Synthesis and biological evaluation of positron emission tomography radiotracers targeting serotonin 4 receptors in brain: [18F]MNI-698 and [18F]MNI-699

Article abstract of DOI:10.1016/j.bmcl.2013.09.097

Two new benzodioxane derivatives were synthesized as candidates to image the serotonin 4 receptors by positron emission tomography (PET) and radiolabeled with fluorine-18 via a two-step procedure. Competition binding assays demonstrated that MNI-698 and M

Full text of DOI:10.1016/j.bmcl.2013.09.097

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6243–6247
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of positron emission tomography
18
radiotracers targeting serotonin 4 receptors in brain: [ F]MNI-698
1
8
and [ F]MNI-699
Fabien Caillé, Thomas J. Morley, Adriana Alexandre S. Tavares, Caroline Papin, Nicole M. Twardy,
David Alagille, H. Sharon Lee, Ronald M. Baldwin, John P. Seibyl, Olivier Barret, Gilles D. Tamagnan
⇑
Molecular NeuroImaging LLC, New Haven, CT 06510, USA
Institute for Neurodegenerative Disorders, New Haven, CT 06510, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new benzodioxane derivatives were synthesized as candidates to image the serotonin 4 receptors by
positron emission tomography (PET) and radiolabeled with fluorine-18 via a two-step procedure. Com-
petition binding assays demonstrated that MNI-698 and MNI-699 had sub-nanomolar binding affinities
Received 21 August 2013
Revised 25 September 2013
Accepted 30 September 2013
Available online 9 October 2013
against rat striatal 5-HT
4 i
receptors (K of 0.20 and 0.07 nM, respectively). PET imaging in rhesus monkey
1
8
18
showed that the regional brain distribution of [ F]MNI-698 and [ F]MNI-699 were consistent with the
known densities of 5-HT in brain. [ F]MNI-698 and [ F]MNI-699 are among the first fluorine-18 radio-
4
1
8
18
Keywords:
PET imaging
tracers developed for imaging the 5-HT
tion in primates for future human use.
4
receptors in vivo and are currently under preclinical investiga-
5
-HT
4
Ó 2013 Elsevier Ltd. All rights reserved.
Serotonin receptors
Fluorine-18
Alzheimer’s disease
Serotonin (5-hydroxytryptamine, 5-HT) is an important neuro-
transmitter known to interact with different receptors and trans-
porters in the brain. Dysregulation of the serotonergic system has
targeting 5-HT
iological changes in 5-HT
psychiatric and neurodegenerative disorders.
4
receptors and also for investigations of pathophys-
receptor density in a variety of neuro-
4
been implicated in multiple neuropsychiatric and neurodegenera-
4
Among the compounds developed to target the 5-HT receptor,
1
tive disorders. The serotonin 4 (5-HT
4
) receptor is a member of the
benzodioxane derivatives, such as SB 204070 (1, Fig. 1), have been
1
1
7
-membrane spanning G-protein coupled receptors positively
shown to be potent and selective antagonists. The iodo analogue,
SB 204710 (2, Fig. 1), showed a high affinity for 5-HT receptors
linked to adenylate cyclase and has been detected in the brain of
mammalian species, including rodents, non-human primates and
4
and after labeling with iodine-123 for SPECT imaging was found
2
–4
humans.
In the central nervous system, 5-HT
4
receptors are pri-
to rapidly (maximum uptake less than 20 min, 2.3% injected dose)
accumulate in 5-HT
4
rich regions in non-human primate brain.1
2,13
marily localized in the limbic and nigrostriatal regions, which are
5
associated with learning and memory functions. Preclinical stud-
Other analogues of compound 1 have been labeled for PET imaging,
1
1
11
ies using animal models of Alzheimer’s disease (AD) have shown
[ C]SB 207145 ([ C]3, Scheme 1) has been evaluated in animal
1
4–16
11
that administration of 5-HT
4
agonists increased acetylcholine re-
and human.
207145 demonstrated its suitability for imaging 5-HT
in brain, we sought to develop tracers incorporating the longer
Although imaging data obtained using [ C]SB
lease, resulting in cognitive improvement on both short and
4
receptors
5
long-term memory. Moreover, post-mortem studies using brain
1
8
11
tissue from AD patients have shown a decrease in 5-HT
4
receptors
lived isotope fluorine-18 ( F t1/2 = 109.7 min; C t1/2 = 20.3 min)
density in these patients’ hippocampus compared with control
to be able to perform large scale imaging studies. Recently, Pike
6
–8
subjects.
neuropsychiatric disorders such as anxiety and depression.
Non-invasive imaging of 5-HT using either positron emission
The 5-HT
4
receptors have also been implicated in other
4
et al. reported the first fluorine-18 radiofluorinated 5-HT radio-
9
,10
17
tracer by radiofluoromethylation of an analogue of 1. However,
4
this radiotracer displayed a low binding affinity towards recombi-
tomography (PET) or single photon emission computed tomogra-
phy (SPECT) is extremely useful for studies evaluating new drugs
4 i
nant human 5-HT receptor (K = 17 nM) and was not further inves-
1
7
tigated. Herein we report the synthesis, in vitro binding affinity,
fluorine-18 labeling, and the first in vivo evaluation in non-human
1
8
18
primate of [ F]MNI-698 and [ F]MNI-699 (Scheme 2).
⇑
Corresponding author. Tel.: +1 203 401 4309; fax: +1 203 789 2119.
E-mail address: gtamagnan@mnimaging.com (G.D. Tamagnan).
Given that compounds 2 and 3 display a high affinity and selec-
tivity for 5-HT receptors, and were found to be suitable agents for
4
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.097

6
244
F. Caillé et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6243–6247
Figure 1. (Top) Representative PET SUV images (sum from 0 to 240 min pi) obtained following a single bolus intravenous injection of [18F]MNI-698 or [18F]MNI-699 in a
female rhesus monkey. (Bottom) SUV time-activity curves obtained following intravenous bolus injection of [18F]MNI-698 (A) and [ F]MNI-699 (B). Note the high uptake in
18
5
4 4
-HT rich regions, such as striatum, caudate, putamen, globus pallidus, hippocampus and substantia nigra, compared to lower uptake in regions with low levels of 5-HT .
imaging of those receptors in vivo, we chose fluorinated analogues
that were structurally similar to these molecules, whereby the
fluorine is incorporated at the terminal end of the N-alkyl chain
of the piperidine moiety. The synthesis of these compounds is
presented in Scheme 2, starting with benzodioxane derivative
reaction was slow and no more than 50% conversion was ob-
tained after 18 hours at reflux in acetonitrile with 1.5 equiv of
fluoroalkyl tosylate. Interestingly, the aniline moiety in position
8 did not need protection during the synthesis, in contrast to
1
0,17
previous reports.
Unfortunately, attempts to synthesize
1
8
4,
which was selectively chlorinated by treatment with N-chloro-
primary tosylate 8, which could be used as precursor for
1
8
succinimide in DMF to afford compound 5. Transesterification of
tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate with com-
pound 5 gave ester 6, which, after removal of the N-Boc group,
afforded the free amine 7. MNI-698 and MNI-699 were obtained
radiolabeling by nucleophilic substitution with [ F]fluoride,
were unsuccessful, owing to degradation during purification.
Therefore, a two-step radiolabeling procedure using amine 7
as precursor was tested.
1
9
Radiolabeled [18F]MNI-698 and [18F]MNI-699 were prepared
by N-alkylation with 2-fluoroethyl 4-methylbenzenesulfonate
20
and 2-fluoropropyl 4-methylbenzenesulfonate, respectively. This
using a GE TRACERlab™ FXF–N automated synthesizer, by
N11CH
3
N
N
O
O
O
O
O
O
O
O
O
O
O
O
Cl
123I
Cl
NH2
NH2
NH2
SB 204070 (1)
[123I]SB 207710 ([123I]2)
[11C]SB 207145 ([11C]3)
receptors.
Scheme 1. Structure of SB 204070 and analogous radiotracers for imaging of 5-HT
4

F. Caillé et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6243–6247
6245
NH
NBoc
O
O
O
O
CO Me
CO Me
2
2
O
O
O
O
O
O
a
b
c
O
Cl
O
Cl
Cl
NH2
NH2
NH2
NH
2
4
5
6
7
OTs
n
F
n
N
N
O
O
O
O
O
O
e
O
O
d
7
Cl
Cl
MNI-698 (n = 1)
MNI-699 (n = 2)
8
a (n = 1)
NH2
NH2
8
b (n = 2)
Scheme 2. Synthesis of MNI-698 and MNI-699. Reagents and conditions: (a) N-chlorosuccinimide, DMF, 70 °C; (b) tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate, n-
BuLi, THF, rt; (c) TFA, CH Cl , reflux.; (d) TsOCH (CH F, Cs CO , CH CN, reflux; (e) TsOCH (CH OTs, Cs CO , CH CN, reflux.
2
2
2
)
2 n
2
3
3
2
)
2 n
2
3
3
alkylation of amine precursor 7 with 2-[18F]fluoroethyl-4-methyl-
l
mol) for [18F]MNI-699. The logDoctanol/pH 7.4 buffer measured by
18
18
benzenesulfonate or 3-[ F]fluoropropyl-4-methylbenzenesulfo-
nate. The reaction was initially carried in a two step process,
the shake-flask method were 1.8 for [ F]MNI-698 and 1.4 for
1
8
[
F]MNI-699, within the range considered optimal for CNS pene-
18
24
with first synthesis, purification and isolation of the [ F]fluoroal-
kyl tosylate intermediates, followed by N-alkylation of precursor
tration. The main drawback of the synthetic process described
above is the low radiochemical yields, which may limit the
applicability of these tracers for routine use, but we are currently
investigating alternative production methods to resolve this
issue.
1
8
7
. Both [ F]fluoroalkyl tosylates were prepared by displacement
of one of the tosylate groups of ethylene or b-propylene glycol
p-ditosylate with dry [18F]fluoride in the presence of potassium
carbonate and Kyptofix-222 in acetonitrile at 90 °C for 10 min
Binding affinity of MNI-698 and MNI-699 for 5HT
was evaluated in vitro using rat striatal membranes in a competi-
tion binding assay with the known 5-HT antagonist
of 0.20 ± 0.05 nM (n = 4)
of 0.07 ± 0.01 nM (n = 2). For comparison, the
d
K of compound 1 in guinea pig distal colon was reported to be
4
receptors
(
Scheme 3). Purification was performed by reverse phase HPLC.
t
Isolation of the radiolabeled intermediates 9a–b by C18 solid
phase extraction (SPE) cartridge resulted in yields of 30–45%. The
SPE cartridge was then eluted into the reaction vessel with solvent
4
3
25
[ H]GR113808. MNI-698 displayed a K
and MNI-699 a K
i
i
(
K
DMF, DMSO, CH
CO , NaOH, Bu
3
CN, or tert-butanol) in presence of base (Cs
NOH) and reacted with intermediate 7 at concen-
2
CO
3
,
2
6
2
3
4
0.02 nM and compounds 2 and 3 were 0.08 nM (in piglet striatal
preparations)
respectively.
2
7
28
trations of 5–50 mM and temperatures between 90 and 160 °C.
Similar to what was observed for the synthesis of MNI-698 and
MNI-699, the alkylation was difficult and only the use of a
and 0.3 nM (in rat striatal membrane),
[
18F]MNI-698 and [18F]MNI-699 were evaluated in vivo, in
2
0 mM solution of compound 7 in DMF (0.6 mL) in the presence
of Cs CO at 130 °C for 30 min was successful to obtain the desired
labeled product, albeit in low yield ([ F]MNI-698: 1.0 ± 0.3%,
anesthetized adult female rhesus monkey (Macaca mulatta). The
radiotracers were injected intravenously as a bolus and images
were acquired over 4 h post-injection. Both radiotracers entered
the monkey brain, with a peak uptake of 6.6% of injected dose
(%ID) for [ F]MNI-698 and 5.6%ID for [ F]MNI-699 at around
20 min post injection (pi). The brain distribution of radioactivity
2
3
18
1
8
18
[
F]MNI-699: 1.7 ± 0.5%, non-decay corrected from [ F]fluoride).
21
18
18
Adding sodium iodide in DMSO in an attempt to catalyze the
reaction failed to yield any product. Alternative alkylating agents,
such as 1-[ F]fluoro-2-bromoethane and 2-[ F]fluoroethyl tri-
fluoromethanesulfonate, were also prepared and tested
failed to yield any of the desired product.
1
8
18
4
was consistent with 5-HT receptor density in monkey (Fig. 1),
2
2,23
but
where the highest uptake is found in the striatum, caudate, puta-
men, globus pallidus, hippocampus and substantia nigra, and mar-
ginal uptake is observed in the rest of the brain. The brain washout
We subsequently investigated the feasibility of a two-step one-
pot reaction and found that the [18F]fluoroalkyl tosylates 9a and
9
purification or isolation to afford [ F]MNI-698 or [ F]MNI-699
with yields similar to those obtained by isolating the intermedi-
ates. The alkyl ditosylates were radiofluorinated in acetonitrile
at 90 °C and, the solvent was evaporated, and a solution of precur-
of [ F]MNI-699 was slower than that of [ F]MNI-698. The stan-
dardized uptake values ratios (SUVr) were calculated using the cer-
18
18
b could be successfully used to alkylate compound 7 without
18
18
ebellum as reference region, (region with lowest 5-HT
expression). Stable SUVr values in target regions were obtained
4
1
8
at around 80 min pi for [ F]MNI-698 and at around 160 min pi
1
8
for [ F]MNI-699. The later pseudo-equilibrium obtained with
1
8
sor 7 in DMF with Cs
2
CO
3
was added to the same reaction vessel
[ F]MNI-699 correlates with the slower brain kinetics observed.
and heated to 130 °C for 30 min. The final products were purified
using a reverse-phased semi-preparative HPLC and formulated
into a physiological solution containing saline, ascorbic acid, Poly-
sorbate-80 and ethanol. The one-pot strategy reduced the total
production time from 120 to 80 min, resulting in yields (non-de-
High SUVr values (3–5) were obtained in target regions for both
radiotracers (striatum, caudate, putamen, globus pallidus > hippo-
campus > substantia nigra). These results compare favorably with
previously reported 5-HT
4
radioligands. The SPECT radiotracer
1
23
[
I]SB207710 presented a striatum-to-cerebellum ratio in non-
18
11
cay corrected from
[
F]fluoride) of 1.3 ± 0.6% (n = 18) for
human primates of around 4; human studies using [ C]SB
207145, reported a binding potential of 3.1 in the caudate
1
8
18
[
F]MNI-698 and 2.0 ± 0.5% (n = 3) for [ F]MNI-699. HPLC analy-
2
9
sis showed that the final radiotracer had a radiochemical purity
greater than 99% with a specific activity greater than 244 GBq/
nucleus.
The signal specificity was demonstrated in a pre-blocking study
using the selective 5-HT antagonist SB 204070. Administration of
lmol (6.6 Ci/
l
mol) for [18F]MNI-698 and 207 GBq/
lmol (5.6 Ci/
4

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F. Caillé et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6243–6247
1
8
F
N
n
O
O
a
b
O
O
OTs
n
OTs
TsO
18F
n
Cl
9
9
a (n = 1)
b (n = 2)
NH2
1
1
8
8
[
[
F]MNI-698 (n = 1)
F]MNI-699 (n = 2)
Scheme 3. Radiosynthesis of [18F]MNI-698 and [18F]MNI-699. Reagents and conditions: (a) [18F]fluoride, K
0 min.
2
3 3 2 3
CO , K 2.2.2, CH CN, 90 °C, 10 min; (b) 7, DMF, Cs CO , 130 °C,
3
Figure 2. (A) SUV images (summed 0–120 min pi) following intravenous bolus injection of [18F]MNI-698 at baseline and pre-blocking conditions using SB 204070 (0.5 mg/kg,
intravenous bolus 30 min before radiotracer injection). (B) SUVr curves obtained at baseline (left graph) and with pre-blocking (right graph). Note the reduction in SUVr
values measured in target regions to levels observed in non-target regions following pre-treatment with 0.5 mg/kg SB 204070.
0
.5 mg/kg resulted in around 93% receptor occupancy in target
experiments using a selective 5-HT
4
antagonist and [18F]MNI-698
regions (Fig. 2).
4
confirmed the radiotracer specific binding to 5-HT receptors in
In summary, two new fluorine-18 fluorinated radiotracers were
monkey brain.
synthesized and evaluated in vitro and in vivo as 5-HT
agents. MNI-698 and MNI-699 possess sub-nanomolar affinity for
-HT receptors, as determined by competition binding assays,
4
imaging
5
4
Supplementary data
and show appropriate lipophilicity for brain imaging. In vivo imag-
ing studies demonstrated that both radioligands successfully enter
the non-human primate brain and displayed a binding pattern con-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.09.
097.
4
sistent with the distribution of 5-HT receptors. Pre-blocking

F. Caillé et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6243–6247
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