Syntheses, radiolabelings, and in vitro evaluations of fluorinated pet radioligands of 5-HT6 serotoninergic receptors

Article abstract of DOI:10.1021/jm500372e

The 5-HT₆ receptors are potent therapeutic targets for psychiatric and neurological diseases (schizophrenia, Alzheimers disease, etc.). However, with lack of specific radiopharmaceuticals, their pharmacology is still incomplete and their exploration is limited to animal models. In this context, we have designed a fluorinated PET radiotracer, [¹⁸F]2FNQ1P, that possesses a high affinity and selectivity for 5-HT₆. In vitro PET autoradiographies in rat brain sections with this radiotracer were in accordance with the 5-HT₆ distribution pattern.

Full text of DOI:10.1021/jm500372e

Brief Article
pubs.acs.org/jmc
Syntheses, Radiolabelings, and in Vitro Evaluations of Fluorinated
PET Radioligands of 5‑HT6 Serotoninergic Receptors
Julie Colomb,†,§ Guillaume Becker,‡,§,∥ Sylvain Fieux,‡,§ Luc Zimmer,‡,§,∥ and Thierry Billard*,†,§
†Institute of Chemistry and Biochemistry (ICBMS-UMR CNRS 5246), University of Lyon, University Lyon 1, CNRS, 43 Boulevard
du 11 Novembre 1918, 69622 Lyon, France
‡Lyon Neuroscience Research Center (UMR 5292, U 1028), University of Lyon, University Lyon 1, CNRS, INSERM, 59 Boulevard
Pinel, 69003 Lyon, France
§CERMEP-In Vivo Imaging, Groupement Hospitalier Est, 59 Boulevard Pinel, 69003 Lyon, France
∥Hospices Civils de Lyon, Neurological Hospital, 59 Boulevard Pinel, 69003 Lyon, France
S
* Supporting Information
ABSTRACT: The 5-HT6 receptors are potent therapeutic targets for psychiatric
and neurological diseases (schizophrenia, Alzheimer’s disease, etc.). However, with
lack of specific radiopharmaceuticals, their pharmacology is still incomplete and
their exploration is limited to animal models. In this context, we have designed a
fluorinated PET radiotracer, [18F]2FNQ1P, that possesses a high affinity and
selectivity for 5-HT6. In vitro PET autoradiographies in rat brain sections with this
radiotracer were in accordance with the 5-HT6 distribution pattern.
of the preclinical and clinical pharmacology10 and of directed
INTRODUCTION
■
brain drug candidates11 and to assess directly the involvement
The 5-HT6 receptor is one of the most recently discovered of
of serotonin 5-HT6 receptors in neuropsychiatric diseases and
the serotonin family of receptors. It was discovered by two
possible therapies. This requires a PET radiotracer labeling 5-
independent groups using molecular cloning technologies and
HT6 receptors with high affinity, high selectivity, high signal-to-
was first isolated from rat striatum.1,2 Three years later, the
noise ratio, lipophilicity sufficient to penetrate the blood−brain
barrier, relatively slow clearance, and a low level of labeled
metabolites in the brain.12 Although several selective 5-HT6
antagonists exist,7,13−17 few have been radiolabeled for
successful use in PET imaging (Figure 1).
human homologue was discovered by Kohen et al.3 It is known
that 5-HT6 receptor density is particularly high in striatal areas
in rats and humans. The first autoradiographic visualizations of
5-HT6 receptor distribution used the 5-HT6 receptor
antagonists [3H]4-amino-N-[2,6-bis(methylamino)pyridin-4-
yl]benzenesulfonamide ([3H]Ro-63-0563) and [125I]30, in
various species.4,5 These studies consistently revealed hetero-
geneous binding throughout the brain, with the high levels in
striatum, moderate levels in cerebral cortex, and low levels in
cerebellum. Cross-species comparison showed similar distribu-
tion patterns between rats and non-human primates and
humans.6 At that stage, however, the role of the 5-HT6 receptor
in the brain was not yet clearly understood. Its specific brain
localization, particularly in the basal ganglia and limbic regions,
and the high affinity shown by several atypical antipsychotics
suggested involvement in the serotonergic control of motor
function, mood-dependent behavior, and related diseases.
Recent studies identified 5-HT6 receptors as promising targets
for cognitive improvement in psychiatric or neurodegenerative
diseases and for antiobesity drugs.7,8
In 2005, a pharmaceutical company patented a new
generation of 5-HT6 receptor antagonists based on the 3-
benzenesulfonyl-8-piperazine-1-ylquinoline scaffold.18 Within
this scaffold, N-methyl derivative 1 presented high binding
affinity for 5-HT6 (Ki = 0.16 nM). [11C]1 was then prepared by
N-methylation of the corresponding desmethyl precursor with
[11C]MeOTf.19 This radioligand readily entered the brain of
anesthetized pigs, with a distribution consistent with reported
5-HT6 receptor density patterns.19,20 Selectivity, however, was
by no means optimal, since [11C]1 also showed non-negligible
affinity toward 5-HT2A receptors (Ki = 0.79 nM), a different
serotoninergic receptor known to be colocalized with 5-HT6
receptors, particularly in the striatum.21 Nevertheless and
because of the lack of other 5-HT6 PET radiotracers, evaluation
was pursued in pigs, non-human primates, and finally in
humans.22 More recently, another compound, 2, was radio-
In vitro imaging (e.g., autoradiography) allows post-mortem
visualization and accurate delineation of receptors in animal
models and humans,9 but in vivo imaging (e.g., positron
emission tomography (PET)) is essential to the development
Received: March 10, 2014
Published: April 22, 2014
© 2014 American Chemical Society
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Brief Article
placed in the ortho or para position of an electron-withdrawing
group to favor such a reaction.
The first envisaged modification concerned only the position
of the fluorine atom in ortho or para position on the
benzenesulfonyl core of 1 to determine a possible “fluorine
position” effect (6, 7, Scheme 1). These molecules have been
previously described in literature; however, their 5-HT6
activities have not been described.29 Some replacements of
the sulfonyl hydrogen acceptor part by bioisosteres, such as the
sulfoxide (8) and sulfonamide (9, 10) groups, were also
considered.30 Modifications to the piperazine substituent were
also envisaged, in accordance with the pharmacophore.
Consequently, the fluorobenzenesulfonyl group, which can be
radiolabeled, could substitute for the methyl substituent (11,
12). A combination of these modifications was also designed,
modifying sulfonamide and the substituent (13, 14). Finally,
the moving of the fluorobenzenesulfonyl part from the
quinoline position 3 to the position 5 was also envisaged (15).
From a retrosynthetic point of view, 6−14 can be obtained
via two coupling reactions from 3-iodo-8-chloroquinoline 17,
easily obtained from the commercially available 8-chloroquino-
line 16, which then constitutes the common starting material
(Scheme 1).
Figure 1. Published 5-HT6 receptor radioligands.
The 8-chloroquinoline 16 was regioselectively iodinated to
furnish 17 with good yield.18 Despite various attempts at direct
sulfonylation of 17,31−34 no efficient methods proved effective,
leading sometimes to desulfitative coupling.35 Consequently, a
two-step strategy was adopted: the corresponding fluoroben-
zene thiols were first introduced by chemoselective copper-
catalyzed coupling reaction,36 and the resulting sulfides (18)
were then oxidized with MMPP (magnesium monoperoxyph-
thalate) into the expected sulfones (19). Buchwald previously
demonstrated that the biphenylphosphine DavePhos facilitated
the coupling reaction of chlorinated aromatic rings with various
amino groups, such as piperazine.37 In the case of 19,
satisfactory yields were obtained with N-methylpiperazine to
afford the expected ligands 6 and 7. A similar synthetic pathway
was applied to obtain 8. More moderate oxidation conditions
(mCPBA) were used to stop at the sulfoxide 20, which was
then coupled to N-methylpiperazine. In this last coupling
reaction, XPhos is preferable to DavePhos to obtain the
expected product 8 in moderate yield (Scheme 1).38
The corresponding sulfonamides 9 and 10 were prepared by
two successive palladium-catalyzed coupling reactions. Sulfo-
namides 21 were obtained by the chemoselective reaction of 17
with the corresponding fluorobenzenesulfonamide in good
yields. A second coupling reaction with N-methylpiperazine,
under conditions similar to those described above, led to the
expected ligands 9 and 10 with satisfactory yields (Scheme 1).
Similar strategies were applied to the synthesis of 11−14
(Scheme 1). After a first coupling between 17 and
benzenesulfinate (Cu-catalyzed) or benzenesulfonamide (Pd-
catalyzed), 22 and 24 were respectively obtained with
satisfactory yields. Then a second coupling reaction with N-
Boc-piperazine afforded 23 and 25 with moderate to good
yields. Finally, Boc deprotection, followed by sulfonylation with
the corresponding fluorobenzenesulfonyl chloride, led to the
expected ligands 11−14.
labeled with carbon-11. Despite subnanomolar 5-HT6 affinity
(Ki = 0.78 nM) and straightforward radiolabeling, [11C]2
showed poor brain entry, limiting its usefulness for in vivo
imaging of 5-HT6 receptors.23 In the same year, another group
proposed various candidate 5-HT6 PET ligands, of which [11C]
3 appeared to be the most promising but was abandoned
because of its poor radiopharmacological properties.24 Very
recently, a structural modification of [11C]1 was patented [11C]
4) as a potential PET radiotracer with subnanomolar affinity for
5-HT6 receptor (Ki = 0.22 nM) and good selectivity toward 5-
HT2A receptors (Ki = 123 nM).25,26 Nevertheless and despite
the interest of several of these radiotracers, the fact that they are
radiolabeled using carbon-11 is a drawback for further
development because of its short radioactive half-life (T1/2
=
20 min). Fluorine-18 is the radionuclide of choice in PET
because of its longer half-life (T1/2 = 110 min), facilitating
transfer between PET imaging centers if it can be successfully
developed as a radiopharmaceutical. This last point justifies
research for fluorinated candidates.
In 2007, we reported a fluorine-18 labeled ligand ([18F]5)
presenting in vitro affinity toward the 5-HT6 receptor (4 nM);
it showed a good brain penetration but no specific binding to 5-
HT6 receptors.27 A new PET radiotracer, with greater
specificity and selectivity for 5-HT6 receptors and ideally with
fluorine-18 radiolabeling, therefore remains an objective. This
article presents the first developments in a new series of
fluorinated 5-HT6 tracer candidates and the emergence of a
potent 5-HT6 radiotracer.
RESULTS AND DISCUSSION
■
When this work started, the best radioligand so far described
and fully validated was [11C]1. Consequently, inspired by this
structure, various structural modifications around the quinoline
core were envisaged by taking care that the envisaged structural
modifications always fit with the 5-HT6 receptor pharmaco-
phore.13,28 The priority was for all these new structures to also
be able to be radiolabeled with the fluoride anion fluorine-18,
which is the only radioisotope source that can be introduced
onto organic compounds by nucleophilic substitution. For our
purposes of aromatic radiolabeling, the fluorine atom must be
The final targeting ligand 15 was synthesized by inverting the
steps of quinoline iodination and coupling with piperazine.
After introduction, mediated by palladium, of N-methylpiper-
azine in position 8, regioselective iodination gave rise to 27,
which was then coupled to fluorobenzenethiol with copper
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a
Scheme 1. Synthesis of Fluorinated Ligands 6−15
a
Reagents and conditions: (a) NIS, AcOH, 80 °C; (b) CuI (2.5%), Cs2CO3, thiol, DMF, 100 °C; (c) MMPP (2.25 equiv), CH2Cl2/MeOH; (d)
t
Pd2dba3 (5%), DavePhos (10%), BuONa (1.4 equiv), N-methylpiperazine, toluene, 110 °C; (e) mCPBA (1.1 equiv), CH2Cl2; (f) Pd2dba3 (5%),
XPhos (10%), tBuONa (1.4 equiv), N-methylpiperazine, toluene, 110 °C; (g) Pd2dba3 (5%), XPhos (10%), Cs2CO3 (1.4 equiv), N-
methylsulfonamide, toluene, 110 °C; (h) Pd2dba3 (5%), XPhos (10%), Cs2CO3 (1.4 equiv), N-methylpiperazine, toluene, 110 °C; (i) PhSO2Na, CuI
(10%), DMEDA (20%), DMSO, 110 °C; (j) (1) TFA, CH2Cl2; (2) ArSO2Cl, Et3N; (k) Pd2dba3 (5%), XPhos (10%), Cs2CO3 (1.4 equiv),
t
PhSO2NHMe, toluene, 110 °C; (l) Pd2dba3 (5%), DavePhos (10%), BuONa (1.4 equiv), N-methylpiperazine, dioxane, 110 °C; (m) CuI (20%),
Cs2CO3, thiol, DMF, 100 °C; (n) Oxone (2.05 equiv), MeOH/H2O, 1/1.
catalysis, and the resultant sulfide 28 was oxidized into the
expected sulfone 15 (Scheme 1).
homology.39 Only molecules with Ki(5-HT6) < 5 nM were
selected. i.e., 6−8, 10 (Table 1).
Affinities of these ligands for 5-HT6 receptors were
determined by binding assays on human recombinant receptors
expressed in CHO cells. 5-HT6 receptor affinity results varied
according to the compound (Table 1). It thus appeared that
replacing the sulfonyl group by the sulfonamide moiety was not
deleterious for the affinity, confirming the bioisostery between
SO2 and NHMeSO2 (6, 7 vs 9, 10) for 5-HT6 receptor. 5-HT6
receptor affinities decreased with increasing of steric hindrance
onto piperazine (11, 12). However, excessive hindrance is
highly deleterious for the affinity with Ki > 1 μM (13, 14). The
drop in the basicity of the piperazine nitrogen substituted by
the sulfonyl group can also contribute to the loss in binding for
5-HT6 receptor. Finally, modifying geometry by changing the
substituent positions also reduces affinity (15).
As expected, only modifying the position of the fluorine atom
onto the benzenesulfonyl part (6, 7) did not bring significantly
improved affinity or selectivity in comparison to the parent 1. A
simple change in the oxidation state of the sulfur atom (8)
decreased 5-HT6 receptor affinity and did not improve 5-HT2A
receptor selectivity. Finally, bioisosteric replacement of sulfone
by sulfonamide 10 led to subnanomolar 5-HT6 receptor affinity
and very low affinity for 5-HT2A receptor. Because lipophilicity
and molecular polar surface (PSA) could be predictive of
blood−brain barrier penetration,40−42 the lipophilicity (log D)
and PSA of 6−8, 10 were also calculated with the ACD/Labs
and Marvin software applications (Table 1). PSA values were
lower than 70 Å2, predicting good brain penetration.42 The
log D values at physiological pH were also compatible with
good brain permeation.
The second receptor target that held our attention was the 5-
HT2A receptor, since it is known that this family is the closest to
5-HT6 receptors, ranging between 33% and 40% in sequence
Consequently, radiolabeling of 10 was envisaged to obtain
the corresponding 18F analogues. To obtain the nitro precursor
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Table 1. Lipophilicity, PSA, 5-HT7. and 5-HT2A Affinities
(Ki) of 6−15
a
Ki (nM)
log D
PSA (pH 7.4)
b
c
ligand
5-HT6
5-HT2A 5-HT2A/5-HT6 (pH 7.4)
(Å2)
6
0.26
0.10
4.4
1.7
14
54
6.5
3.01
2.33
2.00
61.89
61.89
56.85
7
140
12.3
8
9
27
10
11
12
13
14
15
0.9
>1 μM
>1000
1.86
66.33
7.6
30
>1 μM
>1 μM
53
a
b
Determined on CHO cells. Calculated with ACD/Labs, version
7.09, Advanced Chemistry Development, Inc., Toronto, Ontario,
c
Canada, www.acdlabs.com, 2014. Calculator plugins were used for
structure−property prediction and calculation, Marvin 6.0.3, 2013,
ChemAxon (http://www.chemaxon.com).
Figure 2. In vitro autoradiograms of rat brain sections incubated with
[18F]10 and after competition with cold 10 or 5-HT6 antagonist 30
(both at 1 μM). The corresponding anatomic slice shows 5-HT6
receptor locations and density (proportional to gray level) in cortex
and striatum. Histogram: decrease of [18F]10 binding after cold
compound or 5-HT6 antagonist addition (gray and white bars versus
the control black bar).
(29), strategy similar to that described above (Scheme 1) was
applied, with some adjustments probably required because of
the presence of the highly electron-withdrawing nitro
substituent, which would tend to modify substrate reactivity.
In particular, triflate of quinoline had to be used to enable the
final coupling step with N-methylpiperazine (see Supporting
Information).
The nitro precursor 29 has been obtained with an overall
yield of 13% and has been labeled by nucleophilic aromatic
substitution of the nitro group, in classical conditions at 150 °C,
in dimethyl sulfoxide (DMSO), and in presence of Kryptofix
(Scheme 2).43−45
preliminary biological results demonstrated the favorable
properties of [18F]10 as a 5-HT6 radiotracer.
To confirm the potential of [18F]10 as a PET radiotracer,
affinities toward other receptors were explored in vitro:
serotoninergic 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT4,
adrenergic α1B, and dopaminergic D2 and D3 receptors were
selected because of their shared location in the target brain area,
the striatum.47 Compound 10 presented very low affinity
toward the main receptors located in the striatal region and
toward other 5-HT2 family receptors, confirming its excellent
selectivity for the 5-HT6 receptor (Table 2).
a
Scheme 2. Radiolabeling of 29
Table 2. In Vitro Selectivity of Compound 10
a
a
receptor
Ki
receptor
Ki
a
>10−6
>10−6
>10−6
>10−6
M
M
M
M
RCY: radiochemical yield, based on the fluorine-18 activity recovered
5-HT6
0.9 nM
5-HT4
α1B
5-HT1B
5-HT2A
5-HT2B
5-HT2C
>10−6
>10−6
M
from the resin. EOB: end of bombardment. Mean of radiochemical
yields of several radiolabelings (in brackets, number of radiolabeling
experiments). SA: specific activity. EOS: end of synthesis.
M
D2
b
260 nM
>10−6
D3
M
a
Determined on CHO cells.
The radiolabeled compound [18F]10 was produced with
good radiochemical yield, chemical and radiochemical purity of
>90%, and good specific activity of 82 GBq·μmol−1.
CONCLUSION
■
Distributions of [18F]10 were assessed by semiquantitative
autoradiography in rat brain (Figure 2). Autoradiograms
obtained after incubation with a constant in vitro radiotracer
concentration demonstrated the presence of structures able to
concentrate radioactivity (i.e., cortex and striatum). These
binding areas corresponded to those described as rich in 5-HT6
receptors.5 [18F]10 radioactivity levels were markedly reduced
after addition of their respective cold molecules (1 μM).
Furthermore, the binding level of [18F]10 decreased signifi-
cantly after addition of 1 μM 30, a 5-HT6 antagonist.46 These
To our knowledge, this is the first report of a promising
fluorinated PET radioligand for in vivo imaging of the serotonin
5-HT6 receptor. We synthesized 10 compounds according to a
quinoline-based scaffold. Four of these were selected for their
5-HT6 receptor affinity and selectivity toward 5-HT2A receptor.
These nonradioactive fluorinated ligands and their radiolabeling
precursors were obtained from a bis-functionalized quinoline
core followed by coupling reactions in three steps. Radio-
labeling used 18F-nucleophilic aromatic substitution. Three
presented high in vitro binding in the striatum (an area rich in
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slides were dipped in cold buffer (4 °C) for 90 s, in distilled cold water
(4 °C) for 90 s, and then dried and juxtaposed to a phosphor imaging
plate for 60 min (BAS-1800 II, Fujifilm).
5-HT6 receptors), but only 10 specifically presented favorable
in vitro binding for 5-HT6 receptors versus striatal 5-HT2A, 5-
HT2B, 5-HT2C, 5-HT4, α1B, D2, and D3 receptors. In vitro PET
autoradiographies in rat brain sections confirmed the 5-HT6
distribution pattern of [18F]10. In conclusion, this study
enabled selection of a fluorinated radiotracer candidate with
suitable characteristics for PET imaging of 5-HT6 receptors,
justifying further radiopharmacological evaluation in animal
models before considering human brain imaging. For the
following studies, this promising molecule will be named
2FNQ1P.
ASSOCIATED CONTENT
* Supporting Information
Synthesis procedures and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: Thierry.billard@univ-lyon1.fr. Phone: (+33) 472 44
81 29.
■
EXPERIMENTAL SECTION
■
Notes
Chemistry. The purity of the tested compounds and their nitro
precursors was assessed by RP-HPLC and elemental analysis. All
compounds reported are at least 95% pure.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank particularly Didier Le Bars, Christian Tourvieille,
2-Fluoro-N-methyl-N-[8-(4-methylpiperazin-1-yl)quinolin-3-
yl]benzenesulfonamide (10). 10 was obtained in 25% yield. Brown
oil. 1H NMR: δ 8.76 (d, J = 2.6 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H), 7.69
(ddd, J = 7.6 Hz, J = 7.6 Hz, J = 1.7 Hz, 1H), 7.57 (m, 1H), 7.43 (m,
1H), 7.35 (dd, J = 8.2 Hz, J = 1.2 Hz, 1H), 7.23−7.12 (m, 3H), 3.49−
3.45 (m, 7H), 2.86 (m, 4H), 2.47 (s, 3H). 13C NMR: δ 159.28 (d, J =
256.6 Hz), 149.4, 146.9, 141.2, 135.9 (d, J = 8.3 Hz), 134.7, 132.9,
131.9, 129.6, 128.1, 126.1 (d, J = 14.6 Hz), 124.9 (d, J = 3.9 Hz),
122.2, 117.7 (d, J = 21.6 Hz), 117.0, 55.4, 51.9, 46.2, 38.9. 19F NMR: δ
−107.64 (m). Anal. Calcd for C21H23FN4O2S: C, 60.85; H, 5.59; N,
13.52. Found: C, 61.03; H, 5.67; N, 13.69.
■
Fred
́
er
́
ic Bonnefoi, and Franco
̧
is Liger (PET Department of the
CERMEP-Imaging Platform) for their technical assistance. Ora
and particularly Guillaume Villeret are thanked for their
technical assistance. The French Fluorine Network and the
French Radiopharmaceuticals Network are also thanked for
their support. J.C. was supported by a Ph.D. grant from the
Rho
̂
ne-Alpes Region (€32,116).
Radiosynthesis. Fluorine-18 was produced via the 18O(p,n)18F
nuclear reaction (IBA Cyclone 18/9 cyclotron). Nitro/fluoro exchange
was performed on a standard Neptis synthesizer (Ora): after initial
fluoride preparation (collection, drying, and Kryptofix activation),
1.7−2.5 mg of nitro precursor 29 was introduced in 3 mL of DMSO,
and the mixture was heated at 150 °C for 10 min. After dilution with
15 mL of water, the mixture was passed through an activated C18
cartridge for prepurification, and the crude product was eluted from
the cartridge with 1.5 mL of methanol. Pure [18F]10 was obtained after
separation on a preparative high-performance liquid chromatography
(HPLC) (C18 Symmetry Prep Waters, 7 μm, 7.8 mm × 300 mm),
eluting with H2O/CH3CN/TFA, 78/22/0.1%, at 3 mL·min−1 (λ = 254
nm). For biological use, the radiotracer was formulated via SPE
techniques.48 The product was diluted in 20 mL of sterile water and
loaded on a SEP-Pak Light C18 cartridge (Waters, Milford, MA, U.S.).
The loaded cartridge was rinsed with water and eluted with 1 mL of
ethanol, and the final product was diluted with isotonic saline and
sterilized by filtration (sterile filter Millex-GS, 0.22 mm). The
radiochemical purity and specific activity of [18F]10 were assayed by
analytical HPLC (MachereyeNagel EC 250/4.6 Nucleodur 100-5-
C18ec C18 column; mobile phase H3PO4 20 mM/THF, 77/23; flow
rate, 0.9 mL·min−1). The identity of [18F]10 was confirmed by co-
injection with an authentic nonradioactive sample.
ABBREVIATIONS USED
■
5-HT6, serotoninergic receptor 6; 5-HT1B, serotoninergic
receptor 1B; 5-HT2A, serotoninergic receptor 2A; 5-HT2B,
serotoninergic receptor 2B; 5-HT2C, serotoninergic receptor
2C; 5-HT4, serotoninergic receptor 4; α1B, adrenergic receptor
1B; Boc, tert-butyloxycarbonyl; CHO, Chinese hamster ovary;
D2, dopaminergic receptor 2; D3, dopaminergic receptor 3;
DavePhos, 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)-
biphenyl; EOB, end of bombardment; EOS, end of synthesis;
HPLC, high-performance liquid chromatography; mCPBA, m-
chloroperbenzoic acid; MMPP, magnesium monoperoxyph-
thalate; NIS, N-iodosuccinimide; PET, positron emission
tomography; PSA, polar surface area; RCY, radiochemical
yield; RP-HPLC, reverse phase high-performance liquid
chromatography; SA, specific activity; SNAr, nucleophilic
aromatic substitution; SPE, solid-phase extraction; XPhos, 2-
dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl
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SM1850), thaw-mounted on glass slides, and allowed to air-dry before
storage at −80 °C until use. On the day of radiotracer synthesis, the
slides were allowed to reach room temperature and then incubated for
20 min in Tris phosphate buffered saline buffer (138 mM NaCl, 2.7
mM KCl, pH adjusted to 7.6) containing 37 kBq/mL (1 μCi/mL)
[18F]10. For competition experiments, the slides were placed in the
same buffer supplemented with their respective cold compounds (10
at 1 μM) or 30, a selective 5-HT6 antagonist (1 μM). After incubation,
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