioligands. Journal of the American Chemical Society 2017, 139 (24), 8267-8276.
32.
Pharmacol Sci. 2009, 30 (8), 431-440.
33. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E., Relationship Between Lipophilicity and
Brain Extraction of C-11-Labeled Radiopharmaceuticals. J. Nucl. Med. 1983, 24 (11), 1030-1038.
34. Henrottin, J.; Lemaire, C.; Egrise, D.; Zervosen, A.; Van den Eynde, B.; Plenevaux, A.; Franci, X.;
Pike, V. W., PET radiotracers: crossing the blood-brain barrier and surviving metabolism. Trends
Goldman, S.; Luxen, A., Fully automated radiosynthesis of N1-[18F]fluoroethyl-tryptophan and study of its
biological activity as a new potential substrate for indoleamine 2,3-dioxygenase PET imaging. Nucl. Med.
Biol. 2016, 43 (6), 379-389.

O
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O
COOH
NH2
NH2
IDO/TDO
N
NH2
H
Tumour cell
L-kynurenine
Microenvironment
Suppression
O
OH
Increase Immune
Tolerance
NH2
N
H
L-tryptophan
Blocked in G1 phase
Teff cell
T cell
Dendritic cell or Breg cell
Figure 1.
Zheng Qiao et al

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Figure 2.
Zheng Qiao et al

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Scheme 1.
Zheng Qiao et al

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Scheme 2.
Zheng Qiao et al