11C-labeling and preliminary evaluation of vortioxetine as a PET radioligand

Article abstract of DOI:10.1016/j.bmcl.2014.04.044

Vortioxetine is a new multi-modal drug against major depressive disorder with high affinity for a range of different serotonergic targets in the CNS. We report the ¹¹C-labeling of vortioxetine with [¹¹C]MeI using a Suzuki-protocol that allows for the presence of an unprotected amine. Preliminary evaluation of [¹¹C]vortioxetine in a Danish Landrace pig showed rapid brain uptake and brain distribution in accordance with the pharmacological profile, all though an unexpected high binding in cerebellum was also observed. [¹¹C]vortioxetine displayed slow tracer kinetics with peak uptake after 60 min and with limited wash-out from the brain. Further studies are needed but this radioligand may prove to be a valuable tool in unraveling the clinical effects of vortioxetine.

Full text of DOI:10.1016/j.bmcl.2014.04.044

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2408–2411
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
11C-labeling and preliminary evaluation of vortioxetine as a PET
radioligand
Valdemar L. Andersen a,b, Hanne D. Hansen a, Matthias M. Herth a, Gitte M. Knudsen a,
Jesper L. Kristensen a,b,
⇑
a Center for Integrated Molecular Brain Imaging (CIMBI), Rigshospitalet and University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark
b Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Vortioxetine is a new multi-modal drug against major depressive disorder with high affinity for a range of
different serotonergic targets in the CNS. We report the 11C-labeling of vortioxetine with [11C]MeI using a
Suzuki-protocol that allows for the presence of an unprotected amine. Preliminary evaluation of
Received 13 March 2014
Revised 9 April 2014
Accepted 10 April 2014
Available online 19 April 2014
[
11C]vortioxetine in a Danish Landrace pig showed rapid brain uptake and brain distribution in accor-
dance with the pharmacological profile, all though an unexpected high binding in cerebellum was also
observed. [11C]vortioxetine displayed slow tracer kinetics with peak uptake after 60 min and with limited
wash-out from the brain. Further studies are needed but this radioligand may prove to be a valuable tool
in unraveling the clinical effects of vortioxetine.
Keywords:
Cross-coupling
Vortioxetine
PET
Ó 2014 Elsevier Ltd. All rights reserved.
Carbon-11
Palladium
Major depressive disorder (MDD) is a prevalent disease that is
considered by the WHO to be one of the leading causes of disability
worldwide.1 MDD has traditionally been treated with tricyclic
anti-depressants (TCAs) monoamine oxidase inhibitors (MAOIs),
selective serotonin reuptake inhibitors (SSRIs) and serotonin–nor-
epinephrine reuptake inhibitors (SNRI’s).2
version of 1 for PET studies. Access to such a radiotracer would
open the possibility for conducting studies in different patient
groups who respond differently to treatment, in analogy to previ-
ous studies with [11C]Clozapine.12 Based on the in vitro receptor
affinities reported for 1 one would expect to see binding in thala-
mus,7,8 cortex,9 hippocampus9,10 and striatum.8,11
Recently, a new drug for the treatment of MDD made its way to
the market. Vortioxetine (1) is a multi-modal acting drug with high
affinity for a range of serotonergic targets, see Figure 1.3 The anti-
depressant effects of 1 are thought to be mediated through three
serotonergic targets: (1) inhibition of the 5-HTT which leads to
an increase in extracellular 5-HT levels in the brain (in analogy
to previous antidepressants); (2) agonism of 5-HT1AR, which is
believed to shorten the time to onset of clinical effects,4 and (3)
antagonism of 5-HT3R. Preclinical studies indicate that antagonism
of 5-HT3R (among many other effects) could have positive effects
on mood and cognitive dysfunction in patients with depression.5
The recommended starting dose of 1 is 10 mg per day, and PET
studies in humans have shown that the occupancy at the 5-HTT is
ꢀ50% at 5 mg whereas the 5-HT1AR occupancy as measured in a
small sample was not measurable.6 The occupancy at other 5-HTRs
is currently unknown and the exact mode of action of 1 remains to
be fully elucidated. Here, we set out to develop a 11C-labeled
The commonly employed 11C-labeling strategy of O-, N- or
S-methylation is not an option for labeling vortioxetine, whereas
the aromatic methyl-groups are possible sites for labeling via a
Suzuki-coupling with a suitable boronic acid derivate.13 In theory,
two different labeling sites are possible: the ortho- and the para-
position. We chose to focus on the 2-position, as the required pre-
cursor would be readily available.
As 1 contains a secondary amine, one could envisage two label-
ing strategies, see Scheme 1: in route A, a precursor with a suitable
protecting group (Pg) on the secondary amine is first 11C-labeled
and the Pg is subsequently removed. This would appear to be the
safe option as labeling of the precursor using route B (in which
the two steps are reversed) in principle could lead to labeling at
the secondary nitrogen instead. The disadvantage with route A is
that it requires a subsequent deprotection step after the 11C-label-
ing, with concurrent loss of radiochemical yield, whereas Route B
gives [11C]1 directly. We have previously developed procedures
for the Pd-mediated 11C-labeling of aryl boronic derivatives con-
taining unprotected amines14 and decided to try and developed a
radio synthesis based on route B.
⇑
Corresponding author. Tel.: +45 35336487; fax: +45 35306040.
E-mail address: jesper.kristensen@sund.ku.dk (J.L. Kristensen).
http://dx.doi.org/10.1016/j.bmcl.2014.04.044
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

V. L. Andersen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2408–2411
2409
H
N
Boc
N
Boc
N
Target
Mode of action
Affinity (Ki, nM)
5-HTT
5-HT1A
5-HT1B
5-HT3A
5-HT7
inhibitor
agonist
partial agonist
antagonist
antagonist
1.6
15
33
3.7
19
N
Br
N
Br
N
a)
S
SH I
+
S
81 %
2
3
4
Vortioxetine (1)
b,c)
32 %
Figure 1. Structure and selected pharmacological profile of vortioxetine (1) at
various human 5-HT receptors.3
H
N
H
N
O
O
11CH3
N
B
N
The required precursor was synthesized as outlined in Scheme 2.
Starting from 2-bromo-4-methylbenzenethiol (2) and tert-butyl 4-
(2-iodophenyl)piperazine-1-carboxylate (3) the synthesis of the
required precursor was carried out in a three step sequence start-
ing with a Palladium catalysed thioether formation via coupling of
2 and 3 to give 4 in 81% isolated yield.15 The use of dry, degassed
toluene and microwave irradiation at 100 °C proved essential for
an efficient coupling; otherwise debrominated 4 was the main
product. Installment of a boronic ester moiety via a Pd-catalyzed
coupling with bis(pinacolato)diboron and Pd(dppf)Cl2 followed
by liberation of the secondary amine via Boc-deprotection pro-
vided the desired precursor 5 in 32% yield.16
d)
S
S
346 MBq
11C]1
5
[
Scheme 2. Synthesis of precursor and 11C-labeling of vortioxetine (1). Reagents and
conditions: (a) Pd2dba3, DPEphos, t-BuOK, toluene, 100 °C (MW), 30 min; (b)
bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 100 °C, 12 h; (c) TFA,
CH2Cl2; (d) [11C]CH3I, Pd2dba3, P(o-tolyl)3, K2CO3, 60 °C DMF/H2O 9:1, 5 min.
([3H]-(S)-Zacopride).20 Areas with high density of 5-HT1AR include
the cortex and the hippocampus. 5-HT7R density is high in the
thalamus and in the cortex, but based on the affinity of 1 for these
two targets it is most likely that the uptake of [11C]1 in the cortex
arises from [11C]1 binding to the 5-HT1AR.21
The high uptake in the striatum is likely due to the high density
of 5-HTT and possibly also due to binding to the 5-HT1BR. The area
with the largest 5-HT3R density in the pig brain is the spinal cord
(substantia gelatinosa), however this region is too small to be iden-
tified on the PET image and the region is not available in the pig
atlas. It is therefore difficult to analyze whether binding to the 5-
HT3R contributes to the PET signal.
We previously showed that by trapping [11C]CH3I as a [11C]
CH3–PdLn-I complex, it is possible to subsequently label aryl boro-
nic derivatives that contain free amines.14 Using this method 5 was
converted to [11C]1 in 40% radiochemical yield. Following prepara-
tive HPLC-purification it was possible to isolate 346 MBq [11C]1 in
>98% radiochemical purity and a specific activity of 478 GBq/
l
mol.17 Yields were found to be extremely dependent on the qual-
ity of the palladium/ligand catalyst, as N-methylation rather than
cross-coupling occurred in several instances, see Figure 2. Thus, a
freshly prepared catalyst mixture was found to be essential for suc-
cessful synthesis of [11C]1.
[
11C]1 was injected into a Danish Landrace pig (Fig. 3).18 A swift
The uniform uptake of [11C]1 could also indicate that the com-
pound is very lipophilic and therefore has a large fraction of non-
specific binding in the brain. The radio ligand displayed slow tracer
kinetics with peak uptake after 60 min and with limited wash-out
from the brain within the data acquisition time. This complicates
the use of compartment models when performing kinetic modeling
for quantification of binding, however in humans it may be possible
to use reference tissue models although this needs to be validated.
brain uptake was observed and, as expected from the pharmaco-
logical profile, with high binding in cortex, thalamus, hippocam-
pus, striatum. High binding in cerebellum was also observed
which is unexpected as none of the identified targets are reported
as having high densities in this part of the brain. Target densities
have been determined in the pig brain with autoradiography for
the 5-HTT ([3H]escitalopram),19 the 5-HT1A
R
([3H]WAY-
100635),19 the 5-HT7R ([3H]SB-269970),14b and for the 5-HT3R
Pg
N
Pg
N
O
O
Route A
1) 11C-labeling
B
N
11CH3
N
S
S
Route B
1) Deprotection
Route A
2) Deprotection
H
N
H
N
Route B
2) 11C-labeling
O
O
B
N
11CH3
N
S
S
Figure 2. Effect of the state of the palladium–ligand combination on the amount of
N-methylation. (A) Reference spectrum of 1 (with UV-detection). (B) Successful 11C-
labeling of 1. (C) Unsuccessful 11C-labeling of 1—product with retention time ꢀ4
min presumably the N-methylated precursor. (D) No Pd catalyst added—product
with retention time ꢀ4 min presumably the N-methylated precursor.
Competing
N-alkylation?
Scheme 1. Possible radiolabeling approaches to 11C-labeling of vortioxetine.

2410
V. L. Andersen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2408–2411
14. (a) Andersen, V. L.; Herth, M. M.; Lehel, S.; Knudsen, G. M.; Kristensen, J. L.
Tetrahedron Lett. 2013, 54, 213; (b) Hansen, H. D.; Herth, M. M.; Ettrup, A.;
Andersen, V. L.; Lehel, S.; Dyssegaard, A.; Kristensen, J. L.; Knudsen, G. M. J. Nucl.
Med. 2014, 55, 640.
15. Experimental section—synthesis of precursor: tert-butyl 4-(2-((2-bromo-4-
methylphenyl)thio)phenyl)piperazine-1-carboxylate (4): tert-BuOK (311 mg,
2.78 mmol), Pd2dba3 (57.8 mg, 0.063 mmol), DPEphos (136 mg, 0.252 mmol),
2-bromo-4-methylbenzenethiol (512 mg, 2.52 mmol) and tert-butyl 4-(2-
iodophenyl)piperazine-1-carboxylate (980 mg, 2.52 mmol) was dissolved in
dry toluene (4 mL), kept under nitrogen and degassed using a stream of
nitrogen for 10 min. The mixture was then heated to 100 °C (MW). The
resulting crude was purified by dry column vacuum chromatography using
heptane to heptane/EtOAc 10:1 (rf = 0.48 heptane/EtOAc 3:1) yielding 950 mg
of 4 as a slightly yellow oil (81%). 1H NMR (CDCl3, 400 MHz) d: 1.49 (s, 9H) 2.35
(s, 3H) 2.97–3.03 (m, 4 H) 3.50–3.55 (m, 4 H) 6.85 (dd, J = 7.78, 1.51 Hz, 1H)
6.95–6.99 (m, 1H) 7.04–7.08 (m, 2H) 7.17–7.23 (m, 2H) 7.51 (d, J = 1.00 Hz,
1H). 13C NMR (CDCl3, 400 MHz) d: 20.8, 28.5, 51.6, 79.7, 120.4, 124.6, 127.3,
127.7, 129.0, 129.6, 131.7, 132.1, 134.0, 134.2, 139.6, 150.4, 154.9. LC–MS
(m+1): 464 m/z.
16. 1-(2-((4-Methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)thio)phenyl)piperazine (5): Pd(dppf)Cl2 (13.3 mg, 0.018 mmol),
bis(pinacolato)diboron (169 mg, 0.665 mmol), KOAc (178 mg, 1.81 mmol)
and
4 (280 mg, 0.604 mmol) was dissolved in dry 1,4-dioxane (10 mL),
degassed with nitrogen for 10 min and heated to 100 °C for 18 h. The
resulting crude was purified by dry column vacuum chromatography using
heptane to heptane/EtOAc 10:1 (rf = 0.55 heptane/EtOAc 3:1). Yielding 124 mg
of Boc-protected 5 as a slightly yellow oil (40%). 1H NMR (CDCl3, 400 MHz) d:
1.15 (s, 12H) 1.50 (s, 9H) 2.38 (s, 3H) 3.05 (br s, 4 H) 3.58 (br s, 4H) 6.74 (d,
J = 7.83 Hz, 1H) 6.88 (t, J = 7.46 Hz, 1H) 7.00 (m, 1H) 7.06 (m, 1H) 7.22 (d,
J = 7.82 Hz, 1H) 7.35 (d, J = 7.83 Hz, 1H) 7.63 (s, 1H). 13C NMR (CDCl3, 400 MHz)
d: 21.1, 24.6, 28.5 51.4, 79.6, 83.8, 119.5, 124.5, 125.6, 127.2, 132.3, 134.6,
134.8, 136.4, 137.1, 137.4, 155.0. LC–MS (m+1): 511 m/z. This material was
Boc-deprotected in the following way: (90 mg, 0,176 mmol) was dissolved in
CH2Cl2 (10 mL) and TFA (0.5 mL) was added and the mixture was stirred for
30 min. The resulting mixture was then washed with saturated aqueous
NaHCO3 (3 Â 10 mL) and the organic phase was evaporated to dryness yielding
57 mg of 5 as a slightly yellow oil, 0,14 mmol, 79%) 1H NMR (CDCl3, 400 MHz)
d: 1.13 (s, 10H) 2.38 (s, 3H) 3.37 (d, J = 12.23 Hz, 8H) 6.77 (d, J = 7.58 Hz, 1H)
6.93 (s, 1H) 7.02–7.05 (m, 1H) 7.09 (d, J = 7.09 Hz, 1H) 7.23 (d, J = 6.36 Hz, 1H)
7.34 (d, J = 7.82 Hz, 1H) 7.66 (s, 1H). 13C NMR (CDCl3, 400 MHz) d: 21.0, 24.5,
44.2, 48.3, 83.9, 119.8, 125.5, 125.9, 127.4, 132.5, 134.1, 134.7, 136.5, 137.6,
147.2. LC-MS (m+1): 411 m/z.
Figure 3. Top: Coronal, sagittal and transverse (left to right) summed PET images
(0–90 min, 3 mm Gaussian filtering) of [11C]1 in the pig brain. Bottom: Time–
activity curves showing absolute radioligand uptake for the indicated brain regions.
SUV: standardized uptake value.
The protein binding of [11C]1 in pig plasma was determined to 98%
measured after 2.5 h dialysis.22 This level of protein binding is sim-
ilar to that found in humans.3
In conclusion, vortioxetine was 11C-labeled using the Suzuki
reaction and its kinetics was evaluated in the pig brain. The tracer
crossed the blood-brain barrier readily and binding to multiple
sites within the brain was observed. Further investigations are nec-
essary, but [11C]vortioxetine could prove to be an important tool
making it is possible to follow the pharmacokinetics of an antide-
pressant with a new mechanism of action and thus aid uncovering
of the underlying mechanism for 1’s effect on MDD.
17. 11C-labeling of 1: 5, Pd2(dba)3, P(o-tolyl)3 and base were used in a ratio of
40:1:2:4 with appropriate masses calculated from the use of 0.1 mg of P(o-
tolyl)3. [11C]MeI was trapped in 300 L DMF. To the trapped [11C]MeI was added
l
Pd-catalyst and K2CO3 dissolved in 300
60 °C. [11C]MeI was allowed to react for 2 min. 5 was added dissolved in 150
of DMF/water 9:1 and allowed to react for 5 min. Purification was performed by
preparative HPLC using a Luna 5 m C18(2) 100 Å column (Phenomenex Inc.)
lL of DMF/water 9:1 and heated to
lL
l
(250 Â 10 mm, 50:50 citrate buffer pH 4.62: AcN, flowrate: 9 mL/min). The
collected fraction was trapped on a solid-phase C18 sep-pack extraction column
and eluted with 3 ml EtOH. Results were analyzed by HPLC using a Luna 5 lm
Acknowledgments
C18 100 Å column (Phenomenex Inc.) (150 Â 4.6 mm. 50:50 citrate buffer pH
4.62: AcN, flowrate 2 mL/min). Starting activities around 100 GBq EOB as
The Lundbeck Foundation and the University of Copenhagen is
gratefully acknowledged for financial support.
[
11C]CH4. Yields as determined by HPLC: 40%. Isolated yields: 161.4–346 MBq.
Specific activities: 8–478 GBq/lmol. Total synthesis time: 55 min.
18. PET data acquisition: A female Danish Landrace pig (18 kg) was tranquilized by
intramuscular (im) injection of 0.5 mg/kg midazolam. Anaesthesia was
induced by im injection of a Zoletil veterinary mixture (1.25 mg/kg tiletamin,
1.25 mg/kg zolazepam, and 0.5 mg/kg midazolam; Virbac Animal Health,
France). Following induction, anaesthesia was maintained by intravenous (iv)
infusion of 15 mg/kg/h propofol (B. Braun Melsugen AG). During anaesthesia
the pig was endotracheally intubated and ventilated (volume 200 mL,
frequency 16 per min). Venous access was granted through two catheters in
the peripheral milk veins. The Danish Council for Animal Ethics approved the
animal procedures (journal no. 2012-15-2934-00156). [11C]1 was given as an
intravenously (iv) bolus injection and the injected dose was 124 MBq (n = 1).
References and notes
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in the 90-min scan was reconstructed and used for co-registration to
a
standardized MRI-based atlas of the Danish Landrace pig brain. The time–
activity curve was calculated for the following volumes of interest (VOIs):
cerebellum, cortex, hippocampus, lateral and medial thalamus, caudate
nucleus, and putamen. Striatum is defined as the mean radioactivity in
caudate nucleus and putamen. The radioactivity in thalamus is calculated as
the mean radioactivity in the lateral and medial thalamus. Radioactivity in all
VOIs was calculated as the average of radioactive concentration (Bq/mL) in the
left and right sides. Outcome measure in the time–activity curves (TACs) was
calculated as radioactive concentration in VOI (in kBq/mL) normalized to the
injected dose corrected for animal weight (in kBq/kg), yielding standardized
uptake values (g/mL).
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V. L. Andersen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2408–2411
19. Ettrup, A.; Kornum, B. R.; Weikop, P.; Knudsen, G. M. Synapse 2011, 65, 136.
2411
L)
(ꢀ5 MBq) were added to 5 mL plasma sample from the pig. Plasma (500
l
20. Fletcher, S.; Barnes, N. M. Neurosci. Lett. 1999, 269, 91.
was then dialysed at 37 °C against an equal volume of buffer (135 mM NaCl,
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3.0 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, and 2.0 mM KH2PO4, pH 7.4). Counts
per minute in 400 lL of plasma and buffer were determined in a well counter
22. Plasma protein binding assay: The free fraction of [11C]1 in plasma, fp, was
estimated using an equilibrium dialysis chamber method. The dialysis was
conducted in chambers (Harvard Biosciences) separated by cellulose
after various dialysis times (0.5, 1, 2, and 2.5 h), and fp of [11C]1 was calculated
as the ratio of radioactivity in buffer and plasma. Equilibrium in fp was reached
after 1 h of dialysis.
membrane with
a protein cut-off of 10,000 Da. Small amounts of [
11C]1