[18F]-labeled positron emission tomography ligand for the histamine H4 receptor

Article abstract of DOI:10.1002/jlcr.3929

We synthesized 5-[¹⁸F]-fluoro-1H-indol-2-yl)(4-methyl-1-piperazinyl)methanone ([¹⁸F]5) via a Suzuki approach starting from a protected pinacol borane precursor followed by acidic hydrolysis of the t-Boc protecting group. The non-optimized radiochemical yield was 5.7 ± 1.35%, radiochemical purity was over 99%, and molar activity was 100.7 ± 34.5 GBq/μmol (n = 3). [¹⁸F]5 was stable in rat plasma for at least 4 h and was evaluated by μPET imaging and biodistribution using a unilateral quinolinic acid rat model of neuroinflammation. The time-activity curve showed that [¹⁸F]5 entered the brain immediately after intravenous injection and then left it progressively with a very low level reached from 30 min after injection. The biodistribution study showed no difference in the accumulation of [¹⁸F]5 between the lesioned and intact side of the brain and between control rats and animals pretreated with a saturating dose of JNJ-7777120 as a specific H4R antagonist. Hence, despite its in vitro nanomolar affinity for H4R, and its ability to cross the blood–brain barrier in rats, [¹⁸F]5 does not appear suitable to image in vivo the receptor by PET.

Full text of DOI:10.1002/jlcr.3929

Received: 19 May 2020
DOI: 10.1002/jlcr.3929
Revised: 18 May 2021
Accepted: 28 May 2021
R E S E A R C H A R T I C L E
[18F]-labeled positron emission tomography ligand for the
histamine H4 receptor
Agnieszka Zak1 | Lucas Lemaire1 | Sylvie Chalon2
| Gabrielle Chicheri2,3
|
Hamid Marzag1 | Sylvie Bodard2 | Sophie Sérrière2 | Sylvain Routier1
|
Frédéric Buron1
| Johnny Vercouillie2,3,4
1Institut de Chimie Organique et
Analytique, Université d'Orléans, UMR
CNRS 7311, Orléans, France
2UMR 1253, iBrain, Université de Tours,
Inserm, Tours, France
3CERRP, Université de Tours, Tours,
France
4INSERM CIC 1415, University Hospital,
Tours, France
We synthesized 5-[18F]-fluoro-1H-indol-2-yl)(4-methyl-1-piperazinyl)methanone
([18F]5) via a Suzuki approach starting from a protected pinacol borane pre-
cursor followed by acidic hydrolysis of the t-Boc protecting group. The non-
optimized radiochemical yield was 5.7 1.35%, radiochemical purity was
over 99%, and molar activity was 100.7 34.5 GBq/μmol (n = 3). [18F]5 was
stable in rat plasma for at least 4 h and was evaluated by μPET imaging and
biodistribution using
a
unilateral quinolinic acid rat model of
neuroinflammation. The time-activity curve showed that [18F]5 entered the
brain immediately after intravenous injection and then left it progressively
with a very low level reached from 30 min after injection. The biodistribution
study showed no difference in the accumulation of [18F]5 between the
lesioned and intact side of the brain and between control rats and animals
pretreated with a saturating dose of JNJ-7777120 as a specific H4R antagonist.
Hence, despite its in vitro nanomolar affinity for H4R, and its ability to cross
the blood–brain barrier in rats, [18F]5 does not appear suitable to image
in vivo the receptor by PET.
Correspondence
Johnny Vercouillie, UMR 1253, iBrain,
Université de Tours, Inserm, Tours,
France.
Email: vercouillie@univ-tours.fr
Funding information
Agence Nationale de la Recherche, Grant/
Award Number: Labex IRON,
ANR11-LABX-0018-01; FP7 Health,
Grant/Award Number: INMIND
K E Y W O R D S
18F PET, agonism, H4 receptor, indole, radiolabeling
1 | INTRODUCTION
expressed ubiquitously and have a role in inflammation
and allergy.1 Moreover, H3Rs are mainly localized on the
Histamine (2-[1H-imidazol-4-yl]ethanamine) is an endog-
enous short-acting biogenic amine implicated in various
physiological and pathophysiological processes. Besides
its peripheral functions, it plays an important role in the
central nervous system (CNS) as a neurotransmitter and
immunomodulator, with various functions in different
circuitries. Histamine exerts its effects primarily by bind-
ing to four G protein-coupled receptor (GPCR) subtypes:
H1R, H2R, H3R, and H4R.1 Based on results obtained in
the different histamine receptor subtypes of knockout
mice models, it appeared that both H1R and H2R are
histaminergic neuron endings. These receptors are able
to affect behavior.2 Concerning the later discovered sub-
type H4, its precise localization and role are still poorly
elucidated.2,3 However, data showed that H4R has a
strong implication in the regulation of in vivo microglial
activation by histamine.4 In addition, the H4R antagonist
and partial agonist5 JNJ-7777120 were recently shown to
inhibit the pro-inflammatory M1 phenotype of microglia
in a lesion-induced rat model of Parkinson's disease (PD),
with beneficial effects on the survival and functioning of
dopaminergic neurons.6
J Label Compd Radiopharm. 2021;1–10.
wileyonlinelibrary.com/journal/jlcr
© 2021 John Wiley & Sons, Ltd.
1

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ZAK ET AL.
There is increasing evidence that inflammation plays
For this aim, we focused on a non-imidazole selective
H4R antagonist series from which the JNJ-7777120
emerged (Figure 2). JNJ-7777120 was described to bind to
the human H4R with high affinity (4.1 nM) and showed
a greater than 1000-fold selectivity over the other
histamine receptors.13,14 Interestingly the structure–
activity relationship (SAR) described the fluorinated
derivative 5 of JNJ-7777120, which showed a rather good
inhibitory constant of 15 nM.14 We therefore decided
to develop a radiolabeling procedure and prepared the
[18F]5 (Scheme 1), to evaluate its in vivo properties
a crucial role in PD as in other neurodegenerative dis-
eases, in particular through microglia activation.7 In this
context, the imaging of neurionflammation by positron
emission tomography (PET) is now a highly relevant tool
in a number of brain disorders, and the main molecular
target explored to date for this aim is the 18-kDa
translocator protein (TSPO) overexpressed in activated
microglia.8 However, in view of several limitations with
brain TSPO PET exploration, alternative and/or comple-
mentary molecular targets are currently being actively
searched for.9 Among these potential new targets, H4R
appears as a promising candidate.
in
a
well-established rat model of excitotoxic
neuroinflammation.15,16
To date, two carbon-11-labeled analogs (Figure 1) of
the highly potent and subtype-selective H4R antagonists
VUF10558 and JNJ-7777120 have been developed and
investigated in vivo.10,11 It appears that [11C]VUF10558
cannot cross the blood–brain barrier unlike the [11C]JNJ-
7777120; nevertheless, no relevant literature reporting
the usefulness of [11C]JNJ-7777120 has been published
since its radiolabeling few years ago.12 Moreover, the use
of radiopharmaceuticals labeled with 11C is limited by
the short physical half-life of carbon-11 (20 min) and thus
restricted to PET imaging departments closely linked to a
cyclotron.
This constraint is avoided with the fluorine-18 radio-
nuclide due to its relatively long half-life (110 min) and
the possibility of generating fluorine-18 radiotracers,
which can be shipped off-site. These main advantages led
us to develop a fluorine-18 probe for PET imaging of H4R
as a neuroinflammation target in order to improve the
prospects for the understanding, early diagnosis, and
treatment of brain diseases.11
2 | EXPERIMENTAL
2.1 | General
1H NMR and 13C NMR spectra were recorded on a
Bruker DPX 250- or 400-MHz instrument using CDCl3
or DMSO-d6. The chemical shifts are reported in parts
per million (δ scale), and all coupling constant (J) values
are in Hertz (Hz). The following abbreviations were used
to explain the multiplicities: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and dd (doublet of
doublets). Melting points are uncorrected. IR absorption
spectra were obtained on a PerkinElmer Paragon 1000
PC, and values are reported in cmÀ1. HRMS was
recorded on
a Bruker maXis mass spectrometer.
Monitoring of the reactions was performed using silica
gel TLC plates (silica Merck 60 F254). Spots were
visualized by UV light at 254 and 356 nm. Column
FIGURE 1 Tested carbon-11 H4R
radiotracers
FIGURE 2 Structures of the 5-fluoro-1H-
indol-2-yl)(4-methyl-1-piperazinyl)methanone 5,
[18F]5, derived from the JNJ-7777120

ZAK ET AL.
3
SCHEME 1 Synthesis of the protected pinacol borane precursor 4, the reference compound 5, and the radiolabeling of [18F]5. (A) Boc2O
(1.1 eq.), 4-DMAP (0.1 eq.), Et3N (1.1 eq.), THF, r.t., 17 h, 99%. (B) B2pin2 (1.2 eq.), KOAc (0.3 eq.), Pd(dppf)Cl2ÁDCM (0.04 eq.), 1,4-dioxane,
100ꢀC, 17 h, 60%. (C) Boc2O (1.1 eq.), 4-DMAP (0.1 eq.), Et3N (1.1 eq.), THF, r.t., 12 h, 99%. (D) NaOH (10.8 eq.), EtOH, r.t., 48 h, then HCl
1 N, 90%. (E) SOCl2 (6.7 eq.), 1-methylpiperazine (3.1 eq.), DCM, r.t., 1 h 30 min, 91%. (F) [18F], K2.2.2., DMF, pyridine, Cu(OTf)2, 120ꢀC,
10 min. (G) Aqueous 3-N HCl solution, 100ꢀC, 5 min
chromatographies were performed using silica gel
60 (0.063–0.200 mm, Merck). [18F]Fluoride was pro-
duced using a PETtrace 10 cyclotron (GE Healthcare)
via the classical 18O(p,n)18F reaction by bombardment of
18O-enriched (98%) water with 16.5-MeV protons. Auto-
mated radiosyntheses for animal studies were performed
in a sparsely modified TRACERlab Fx-FN Pro module
(GE Medical Systems) including a semi-preparative
HPLC equipped with UV and radiodetectors. The minor
change made to the synthesizer consisted in connecting
valve VZ1 to a waste vial and not to the second reactor.
A ZORBAX Eclipse XDB-C18 column (9.4 Â 250 mm
5 μm, Agilent) was used for semi-preparative purification
with a wavelength at 254 nm for UV detection. HPLC
quality controls were performed on an Ultimate 3000
system equipped with a UV detector and a radioactivity
detector (PET metabolite, Bioscan). The analytical HPLC
analyses were performed on an EVO column
(150 Â 4.6 mm, 2.6 μm) at 291-nm wavelength and
1-ml/min flow.

4
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2.2 | Chemistry
2.24 (s, 3H, N─CH3), 1.32 (s, 12H, 4xCH3); 13C NMR
80ꢀC (63 MHz, DMSO-d6) δ 161.7 (C═O), 137.6 (Cq),
130.1 (CHAr), 128.8 (CHAr), 128.3 (Cq), 126.4 (Cq), 111.1
(CHAr), 103.9 (CHAr), 83.8 (2xCq), 54.3 (2xCH2), 45.2
(N─CH3), 44.1 (2xCH2), 24.4 (4xCH3); HRMS (EI-MS):
calcd for C20H29BN3O3 m/z 370.2297 [M + H]+, found
370.2301.
2.2.1 | tert-Butyl 5-bromo-
2-(4-methylpiperazine-1-carbonyl)-1H-indole-
1-carboxylate (2)
The solution of (5-bromo-1H-indol-2-yl)(4-methyl-
piperazin-1-yl)methanone (0.220 g, 0.70 mmol) in anhy-
drous THF (5 ml) was cooled down to 0ꢀC under argon
atmosphere. Et3N (0.11 ml, 0.77 mmol, 1.1 eq.) and
4-DMAP (0.008 g, 0.07 mmol, 0.1 eq.) were added to the
reaction mixture followed by Boc2O (0.168 g, 0.77 mmol,
1.1 eq.). The reaction was allowed to warm up to r.t. and
continued for 15 h. The mixture was then concentrated
under vacuum, and the residue was purified by flash
chromatography using DCM:MeOH (95:5) to afford the
pure product as a pale brown oil (0.290 g, 99%). Rf = 0.28
(DCM:MeOH 95:5); IR (ATR diamond, cmÀ1) ν 2978,
1739, 1635, 1478, 1434, 1350, 1336, 1250, 1234, 1201,
1141, 1093, 1075, 853; 1H NMR (250 MHz, CDCl3) δ
8.11–8.00 (m, 1H, HAr), 7.96 (s, 1H, HAr), 7.70 (dd,
J = 8.4, 1.1 Hz, 1H, HAr), 6.52 (s, 1H, HAr), 3.79–3.61
(m, 2H, CH2), 3.38–3.23 (m, 2H, CH2), 2.49–2.34 (m, 2H,
CH2), 2.33–2.14 (m, 5H, CH2 and N─CH3), 1.26 (s, 9H,
3xCH3); 13C NMR (63 MHz, CDCl3) δ 163.0 (C═O), 148.7
(C═O), 137.7 (Cq), 132.3 (Cq), 131.5 (CHAr), 128.4 (CHAr),
128.0 (Cq), 114.6 (CHAr), 108.4 (CHAr), 84.7 (Cq), 54.4
(CH2), 54.1 (CH2), 53.3 (Cq), 46.6 (CH2), 45.8 (N─CH3),
41.5 (CH2), 24.7 (3xCH3); HRMS (EI-MS): calcd for
C19H25BrN3O3 m/z 421.1001 [M + H]+, found 421.1008.
2.2.3 | tert-Butyl 2-(4-methylpiperazine-
1-carbonyl)-5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-indole-
1-carboxylate (4)
The solution of 3 (0.104 g, 0.3 mmol) in anhydrous THF
(5 ml) was cooled down to 0ꢀC under argon atmosphere.
Et3N (0.08 ml, 0.6 mmol, 1.1 eq.) and 4-DMAP (0.004 g,
0.03 mmol, 0.1 eq.) were added to the reaction mixture
followed by Boc2O (0.072 g, 0.33 mmol, 1.1 eq.). The reac-
tion was allowed to warm up to r.t. and continued for
12 h. The mixture was then concentrated under vacuum,
and the residue was purified by flash chromatography
using DCM:MeOH (95:5) to afford the pure product as a
yellow oil (0.075 g, 59%). Rf = 0.51 (DCM:MeOH 95:5);
IR (ATR diamond, cmÀ1) ν 2978, 1738, 1644, 1434, 1350,
1336, 1292, 1251, 1233, 1162, 1141, 1092, 1074, 1001,
853, 729; 1H NMR (400 MHz, CDCl3)
δ
8.13
(d, J = 8.4 Hz, 1H), 8.03 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H),
6.60 (s, 1H), 3.78 (t, J = 9.1 Hz, 2H, CH2), 3.41–3.34
(m, 2H, CH2), 2.49 (t, J = 4.9 Hz, 2H, CH2), 2.36–2.31
(m, 5H), 1.61 (s, 9H, 3xCH3), 1.35 (s, 12H, 4xCH3); 13C
NMR (63 MHz, CDCl3) δ 163.3 (C═O), 149.0 (C═O),
138.0 (Cq), 132.7 (Cq), 131.7 (CHAr), 128.7 (CHAr), 128.3
(Cq), 114.9 (CHAr), 108.7 (CHAr), 85.0 (Cq), 83.9 (2xCq),
54.8 (CH2), 54.5 (CH2), 46.9 (CH2), 46.6 (N─CH3), 41.8
(CH2), 28.2 (3xCH3), 25.0 (4xCH3); HRMS (EI-MS): calcd
for C25H37BN3O5 m/z 470.2821 [M + H]+, found
470.2823.
2.2.2 | (4-Methylpiperazin-1-yl)
(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-indol-2-yl)methanone (3)
The solution of 2 (0.2 g, 0.47 mmol), (Bpin)2 (0.142 g,
0.56 mmol, 1.2 eq.), and KOAc (0.138 g, 1.41 mmol,
3.0 eq.) in 1,4-dioxane (3 ml) was flushed with argon for
15 min. Pd(dppf)Cl2ÁDCM (0.015 g, 0.02 mmol, 8.0 mol%)
was added to the solution, and the reaction mixture was
sealed in a tube and heated up to 130ꢀC for 17 h. The
resulting solution was filtered through a pad of Celite®
and concentrated under reduced pressure. The crude
product was purified by flash chromatography using
DCM:MeOH (95:5) as an eluent resulting in the desired
product as a beige solid (0.101 g, 60%). Rf = 0.33 (DCM:
MeOH 95:5); mp 220–222ꢀC; IR (ATR diamond, cmÀ1) ν
3265, 2786, 1595, 1572, 1478, 1442, 1380, 1355, 1327,
2.2.4 | (5-Fluoro-1H-indol-2-yl)(4-methyl-
1-piperazinyl)methanone (5)
The commercially available compound
6 (0.607 g,
2.93 mmol), NaOH (1.27 g, 31.78 mmol), and ethanol
(20 ml) were mixed at r.t. for 48 h. The mixture was then
concentrated under vacuum, and the residue was
adjusted to pH 1 with HCl 1 N. EtOAc (20 ml)
was added, and the organic layer was extracted, washed
with water (10 ml), dried over MgSO4, filtered, and
concentrated under vacuum. The crude mixture was dis-
solved in DCM (20 ml), and SOCl2 (1.28 ml, 3.1 eq.) was
slowly added. The mixture was stirred at r.t. for 1 h, and
1
1287, 1254, 1205, 1141, 750; H NMR (250 MHz, DMSO-
d6) δ 11.48 (s, 1H, NH), 8.02 (s, 1H, HAr), 7.50
(d, J = 8.3 Hz, 1H, HAr), 7.42 (d, J = 8.3 Hz, 1H, HAr),
6.79 (s, 1H, HAr), 3.74 (t, 4H, 2xCH2), 2.39 (t, 4H, 2xCH2),

ZAK ET AL.
5
the amine (0.87 ml, 3.1 eq.) was added. The solution was
stirred at r.t. for 1 h 30 min and then concentrated under
vacuum. EtOAc (20 ml) was added, and the solution was
adjusted to pH 9 with NaOH 1 N. The organic phase
was extracted, washed with water (10 ml), dried over
MgSO4, filtered, and concentrated under vacuum to
afford the pure product as a yellow solid (0.627 g, 82%).
1H NMR (250 MHz, DMSO-d6): δ 11.71 (s, NH), 7.39
(m, 2H, HAr), 7.05 (m, 1H, HAr), 6.80 (m, 1H, HAr), 3.93–3.74
(br, 4H, 2xCH2), 2.68 (m, 4H, 2xCH2), 2.42 (s, 3H, CH3).
The physical data are in accordance with the literature.14
modified TRACERlab FX-FN Pro (GE) synthesizer and
passed through an anion-exchange resin (Waters,
Sep-Pak Accell Light QMA cartridge in the carbonate
form). Using a boronic ester to afford the nucleophilic
aromatic substitution, the trapped [18F]fluoride was
released from the cartridge with the Mossine et al.19
eluent solution consisting of 550 μl of aqueous solution
containing 5 mg of potassium trifluoromethanesulfonate
and 50 μg of potassium in water. Azeotropic distillation
was performed twice using each time 1 ml of acetonitrile
at 100ꢀC under He flow and vacuum. To the anhydrous
[18F]KF solution, precursor
4
(2.0 mg), Cu(OTf)2
(3.5 mg), and pyridine (40 μl) were added and dissolved
in DMF (960 μl). Then, the mixture was heated at 120ꢀC
for 10 min and cooled to 100ꢀC to perform hydrolysis
with an aqueous 3-N HCl solution (1 ml) for 5 min. The
solution was cooled to 40ꢀC and neutralized with an
aqueous 0.5-M NaOH solution (7 ml). The crude solution
was passed through a t-C18 Sep-Pak Plus cartridge
(Waters). The reactor and cartridge were rinsed with
water (5 ml), and the crude compound was eluted from
the cartridge with acetonitrile (1 ml). Prior to HPLC puri-
fication, the crude solution was diluted with 2 ml of the
mobile phase (acetonitrile/(ammonium acetate, 0.1 M
+ 0.2% Et3N) 25/75. Purification occurred on a ZORBAX
Eclipse XDB-C18 (9.4 Â 250 mm, 5 μm, Agilent) at a
4 ml/min flow (Figure 3). The collected peak was then
diluted in water (20 ml), and the solution was trapped on
2.3 | Radiochemistry
2.3.1 | 5-[18F]-Fluoro-1H-indol-2-yl)
(4-methyl-1-piperazinyl)methanone (5)
Prior to the automation on the TRACERlab FX-FN Pro
(GE) synthesizer, attempts in warm conditions were
performed to optimize temperature and reaction time
reaction based on a similar previous approach.17 The
optimized conditions defined during this development
phase were applied for the production on the synthesizer.
As previously reported,18 [18F]fluoride ion was produced
on a cyclotron (PET trace, GE Healthcare) by irradiation
of enriched 18O H2O with protons via the 18O(p,n)18F
nuclear reaction. [18F]fluoride was transferred to a
FIGURE 3 Semi-preparative purification of [18F]5 on a ZORBAX Eclipse XDB-C18 (5 μm, 9.4 Â 250 mm) with a 4-ml/min flow using
acetonitrile/(ammonium acetate 0.1 M + 0.2% Et3N): 25/75 as mobile phase. Top, radioactivity signal; bottom, UV signal

6
ZAK ET AL.
a tC18 cartridge (Waters Sep-Pak Accell Light tC18
cartridge). The cartridge was rinsed with water (5 ml),
and [18F]5 was eluted from the tC18 with EtOH (0.5 ml).
The formulation was completed by adding 0.9% NaCl
solution (4.5 ml).
500 μl of acetonitrile were added, the organic layer
was removed and filtered, 20 μl was loaded onto the
HPLC, and the stability was checked at 4-h time point
after production.
2.5 | Biological studies
2.4 | Quality control and stability
2.5.1 | In vitro experiments
The chemical and radiochemical purity of the final
product for clinical use was determined by HPLC anal-
ysis using a Kinetex EVO column (4.6 Â 150 mm,
2.6 μm, Phenomenex, USA) and eluted with acetoni-
trile/(ammonium acetate, 0.1 M + 0.2% Et3N) 30/70 at
a flow rate of 1.0 ml/min (Figure 4). The stability of
the injectable solution was checked by HPLC up to
4 h after the end of preparation. To operate this con-
trol, 100 μl of the radiotracer solution was mixed with
1 ml of rat plasma and incubated at 37ꢀC. Then,
The affinity of 5 towards H4R was determined by CEREP
(Le Bois l'Eveque, 86600 Celle-Lévescault, France)
according to their standard assay protocols, using HEK-
293 cells expressing the human recombinant H4R and
[3H]histamine as a reference tracer (see http://www.
cerep.fr/cerep/users/pages/catalog/search/catalog.asp).
For the biological experiments, only one production
of [18F]5 was used with
132.64 GBq/μmol.
a
molar activity of
FIGURE 4 Analytical HPLC chromatogram of the final product [18F]5 after HPLC purification. Chromatograms were obtained on a
Kinetex EVO C18 (2.6 μm, 4.6 Â 150 mm). HPLC was operated in isocratic mode at 1-ml/min flow rate using acetonitrile/(ammonium
acetate 0.1 M + 0.2% Et3N): 30/70 as mobile phase

ZAK ET AL.
7
2.5.2 | In vivo experiments
using the unprotected indole, but assays of boronylation
of 1 failed. We therefore protected the indolic nitrogen
atom by a t-Boc group in a nearly quantitative manner,
and derivative 2 was subjected to the expected palladium
catalyzed boronylation. Optimized conditions involved
the use of bis(pinacolo)diborane, Pd(dppf)Cl2ÁDCM, as
catalyst in the presence of KOAc as base in dioxane at
100ꢀC for 17 h. However, despite efforts, the t-Boc group
did not survive the high temperature reaction and long
reaction time. Purification led directly to the pure dep-
rotected derivative 3 in 60% yield. A further protection of
the indolic nitrogen atom was performed using a slight
excess of Boc2O in basic media to afford 4 in a satisfying
yield. The reference compound 5 was obtained from the
commercially ethyl 5-fluoroindole-2-carboxylate 6 in two
steps. The first one consisted in a saponification with
NaOH, to afford the corresponding carboxylic acid. Then,
the crude carboxylic acid is transformed into its
corresponding acyl chloride via thionyl chloride followed
by amidation with N-methylpiperazine. Compound 5 was
obtained in 82% overall yield.
A total of 14 male adult Wistar rats were included (CERJ,
Le Genest-Saint-Isle, France). Animals were bred, and
experiments were conducted according to the European
Community Council Directive 2010/63/EU for
laboratory animal care and French rules (Authorization
No. 2015022011523044). All rats were subjected to a
stereotaxic injection of quinolinic acid (150 nmol;
Sigma-Aldrich, Lyon, France) in the right striatum using
a previously described procedure.20 At 7 days after lesion,
they were randomly divided into two groups of six ani-
mals, that is, the control and JNJ groups. The latter
received an intravenous injection of the H4R-specific
antagonist JNJ-7777120 (3 mg/kg; Tocris, R&D Systems
Europe, Lille, France) 15 min before the tracer injection.
Animals of both groups were then intravenously injected
with [18F]5 (2–5 MBq in 0.15 ml of saline) and sacrificed
1 h after injection. The blood, brain regions, and periph-
eral tissue samples were then removed and weighed, and
their radioactivity was measured using a γ-counter (2480
Gamma counter Wizard, PerkinElmer, France). The
percentage of injected dose per gram of tissue (%ID/g)
was calculated by comparison with samples of the
injected solution.
3.2 | Radiochemistry
To radiolabel compound 5 with [18F]fluoride, we adopted
a two-step procedure, that is, fluorination from a pinacol
borane entity followed by acidic deprotection of the
indole (Scheme 1). As previously reported19,21 and based
on previous experiments,17 we achieved fluorination
using an eluent solution containing the fluorine-18 from
QMA and KOTf and K2CO3 in CH3CN/water. To this
medium was added a solution containing the precursor,
Cu(OTf)2, a low amount of pyridine and the whole
dissolved in DMF. The condition of the first step was
optimized by a warm approach as already performed by
our team for a COX-2 tracer.17 The first experiments
conducted at 100ꢀC for 5 min did not enable the incorpo-
ration of fluorine-18 onto precursor 4. A longer reaction
time of 20 min resulted in 16.5% incorporation. To
increase the radiochemical yield, we also increased the
reaction temperature up to 130ꢀC. Incorporation yields at
120ꢀC and 130ꢀC gave similar results of 28% and 30%,
respectively. Nevertheless, at 130ꢀC, we observed that an
additional radioactive peak appeared and moreover that
a prolonged reaction time resulted in a decrease in
incorporation yield, an increase of the side-product
radioactive peak, and a degradation of the precursor.
Then, the optimized conditions were applied on the
synthesizer as follow with a fluorination step performed
at 120ꢀC during 10 min immediately followed by the
hydrolysis at 100ꢀC for 5 min using a 3-N HCl aqueous
solution. The non-optimized production afforded [18F]5
2.5.3 | μPET imaging
In parallel, two rats were intravenously injected with
[18F]5 (47 MBq), and their brains were scanned with a
μPET/CT system (SuperArgus, Sedecal, Madrid, Spain)
during 120 min under continuous gas anesthesia
(isoflurane, Baxter, France), according to a previously
described procedure.18 The time-activity curve was
expressed as the average of % injected dose/g measured
in the whole brain of both animals.
3 | RESULTS AND DISCUSSION
3.1 | Chemistry
The synthesis of precursor 4 for the radiosynthesis and
the reference compound 5 is depicted in Scheme 1. The
synthesis of bromo derivative 1 was achieved in high
yield according to the reported amidification procedure,
which involved 5-bromo-1H-indole-2-carboxylic acid
and N-methyl-piperazine in the presence of
carbonyldiimidazole. As the purpose was to determine
the potency of the tracer candidate to target the H4R, and
the radiochemical (RCY) was not the primary objective,
we initially planned a one-step radiosynthesis strategy

8
ZAK ET AL.
in a 5.7 1.35% radiochemical yield, in 78 min with a
molar activity of 100.7 34.5 GBq/μmol (n = 3). In com-
parison to results obtained with the COX-2 tracer,17 we
observed that the preparation of [18F]5 resulted in much
lower yields in warm conditions (30% vs. 92%) as with
the synthesizer (5.7% vs. 49%). The differences in RCY
could be due to a hydrolysis of the Boc group by [18F]
fluoride ions making the nucleophilic substitution less
conducive and entering directly in competition with the
desired reaction.22 The radiochemical stability was
checked up to 4 h after production without observing
degradation of the radiotracer, and radiochemical purity
remained greater than 99%. The stability of [18F]5 in rat
plasma at 37ꢀC was checked up to 4 h, and no degrada-
tion of the radiotracer was observed.
These literature data also highlighted that these affinities
were quite similar between human and rodent H4 receptors,
although species differences in these receptors have been
described.24,25 In addition, although contradictory data have
been found, an extensive analysis of them indicated that the
presence of H4R on neurons is unlikely, whereas its specific
expression by microglia is recognized.3,26,27 Indeed, if the
presence of H4 mRNA is recognized, the expression of
protein is still questionable due to the lack of specificity of
the available antibodies.28 In this context, we evaluated the
in vivo binding of our new tracer in a well-known rat model
of microglial activation.15
As illustrated, we observed that [18F]5 entered the rat
brain just after intravenous injection, with a peak around
5 min after injection and then a progressive decrease
followed by a low level from 30 min after injection. As
the recorded activity was homogeneous across the whole
brain, it is represented as a single curve (Figure 5).
As reported in Table 1, the uptake of [18F]5 1 h after
injection was homogeneous in different regions and was
similar in both sides of the brain, despite the microglial
activation we induced in the right side.15 In addition, the
preinjection of a saturating dose of the H4R antagonist
JNJ-7777120 did not induce any modification in the
tracer accumulation, both in central and in peripheral
tissues (Tables 1 and 2).
3.3 | Biological studies
We observed that compound 5 displayed a Ki of 1.5 nM in
the condition tested by the CEREP for human H4R. This
affinity was in agreement with the previously published
data14 and in the same range as the affinity obtained for
the first described H4R antagonist, that is, JNJ-7777120,
which is the chlorinated analog of 5,13 and also for
A-987306, which has a different chemical structure.23
FIGURE 5 Time-activity curve of [18F]5 in the rat whole brain. Mean SEM; n = 2

ZAK ET AL.
9
TABLE 1 Rat brain biodistribution of [18F]5
Right Cx
Left Cx
Right Stri
0.15 0.02
0.18 0.04
Left Stri
Right Hippoc
0.16 0.02
Left Hippoc
0.16 0.02
0.18 0.02
Cereb
Control group
JNJ group
0.10 0.01
0.13 0.02
0.10 0.01
0.13 0.02
0.14 0.02
0.17 0.02
0.12 0.01
0.14 0.03
0.18 0.03
Note: Results are expressed as mean % of injected dose/g tissue SD; n = 6 per group. All animals received an injection of QA in the right striatum 7 days
before the biodistribution experiment. Rats from the JNJ group received an intravenous injection of JNJ-7777120 (3 mg/kg) 15 min before the tracer injection.
All animals were sacrificed 1 h after [18F]5 injection.
Abbreviations: Cereb, cerebellum; Cx, cortex; Hippoc, hippocampus; Stri, striatum.
TABLE 2 Rat biodistribution of [18F]5 in peripheral tissue
Bl
He
Lu
Liv
Sp
Kid
Int
Mus
Bo
Control group
JNJ group
0.11 0.01 0.10 0.02 0.31 0.06 0.96 0.14 0.15 0.01 0.73 0.13 0.10 0.07 0.07 0.01 0.10 0.01
0.11 0.02 0.11 0.02 0.35 0.15 0.89 0.24 0.20 0.03 0.67 0.13 0.12 0.02 0.09 0.04 0.11 0.02
Note: Results are expressed as mean of % injected dose/g tissue SD; n = 6 per group. Rats from the JNJ group received an intravenous injection of JNJ-
7777120 (3 mg/kg) 15 min before the tracer injection. All animals were sacrificed 1 h after [18F]5 injection.
Abbreviations: Bl, blood; Bo, bone; He, heart; Int, intestine; Kid, kidney; Liv, liver; Lu, lung; Mus, muscle; Sp, spleen.
To our knowledge, no useful PET tracer is to date
available for H4R.11 The present study shows that,
despite a nanomolar affinity for the target and appropri-
ate molar activity, the fluorinated derivative of JNJ-
7777120, that is, [18F]5, did not colocalize with the brain
activated microglia expressed in the rat model of acute
neuroinflammation that we used. This could be linked to
an in vivo nonspecific binding of the tracer to H4R
and/or to a low expression of this receptor in this model.
In any case, this highlights the need to develop new
chemical structures as ligands for this promising target.
ORCID
Sylvie Chalon https://orcid.org/0000-0003-1865-8380
Sylvain Routier https://orcid.org/0000-0002-2823-0591
Frédéric Buron https://orcid.org/0000-0001-5773-1515
Johnny Vercouillie https://orcid.org/0000-0002-7474-
7778
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We successfully prepared the fluorinated derivative of
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