Radiosynthesis and Biological Evaluation of [18F]R91150, a Selective 5-HT2AReceptor Antagonist for PET-Imaging

Article abstract of DOI:10.1021/acsmedchemlett.0c00658

Serotonergic 5-HT2A receptors in cortical and forebrain regions are an important substrate for the neuromodulatory actions of serotonin in the brain. They have been implicated in the etiology of many neuropsychiatric disorders and serve as a target for an

Full text of DOI:10.1021/acsmedchemlett.0c00658

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Letter
Radiosynthesis and Biological Evaluation of [18F]R91150, a Selective
5‑HT2A Receptor Antagonist for PET-Imaging
Michael Willmann, Julian Hegger, Bernd Neumaier,* and Johannes Ermert
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ABSTRACT: Serotonergic 5-HT2A receptors in cortical and
forebrain regions are an important substrate for the neuro-
modulatory actions of serotonin in the brain. They have been
implicated in the etiology of many neuropsychiatric disorders and
serve as a target for antipsychotic, antidepressant, and anxiolytic
drugs. Positron emission tomography imaging using suitable
radioligands can be applied for in vivo quantification of receptor
densities and receptor occupancy for therapy evaluation. Recently,
the radiosynthesis of the selective 5-HT2AR antagonist [18F]-
R91150 was reported. However, the six-step radiosynthesis is
cumbersome and time-consuming with low radiochemical yields
(RCYs) of <5%. In this work, [18F]R91150 was prepared using late-stage Cu-mediated radiofluorination to simplify its synthesis.
The detailed protocol enabled us to obtain RCYs of 14 1%, and the total synthesis time was reduced to 60 min. In addition,
autoradiographic studies with [18F]R91150 in rat brain slices revealed the typical uptake pattern of 5-HT2A receptor ligands.
KEYWORDS: 5-HT2A, autoradiography, PET imaging, fluorine-18, [18F]R91150
erotonin, or 5-hydroxytryptamine (5-HT), is an important
neurotransmitter in the central nervous system and
[11C]MDL100907,14,15 or [18F]MDL10090716,17 and radio-
labeled agonists such as [11C]CIMBI-3618−20 or [18F]-
FETCIMBI-36.21 Likewise, several 123I-labeled compounds
have been developed for SPECT imaging, although only the
antagonist [123I]iodo-R91150, a radiolabeled analog of the
Janssen Research Foundation compound R91150 (Figure 1),
proved to exhibit suitable binding properties, selectivity, and
brain uptake for imaging of 5-HT2ARs in rats.22,23 Subsequent
animal and human studies with [123I]iodo-R91150 revealed a
binding pattern consistent with the distribution of 5-HT2ARs
observed in previous autoradiographic studies,24−27 and the
tracer has since been used for imaging in patients with
schizophrenia,28 depression,29 Parkinson’s disease30 or Asperg-
er’s syndrome.31 A disadvantage of [123I]iodo-R91150,
especially with regard to preclinical imaging in small animal
models, is that radioiodination increases the lipophilicity
relative to the parent compound R91150 more than 10-
fold32 (Figure 1), which results in a relatively low signal-to-
noise ratio. Exploiting the presence of an aromatic fluorine
atom in the parent compound (Figure 1), this aspect has been
addressed by preparation of the 18F-labeled analog [18F]-
S
peripheral tissues that exerts its effects through a family of
seven 5-HT receptors (5-HT1−7). In the brain, 5-HT release
from serotonergic fibers that originate in the raphe nuclei and
innervate cortical and limbic structures of the medial temporal
lobe system has long been recognized to modulate cognition,
emotional processes, and various forms of learning and
memory.1,2 More recent findings indicate that serotonergic 5-
HT2A receptors (5-HT2ARs), which are widely expressed in
cortical and forebrain regions, may be an important substrate
for these neuromodulatory actions.3 In support of this
assumption, changes in 5-HT2AR expression or function have
been implicated in the etiology of many neuropsychiatric
disorders such as depression, anxiety, or schizophrenia and
cognitive disturbances associated with Alzheimer’s or Parkin-
son’s disease.4−7 Based on these findings, pharmacological
manipulation of 5-HT2ARs has emerged as a promising
approach for the treatment of mental or cognitive disorders,
as evidenced by various studies on the antidepressant,8
anxiolytic,9 and antipsychotic10 effects of 5-HT2AR antagonism.
Molecular imaging techniques such as positron emission
tomography (PET) or single photon emission computed
tomography (SPECT) can be used for in vivo quantification of
(changes in) receptor densities and measurement of receptor
occupancy by novel therapeutic drugs. To date, various
radioligands for PET-based visualization of 5-HT2ARs have
been developed that can be broadly classified into radiolabeled
antagonists such as [18F]setoperone,11,12 [18F]altanserin,13
Received: December 20, 2020
Accepted: March 30, 2021
Published: April 1, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Structure and properties of the selective 5-HT2AR antagonist R91150 and its radiolabeled analogs. IC50 values refer to inhibition of
[3H]ketanserin binding to rat frontal cortex membranes. Selectivity factors refer to 5-HT2AR over serotonergic 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2C,
and 5-HT3, adrenergic α1 and α2, histaminergic H1, and dopaminergic D2 receptors. Capacity factors k′ were determined at pH 5 and provide a
measure for lipophilicity with excellent correlation to logP octanol/buffer values. Data from refs 22, 23, 32, and 34.
a
Scheme 1. Synthesis of R91150
a
Reaction conditions: (i) MeLi, Et2O, −78 °C to r.t., 2 h. (ii) conc H2SO4, MeCN, r.t., 2 h. (iii) conc HCl, 100 °C, 48 h. (iv) PyBOP, Et3N, 4,
DCM, r.t., 18 h. (v) NH4HCO3, palladium black, MeOH, 65 °C, 3.5 h. (vi) K2CO3, KI, DMF, 80 °C, 12 h. (vii) DCM/TFA (3:1), 80 °C, 2 h.
R91150 for use in PET measurements.32 Preliminary in vitro
and ex vivo experiments indicated that [18F]R91150 may have
certain advantages over established 5-HT2A antagonist radio-
ligands for human use.32 This includes a higher selectivity and
lack of lipophilic, brain-permeant radiometabolites, if com-
pared to [18F]altanserin33 or [18F]setoperone,12 an increased 5-
HT2A affinity, reduced lipophilicity, and improved signal-to-
background ratio, if compared to [123I]iodo-R91150,32,34 and a
longer-lived 18F-label resulting in a better match between the
radionuclide and the biological half-life, if compared to
[11C]MDL100907.35,36 On the other hand, a major disadvant-
age of [18F]R91150 is the time-consuming six-step radiosyn-
thesis providing [18F]R91150 in low RCYs of <5%.
Furthermore, the radiosynthesis is not amenable to automa-
tion32 and has prevented a more widespread use of this
radioligand. However, a number of Cu-mediated radio-
fluorination methods developed in the past decade (for
overview37−41) have provided new approaches for facile
introduction of 18F into aromatic compounds. In the present
work, we describe a novel synthetic strategy for production of
[18F]R91150 via copper-mediated late-stage radiofluorination
and present the results from autoradiographic studies
performed with the tracer.
For competition studies and to confirm the identity of
[18F]R91150, the reference compound R91150 was prepared
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a
Scheme 2. Synthesis of Labeling Precursor 8
a
Reaction conditions: (i) K2CO3, KI, DMF, r.t., 12 h.
Scheme 3. Two-Step Radiosynthesis of [18F]R91150
according to the total synthesis described by Mu
(Scheme 1).
̈
lhausen et al.32
HPLC, followed by solid phase extraction (SPE) using a C18
cartridge.
To this end, commercially available N-benzyl-piperid-4-one
was reacted with methyllithium (MeLi) to introduce the
quaternary methyl bearing carbon atom, which provided
benzyl-4-methyl-piperidin-4-ol (1) in good yields (75%). The
hydroxyl group was almost quantitatively (98%) converted into
the acetyl-protected amine by a Ritter-Reaction of 1 with
MeCN and concentrated H2SO4 to obtain N-(1-benzyl-4-
methylpiperidin-4-yl)acetamide (2), which was directly de-
protected to 1-benzyl-4-methyl-piperidin-4-amine (3) in 86%
yield by refluxing in concentrated hydrochloric acid (aq HCl)
for at least 2 days. Intermediate 3 was conjugated with 4-(tert-
butoxycarbonylamino)-2-methoxybenzoic acid 4 (prepared
from commercially available 4-amino-2-methoxybenzoic acid)
using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP) for carboxylic acid activation,
which provided tert-butyl 4-(1-benzyl-4-methylpiperidin-4-
ylcarbamoyl)-3-methoxyphenylcarbamate (5) in fair yield
(57%) but still contaminated with pyrrolidine byproducts
The radiolabeling precursor 8 was synthesized in an
analogous manner in 30% yield by employing commercially
available 2-(4-(3-bromopropoxy)phenyl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane in the amination reaction (Scheme 2).
Note: Unless noted otherwise, all radiochemical yields
(RCYs) described herein have been corrected for decay to the
start of the synthesis, as recommended in the consensus
nomenclature rules for radiopharmaceutical chemistry.42
[18F]R91150 was prepared in a two-step synthesis by Cu-
mediated radiofluorination of the N-Boc-protected precursor 8
with subsequent deprotection followed by HPLC-purification
(Scheme 3). The tracer was prepared within 60 min. Initially
we attempted a direct labeling approach that involved
deprotection of 8 (33% TFA in DCM at 80 °C for 5 min)
with subsequent labeling in the same vial (after removal of
TFA/DCM). Unfortunately, these attempts were not success-
ful, potentially due to fast protodeboronation43 of the
precursor under the applied conditions. Assuming that the
deprotected boronic acid pinacol ester might be prone to
degradation upon long-term storage, we did not further
attempt to implement a one-step radiosynthesis of [18F]-
R91150.
1
according to H-/13C-NMR. Subsequent benzyl deprotection
of 5 with palladium black was complete after 3.5 h (as
indicated by TLC), and purification by flash column
chromatography effectively removed all pyrrolidine from the
previous step, affording 6 in 31% yield. The following
amination of 6 with commercially available 1-(3-bromopro-
poxy)-4-fluorobenzene gave the N-Boc-protected reference
compound 7 in 34% yield. R91150 was finally obtained in 67%
yield by deprotection of 7 in a 3:1 mixture of dichloromethane
(DCM) and trifluoroacetic acid (TFA) at 80 °C for 2 h. For
optimal purity, the compound was purified by semipreparative
Therefore, the N-Boc-protected boronic acid pinacol ester 8
was radiofluorinated using the Cu(II)-mediated radiofluorina-
tion protocol according to Zischler et al.44 To this end,
[18F]fluoride was trapped on an anion-exchange (QMA)
cartridge and eluted with tetraethylammonium bicarbonate
(Et4NHCO3) in n-butanol with an elution efficacy of 95 4%
(n = 10). A solution of precursor 8 and Cu(OTf)2(py)4 in
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Table 1. Comparison of the Radiosynthesis of [18F]R91150 as Published by Mu
Radiosynthesis in This Work
̈
lhausen et al.32 with the Two-Step
reaction steps
synthesis time
RCY
n.d.c. yield
automation
possible
not applicable
This work
2 (facile)
6 (complex)
60 min
190 min
14 1%
1.8−5.7%
10 1%
0.5−1.7%
Mu
̈
lhausen et al.32
DMA was added, and the mixture was stirred at 110 °C for 10
min, which afforded [18F]7 in a RCY of 15 1% (n = 3) after
isolation by SPE (Sep-Pak C18). Quantitative deprotection of
[18F]7 was achieved with TFA in DCM at 80 °C for 5 min.
Subsequently, the 5-HT2A-radioligand [18F]R91150 was
isolated using an analytical HPLC column with 14
1%
RCY (aliquots, n = 3) or in 13% RCY (n = 1) using a
semipreparative HPLC column. For in vitro studies, [18F]-
R91150 was isolated using an analytical HPLC column and the
collected fraction (2−5 MBq/mL) was diluted in Tris HCl
(pH 7.4) buffer (∼2.5 kBq/mL).
The molar activity was determined to 70 GBq/μmol with a
radiochemical purity of 97%, based on a starting activity of 4.4
GBq and a synthesis time of approximately 60 min. Cu-content
was determined by ICP-MS of the isolated fraction (using
semipreparative HPLC) containing the tracer (10 mL, 174
MBq) and found to be 2.6 μg/L. This is far below any level of
concern according to the ICH Guideline of Elemental
Impurities (Q3D).45
Figure 3. In vitro autoradiography of sagittal rat brain slices with
[18F]R91150 (A and C: total binding) and in competition with
R91150 (B and D: nonspecific binding). Abbreviations: cerebellum
(cb), choroid plexus (Ph. ch.), lamina V of the cortex (il.V), olfactory
bulb (ob), olfactory tubercle (ot), lateral reticular nucleus (LRt),
striatum (str), trigeminal nerve (V). Each column shows different sets
of experiments.
In summary, the radiosynthesis of [18F]R91150 was
considerably facilitated by reduction of synthesis time and
improvement of radiochemical yield (Table 1). This enabled
the first in vitro studies of this radioligand and finally also
allows for automation of the radiosynthesis.
In addition, the specific binding pattern of [18F]R91150 was
verified in competition assays with the 5-HT2A targeting drugs
altanserin, (+)-lisuride,46 and (-)-lisuride (Figure 4). In three
independent experiments with molar activities between 1.5 and
70 GBq/μmol, [18F]R91150 showed distinct specific binding
throughout the cortex, with a higher binding in frontal
compared to caudal cortical regions (Figure 2). Binding was
displaced by R91150, altanserin, and (+)-lisuride to approx-
imately the same extent, validating the 5-HT2A specificity of
[18F]R91150. In direct comparison with other specific 5-HT2A
ligands (e.g., (R)-[18F]MH.MZ,47 [18F]MDL100,90716, [18F]-
altanserin48,49), a high degree of regional correspondence was
observed for binding in the neocortex (strongest labeling in
lamina V), the striatum (stronger signal in caudal parts of the
nucleus), and the olfactory tubercle along with several of the
brainstem nuclei (see Figure 3).50 These regions of high 5-
HT2AR expression, as determined in previous in situ hybrid-
ization studies,51 are clearly visualized in the autoradiographic
images (see Figures 2 and 3).
The suitability of radioligand candidates for in vivo
experiments strongly depends on a high ratio of specific to
nonspecific binding and a lack of affinity for other, nontarget
receptors (unspecific binding). Here, in vitro autoradiography
was used in competition with nonradioactive R91150 to
determine nonspecific binding of the radioligand. To this end,
transaxial and sagittal rat brain slices were incubated either
with [18F]R91150 alone (total binding, Figure 2A, Figure 3A
and C, Figure 4A) or with [18F]R91150 and an excess of the
corresponding nonradioactive reference compound (non-
specific binding, Figure 2B, Figure 3B and D, Figure 4B−D).
Almost no specific binding was observed throughout the
cerebellum, which is consistent with several previous auto-
radiographic and immunohistological studies which confirmed
low 5-HT2AR abundance in the cerebellum.52−54 Nonspecific
binding was determined as the nondisplaceable radioligand
binding in the presence of 1 μM R91150, pointing to a low
degree of nonspecific binding of 29% (Figure 2B, Table 2). A
high [18F]R91150 accumulation was found in the anterior
forebrain, where tracer accumulation displayed a strong
laminar pattern, similar to that observed in immunohistochem-
ical localization studies on 5-HT2AR expression in the middle
layers of the cortex, that identified layer V as the region with
the most conspicuous 5-HT2AR densities.55 In the competition
assays with (−)-lisuride, (+)-lisuride, and altanserin, (−)-li-
suride displaced [18F]R91150 binding in the cortex to a lower
degree (53%) than (+)-lisuride (76%, Figure 4), indicating a
Figure 2. In vitro autoradiography of transaxial rat brain slices with
[18F]R91150 (A: total binding) and in competition with R91150 (B:
nonspecific binding). Major binding was detected in the lamina V
(il.V) of the cortex and in the caudal parts of the striatum (str).
Abbreviations: cerebellum (cb), lamina V of the cortex (il.V), orbital
cortex, striatum (str).
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Figure 4. Competition assay of [18F]R91150 (A) with (−)-lisuride (B), (+)-lisuride (C), and altanserin (D). Only (−)-lisuride (B) could not
completely block the binding of [18F]R91150. Transaxial and sagittal orientations are shown for each set.
a
Table 2. Quantification of [18F]R91150 Binding
b
%-Displacement of [18F]R91150 binding
1 μM
2 μM
R91150
altanserin
(+)-lisuride
(−)-lisuride
cortex
striatum
cerebellum
70.5 3.8
34.3 12.5
0
70.6 3.2
32.9 12.6
0
75.6 5.5
49.9 13.7
0
52.7 2.6
32.4 4.5
0
c
a
b
Displacement by known 5-HT2A targeting drugs indicates specific binding behavior of [18F]R91150. Corrected for background, n = 4. Regions of
c
interest and data used for quantification are described in the Supporting Information. Stronger displacement compared to R91150 due to higher
concentration of the ligand.
lower 5-HT2A affinity of this enantiomer as compared to
(+)-lisuride.
orcid.org/0000-0001-5425-3116; Phone: +49 2461
614141; Email: b.neumaier@fz-juelich.de
In conclusion, the radiosynthesis of [18F]R91150 was
simplified by using late stage Cu-mediated radiofluorination,
facilitating the production of [18F]R91150 in fair RCY of 14
1% within 60 min. The in vitro evaluation of [18F]R91150
revealed the potential of this tracer for PET imaging of 5-
HT2AR status in the human brain. Future studies should focus
on the in vivo behavior of [18F]R91150 and the transfer of its
synthesis to an automated module for clinical applications.
Authors
Michael Willmann − Forschungszentrum Ju
Institute of Neuroscience and Medicine, INM-5, 52425 Ju
Germany
Julian Hegger − Forschungszentrum Ju
̈
lich GmbH,
̈
lich,
̈
lich GmbH, Institute of
Neuroscience and Medicine, INM-5, 52425 Ju
Johannes Ermert − Forschungszentrum Julich GmbH,
Institute of Neuroscience and Medicine, INM-5, 52425 Ju
̈
lich, Germany
̈
̈
lich,
Germany; orcid.org/0000-0002-2561-7766
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00658.
■
sı
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00658
Author Contributions
Experimental details for all compounds synthesized
including radiosynthesis; determination of molar activ-
ity; spectroscopic data for standards and precursors;
protocols for animal imaging studies; 1H-NMR and 13C-
NMR spectra of all compounds; HRMS spectra of
compound 8 and R91150; in vitro autoradiography of
[18F]R91150; semipreparative and analytical (Radio)-
HPLC chromatogram of [18F]R91150 (PDF)
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Annette Schulze, Dirk Bier
and Marcus Holschbach (all INM-5), and Sabine Willbold
AUTHOR INFORMATION
Corresponding Author
Bernd Neumaier − Forschungszentrum Ju
of Neuroscience and Medicine, INM-5, 52425 Ju
■
(Zentralinstitut fu
3), all Forschungszentrum Ju
̈
r Engineering, Elektronik und Analytik, ZEA-
lich, for recording the spectro-
̈
lich GmbH, Institute
lich,
̈
̈
scopic data and conducting the biological studies. Writing
assistance was provided by Felix Neumaier, Forschungszen-
Germany; Uniklinik Köln, Institute of Radiochemistry and
Experimental Molecular Imaging, 50937 Köln, Germany;
trum Julich, INM-5.
̈
742
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Letter
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ABBREVIATIONS
■
5-HT, 5-hydroxytryptamine; 5-HT2AR, serotonergic 5-HT2A
receptor; Boc, tert-butyloxycarbonyl; Cu(OTf)2(py)4, tetrakis-
(pyridine)copper(II) triflate; DCM, dichloromethane; DMA,
N,N-dimethylacetamide; DMF, N,N-dimethylformamide; HCl,
hydrochloric acid; HPLC, high performance liquid chromatog-
raphy; ICP-MS, inductively coupled plasma mass spectrome-
try; MeLi, methyllithium; NMR, nuclear magnetic resonance;
n.d.c., nondecay corrected; QMA, quaternary methylammo-
nium; PET, positron emission tomography; PyBOP, benzo-
triazol-1-yl-oxytripyrrolidinophosphonium hexafluorophos-
phate; R91150, 4-amino-N-[1-[3-(4-fluorophenoxy)propyl]-4-
methyl-4-piperidinyl]-2-methoxybenzamide; RCY, radiochem-
ical yield; r.t., room temperature; SPECT, single photon
emission computed tomography; SPE, solid phase extraction;
TFA, trifluoroacetic acid; TLC, thin layer chromatography.
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