Looking for a 5-HT7 radiotracer for positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2010.04.076

In search of a serotonin 5-HT₇ radiotracer for positron emission tomography, we developed 1-{2-[(2S)-1-(phenylsulfonyl)pyrrolidin-2-yl]ethyl} piperidin-4-yl 4-fluorobenzoate. After labeling in good yield with fluorine-18 via nitro for fluorine exchange, preliminary biological experiments with autoradiographies failed to evidence any specific 5-HT₇ receptor delineation.

Full text of DOI:10.1016/j.bmcl.2010.04.076

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3730–3733
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Looking for a 5-HT7 radiotracer for positron emission tomography
Julien Andriès a, Laëtitia Lemoine b,c, Alice Mouchel-Blaisot a, Sandrine Tang a,c, Mathieu Verdurand b,c
,
Didier Le Bars a,c, Luc Zimmer b,c, Thierry Billard a,c,
*
a Université de Lyon, Université Lyon 1, CNRS, ICBMS (UMR CNRS 5246), Lyon, France
b Université de Lyon, Université Lyon 1, CNRS, Neuropharmacology (EAC CNRS 5006), Lyon, France
c CERMEP-Imagerie du Vivant, PET Department, Lyon, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In search of a serotonin 5-HT7 radiotracer for positron emission tomography, we developed 1-{2-[(2S)-1-
(phenylsulfonyl)pyrrolidin-2-yl]ethyl}piperidin-4-yl 4-fluorobenzoate. After labeling in good yield with
fluorine-18 via nitro for fluorine exchange, preliminary biological experiments with autoradiographies
failed to evidence any specific 5-HT7 receptor delineation.
Received 16 March 2010
Revised 16 April 2010
Accepted 16 April 2010
Available online 24 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
5-HT7
Serotonin
PET
Radiolabeling
Fluorine
Serotonin (5-hydroxytryptamine, 5-HT) is a central neurotrans-
mitter involved in a great variety of physiological functions, as well
as neurological and pathological disorders such as depression, eat-
ing disorders and Alzheimer’s disease.1 Pharmacological studies al-
lowed identification of numerous serotoninergic receptors families
and subtypes. These receptors have been classified by structural,
functional and pharmacological criteria into seven distinct recep-
tor classes (5-HT1–7).2 The 5-HT7 receptor is one of the most re-
cently identified serotonin receptors.3 The recent availability of
selective 5-HT7 receptor antagonists4 and of 5-HT7 receptor knock-
out mice has considerably advanced the understanding of the
physiological function of this receptor. The 5-HT7 receptor is cou-
pled to Gs proteins and, like 5-HT4 or 5-HT6 receptors, is positively
linked to adenylate cyclase.5 Recent results indicate that this
receptor might be involved in mood regulation. For instance, 5-
HT7 receptor knockout mice exhibit antidepressant-like behaviors6
and 5-HT7 receptors are downregulated in hypothalamus after
chronic antidepressant treatments.7 Therefore, it is crucial for the
therapeutic research to gain access to in vivo studies in order to
understand the implication of this receptor in neurological and
psychiatric diseases.
implying the intravenous administration of a molecule at a very
low amount (3–10 g). At these concentrations the in vivo mea-
surements are usually far below levels at which pharmacological
l
effects might occur.
An ability to image serotonin 5-HT7 receptors in human brain
in vivo is needed to assess directly 5-HT7 receptors involvement
in neuropsychiatric diseases and their possible therapies. By study-
ing receptors in vivo, firstly in animal models and secondly in
humans, many of the uncertainties attached to studies of post-
mortem brain can be avoided,8 helping to understand the role of
the 5-HT7 receptors particularly in mood disorders. Quantitative
imaging of 5-HT7 receptors would also allow to study of the inter-
actions of established and new drugs with these receptors. This im-
plies the availability of a PET radiotracer which specifically labels
the 5-HT7 receptor. Our objective is therefore to develop the re-
quired 5-HT7 radioligands which will open new research directions
in clinical research and in drug development.
According to the literature, several pharmacological compounds
show good selectivity and affinity toward 5-HT7 receptors.9 In par-
ticular, arylsulfonamidoalkylamines exhibit one of the most potent
and selective antagonist properties.9c,10
With the development of PET (positron emission tomography)
as a molecular imaging method, the opportunity has evolved to
perform in vivo observations both in animal models and in hu-
mans.8 These studies can be performed at ‘tracer concentrations’
Because of the radioactive decay of positron emitters, the half-
lives of the radioisotopes constitute crucial parameters in the syn-
thesis of radiolabeled compounds. Therefore, we envisaged to use
specifically fluorine-18 (T1/2 = 109.7 min), more convenient for pre-
clinical and clinical uses than carbon-11 radiolabeling. Because of
its high specific radioactivity and its easy obtention via the
18O(p,n) reaction, 18FÀ anion constitutes the most common source
* Corresponding author.
E-mail address: Billard@univ-lyon1.fr (T. Billard).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.076

J. Andriès et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3730–3733
3731
The first one ([18F]-1a) is a piperazine derivative adapted from
recent literature results.11b The second ([18F]-2a) is based on SB-
269970 structure bearing an ester moiety in four-position of the
piperidine core.11a In each compounds, the fluorine atom is in para
position of electron-withdrawing group (required for SNAr reac-
tion) and can be inserted in the last step by the substitution of a
nitro group by activated fluoride anion.
Non-radioactive compound 1a and its nitro precursor for radio-
labeling (1b) have been synthesized in three steps starting from 3-
amino-propanol (Scheme 1).
Concerning the second ligand (2), a divergent strategy has been
elaborated to obtain the precursor for radiolabeling (2b) or the
non-radioactive fluorinated compound 2a via a common synthetic
way, with a discrimination step at the last stage (Scheme 2).
The N-phenylsulfonyl homoproline 7 has been easily synthe-
sized from proline by modified literature processes.12 The subse-
quent coupling between chlorhydrate of piperidone hydrate and
6 with CDI13 and the over reduction with LiAlH4 gave 8 with good
yield. Finally, the expected compounds 2a and 2b were obtained
after esterification, in nine steps with an overall yield of 9% and
7%, respectively (Scheme 2).
O O
S
N
N
OMe
H
N
18F
[
18F]-1a
O
N
N
S
O
O
18F
O
[
18F]-2a
Figure 1. Targets for potential radiolabeled 5-HT7 ligand.
for 18F introduction into molecules. Consequently, we chose to
introduce the radioisotope at the last step, to conserve the maxi-
mum of radioactivity, by a SNAr reaction using radioactive fluoride
anions.
With this constraint in mind, two structures have been envis-
aged, inspired by the literature results (Fig. 1).9c,10,11
O O
S
H
ClO2S
X
HO
N
HO
NH2
X
X = F : 3a (80%)
X = NO2 : 3b (66%)
DMAP / Et3N
TsCl
CH2Cl2 / rt
MeO
N
O O
S
O O
S
HN
OMe
N
N
TsO
N
H
H
K2CO3
CH3CN / 80ºC
N
X
X
X = F : 1a (90%)
X = NO2 : 1b (22%)
X = F : 4a (80%)
X = NO2 : 4b (66%)
Scheme 1. Synthesis of 1a and its nitro precursor for radiolabeling 1b.
1) LiAlH4
1) SOCl2 / MeOH
CN
2) MsCl / Et3N
CO2H
CO2Me
N
H
N
N
2) PhSO2Cl / Et3N
3) NaCN / DMF / 100ºC
SO2Ph
SO2Ph
(61%)
6
(85%)
5
O
N
EDCI / HOBt / Et3N
DMF
AcOH / HCl
95ºC
CO2H
N
N
O
Cl
SO2Ph
SO2Ph
H
LiAlH4
67°C
OH
OH
N
(86%)
8
(60%)
7
H
O
DIEA
N
N
N
S
O
O
O
N
OH
X
O
SO2Ph
Cl
X = F : 2a (54%)
X = NO2 : 2b (40%)
(65%)
9
X
Scheme 2. Synthesis of 2a and its nitro precursor for radiolabeling 2b.

3732
J. Andriès et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3730–3733
Table 1
were observed and HPLC conditions chosen ensured good separa-
tion of [18F]-2a from its nitro precursor 2b.
Lipophilicity (log D) and 5-HT7 affinity (pKB) of 1a and 2a compounds
Since the radiolabeled compound [18F]-2a could be obtained in
an efficient manner, its biological evaluation in rodents has been
then envisaged. Firstly, [18F]-2a binding to 5-HT7 receptors was
evaluated by in vitro autoradiographies using control rat brain.
log D (pH = 7.4)a
pKB
b
Compound
1a
2a
3.27
4.13
7.26
7.10
a
Calculated with ACD/Labs V. 7.09 software.
Determined by CEREP (www.cerep.com).
[
18F]-2a binding was homogenous throughout the brain, with a
b
lack of specific binding in regions rich in 5-HT7 receptors like hip-
pocampus in comparison to other regions devoided of 5-HT7 recep-
tors like cerebellum.14 No significant binding difference was found
between control sections and sections pre-incubated with seroto-
18F]-1a : 3% (EOB)
1b
or
2b
[
K18F / K[2.2.2]
or
nin (10 lM) (results not shown). Therefore, these initial in vitro
DMSO / 150ºC
10 min.
[
18F]-2a : 15% (EOB)
autoradiographies suggested a lack of selectivity of the [18F]-2a
compound in this animal model.
Since in vitro studies cannot be directly extrapolated to in vivo
studies, it was crucial to perform ex vivo studies. Ex vivo autorad-
iographies experiments were conducted with the aim to evaluate
the cerebral distribution of [18F]-2a in alive animals. They imply
the injection of the compound in the alive animal and subsequent
brain removal and autoradiography study. The general observation
was that [18F]-2a showed a very low brain penetration. Hippocam-
pus, the region known to be rich in 5-HT7 receptors, displayed a
low [18F]-2a labeling. A similar low radiotracer uptake was seen
in cerebellum suggesting a high level of non specific binding. The
pre-injection of unlabeled compound 1 suppressed significantly
the already low binding of [18F]-2a in all regions of the brain stud-
ied showing that its fixation is saturable (Fig. 2).
Scheme 3. Radiolabeling of 1b and 2b.
Lipophilicity (log D) of compounds 1a and 2a have been then
calculated with the ACD/Labs software and their respective affinity
toward 5-HT7 receptors (pKB) have been determined by binding as-
says on expressed in CHO cells (Table 1).
The 5-HT7 affinity values are intermediate although satisfactory
and the calculated log D values at physiological pH suggest a rela-
tively high lipophilicity for compounds. Since the lipophilicity of a
compound is predictive of its blood–brain barrier penetration,
these initial results were encouraging and led us to envisage the
radiolabeling of 1a and 2a compounds and their first PET studies.
Fluorine-18 was obtained via the 18O(p,n) 18F nuclear reaction
(IBA Cyclone 18/9 cyclotron). The nitro/fluoro exchange was real-
ized by using a reprogrammed automated fluorination module
(coincidence). Labeling of 1b and 2b, by nucleophilic aromatic sub-
stitution of nitro group, was realized in classical conditions at
150 °C, in presence of kryptofixÒ (Scheme 3).
Radiolabeling of 1b did not give satisfactory results in term of
radiochemical yield. This could be explained by a fast deprotona-
tion of acidic hydrogen of sulfonamide by the very basic, naked
fluoride anion. This deprotonation would quench the fluoride,
which would not be further available for SNAr reaction. On the
other hand, the expected radiolabeled compound [18F]-2a was ob-
tained with a satisfactory radiochemical yield of 15% corrected for
decay and 50 min radiosynthesis time (including purification
The apparent discrepancy between the high value of log D (lipo-
philicity) and the low brain penetration measured ex vivo could be
explained by the metabolic hydrolysis of ester moiety, generating
[
18F]fluorobenzoate which should be eliminated by urinal way.
Such reasonable hypothesis seemed to be confirmed by the high le-
vel of radioactivity we detected into rat kidneys suggesting a rapid
renal elimination.
In summary, we reported in this study the radiolabeling of a
new 5-HT7 receptor antagonist. Our preliminary biological results,
obtained with complementary in vitro and ex vivo approaches,
demonstrate the inability of [18F]-2a to visualize 5-HT7 receptors
in vivo. Because the discovery of a potent 5-HT7 radiotracer is cru-
cial for the exploration of brain serotoninergic function, other 5-
HT7 leads, without easy metabolisable parts, will be evaluated for
future PET investigations.
step); the specific activities was in the range of 56–95 GBq/
corrected at the end of synthesis. No major radioactive by-products
lmol
Figure 2. Ex vivo autoradiographies of rat brain sections (Hip, hippocampus; Cereb, cerebellum) in control rats (control; 20 min after the iv injection of 2 mCi (SA = 2 Ci/
mol) of [18F]-2a) and in pretreated rats with the unlabeled compound 2a (compound 2a; 5 mg/kg iv, injected 30 min before the [18F]-2a compound). On the left is reported
the corresponding regions according to rat brain stereotaxic atlas.
l

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The authors thank Gerald Hun for his technical assistance on
the radiosynthesis. This work is supported by an ANR Grant
(ANR-07-JCJC-0027 ‘SyRIM’). Laëtitia Lemoine was supported by a
CIFRE grant with Advanced Accelerator Applications.
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